Investigation of the Effect on the Albumin Binding Moiety for the Pharmacokinetic Properties of 68Ga-, 205/206Bi-, and 177Lu-Labeled NAPamide-Based Radiopharmaceuticals

Although radiolabeled alpha-melanocyte stimulating hormone-analogue NAPamide derivatives are valuable melanoma-specific diagnostic probes, their rapid elimination kinetics and high renal uptake may preclude them from being used in clinical settings. We aimed at improving the pharmacokinetics of radiolabeled DOTA-NAPamide compounds by incorporating a 4-(p-iodo-phenyl)-butanoic acid (IPB) into the molecules. Followed by 68Ga-, 205/206Bi-, and 177Lu-labelling, the radiopharmaceuticals ([68Ga]Ga-DOTA-IPB-NAPamide, [205/206Bi]Bi-DOTA-IPB-NAPamide, [177Lu]Lu-DOTA-IPB-NAPamide) were characterized in vitro. To test the imaging behavior of the IPB-containing probes, B16F10 tumor-bearing C57BL/6 mice were subjected to in vivo microPET/microSPECT/CT imaging and ex vivo biodistribution studies. All tracers were stable in vitro, with radiochemical purity exceeding 98%. The use of albumin-binding moiety lengthened the in vivo biological half-life of the IPB-carrying radiopharmaceuticals, resulting in elevated tumor accumulation. Both [68Ga]Ga-DOTA-IPB-NAPamide (5.06 ± 1.08 %ID/g) and [205/206Bi]Bi-DOTA-IPB-NAPamide (4.50 ± 0.98 %ID/g) exhibited higher B16F10 tumor concentrations than their matches without the albumin-binding residue ([68Ga]Ga-DOTA-NAPamide and [205/206Bi]Bi-DOTA-NAPamide: 1.18 ± 0.27 %ID/g and 3.14 ± 0.32; respectively), however; the large amounts of off-target radioactivity do not confirm the benefits of half-life extension for short-lived isotopes. Enhanced [177Lu]Lu-DOTA-IPB-NAPamide tumor uptake even 24 h post-injection proved the advantage of IPB-based prolonged circulation time regarding long-lived radionuclides, although the significant background noise must be addressed in this case as well.


Introduction
Malignant melanoma (MM) derived from melanocytes is the fifth most common cancer among adults in the United States.In addition, being a highly aggressive neoplasm, MM is responsible for 75% of deaths caused by skin cancer [1].Therefore, the development of effective melanoma specific diagnostic and therapeutic vectors is of paramount importance to increase the survival rate of patients.Besides providing an essential contribution to the growth of melanoma cells, the melanocortin type 1 receptor (MC1-R)-also named melanocytestimulating hormone receptor (MSHR)-is abundantly expressed on the surface of melanin positive primary MM and related metastatic lesions [2].Consequently, MC1-R seems to be a useful target for selective melanoma imaging and therapy.Beyond being stimulated by α-Melanocyte-stimulating hormone (α-MSH), the MC1-R receptor can also exert its effects via activation by the specific binding of various α-MSH analogues [3].Previous studies proved that radiolabeled α-MSH analogue NAPamide (Ac-Nle-Asp-His-D-Phe-Arg-Trp-Gly-Lys) derivatives serve as effective molecular probes for the determination of the MC1-R expression pattern of the melanoma cells [4,5].Although the existing radiolabeled NAPamide analogues are characterized by relatively high tumor uptake, their rapid elimination kinetics along with the high renal uptake must be addressed [6].
As peptides exert meaningful binding affinity and specificity to various tumor biomarkers, coupling them with radioisotopes to transport cytotoxic radiation to tumor cells appears to be a valuable strategy for both imaging and therapeutic purposes [7].From a clinical point of view, the labelling of the chelator-target molecule conjugate with both diagnostic and therapeutic radionuclides has its clear advantages.For this purpose, the application of chelator DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) is appropriate because it forms a stable complex with different diagnostic and therapeutic radiometals including gallium-68 ( 68 Ga), lutetium-177 ( 177 Lu) and bismuth-213 ( 213 Bi).
