A reversed-phase HPLC (RP-HPLC) analysis was performed with a C18 column (Capcell Pak C18 AQ, 4.6 × 250 mm; Shiseido Co., Tokyo, Japan) at a flow rate of 1 ml/min eluted with a linear gradient of 0.1% aqueous trifluoroacetic acid (TFA) and acetonitrile with 0.1%TFA from 90:10 to 0:100 for 30 min. Thin-layer chromatography (TLC) was developed with a mixture of 1-butanol, acetic acid and water (4:1:1, v/v/v). The TLC was visualized and quantified using an imaging scanner (Typhoon FLA7000, GE Healthcare Japan, Tokyo, Japan). The stannyl precursor of [211At]-2-AAMP was synthesized according to the procedure described previously . 2-Iodo-a-methyl-L-phenylalanine (2-IAMP) was supplied from Nagase Sangyo (Tokyo, Japan)
211At was produced via the 209Bi(a,2n)211At reaction and isolated by the dry distillation . The detailed procedures were described in Detailed Materials and Methods (see Online Resource). For preparation of [211At]-2-AAMP, the protected compound of [211At]-2-AAMP (Prot-[211At]-2-AAMP) was prepared and then used for the following deprotection reaction without purification. (Fig. 1). To a solution of the stannyl precursor in methanol containing 1% acetic acid (100 μg/450 μl) was added an aqueous solution of 211At (150 μl) and subsequently a solution of N-chlrosuccinimide (NCS) in methanol (100 μg/10 μl). After the mixture was allowed to stand for 15 min at room temperature, an aqueous solution of sodium bisulfite (200 μg/10 μl) and subsequently 6 N NaOH (620 μl) was added. After the reaction for 1 h at 70°C, 6 N HCl was added to the mixture at 0°C to adjust pH between 2 and 7. The mixture was analyzed by TLC before RP-HPLC purification to determine the radiochemical yield. The eluent of RP-HPLC was collected at 1 min intervals in each tube containing 10% aqueous solution of ascorbic acid (10 μl). The fractions containing the product was identified by comparing the retention time of 2-IAMP, and was concentrated in vacuo. Then, remained solution was applied to a Sep-pak C18 cartridge preconditioned with methanol and water. The cartridge was washed with water (2 ml), and eluted with methanol (1 ml). After the eluent was added 10% aqueous solution of ascorbic acid (10 μl), the solvent was removed in vacuo. Radiochemical purity was determined by TLC and RP-HPLC.
Stability assessments of [211At]-2-AAMP
Animal experimental protocol was approved by the Institutional Animal Care and Use Committee at our facility (19-T001-1), and all animal experiments were conducted in accordance with the institutional guidelines regarding animal care and handling. For the evaluation of in vitro stability, each 50 μl aliquot of [211At]-2-AAMP in phosphate buffered saline (PBS) (2.4 MBq/ml) was added to a freshly prepared murine plasma (450 μl). After the mixture was incubated for 6 h at 37°C, a 2 μl aliquot of each sample was analyzed by TLC. For the evaluation of in vivo stability, urine was collected up to 1 h after intravenous injection of [211At]-2-AAMP (100 kBq) in 100 μl of PBS into six-week-old male ICR normal mice (CLEA Japan, Tokyo, Japan). A 2 μl aliquot of each urine sample was analyzed by TLC.
Cellular uptake and release of [211At]-2-AAMP
SKOV3, a human ovarian adenocarcinoma cell line, was obtained from American Type Culture Collection (Manassas, VA, USA). The procedure for cell culture was shown in Detailed Materials and Methods (see Online Resource). Cells (1.0×105 cells/well) were seeded in the 24-well plates and incubated in the growth medium for 24 h. For a time-course study, cells were washed with Hanks' balanced salt solution (HBSS) two times, then incubated in HBSS containing [211At]-2-AAMP at 37oC for 10, 20, 30, 60 and 90 min. For examining sodium-independency, SKOV3 was incubated with [211At]-2-AAMP in HBSS or sodium-free HBSS at 37oC for 10 min. To investigate uptake pathway of [211At]-2-AAMP, cells were incubated with [211At]-2-AAMP containing various inhibitors for 10 min. [211At]-2-AAMP uptake was terminated by removing the [211At]-2-AAMP solution, followed by washing cells three times with ice-cold PBS. Cells were solubilized with 0.1 N NaOH and the radioactivity was measured with a well-type g-counter (ARC-7001, Hitachi-Aloka Medical, Tokyo, Japan). For examining extracellular release, SKOV3 cells were incubated in HBSS containing [211At]-2-AAMP for 10 min. After 2 times washes with HBSS, cells were incubated in HBSS at 37oC for 10, 20, 30, and 60 min. Then, supernatant was collected, and the radioactivity was measured with a well-type g-counter.
