Evaluation of Radioiodinated Fluoronicotinamide/Fluoropicolinamide-Benzamide Derivatives as Theranostic Agents for Melanoma

Malignant melanoma is the most harmful type of skin cancer and its incidence has increased in this past decade. Early diagnosis and treatment are urgently desired. In this study, we conjugated picolinamide/nicotinamide with the pharmacophore of 131I-MIP-1145 to develop 131I-iodofluoropicolinamide benzamide (131I-IFPABZA) and 131I-iodofluoronicotiamide benzamide (131I-IFNABZA) with acceptable radiochemical yield (40 ± 5%) and high radiochemical purity (>98%). We also presented their biological characteristics in melanoma-bearing mouse models. 131I-IFPABZA (Log P = 2.01) was more lipophilic than 131I-IFNABZA (Log P = 1.49). B16F10-bearing mice injected with 131I-IFNABZA exhibited higher tumor-to-muscle ratio (T/M) than those administered with 131I-IFPABZA in planar γ-imaging and biodistribution studies. However, the imaging of 131I-IFNABZA- and 131I-IFPABZA-injected mice only showed marginal tumor uptake in A375 amelanotic melanoma-bearing mice throughout the experiment period, indicating the high binding affinity of these two radiotracers to melanin. Comparing the radiation-absorbed dose of 131I-IFNABZA with the melanin-targeted agents reported in the literature, 131I-IFNABZA exerts lower doses to normal tissues on the basis of similar tumor dose. Based on the in vitro and in vivo studies, we clearly demonstrated the potential of using 131I-IFNABZA as a theranostic agent against melanoma.


Introduction
Among skin cancers, malignant melanoma is the deadliest form and results in the majority of skin cancer deaths around the world [1,2]. The annual incidence of malignant melanoma has risen rapidly, especially in North America, owing to genetic defects and increased ultraviolet light exposure caused by global environmental change and destruction of the ozonosphere [3]. After surgery, the 5-year survival rate is >90% in patients with localized and noninvasive melanoma [4]. Once metastasized, melanoma becomes uncontrollable, and the survival rate dramatically decreases to less than 20% due to limited therapeutic options [5,6].
Melanin is composed of two biogenetically related pigments, eumelanin and pheomelanin [7]. The primary function of melanin is to protect skin against UV-induced damage. Most of the malignant melanoma cells overexpressed melanin because of increased tyrosinase activity [8,9]. Therefore, melanin is thought to be a potential target for early detection and treatment for advanced malignant melanoma. Michelot et al. first indicated a significant accumulation of 125 I-iodobenzamide ( 125 I-IBZA) in B16 murine melanoma (6.75%ID/g) and liver (6.04%ID/g) at 1 h post injection [10,11]. In 2010, Joyal et al. discovered a superior melanin-targeting ability of an iodine-123/131 labeled fluorobenzoate-benzamide derivative ( 123/131 I-MIP-1145) [12]. The uptake of 131 I-MIP-1145 in melanin-expressed SK-MEL-3 human melanoma xenograft (8.82 ± 3.55%ID/g) was significantly higher than that in the amelanotic A375 one (1.19 ± 0.07%ID/g) at 4 h after intravenous injection [12]. The growth inhibition effect on the SK-MEL-3 xenograft was also noticed when the tumor-bearing mice received 131 I-MIP-1145 administration (1 × 25, 2 × 25, and 3 × 25 MBq) [12]. However, the high lipophilicity of 123/131 I-MIP-1145 (logP = 4.5) led to apparent radioactivity accumulation in liver and gastrointestinal tract and resulted in high radiation absorption dose in normal tissues [12]. Several studies reported that nicotinamide ( 18 F-MEL050) [13,14] and picolinamide derivatives ( 18 F-5-FPN) [15] showed a prolonged retention in melanotic melanoma and elevated clearance rate in normal tissues when compared with benzamides analogues. In our previous study, we successfully conjugated an 131 I-labeled benzamide derivative with a nicotinamide to give 131 I-ICNA and demonstrated its potency in remarkable melanoma targeting along with low retention in normal tissues [16].
Based on the previous results of 131 I-MIP-1145 and 131 I-ICNA, we aim to conjugate picolinamide and nicotinamide with the pharmacophore of 131 I-MIP-1145 to determine the feasibility of 131 I-iodofluoropicolinamide benzamide ( 131 I-IFPABZA) and 131 I-iodofluoronicotiamide benzamide ( 131 I-IFNABZA) as a theranostic agent to target melanin-expressed melanoma in this study. To the best of our knowledge, this study should be the first one to investigate the biological characteristics of fluoropicolinamide-and fluoronicotinamide-benzamide derivatives and to evaluate their clinical potential as a theranostic agent against melanoma.