The short biological half-life, rapid clearance and related lower tumor uptake and retention, as well as the high renal uptake of peptide-based radiocomplexes, constitute major drawbacks for both PET imaging and therapeutic applications [8].To overcome these shortcomings, the optimization of the structure of the radioligands is often required for their clinical usage regarding the linker unit between the chelator and vector molecules, which affects the pharmacokinetic properties of the labeled compounds.Therefore, the modification of the linker may improve the diagnostic and the therapeutic potential of the radiopharmaceutical.The introduction of an albumin-binding moiety is an increasingly investigated method for the prolongation of the blood circulation time of the radiolabeled peptides, and subsequent increase in tumor accumulation and a decrease in renal uptake.In addition, using an albumin-binding unit containing radiopharmaceuticals, a smaller amount of radioactivity is sufficient for successful therapy compared to their unmodified counterparts.Hence, the presence of the albumin-binding motif has a meaningful effect on the tumor-targeting capability and the pharmacokinetic profile of the radioligands, that are crucial for the achievement of improved image quality and effective targeted radiation therapy as well.In addition, some studies revealed the albumin-binding ability of the 4-(p-iodophenyl) butyric acid-discovered from a DNA-encoded chemical library-and its successful use for the lengthening of the circulation time of folate-and prostate specific membrane antigen (PSMA)-based radiopharmaceuticals [9][10][11].
During targeted radionuclide therapy (TRNT), it is of utmost importance that the cytotoxic radiation reaches the cancer cells as specifically as possible without exerting adverse toxic effects on the surrounding healthy tissues.Thus, to achieve successful TRNT, data derived from biodistribution studies could be used for the determination of the localization and the retention of the radiopharmaceutical in the targeted organs and tissues.Although β-particle emitting radionuclides-for example, 177 Lu isotope-are applied most often in TRNT settings, their low linear energy transfer (LET 0.2 keV/µm) value as well as long path in tissue (1-5 mm) lead to the destruction of healthy cells and hamper their widespread clinical usage [12].Given the short-range (50-100 µm) and high LET (50-230 keV/µm) of α-emitting radionuclides such as 213 Bi (t 1/2 = 46 min, alpha energy of 8.4 MeV, tissue penetration: 85 µm) that ensure a selective tumor cell killing effect sparing the nearby intact tissues, targeted alpha therapy (TAT) has become the focus of increasing scientific and clinical interest [13].
Based on the considerations mentioned above, we designed the synthesis and performed the preclinical evaluation of 68 Ga-, 177 Lu-and 213 Bi-labeled, DOTA-conjugated NAPamide radioligands containing 4-(p-iodophenyl)butyryl group (IPB) as an albumin binding moiety in order to investigate their diagnostic and therapeutic feasibility.

Results and Discussion
As for metal-based tracers, the radiometal and the targeting vector are combined with the chelating agent and the linker in an inert and stable complex.The usefulness of the integration of an albumin-binding unit in the reduction of the rapid clearance of 177 Lulabeled radiofolate was first realized by Müller et al. [9] who used 4-(p-iodophenyl)butyryl moiety (IPB) as an albumin-binder.Later, this method was applied to reduce the renal and the hepatic accumulation of some PSMA-based radiopharmaceuticals using different albumin binding moieties [10,11].Currently, we aimed at incorporating the IPB motif into MM targeting and radiolabeled the DOTA-NAPamide compound to reduce its rapid elimination and high renal uptake.

Radiochemistry
Based on their decay properties, radiometals can be used for both diagnostic and therapeutic purposes.The radiation emitted by diagnostic radiometals interacts weakly with the examined tissues, and is able to reach the external detector.On the contrary, however, radioisotopes with therapeutic rays emit locally cytotoxic radiation. 68Ga-labeled peptides seem to be strong applicants for the PET-based detection of different types of tumors providing an auspicious opportunity to plan an effective therapeutic strategy and to monitor the therapeutic response.Therefore, we first performed the radiolabeling of DOTA-IPB-NAPamide (10) using diagnostic 68 Ga (t 1/2 = 68 min, I = 89%, E max (β + ) = 1.92 MeV) that was produced in a cyclotron via 68 Zn(p,n) 68 Ga nuclear reaction with a high labeling yield (>98%).For the accomplishment of preliminary preclinical studies, compound 10 was labeled with 205/206 Bi isotope-a surrogate of alpha-emitting 213 Bi isotope.Identically to the incorporation of [ 68 Ga]Ga 3+ , high labeling yield (>98%) was experienced in case of the 205/206 Bi isotopes.Finally, the radiolabeling of precursor 10 was carried out with beta-emitting, long-lived 177 Lu isotope (t 1/2 = 6.647 days).Furthermore, we conducted the radiolabeling of the commercially available DOTA-NAPamide with 68 Ga, 205/206 Bi and 177 Lu radiometals and used these radiopharmaceuticals as references.The labeled complexes were purified by solid-phase extraction on a Sep-Pak C18 Plus Light column.The 68 Ga-labeled radiopharmaceuticals were analyzed using radio-HPLC (Figure 1), while radio instant thin-layer chromatography (iTLC) was applied for the characterization of the 205/206 Bi-and 177 Lu-labeled complexes (Figure 2).In all cases, the radiochemical purity of the radiotracers exceeded 98%.

