The Synthesis and Preclinical Investigation of Lactosamine-Based Radiopharmaceuticals for the Detection of Galectin-3-Expressing Melanoma Cells

Given that galectin-3 (Gal-3) is a β-galactoside-binding lectin promoting tumor growth and metastatis, it could be a valuable target for the treatment of Gal-3-expressing neoplasms. An aromatic group introduced to the C-3′ position of lactosamine increased its affinity for Gal-3. Herein, we aimed at developing a radiopharmaceutical for the detection of Gal-3 positive malignancies. To enhance tumor specificity, a heterodimeric radiotracer capable of binding to both Gal-3 and αvβ3 integrin was also synthetized. Arginine-glycine-asparagine (RGD) peptide is the ligand of angiogenesis- and metastasis-associated αvβ3 integrin. Following the synthesis of the chelator-conjugated (2-naphthyl)methylated lactosamine, the obtained compound was applied as a precursor for radiolabeling and was conjugated to the RGD peptide by click reaction as well. Both synthetized precursors were radiolabeled with 68Ga, resulting in high labeling yield (>97). The biological studies were carried out using B16F10 melanoma tumor-bearing C57BL6 mice. High tumor accumulation of both labeled lactosamine derivatives—detected by in vivo PET and ex vivo biodistribution studies—indicated their potential for melanoma detection. However, the heterodimer radiotracer showed high hepatic uptake, while low liver accumulation characterized chelator-conjugated lactosamine, resulting in PET images with excellent contrast. Therefore, this novel carbohydrate-based radiotracer is suitable for the highly selective determination of Gal-3-expressing melanoma cells.


Introduction
Positron emission tomography (PET) is an effective diagnostic method that uses radioactive agents and plays a vital role in the detection of various cancers even in the early stages of the disease. Since early diagnostic assessment significantly improves the chance of survival, its oncological importance cannot be overemphasized enough. Today, the most commonly applied radiopharmaceutical for the staging of tumor lesions and for the determination of response to anticancer treatment in clinical practice is 2-deoxy-2-[ 18 F]fluoroβ-D-glucose ([ 18 F]FDG). However, increased [ 18 F]FDG uptake is also characteristic for infections and inflammations, which can be misdiagnosed as malignancies [1]. Therefore, there is an urgent need for the development of more tumor-specific radiopharmaceuticals. Radiotracers based on receptor-ligand interaction are capable of binding with high affinity and selectivity to receptors overexpressed in neoplastic cells, which reduces their accumulation in healthy tissues. For example, radiolabeled peptides could be promising candidates Gal-3 affinity, Sörme and co-workers [26] found that three compounds bearing an aromatic amide in the C-3 position showed significantly higher inhibitory activity. Later, they evaluated the human Gal-3 CRD in complex with the ligands containing the 3 -benzamide and 3 -p-methoxy-2,3,5,6-tetrafluorobenzamide groups by X-ray crystallography [27]. Based on these measurements, they established that the benzamide group can interact with the Arg-144 side chain in subunit B of the CRD and causes a conformational change in the side chain. Furthermore, the benzamido ring sits in a nonpolar pocket formed by the side chains of Arg-144, Ala-146 and Asn-160. In addition, a third interaction is in favor of the formation of strong binding, as the guanidino group of Arg-144 can interact with the benzamide group via the cation-Π interaction (also called arginine-arene inter action) [27]. These findings support that the affinity of N-acetyl-lactosamine-based galectin-3 inhibitors can be effectively increased by introducing an aromatic substituent at the C-3 position of the galactose unit.
Despite the fact that many Gal-3 ligands have been prepared, only a few radiolabeled derivatives are known from the available literature. Deutscher et al. [28,29] reported about the synthesis of the radiolabeled analog of G3-C12 peptide, named 111 In-DOTA-(GSG)-G3-C12 and its preclinical investigation. This radiolabeled peptide-assessed in the following two different xenograft tumor models: MDA-MB-435 human breast carcinoma [28] and PC3-M human prostate carcinoma cells [29]-appeared to be a valuable radiopharmaceutical for the imaging of Gal-3 positive tumors by SPECT. D'Alessandria et al. developed the 89 Zr-DFO-mAb to Gal-3 radiotracer, which was used to evaluate Gal-3 expression in thyroid carcinoma models by in vivo immunoPET imaging and served specific tumor accumulation [30]. Moreover, radiofluorinated analogs of established Gal-3 inhibitors, namely 1,1 -sulfanediyl-bis-{3-deoxy-3-[4-(3-fluorophenyl)-1H-1,2,3-triazol-1-yl]β-D-galactopyranoside} (TD139) and GB1107, have been synthetized and applied in PET studies [31]. According to the in vivo distribution studies, the TD139 surrogate showed rapid clearance from the blood, whereas the GB1107 surrogate was characterized by slow elimination; consequently none of the 18 F-labeled compounds proved to be a suitable PET tracer for the detection of Gal-3 level in pancreatic carcinoma [31]. Hence, to the best of our knowledge, a carbohydrate-based PET radiotracer capable of detecting Gal-3 expression in tumor cells has not yet been described so far. Therefore, we intended to accomplish the synthesis of a 68 Ga-labeled lactosaminebased radiopharmaceutical and investigate its Gal-3-targeting properties by in vivo PET imaging and biodistribution studies. In addition, a heterodimeric labeled compound capable of binding to both galectin-3 and α v β 3 integrin was developed to enhance tumor specificity. α v β 3 integrin is a known biomarker of tumor angiogenesis that increases tumor growth and the metastatic potential of tumor cells as well [32]. Peptide analogue containing arginine-glycine-asparagine (RGD) tripeptide sequence binds with high affinity and selectivity to α V β 3 integrin receptor [33]. Consequently, we used a cyclic RGD analog as a vector molecule for the synthesis of the heterodimer radiotracer.

