Preclinical evaluation of new α-radionuclide therapy targeting LAT1: 2-[211At]astato-α-methyl-L-phenylalanine in tumor-bearing model CURRENT STATUS: POSTED

PURPOSE: Targeted α-radionuclide therapy has growing attention as a promising therapy for refractory cancers. However, the application is limited to certain types of cancer. Since L-type amino acids transporter 1 (LAT1) is highly expressed in various human cancers, we prepared LAT1-selective α-emitting amino acid analog, 2-[211At]astato-α-methyl-L-phenylalanine ([211At]-2-AAMP), and evaluated its potential as a therapeutic agent. METHODS: [211At]-2-AAMP was prepared from the stannyl precursor. Stability of [211At]-2-AAMP was evaluated by both in vitro and in vivo. In vitro studies using LAT1 expressing human ovary cancer cell line, SKOV3, were performed for evaluating cellular uptake and cytotoxicity of [211At]-2-AAMP. Biodistribution and therapeutic studies in SKOV3 bearing mice were performed after intravenous injection of [211At]-2-AAMP. RESULTS: [211At]-2-AAMP was stable in murine plasma in vitro and excreted into urine as intact. Cellular uptake of [211At]-2-AAMP was inhibited by treatment with LAT1-selective inhibitor. After 24 hours of incubation, [211At]-2-AAMP suppressed clonogenic growth at 10 kBq/ml, and induced cell death and DNA double strand break at 25 kBq/ml. When injected to mice, [211At]-2-AAMP exhibited the peak accumulation in the tumor at 30 min postinjection, and the radioactivity levels in the tumor retained up to 60 min. The majority of the radioactivity was rapidly eliminated from the body into the urine as an intact form immediately after injection. [211At]-2-AAMP significantly improved the survival of mice (P<0.05) without serious side effects. CONCLUSION: [211At]-2-AAMP showed α-radiation-dependent cellular growth inhibition after taking up via LAT1. Furthermore, [211At]-2-AAMP provided a beneficial effect on survival in vivo. These findings suggest that [211At]-2-AAMP might be useful for treatment of LAT1-positive significantly improved survival rates of mice in vivo. These results suggest that [ 211 At]-2-AAMP is possibly beneficial for treatment of LAT1-positive cancer. However, significant delay or reduction of tumor size was not observed by treatment with [ 211 At]-2-AAMP in vivo partially because small number of mice. Since the body weight loss after 2 MBq of [ 211 At]-2-AAMP injection was not large and the present administration dose did not reach the maximum tolerated dose, suggesting that higher doses would be safely administered to


Introduction
Targeted radionuclides therapy with α-emitters has drawn global attention. Linear energy transfer (LET) of α-particle is approximately 80 keV/μm, and thus α-radiation is considered to be high-LET radiation [1,2]. According to the lots of studies with external beam irradiation, high-LET radiation can induce irreparable DNA double-strand breaks and effectively lead cell death compared to low-LET 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 [ 211 At]-2-AAMP for 10 min. After 2 times washes with HBSS, cells were incubated in HBSS at 37 o C for 10, 20, 30, and 60 min. Then, supernatant was collected, and the radioactivity was measured with a well-type g-counter.

Colony assay
Cells (1×10 6 cells/dish) were pre-incubated in the growth medium for 24 h, and treated with 0, 10, 25, and 50 kBq/ml of [ 211 At]-2-AAMP for 24 h. After [ 211 At]-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 37 o C 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.
Competitive inhibition of [ 211 At]-2-AAMP with various inhibitors showed that the uptake of [ 211 At]-2-AAMP was significantly inhibited by treatment with AMT, a selective inhibitor of LAT1, as well as substrates of LAT1, such as branched-chain amino acids and aromatic amino acids (Fig. 3d). As shown in Fig. 3e, [ 211 At]-2-AAMP was gradually released to extracellular space, and the released fraction reached more than 90% within 60 min after exchanging the buffer.  Table 1

Therapeutic effects in tumor-bearing mice
In our preliminary examination with small number of mice, weight loss more than 20% was not However, there was no significant difference of tumor volume between 2 groups ( Fig. 5a and b). Body weight was transiently reduced after treatment with [ 211 At]-2-AAMP ( Fig. 5c and d). The peak of weight loss was at 3 days after injection, and the maximum reduction was 14.5%. Kaplan-Meyer survival analysis showed that the survival of mice was significantly improved with [ 211 At]-2-AAMP treatment (Fig. 6, P<0.05).

Discussion
In the present study, we newly synthesized [ 211 At]-2-AAMP and evaluated its potential as an α- and its therapeutic effect in vivo [17,18]. However, L-phenylalanine is a substrate for not only LAT1, but also LAT2 [19]. In this regard, this is the first report of a LAT1-specific 211 At-labeled amino acid derivative. In general, astatinated aryl compounds can be prepared by the procedure similar to be used for radioiodinated and radiobrominated compounds. in pancreas compared to the other organs [21]. No significant uptake of [ 123 I]-3-iodo or [ 18 F]-3-fluoroα-methyltyrosine, other LAT1 specific tracers, was not observed in the human pancreas [22,23].
Therefore, the high distribution in the pancreas will not be expected in patients. Dehalogenation of