Radiochemistry
Based on their decay properties, radiometals can be used for both diagnostic and therapeutic purposes.The radiation emitted by diagnostic radiometals interacts weakly with the examined tissues, and is able to reach the external detector.On the contrary, however, radioisotopes with therapeutic rays emit locally cytotoxic radiation. 68Ga-labeled peptides seem to be strong applicants for the PET-based detection of different types of tumors providing an auspicious opportunity to plan an effective therapeutic strategy and to monitor the therapeutic response.Therefore, we first performed the radiolabeling of DOTA-IPB-NAPamide (10) using diagnostic 68 Ga (t1/2 = 68 min, I = 89%, Emax(β + ) = 1.92 MeV) that was produced in a cyclotron via 68 Zn(p,n) 68 Ga nuclear reaction with a high labeling yield (>98%).For the accomplishment of preliminary preclinical studies, compound 10 was labeled with 205/206 Bi isotope-a surrogate of alpha-emitting 213 Bi isotope.Identically to the incorporation of [ 68 Ga]Ga 3+ , high labeling yield (98%) was experienced in case of the 205/206 Bi isotopes.Finally, the radiolabeling of precursor 10 was carried out with beta-emitting, long-lived 177 Lu isotope (t1/2 = 6.647 days).Furthermore, we conducted the radiolabeling of the commercially available DOTA-NAPamide with 68 Ga, 205/206 Bi and 177 Lu radiometals and used these radiopharmaceuticals as references.The labeled complexes were purified by solid-phase extraction on a Sep-Pak C18 Plus Light column.The 68 Ga-labeled radiopharmaceuticals were analyzed using radio-HPLC (Figure 1), Scheme 1. Synthesis of DOTA-IPB-NAPamide (10).while radio instant thin-layer chromatography (iTLC) was applied for the characterization of the 205/206 Bi-and 177 Lu-labeled complexes (Figure 2).In all cases, the radiochemical purity of the radiotracers exceeded 98%.The stability test of the purified labeled derivatives was performed in the presence of rat serum, 0.01 mM EDTA solution and metal ions (Mg 2+ : 0.51 mM, Ca 2+ : 1.14 mM, Zn 2+ : 0.01 mM, Cu 2+ : 0.001 mM) separately.After a 2 h incubation, the radiocomplexes showed high stability in the serum and inertness towards transchelation and transmethylation under the examined conditions.The stability of the 177   The stability test of the purified labeled derivatives was performed in the presence of rat serum, 0.01 mM EDTA solution and metal ions (Mg 2+ : 0.51 mM, Ca 2+ : 1.14 mM, Zn 2+ : 0.01 mM, Cu 2+ : 0.001 mM) separately.After a 2 h incubation, the radiocomplexes showed high stability in the serum and inertness towards transchelation and transmethylation under the examined conditions.The stability of the 177   To assess the tumor targeting potential and the organ distribution of [ 68 Ga]Ga-DOTA-NAPamide and [ 68 Ga]Ga-DOTA-IPB-NAPamide, MC1-R overexpressing B16F10 melanoma tumor-bearing mice were subjected to miniPET examination and post imaging uptake studies.It is well established from the literature that the B16F10 tumor of mouse origin is a competent model for the research and the development of radiopharmaceuticals targeting malignant melanoma due to the fact that it exhibits high MC1-R expression with more than 20,000 MC1-R per melanoma cell [14].