Partition Coefficient and In Vitro Stability of 131 I-IFPABZA and 131 I-IFNABZA
The determined log P value of 131 I-IFPABZA and 131 I-IFNABZA was 2.01 ± 0.12 and 1.19 ± 0.07, respectively, indicating both of them were considerably hydrophobic compounds. In the stability tests, the percentage of intact 131 I-IFNABZA was 92.8 ± 1.6% after a 24 h incubation in FBS at 37 °C , but that of 131 I-IFPABZA was comparatively lower (89.4 ± 1.1%) ( Figure 3A,B). In fact, at the first time point, the percentage of intact 131 I-IFNABZA incubated in PBS was below 90% at either 4 or 37 °C ( Figure 3A), suggesting a relatively poor stability.

Partition Coefficient and In Vitro Stability of 131 I-IFPABZA and 131 I-IFNABZA
The determined log P value of 131 I-IFPABZA and 131 I-IFNABZA was 2.01 ± 0.12 and 1.19 ± 0.07, respectively, indicating both of them were considerably hydrophobic compounds. In the stability tests, the percentage of intact 131 I-IFNABZA was 92.8 ± 1.6% after a 24 h incubation in FBS at 37 • C, but that of 131 I-IFPABZA was comparatively lower (89.4 ± 1.1%) ( Figure 3A,B). In fact, at the first time point, the percentage of intact 131 I-IFNABZA incubated in PBS was below 90% at either 4 or 37 • C ( Figure 3A), suggesting a relatively poor stability.

Partition Coefficient and In Vitro Stability of 131 I-IFPABZA and 131 I-IFNABZA
The determined log P value of 131 I-IFPABZA and 131 I-IFNABZA was 2.01 ± 0.12 and 1.19 ± 0.07, respectively, indicating both of them were considerably hydrophobic compounds. In the stability tests, the percentage of intact 131 I-IFNABZA was 92.8 ± 1.6% after a 24 h incubation in FBS at 37 °C , but that of 131 I-IFPABZA was comparatively lower (89.4 ± 1.1%) ( Figure 3A,B). In fact, at the first time point, the percentage of intact 131 I-IFNABZA incubated in PBS was below 90% at either 4 or 37 °C ( Figure 3A), suggesting a relatively poor stability.

In Vitro Binding of 131 I-IFPABZA and 131 I-IFNABZA to Melanin
Both 131 I-IFPABZA and 131 I-IFNABZA showed a high binding affinity to melanin. More than 98% radioactivity of 131 I-IFPABZA and 131 I-IFNABZA bound to melanin at a concentration ranging from 3 to 200 ppm at 1 h post incubation at 37 • C ( Figure 4A). At a concentration of 50 ppm melanin suspension, the bound percentage of these two compounds rapidly reached 98% after initial 30 min incubation and remained high for 2 h ( Figure 4B).

In Vitro Binding of 131 I-IFPABZA and 131 I-IFNABZA to Melanin
Both 131 I-IFPABZA and 131 I-IFNABZA showed a high binding affinity to melanin. More than 98% radioactivity of 131 I-IFPABZA and 131 I-IFNABZA bound to melanin at a concentration ranging from 3 to 200 ppm at 1 h post incubation at 37 °C ( Figure 4A). At a concentration of 50 ppm melanin suspension, the bound percentage of these two compounds rapidly reached 98% after initial 30 min incubation and remained high for 2 h ( Figure 4B).