DBCO-PEG 4 -cRGDfK (7)
Compound 5 (4.65 mg, 0.0077 mmol) was dissolved in dimethylsulfoxide (200 µL). Then, N,N-diisopropylethylamine (6.8 µL, 0.0385 mmol) and a solution of compound 6 (5 mg, 0.0077 mmol) in dimethyl sulfoxyde (200 µL) was added. The reaction mixture was stirred overnight at room temperature. Then water was added to the reaction mixture and concentrated by lyophilization. The purification of the residue was carried out with semipreparative RP-HPLC using the analysis method described for compound 4. The t R was 17.85 min and the obtained fraction was concentrated by lyophilisation to yield compound 7 ( Figure S9).

DOTAGA-LacN(NAP)-cRGDfK (8)
Compound 7 (2 mg, 0.0017 mmol) was dissolved in dimethyl sulfoxide (150 µL). Subsequently, compound 4 (2.5 mg, 0.002 mmol) was also dissolved in dimethyl sulfoxide (150 µL) and added to the solution of compound 7. The reaction mixture was stirred over night at room temperature. After that, water was added to the reaction mixture and concentrated by lyophilization. The purification of the residue was carried out with semipreparative RP-HPLC using the analysis method described for compound 4. The t R was 18 min and the obtained fraction was concentrated by lyophilization to yield compound 8 ( Figure S10).

Animal Housing
Twelve-week-old male C57BL/6 mice (n = 30) were kept in individually ventilated cages (IVC) (Techniplast, Akronom Ltd. Budapest, Hungary) at regulated temperature (26 ± 2 • C) and controlled humidity (55 ± 10%). Artificial lighting was assured in mechanically moderated 12-h circadian cycles. Tap water and semi-synthetic rodent chow (SDS VRF, Animalab Ltd., Budapest, Hungary) were administered ad libitum for the enrolled experimental small animals to maintain their physiological nutrient requirements. All applicable paragraphs of the Hungarian Laws and directions and the regulations of the European Union were taken into account regarding both the maintenance and the treatment of the mice. Experimental animals received human care and authorized by the Ethical Committee for Animal Research, University of Debrecen, Hungary (ethical licence number: 16/2020/DEMÁB).

In Vivo PET Imaging and Image Analysis
In a bid to investigate the tumor-targeting capability of the radiopharmaceuticals concerned, in vivo PET imaging was accomplished 10 ± 1 days post-tumor cell implantation. Both normal control and B16-F10 tumorous mice were iv. administered with 6.8 ± 0.

Statistical Analysis
MedCalc 18.5 commercial software package (MedCalc 18.5, MedCalc Software, Mariakerke, Belgium) was applied for the statistical analyses. To determine the significance, the following tests were utilized: Student's two-tailed test, two-way ANOVA, and Mann-Whitney U test. Figures are expressed as mean ± SD. The significance was set to 0.05 (p < 0.05) except for otherwise indicated.