Upon qualitative PET image assessment, the sc.growing tumors were unequivocally identifiable using both MC1-R specific radiopharmaceuticals; however, higher backgroundactivity was observed for [ 68 Ga]Ga-DOTA-IPB-NAPamide (as displayed in Figure 3).The sufficient tumor accumulation of the 68 Ga-labeled probes strengthened their MC1-R binding ability as well as their tumor-homing potential.This was consistent with the findings of Nagy et al., who tested the MC1-R selectivity of DOTA chelated NAPamide molecules labeled with 68 Ga and 44 Sc in receptor positive B16F10 cells and A375 cell lines with low receptor expression, and pointed out that the tracer accumulation was notably higher in the B16F10 cells with high receptor density relative to the A375 cells without significant MC1-R presence (p < 0.01) [15].Corresponding to our results, Cheng et al. and Tafreshi et al. also registered increased uptake of radiolabeled α-MSH analogues in B16F10 mouse melanoma cells [14,16,17].Based on the evaluation of the decay-corrected PET scans, a notable difference was encountered between the two 68 Ga-labeled probes with respective SUV mean values being 0.43 ± 0.09 and 1.31 ± 0.24 for [ 68 Ga]Ga-DOTA-NAPamide and [ 68 Ga]Ga-DOTA-IPB-NAPamide (p ≤ 0.05).This is in line with previous literature data, according to which using different albumin-binding unit such as 4-(p-iodo-phenyl)-butanoic acid moiety (IPB) to lengthen the half-life of small, tumor-specific molecules (for example PSMA) results in elevated uptake in malignant lesions [18,19].
In agreement with the in vivo data, the increased circulation time was also reflected in the results of the post imaging biodistribution studies.Using [ 68 Ga]Ga-DOTA-IPB-NAPamide, notably higher radiotracer accretion was registered in most of the selected organs and tissues compared with that of [ 68 Ga]Ga-DOTA-NAPamide lacking in the albuminbinding motif (p ≤ 0.01; seen in Table 1).Besides the presence of the albumin-binding unit, however, the amount of the injected peptide and related saturation of the receptor binding sites as well as the distinct activity of the radiopeptide could also exert a meaningful impact on the uptake pattern [20,21].The prolonged circulation time of [ 68 Ga]Ga-DOTA-IPB-NAPamide was further validated by its prominently high activity in the blood (%ID/g: 14.21 ± 1.87 at 90 min post injection).In addition, the currently presented ex vivo %ID/g data as well as the in vivo SUV mean values of [ 68 Ga]Ga-DOTA-NAPamide correlated well with previous studies, where the distribution and the tumor homing properties of receptoraffine [ 68 Ga]Ga-DOTA-NAPamide and [ 44 Sc]Sc-DOTA-NAPamide probes were assessed in B16F10 experimental tumor model, and identical organ distribution pattern and tumor uptake were observed [15].Upon the evaluation of the uptake pattern of [ 68 Ga]Ga-DOTA-NAPamide-except for the kidneys-faint radioactivity was detected in the investigated abdominal and thoracic organs.Consistent results were reported in earlier works with 111 In-, 67 Ga-and 68 Galabeled DOTA-NAPamide compounds that also demonstrated negligible abdominal and thoracic activity [15,20,22,23].On the contrary, however, Cheng et al. found considerable uptake of 18 F-and 64 Cu-labeled NAPamide in the majority of the abdominal and thoracic organs including the lungs, the heart, the stomach, the intestines, the spleen, the pancreas as well as the liver [14,16].For the assessment of the MC1-R specificity and the tissue uptake pattern of [ 205/206 Bi]Bi-DOTA-NAPamide and [ 205/206 Bi]Bi-DOTA-IPB-NAPamide, ex vivo biodistribution studies were performed applying C57BL/6 male mice bearing B16F10 tumors.The %ID/g data acquired from the 205/206 Bi-based experiments were in strong correlation with the uptake values of the 68 Galabeled PET diagnostic probes (demonstrated in Table 2).Comparing the two 205/206 Bi-labeled tracers, the radiopharmaceutical concentration of the evaluated organs and tissues was markedly higher (p ≤ 0.05 and p ≤ 0.01) upon [ 205/206 Bi]Bi-DOTA-IPB-NAPamide administration than in the case of [ 205/206 Bi]Bi-DOTA-NAPamide. Due to its lipophilic property-that was in line with the LogP value-the albumin moiety containing compound displayed higher hepatic and lower renal accumulation (presented in Table 2).This indicated that the elimination of the [ 205/206 Bi]Bi-DOTA-IPB-NAPamide occurred mainly via the hepatobiliary system.Moreover, the elevated IPB-NAPamide uptake of the liver was in correlation with results of previous experiments dealing with 18 F-labeled NAPamide and 213 Bi-labeled FOLDamide compounds [14,24].In addition, different biodistribution profiles between the two 213 Bi-labeled probes were observed specifically in the blood, and in organs with congestion (e.g., lung, heart) with higher radiotracer concentration in the [ 205/206 Bi]Bi-DOTA-IPB-NAPamide administered group that further strengthened the increased circulatory half-life of the radiopharmaceutical induced by the albumin-binding sequence.Upon the assessment of the uptake of [ 205/206 Bi]Bi-DOTA-NAPamide without the IPB motif, a moderate radiotracer concentration was depicted in all organs and tissues, which was in agreement with earlier research findings on 213 Bi-labeled NAPamide compound [24].A rapid renal clearance of [ 205/206 Bi]Bi-DOTA-NAPamide was evidenced by the low blood activities and the obvious kidney uptake (8.78 ± 3.61 %ID/g).