Assessment of In Vitro Cellular Uptake
The cellular uptake of 131 I-IFPABZA and 131 I-IFNABZA (expressed as %AD/10 6 cells) increased with time, reaching a maximum accumulation of 67.8 ± 0.2%AD/10 6 cells at 240 min post incubation, and 62.6 ± 0.2%AD/10 6 cells at 480 min post incubation, respectively, and remained high throughout the 8 h study period ( Figure 5A,B). However, the accumulation of both radiotracers in A375 cells was significantly lower than that in B16F10 cells. The peak uptake of 131 I-IFPABZA in A375 cells was 42.8 ± 0.3%AD/10 6 cells (at 120 min post incubation), while that of 131 I-IFNABZA was below 15%AD/10 6 cells at all time points. 131 I-IFNABZA possessed a noted higher melanotic-to-amelanotic ratio than 131 I-IFPABZA.
The washout studies showed that the efflux out of the B16F10 of 131 I-IFPABZA and 131 I-IFNABZA was not obviously even at 8 h post incubation with fresh medium, implying their high specificity toward melanin ( Figure 5C,D). The percentage of these two drugs retained in B16F10 cells remained >95% throughout the experiment period. However, when incubated with fresh medium, the accumulation of 131 I-IFPABZA and 131 I-IFNABZA in A375 cells decreased with time, and the intracellular radioactivity percentage reached 44.2 ± 0.3 and 34.0 ± 0.4%AD/10 6 cells, respectively, suggesting the majority of their cellular uptake in A375 cells accounted for non-specific binding.

Assessment of In Vitro Cellular Uptake
The cellular uptake of 131 I-IFPABZA and 131 I-IFNABZA (expressed as %AD/10 6 cells) increased with time, reaching a maximum accumulation of 67.8 ± 0.2%AD/10 6 cells at 240 min post incubation, and 62.6 ± 0.2%AD/10 6 cells at 480 min post incubation, respectively, and remained high throughout the 8 h study period ( Figure 5A,B). However, the accumulation of both radiotracers in A375 cells was significantly lower than that in B16F10 cells. The peak uptake of 131 I-IFPABZA in A375 cells was 42.8 ± 0.3%AD/10 6 cells (at 120 min post incubation), while that of 131 I-IFNABZA was below 15%AD/10 6 cells at all time points. 131 I-IFNABZA possessed a noted higher melanotic-to-amelanotic ratio than 131 I-IFPABZA.
The washout studies showed that the efflux out of the B16F10 of 131 I-IFPABZA and 131 I-IFNABZA was not obviously even at 8 h post incubation with fresh medium, implying their high specificity toward melanin ( Figure 5C,D). The percentage of these two drugs retained in B16F10 cells remained >95% throughout the experiment period. However, when incubated with fresh medium, the accumulation of 131 I-IFPABZA and 131 I-IFNABZA in A375 cells decreased with time, and the intracellular radioactivity percentage reached 44.2 ± 0.3 and 34.0 ± 0.4%AD/10 6 cells, respectively, suggesting the majority of their cellular uptake in A375 cells accounted for non-specific binding. and A375 cells All treatments were repeated in six times (n = 6), and data were expressed as mean ± SD.