Chemistry
Our research team previously established a method for the synthesis of the dualtargeting 68 Ga-NODAGA-LacN-E[c(RGDfK)] 2 radiotracer [34]. In the present study, a similar synthetic sequence was utilized for the production of the precursor molecules.
First, a lactosamine derivative that was able to bind to the Gal-3 receptor with higher affinity than lactosamine itself was prepared. According to the previously mentioned X-ray crystallographic studies carried out by Sörme and colleagues [27], we designed the synthesis of a lactosamine containing an aromatic group at the C-3 position. A (2-naphthyl)methyl group that could be easily formed via stanylene acetal was chosen as an aromatic group. This method was suggested for the synthesis of Gal-3 inhibitors by Sörme et al. [35].
Another building block of the heterodimer was a cRGDfK peptide functionalized with a pegylated DBCO unit for the click reaction. Accordingly, the cRGDfK (7) peptide was coupled with PEG4-DBCO-NHS (6) in a mixture of DMSO and DIPEA to yield compound 8 (Figure 3). After that, a thiourea bond was formed between the amino group of the (2-naphthyl)methylated lactosamine derivative 3 and the isothiocyanate group of p-SCN-Bn-DOTAGA (4) in a mixture of DMSO and 0.1 M sodium carbonate buffer, resulting in compound 5 ( Figure 2). matic group. This method was suggested for the synthesis of Gal-3 inhibitors by Sörme et al. [35].
Another building block of the heterodimer was a cRGDfK peptide functionalized with a pegylated DBCO unit for the click reaction. Accordingly, the cRGDfK (7) peptide was coupled with PEG4-DBCO-NHS (6) in a mixture of DMSO and DIPEA to yield compound 8 (Figure 3). Chelator-conjugated lactosamine derivative 5-labeled with 68 Ga isotope-on the one hand was used for biological studies, whereas on the other hand, compound 5 provided one of the building blocks of the desired heterodimer radiopharmaceutical.
Another building block of the heterodimer was a cRGDfK peptide functionalized with a pegylated DBCO unit for the click reaction. Accordingly, the cRGDfK (7) peptide was coupled with PEG4-DBCO-NHS (6) in a mixture of DMSO and DIPEA to yield compound 8 ( Figure 3).
Afterwards, the following procedure was applied for the synthesis of heterodimer 9: the previously prepared chelator-conjugated compound 5 was attached to the compound 8 containing PEG4 and DBCO unit by a copper-free strain-promoted azide-alkyne cycloaddition (SPAAC) (Figure 4).
Finally, a reference compound was prepared for the biological studies. The cRGDfK (7) was directly conjugated to the p-SCN-Bn-DOTAGA (4) in a mixture of DMSO and 0.1 M sodium carbonate buffer to form DOTAGA-cRGDfK (10) ( Figure 5).  Afterwards, the following procedure was applied for the synthesis of heterodimer 9: the previously prepared chelator-conjugated compound 5 was attached to the compound 8 containing PEG4 and DBCO unit by a copper-free strain-promoted azide-alkyne cycloaddition (SPAAC) (Figure 4).   Afterwards, the following procedure was applied for the synthesis of heterodimer 9: the previously prepared chelator-conjugated compound 5 was attached to the compound 8 containing PEG4 and DBCO unit by a copper-free strain-promoted azide-alkyne cycloaddition (SPAAC) (Figure 4). Finally, a reference compound was prepared for the biological studies. The cRGDfK (7) was directly conjugated to the p-SCN-Bn-DOTAGA (4) in a mixture of DMSO and 0.1 M sodium carbonate buffer to form DOTAGA-cRGDfK (10) ( Figure 5).

Radiochemistry
Precursors 5, 9 and 10 were radiolabeled with 68 Ga isotope with the application of the same labeling procedure. 68 Ga nuclide was produced using the 68 Zn(p, n) 68 Ga nuclear reaction in a cyclotron, and after solid-phase extraction (SPE) it was applied for radiolabeling processes. The obtained [ 68 Ga]GaCl 3 solution (100-200 MBq) was mixed with 3 M NH 4 OAc buffer (pH 4), then a solution of compound 5, 9 and 10 (concentration: 1 mg/mL) were added to, respectively. These mixtures were incubated at 95 • C for 15 min. In all three cases, high labeling yield was detected (>97%). After SPE purification of the reaction mixtures, the radiochemical purity of the labeled compounds was analyzed by analytical radioHPLC and found to be better than 95% for all radiotraces. In addition, the molar activities were 4.75 ± 0.057 GBq/µmol, 11.63 ± 0.17 GBq/µmol and 5.49 ± 0.19 GBq/µmol