Ex Vivo
Within the framework of the ex vivo organ distribution experiments, 9 ± 1 days post B16F10 cell implantation, the MC1-R selectivity of the 205/206 Bi-labeled DOTA conjugated NAPamide derivatives was evaluated in the corresponding melanoma tumor-bearing mice 90 minutes after the iv.injection of 2.39 ± 0.  206 Bi]Bi-DOTA-NAPamide coupled with much lower hepatic activity (%ID/g: 1.09 ± 0.17) and negligible uptake in the other assessed organs and tissues-apart from the kidneys-leads to elevated tumorto-non-target ratios that allows for the acquisition of high contrasted images.In a similar manner, Kálmán-Szabó et al. [24] has recently published comparable ex vivo %ID/g data assessing the biodistribution and the MC1-R overexpressing tumor homing potential of a NAPamide-based radiotracer labeled with another isotope of bismuth, the alpha emitter 213 Bi ( 213 Bi]Bi-DOTA-NAPamide). Applying MC1-R positive melanoma tumor models, Kálmán-Szabó and co-workers reported elevated [ 213 Bi]Bi-DOTA-NAPamide uptake in the B16F10 tumors 90 min post administration (%ID/g: 3.76 ± 0.96).Apart from [ 213 Bi]Bi-DOTA-NAPamide, they also proposed the synthesis and the radiolabeling of the following novel MC1-R selective amide derivatives: HOLDamide, FOLDamide and MARSamide [24].Similarly to the NAPamide molecule, the other probes also seemed to be feasible in the imaging of receptor expressing B16F10 tumors.Although the neoplasms could be visualized using all investigated tracers, the highest radioactivities were recorded in case of the 213 Bi-labeled NAPamide and FOLDamide compounds with respective accumulation figures being 3.76 ± 0.96 %ID/g and 3.28 ± 0.95 %ID/g.In the last part of our experiments, in vivo imaging and post imaging organ distribution experiments were performed for the determination of the uptake pattern and the tumor targeting potential of MC1-R specific, β-emitter 177 Lu-labeled NAPamide probes either in the presence or in the absence of the IPB albumin-binding sequence ([ 177 Lu]Lu-DOTA-NAPamide and [ 177 Lu]Lu-DOTA-IPB-NAPamide).The in vivo SPECT/CT imaging studies revealed that the sc.growing B16F10 tumors could be clearly identified 24 h post injection of [ 177 Lu]Lu-DOTA-IPB-NAPamide; however, using the radiopharmaceutical without the albumin-binding IPB, the malignancy was not detectable at the same investigation time point (Figure 4).This was in accordance with the ex vivo figures that also confirmed significantly more elevated (p ≤ 0.01) tumor uptake of [ 177 Lu]Lu-DOTA-IPB-NAPamide in comparison with [ 177 Lu]Lu-DOTA-NAPamide. Identically to the results with the 68 Ga-and the 205/206 Bilabeled counterparts, the incorporation of the albumin binding moiety could be responsible for the more increased tumor uptake as well as tumor retention.In correlation with the results on the 68 Ga-labeled probes (Table 1), and in accordance with the LogP values, higher liver accumulation was observed for the IPB-containing 177 Lu-labeled radiopharmaceutical; however, interestingly, the kidney activity was also elevated (Table 3).This may indicate that the probe is cleared through the kidneys as well as the hepatobiliary system.Comparing the distribution data of the currently assessed 177 Lu-labeled molecules with the observations of other research studies, we also found that the kidney activity was the highest 24 h post injection of the radiopharmaceuticals. Similarly to our result, Guo and Miao-who explored the post-imaging distribution of MC1-R specific 177 Lu-DOTA-GGNle-CycMSHhex radiopharmaceutical using a B16/F1 tumor model-also reported meaningful tumor accumulation (8.24 ± 1.51 %ID) one day post tracer administration; however, faint blood activity (0.23 ± 0.41 %ID) was registered at the same time point, which contradicted our observations [25].In addition, the relatively high renal 177 Lu-DOTA-GGNle-CycMSHhex accretion (4.75 ± 1.03 %ID) 24 h post tracer injection was in line with the current results [25].The ex vivo biodistribution data of [ 177 Lu]Lu-DOTA-NAPamide and [ 177 Lu]Lu-DOTA-IPB-NAPamide are displayed in Table 3.