Scintigraphic Imaging
The scintigraphic imaging showed that both 131 I-IFPABZA and 131 I-IFNABZA were retained in B16F10 tumors ( Figure 6A), but not in A375 xenografts ( Figure 6B). The T/M in 131 I-IFPABZA-injected B16F10-bearing mice increased from 2.96 ± 0.54 at 1 h p.i. to 7.59 ± 1.76 at 48 h p.i., while that of 131 I-IFNABZA increased from 2.97 ± 1.68 to 10.10 ± 1.18. At 96 h post administration, the T/M of 131 I-IFPABZA and 131 I-IFNABZA in B16F10-bearing mice was 5.41 ± 0.86 and 6.09 ± 2.60, respectively. In contrast to B16F10 melanoma, the results of the scintigraphic imaging demonstrated that the retention of 131 I-IFPABZA and 131 I-IFNABZA was only negligible in tumor throughout the experiment period. These results suggested that both 131 I-IFPBZA and 131 I-IFNABZA exhibited superior in vivo melanin targeting ability. Except for tumor, high radioactivity accumulation in the abdomen was also noticed at 1 h p.i., but most of the radioactivity of the two radiotracers retained in the abdomen was washed out at 24 h p.i.. The clearance rate of 131 I-IFNABZA was slightly higher than that of 131 I-IFPBZA.
In contrast to B16F10 melanoma, the results of the scintigraphic imaging demonstrated that the retention of 131 I-IFPABZA and 131 I-IFNABZA was only negligible in tumor throughout the experiment period. These results suggested that both 131 I-IFPBZA and 131 I-IFNABZA exhibited superior in vivo melanin targeting ability. Except for tumor, high radioactivity accumulation in the abdomen was also noticed at 1 h p.i., but most of the radioactivity of the two radiotracers retained in the abdomen was washed out at 24 h p.i. The clearance rate of 131 I-IFNABZA was slightly higher than that of 131 I-IFPBZA.

Biodistribution Study
Considering the binding affinity and specificity determined by in vitro and scintigraphic imaging, we only performed the biodistribution studies of 131 I-IFNABZA in B16F10 melanoma-and A375 amelanotic melanoma-bearing mice (Table 1 and Table S1). At 5 min p.i., high radioactivity retention was noticed in lung (23.49 ± 2.20%ID/g), kidneys (26.80 ± 2.32%ID/g), and liver (11.91 ± 0.93%ID/g), accompanied with low radioactivity in blood (1.70 ± 0.15%ID/g), suggesting that a fast distribution of 131 I-IFNABZA to the organs from the blood (Table 1). Although noted uptake in normal organs at the initial time points was observed, most of the radioactivity in normal organs was rapidly washed out and eliminated through urinary and intestinal routes within 24 h, which was consistent with those noticed in the scintigraphic images. A rapid and significant tumor accumulation of 131 I-IFNABZA at 5 min p.i. (4.29 ± 0.93%ID/g) was observed and the tumor uptake was 5.84 ± 1.80, 5.19 ± 2.84, 5.06 ± 2.09, 5.17 ± 1.53, and 1.51 ± 0.34%ID/g at 1, 4, 24, 48, and 96 h p.i., respectively, in B16F10 melanoma-bearing mice. The T/M was 1.48 ± 0.33, 9.90 ± 3.66, 34.60 ± 19.49, 168.70 ± 89.52, and 258.50 ± 76.50, at 5 min, 1, 4, 24, 48 h p.i., respectively, and then, declined to 151.0 ± 34.0 at 96 h p.i. The apparent uptake in black eyeballs, another tissue with a high amount of melanin in C57BL/6 mice, was also noticed ( Table 1). The uptake in eyeballs and A375 tumors was 1.20 ± 0.26 and 1.66 ± 0.39%ID/g at 5 min p.i., respectively, and decreased to 0.04 ± 0.01 and 0.10 ± 0.05 at 24 h p.i., respectively (Table S1). The distribution of 131 I-IFNABZA in A375 amelanotic melanoma-bearing mice was similar to those observed in B16F10 melanoma-bearing mice, except for low tumor and eyeball uptake. High accumulation of 131 I-IFNABZA in lung, kidney, and liver was also noticed at initial time points, and the radioactivity rapidly eliminated within 24 h (Table S1). Results were expressed as the percentage of injected dose per gram of organ/tissue (%ID/g). Each value represented mean ± SD (n = 4). Small int., small intestine; large int., large intestine.

Estimated Absorption Dose Calculation
The results of the biodistribution study were further applied to calculate the estimated absorption dose in humans. As shown in Table 2, liver exhibited the highest absorbed dose (0.122 mSv/MBq), followed by spleen (0.151 mSv/MBq). Even though high accumulation of 131 I-IFNABZA in lung was noticed at initial time points, the estimated absorbed dose was only 2.25 × 10 −2 mSv/MBq. Overall, the estimated effective dose for the whole body in a 73 kg adult human was 3.02 × 10 −2 mSv/MBq. Radiation-absorbed dosimetry was converted from the biodistribution of 131 I-IFNABZA in 0.025 kg tumor-bearing mice to a 73 kg adult human.