Radiochemistry
Precursors 5, 9 and 10 were radiolabeled with 68 Ga isotope with the application of the same labeling procedure. 68 Ga nuclide was produced using the 68 Zn(p, n) 68 Ga nuclear reaction in a cyclotron, and after solid-phase extraction (SPE) it was applied for radiolabeling processes. The obtained [ 68 Ga]GaCl3 solution (100-200 MBq) was mixed with 3 M NH4OAc buffer (pH 4), then a solution of compound 5, 9 and 10 (concentration: 1 mg/mL) were added to, respectively. These mixtures were incubated at 95 °C for 15 min. In all three cases, high labeling yield was detected (>97%). After SPE purification of the reaction mixtures, the radiochemical purity of the labeled compounds was analyzed by analytical radioHPLC and found to be better than 95% for all radiotraces. In addition, the molar activities were 4.75 ± 0.057 GBq/µmol, 11.63 ± 0. Furthermore, these log P values showed that conjugation with (2-naphthyl)methylated lactosamine 3 and pegylation did not increase but rather decreased the hydrophilicity of the labeled heterodimeric compound.
Furthermore, the labeled complexes were mixed with human serum, 0.01 M oxalic acid and 0.01 M Na2EDTA and incubated at room temperature, respectively. Samples from the solutions at 0, 60 and 120 min were analyzed by radio-HPLC. All three radiotracers remained stable under the tested conditions for two hours.  Figure 6. Following the qualitative assessment of the PET images, the urinary system (urinary bladder Furthermore, these log P values showed that conjugation with (2-naphthyl)methylated lactosamine 3 and pegylation did not increase but rather decreased the hydrophilicity of the labeled heterodimeric compound.

Biology
Furthermore, the labeled complexes were mixed with human serum, 0.01 M oxalic acid and 0.01 M Na 2 EDTA and incubated at room temperature, respectively. Samples from the solutions at 0, 60 and 120 min were analyzed by radio-HPLC. All three radiotracers remained stable under the tested conditions for two hours.  Figure 6. Following the qualitative assessment of the PET images, the urinary system (urinary bladder with urine) was clearly visualized ( Figure 6 red and black arrows) due to the log P values, which confirmed the highly hydrophilic properties of the investigated radiotracers. The high lipophilicity of the radiopharmaceutical causes hepatobiliary excretion and high nonspecific uptake in healthy tissues, which limits its use in imaging and therapy. Therefore, it is important to reduce the lipophilicity of radiolabeled peptides, which increases the tumor-to-background ratio. However, too high hydrophilicity may result in short circulation half-life of the radiopharmaceutical, leading to low accumulation in tumors. However, despite the low log P values, as the lower row of Figure 6 demonstrates, increased hepatic radiopharmaceutical accumulation was observed with the RGD-containing radiotracers. Further, comparing the liver tracer uptakes of the three different radioisotopes, the most elevated accumulation was registered in connection with radiotracer [ 68 Ga]Ga-DOTAGA-LacN(NAP)-cRGDfK. nonspecific uptake in healthy tissues, which limits its use in imaging and therapy. Therefore, it is important to reduce the lipophilicity of radiolabeled peptides, which increases the tumor-to-background ratio. However, too high hydrophilicity may result in short circulation half-life of the radiopharmaceutical, leading to low accumulation in tumors. However, despite the low log P values, as the lower row of Figure 6 demonstrates, increased hepatic radiopharmaceutical accumulation was observed with the RGD-containing radiotracers. Further, comparing the liver tracer uptakes of the three different radioisotopes, the most elevated accumulation was registered in connection with radiotracer [ 68 Ga]Ga-DOTAGA-LacN(NAP)-cRGDfK. The above-detailed in vivo PET results were in line with the ex vivo data (as demonstrated in Figure 7). As part of the ex vivo biodistribution studies-after PET imagingthe sacrifice of the experimental animals occurred followed by the gamma counter-based measurement of the radioactivity of the organs and tissues (Figure 7). In line with the in vivo SUV data, in the case of all three radiotracers, remarkable accumulation was observed in the kidneys (approx. %ID/g: 2-8) and in the urine (approx. %ID/g: 300). Comparing the %ID/g data of the abdominal, thoracic, and other organs, while 68 Ga-labeled DOTAGA-LacN(NAP) showed (p ≤ 0.01) the lowest values, [ 68 Ga]Ga-DOTAGA-LacN(NAP)-cRGDfK accumulation was the highest in most of the examined tissues. This elevated accumulation identified in the stomach (%ID/g: 1.70 ± 0.71), liver (%ID/g: 2.30 ± 0.73), gall bladder (%ID/g: 3.07 ± 2.10), and intestines (%ID/g: approx. 1.70 ± 0.70) indicates that there is an elimination route of LacN(NAP)-cRGDfK-targeted radiopharmaceutical through the digestive system (Figure 7). The above-detailed in vivo PET results were in line with the ex vivo data (as demonstrated in Figure 7). As part of the ex vivo biodistribution studies-after PET imaging-the sacrifice of the experimental animals occurred followed by the gamma counter-based measurement of the radioactivity of the organs and tissues (Figure 7). In line with the in vivo SUV data, in the case of all three radiotracers, remarkable accumulation was observed in the kidneys (approx. %ID/g: 2-8) and in the urine (approx. %ID/g: 300). Comparing the %ID/g data of the abdominal, thoracic, and other organs, while 68 Ga-labeled DOTAGA-LacN(NAP) showed (p ≤ 0.01) the lowest values, [ 68 Ga]Ga-DOTAGA-LacN(NAP)-cRGDfK accumulation was the highest in most of the examined tissues. This elevated accumulation identified in the stomach (%ID/g: 1.70 ± 0.71), liver (%ID/g: 2.30 ± 0.73), gall bladder (%ID/g: 3.07 ± 2.10), and intestines (%ID/g: approx. 1.70 ± 0.70) indicates that there is an elimination route of LacN(NAP)-cRGDfK-targeted radiopharmaceutical through the digestive system (Figure 7).