In Vivo and Ex Vivo Biodistribution
Summarizing the in vivo imaging and ex vivo biological findings, we can draw the conclusion that the albumin-binding sequence containing 68 Ga-, 205/206 Bi-and 177 Lu-labeled MC1-R specific NAPamide radiopharmaceuticals compared with the ones without the IPB motif exhibited significantly higher accumulation in the examined organs and in the B16F10 tumors as well due to their increased circulation time.Given the selective MC1-R binding ability of all assessed probes-regardless of the imaging label-these radiolabeled, amide-based derivatives could serve as a magic bullet for the in vivo molecular diagnostics of MCR-1-overexpressing malignant melanoma.Finally, the NAPamide-based molecules labeled with α-emitter 213 Bi or β-emitter 177 Lu could play a pivotal role in the targeted radiotherapy of MC1-R-positive cancers.Therefore, these radiopharmaceuticals display meaningful potential for theranostic applications as well.

PET Data Assessment
Three-dimensional ordered-subsets expectation-maximization algorithm (3D OSEM) was used for PET image reconstruction.Three-dimensional ellipsoidal volume of interests (VOI) were manually placed around the border of the organ/tissue activity to calculate their radiotracer concentration.The radiopharmaceutical accumulation was presented as standardized uptake values (SUVs).The following formula was applied for the determination of the SUV parameter: SUV (g/mL) = [VOI activity (Bq/mL)]/[administered activity (Bq)/animal weight (g)] 3.4.6.In Vivo Single-Photon Emission Computed Tomography/Computed Tomography (SPECT/CT) Acquisition For the evaluation of the tumor-targeting capacity of [ 177 Lu]Lu-DOTA-NAPamide and [ 177 Lu]Lu-DOTA-IPB-NAPamide, SPECT/CT images were acquired at 24 h postinjection using a nanoSPECT/CT system (Mediso Ltd., Budapest, Hungary).The experimental small animals were iv.administered with 6.34 ± 1.07 MBq of [ 177 Lu]Lu-DOTA-NAPamide or [ 177 Lu]Lu-DOTA-IPB-NAPamide through the lateral tail vein.Followed by a 24 h long uptake period, SPECT data were gathered, and images were reconstructed according to the manufacturer's instructions applying the Nucline acquisition software and the InterView™ FUSION 1.2 (Mediso Ltd., Budapest, Hungary) image analysis software.
3.4.8.Statistical Analysis MedCalc 18.5 commercial software package (MedCalc 18.5, MedCalc Software, Mariakerke, Belgium) was used to perform the statistical analyses.Statistical differences were defined using Student's two-tailed t test, two-way ANOVA and Mann-Whitney U-test.The data are displayed as the mean ± SD, and the significance was set at p < 0.05, unless otherwise indicated.

Conclusions
We successfully synthetized 4-(p-iodophenyl)butanoic acid-modified DOTA-conjugated NAPamide derivative 10 and radiolabeled with 68 Ga, 205/206 Bi and 177 Lu isotopes with high labeling yield.According to the results of the serum stability tests, no significant changes in the stability of the labeled complexes were observed after two hours, moreover in the case of [ 177 Lu]Lu-DOTA-IPB-NAPamide even after 24 h.
From the PET image assessment of the 68 Ga-labeled DOTA-IPB-NAPamide probe, the B16F10 melanoma tumors could be well identified that indicated the target specificity