Discussion
Several radioactive melanin-targeting probes have been developed for the diagnosis and treatment of melanoma. The drugs that received the most attention were benzamide analogues. Bonnot-Duquennoy et al. found that two doses of 131 I-ICF01012 injection (18.5 × 2 MBq) can extend the tumor doubling time from 2.41 to 5.97 d, but only slightly improved the survival time from 32 to 39 d [17]. Joyal et al. also showed that tumor growth inhibition was significant after three doses of 131 I-MIP-1145 administration (25 × 3 MBq) [12]. Unfortunately, the high accumulation in the gastrointestinal tract raised a concern about normal tissue injury. Xu et al. developed 131 I-5-IPN as a therapeutic agent against murine B16F10 melanoma. However, its therapeutic efficacy was not noted, even though the tumor uptake reached 16.37%ID/g at 1 h p.i. [18]. In the present study, we prepared a radioiodine-labeled fluoropicolinamide-benzamide derivative ( 131 I-IFPABZA) and fluoronicotinamide-benzamide derivative ( 131 I-IFNABZA) and determined their in vitro and in vivo biological characteristics. The radioiodination of aromatic compound ( 131 I-IFPABZA and 131 I-IFNABZA) was performed through radioiododothallation instead of the electrophilic substitution mechanism because the amide linkage, an electron withdrawing group, is located in the ortho position on the aromatic ring [12,19,20].
A series of in vitro melanin-binding studies revealed that both 131 I-IFPABZA and 131 I-IFNABZA could rapidly and strongly bind to melanin (Figure 3). In the cellular uptake studies, the accumulation of 131 I-IFNABZA in melanotic B16F10 cells was 4.6-fold higher than that in amelanotic A375 cells after 2 h incubation, while the ratio was only 1.6 for 131 I-IFPABZA (Figure 4). Although the scintigraphic imaging of B16F10 melanoma-bearing mice clearly showed a significant accumulation and prolonged retention of 131 I-IFPABZA and 131 I-IFNABZA in the tumor region, the T/M of 131 I-IFNABZA at 48 h p.i. (10.10 ± 1.18) was superior to that of 131 I-IFPABZA (7.59 ± 1.74) ( Figure 5). Taken together, our results suggested that 131 I-IFNABZA should be a potential melanin-targeting agent rather than 131 I-IFPABZA.
Compared with B16F10 melanoma, biodistribution studies clearly revealed that 131 I-IFNABZA was rarely found in A375 amelanotic melanoma, which was consistent with the results derived from cellular uptake studies and scintigraphic imaging. Except for melanotic tumor, we noticed the lung uptake at 5 and 15 min p.i. was 23.49 ± 2.20 and 13.87 ± 2.64%ID/g, respectively, which were significantly higher than blood activity at the same time points. In order to rule out that this was caused by the precipitation in lung, we assessed the experiments of 131 I-IFNABZA dissolved in various excipients (Table S2), and found there was no apparent difference in radioactivity accumulated in lung (ranging from 14 to 16%ID/g) and other organs, suggesting the precipitation that originated from relatively poor solubility was not the reason for unexpected lung retention. Another possible explanation is that 131 I-IFNABZA may specifically bind to the sigma receptor, which is highly expressed in lung. In fact, previous literature reported that benzamide derivatives showed high-affinity binding to the sigma receptor [21,22].
To evaluate the potential of 131 I-IFNABZA in melanoma treatment, dosimetry was estimated by OLINDA software. The low absorbed dose in the normal organ was attributed to the fast elimination rate of 131 I-IFNABZA. The estimated absorbed dose of liver was 0.112 mSv/MBq, which was similar to that receiving 131 I-MIP-1145 (0.086 mSv/MBq) and 131 I-ICNA (0.13 mSv/MBq) injection, but the estimated effective dose for the whole body was 46.3-fold and 1.5-fold lower than that of 131 I-MIP-1145 and 131 I-ICNA, respectively.