Biology
The high liver uptake and hepatobiliary excretion of the labeled RGD-containing compounds can be explained by the following. According to prior literature data, due to the size and physicochemical properties of the RGD-containing radiotracers, the reticuloendothelial cells of the liver and the spleen, the vascularization and hepatic metabolism can also increase their accumulation in these organs. In addition, the elevated intestinal uptake may be related to the physiological α v β 3 expression of the intestinal smooth muscle cells [36].
However, low galectin-3 expression was found in the healthy kidney and liver by Chen et al. [37] and Hsu et al. [38]. In accordance with these observations, higher liver accumulation was recorded when α v β 3 -specific RGD-containing radioactive tracers were used, while this value was significantly lower in the case of the galectin-3-specific 68 Galabeled DOTAGA-LacN(NAP) probe. The high liver uptake and hepatobiliary excretion of the labeled RGD-containing compounds can be explained by the following. According to prior literature data, due to the size and physicochemical properties of the RGD-containing radiotracers, the reticuloendothelial cells of the liver and the spleen, the vascularization and hepatic metabolism can also increase their accumulation in these organs. In addition, the elevated intestinal uptake may be related to the physiological αvβ3 expression of the intestinal smooth muscle cells [36].
However, low galectin-3 expression was found in the healthy kidney and liver by Chen et al. [37] and Hsu et al. [38]. In accordance with these observations, higher liver accumulation was recorded when αvβ3-specific RGD-containing radioactive tracers were used, while this value was significantly lower in the case of the galectin-3-specific 68 Galabeled DOTAGA-LacN(NAP) probe.

Conclusions
We successfully synthesized a 68 Ga-labeled radiopharmaceutical containing (2-naphtyl)methylated lactosamine for the PET detection of Gal-3 expression in melanoma cells. Furthermore, we accomplished the preparation of a 68 Ga-labeled heterodimeric compound that can target both galectin-3 and α v β 3 integrin.
Following the radiolabeling processes, the pharmacokinetics and the tumor targeting properties of the synthetized radiotracers were investigated in B16-F10 melanoma tumor-bearing mice applying in vivo PET and ex vivo biodistribution studies. Satisfactory tumor accumulation was experienced in case of both novel (2-naphtyl)methylated lactosaminecontaining radiopharmaceuticals. However, despite the glycosylation and pegylation of the RGD peptide, the labeled heterodimer showed high liver uptake and hepatobiliary excretion. Furthermore, despite dual targeting, its tumor uptake was almost identical to that of the Gal-3-specific radiotracer. More enhanced tumor-to-background ratio and higher resolution PET images of the Gal-3 positive tumor cells acquired with the application of the [ 68 Ga]Ga-DOTAGA-LacN(NAP) will strengthen the selectivity and imaging properties of this 68 Ga-labeled carbohydrate derivative compared with those of the heterodimer.