Cells were dissolved in sample buffer (25% glycerin, 1% SDS, 62.5 mM Tris-Cl, 10 mM dithiothreitol) and incubated at 65oC (LAT1) or 95oC (CD98) for 15 min. Aliquots of samples containing 20 μg of protein were analyzed by 10% SDS-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane. Blots were incubated at 4oC overnight in 10 mM Tris–HCl, 100 mM NaCl, 0.1% Tween 20, pH 7.5 (TBST), with 5% skim milk. Then, blots were incubated with rabbit anti-LAT1 carboxyl-terminal antibody (1:5000 dilution), rabbit anti-LAT1 amino-terminal antibody (1:5000 dilution), or rabbit anti-CD98 antibody (1:200 dilution, Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA) at 4oC overnight [9, 16]. After washing with TBST, the blots were incubated with horseradish peroxidase conjugated anti-rabbit IgG antibody (1:20000 dilution, Cell Signaling Technology, Beverly, MA, USA) for 1.5 h at room temperature. The blots were further washed with TBST, and specific proteins were visualized by using enhanced chemiluminescence western blotting detection reagents (GE Healthcare).
Cells (1×106 cells/dish) were pre-incubated in the growth medium for 24 h, and treated with 0, 10, 25, and 50 kBq/ml of [211At]-2-AAMP for 24 h. After [211At]-2-AAMP treatment, cells were washed with PBS, suspended in growth medium, and seeded at 200 cells/well in 6-well plate. Then, cells were incubated at 37oC for 14 days. After incubation, cells were washed with PBS twice and stained with crystalviolet solution (6% glutaraldehyde, 0.5% crystalviolet). After washing cells with tap-water, the number of colonies (> 50 cells) were counted.
Lactate dehydrogenase (LDH) release assay
Cells (1×104 cells/well) were pre-incubated for 24 h in a 96-well culture plate and treated with 0, 10, 25 and 50 kBq/ml of [211At]-2-AAMP for 24 h. At the end of incubation, supernatants were collected and the LDH content was measured by using a Cytotoxicity Detection Kit (Roche Applied Sciences, Laval, Quebec, Canada). LDH release is expressed as percent of total content, which was determined by lysing an equal amount of cells with 1% Triton X-100.
DNA double-strand break (DSB) assay
Cells (1x106 cells/dish) treated with [211At]-2-AAMP (25 kBq/ml) for 24 h. Then neutral comet assay was applied to detect DNA-DSB using a CometAssay Kit (Trevigen, Gaithersburg, MD, USA) according to the manufacturer's instructions. Comet tails were stained with SYBR Green and analyzed using a fluorescent microscope.
Cells (5×106 cells/head) were implanted into the right thigh of the five-week-old female BALB/c nude mice (CLEA Japan). When palpable tumors developed, [211At]-2-AAMP (100 kBq) in 100 μl of PBS was intravenously injected. At selected time points after administration, the mice were euthanized, and the tissues of interest were dissected and weighed. The radioactivity was measured by a well-type g-counter. The uptake of [211At]-2-AAMP was expressed as a percentage of the injected dose per gram of organ.
Therapeutic study in vivo
Tumor-bearing mice were prepared in the same manner to biodistribution study. After tumor volumes had reached approximately 200 mm3, [211At]-2-AAMP therapy was conducted. PBS or [211At]-2-AAMP (2 MBq/head) was intravenously injected to the mice once at Day 0. The body weight and tumor size were measured at least twice a week for a month. Tumor volume (mm3) was calculated as (length×width2)/2. In the case of weight loss more than 20%, appearing moribund state signs, or the tumor size greater than 800 mm3, the mouse was euthanized humanely using isoflurane inhalation. Survival proportion was analyzed by Kaplan-Meier analysis (GraphPad Prism 6, Graph Pad Software, San Diego, CA, USA).
Results are expressed as mean±standard error (SEM). The statistical significance of differences between two groups was calculated using the unpaired Student's t-test. The significance of differences was determined by a one-way analysis of variance (ANOVA), followed by Dunnett’s test for multigroup comparisons. The survival curves for [211At]-2-AAMP treatment were compared with that for the control group using the log-rank test. The criterion of significance was P<0.05, as determined with GraphPad Prism 6 software.