Reagents and Instruments
All reagents and solvents were purchased from commercial suppliers and used without further purification A solution of 5-fluoropyridine-2-carboxylic acid (60.3 mg, 0.427 mmol) in thionyl chloride (0.904 mL, 12.4 mmol) was reacted at 74 • C for 3 h. After the reaction, excess thionyl chloride was removed under reduced pressure. The residue was redissolved in anhydrous THF (2.0 mL). Compound 2 (0.21 g, 0.54 mmol) and potassium carbonate (114.7 mg, 0.83 mmol) were added to the mixture and allowed to stir at r.t. for 16 h. After evaporation, the residue was redissolved in dichloromethane, and washed with brine and ddH 2 O. The collected organic layer was dried over MgSO 4 , and then concentrated. The crude product was purified by column chromatography on silica gel using MeOH/CH 2 Cl 2 (1:10, v/v) as the mobile phase to afford compound 3a (0.048 g, in 22% yield) as a yellowish solid. The compound 3b was prepared with the same procedure described above, except for using 6-fluoronicotinic acid (60.2 mg, 0.427 mmol) as the starting material. The chemical yield of compound 3b (0.029 g, yellowish solid) was around 13%. Compound 3a: 1

Partition Coefficient of 131 I-IFPABZA and 131 I-IFNABZA
The partition coefficient of 131 I-IFPABZA and 131 I-IFNABZA was assessed by determining their distribution between 1-octanol and PBS. Approximately 7.4 kBq of 131 I-IFPABZA or 131 I-IFNABZA was added to a tube containing 1 mL of 1-octanol and 1 mL of PBS. The tube was vortexed for 5 min and centrifuged at 3000× g for 5 min. Aliquots (0.5 mL) of 1-octanol were taken and added into the next tube containing 0.5 mL of 1-octanol and 1 mL of fresh PBS. These procedures were repeated 5 times. Finally, counting samples (100 µL) of each layer were aspirated and the radioactivity was counted by a gamma counter. The partition coefficient was expressed as log P, which was calculated as follows.
Log P = Log activity in 1 − Octanol activity in PBS (1)

In Vitro Stability of 131 I-IFPABZA and 131 I-IFNABZA
Purified 131 I-IFPABZA and 131 I-IFNABZA were incubated either in 1 mL of PBS at 4 • C or in FBS at 37 • C. The percentage of intact radioactive compound, determined by radio-TLC, in different incubation conditions, was considered as an index of in vitro stability at designated time points (1,2,4,24, and 48 h).

Binding Affinity to Melanin
To assess the affinity of 131 I-IFPABZA and 131 I-IFNABZA to melanin, 10 mCi of 131 I-IFPABZA and 131 I-IFNABZA was incubated with various concentrations of melanin solution (0~200 ppm) at 37 • C for 1 h. After incubation, the mixture was centrifuged at 20,000× g for 5 min, and the supernatant was filtered by a 0.22 mm membrane filter. The radioactivity of the pellet and the filtrate was measured by the g-counter. To investigate the time effect on the binding affinity to melanin, both 131 I-IFPABZA and 131 I-IFNABZA (10 mCi) were incubated with 50 ppm of melanin solution at 37 • C for 0.5, 1, 1.5, and 2 h. The binding affinity was calculated as follows.

Cell Cultures and Xenograft Inoculation
The B16F10 murine melanoma cells and A375 human amelanotic melanoma cells were grown in Dulbecco's modified Eagle high-glucose medium (Gibco Life Sciences) supplemented with 10% FBS in a humidified atmosphere containing 5% CO 2 . To conduct subcutaneous tumor inoculation, 5 × 10 5 of B16F10 and 1 × 10 6 of A375 cells in 100 µL of serum-free medium were implanted into the right flank of 6-week-old male C57BL/6 mouse and male BALB/c nude mouse, respectively. When the tumor reached 150 ± 50 mm 3 , the mice were selected for the following biological studies. All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of National Yang-Ming University (No. 1060604).

In Vitro Cellular Uptake Assay
Cells were seeded in a 6-well plate at a density of 1 × 10 6 cells/well and cultured in 3 mL of culture medium containing 10% FBS at 37 • C overnight. The medium was replaced by fresh medium containing 131 I-IFPABZA or 131 I-IFNABZA (3 mL, 1 µCi/mL). At 15, 30, 60, 120, 240, and 480 min post incubation at 37 • C, the medium was aspirated, and cells were washed twice with 0.5 mL of PBS. The medium and washing PBS were collected in a counting tube. Cells were treated with 0.5 mL of 0.25% trypsin for 5 min for detachment. The cells were harvested and the well was washed twice with 0.5 mL of PBS. The suspension and washing PBS were collected in an anther counting tube and the number of viable cells was determined using hemocytometry. Radioactivity was measured by the g-counter and the cellular uptake of 131 I-IFNABZA and 131 I-IFPABZA was expressed as the percentage of administrated dose per one million cells (%AD/10 6 cells).

Scintigraphic Imaging
The dual head gamma camera (e. cam Signature, Siemens, Germany) equipped with a 4-mm pinhole collimator was applied for the scintigraphic imaging. Both melanotic B16F10 melanoma and amelanotic A375 melanoma-bearing mouse models (n = 3 in each group) were treated with Lugol's solution before imaging. The scintigraphic imaging was conducted 1, 4, 24, 48, and 96 h after intravenous injection of 200 mCi of 131 I-IFPABZA or 131 I-IFNABZA. During imaging, mice were anesthetized with 1-3% isoflurane in 2L of oxygen in the prone position. For quantitative data analysis, a region of interest (ROI) was placed on the tumor and contralateral side of muscle. The tumor-to-muscle ratios (T/M) were calculated on the basis of counts per pixel in the ROIs.

Biodistribution Study
The biodistribution of 131 I-IFNABZA in both B16F10 melanoma-and A375 amelanotic melanoma-bearing mice was conducted. Mice were treated with Lugol's solution before receiving an injection of 131 I-IFNABZA (20 mCi/mice). Four mice in each group were sacrificed at 5, 15 min, 1, 4, 24, 48, and 96 h post injection of 131 I-IFNABZA. The tissues/organs were harvested, weighted, and subjected to radioactivity measurement using the g-counter. The results were expressed as percentage of injected dose per gram of tissue/organ (%ID/g).

Calculation of the Estimated Absorption Dose of Normal Organs
For estimating the radiation absorption dose of various tissues/organs in the human body, we followed the methods reported in our previous studies [16,30]. The radioactive compound and its radioactive metabolites were assumed to distribute homogeneously in tissues/organs, and the relative tissue/organ weight was calculated based on previous studies [31,32]. Based on the results of the biodistribution study in mice, the calculated mean value of the percentage of injected activity per gram of tissue/organ (%IA/g) was extrapolated to the uptake in organs of a 73 kg adult using the following formula.
[(%IA/gorgan)mice × (kgtotal body weight)mice]× (gorgan/(Kgtotal body weight)human) = (%IA/organ)human The extrapolated values (%IA) in the human organs at 5, 15 min, 1, 4, 24, 48, and 96 h were exponentially fitted and integrated to obtain the number of disintegrations in the source organs. The value of each source organ was input into the OLINDA/EXM 1.0 computer program for the dosimetry estimation.

Conclusions
Two novel benzamide derivatives, radioiodinated fluoropicolinamide-benzamide derivative ( 131 I-IFPABZA) and fluoronicotinamide-benzamide derivative ( 131 I-IFNABZA), have been successfully prepared with acceptable radiochemical yield and high radiochemical purity. The cellular uptake study and scintigraphic imaging revealed that 131 I-IFNABZA exhibited better melanoma-targeting ability than 131 I-IFPBZA. A prolonged retention in B16F10 melanoma of 131 I-IFNABZA was demonstrated in the biodistribution study. The estimated effective dose for the whole body after intravenous injection of 131 I-IFNABZA was much lower than that of previous studies. Overall, we believe that 131 I-IFNABZA could be a potential theranostic agent for melanoma.

Acknowledgments:
The authors appreciate the technical support from the Department of Nuclear Medicine in Taipei Veterans General Hospital.

Conflicts of Interest:
The authors declare no conflict of interest.