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Objective We aimed to estimate in vivo 211At-labeled meta-benzylguanidine (211At-MABG) absorbed doses by the two dose conversion methods, using 131I-MIBG biodistribution data from a previously reported neuroblastoma xenograft model. Additionally, we examined the effects of different cell lines and time limitations using data from two other works. Methods We used the framework of the Monte Carlo method to create 3200 virtual experimental data sets of activity concentrations (kBq/g) to get the statistical information. Time activity concentration curves were produced using the fitting method of a genetic algorithm. The basic method was that absorbed doses of 211At-MABG were calculated based on the medical internal radiation dose formalism with the conversion of the physical half-life time of 131I to that of 211At. We have further improved the basic method, that is a novel dose conversion method, RAP (RAtio of Pharmacokinetics), using percent injected dose/g. Results Virtual experiments showed that 211At-MABG and 131I-MIBG had similar properties of initial activity concentrations and biological components, but the basic method did not simulate the 211At-MABG dose. Simulated 211At-MABG doses from 131I-MIBG using the RAP method were in agreement with those from 211At-MABG, so that their boxes overlapped in the box plots. The RAP method showed applicability to the different cell lines, but it was difficult to predict long-term doses from short-term experimental data. Conclusions The present RAP dose conversion method could estimate 211At-MABG absorbed doses from the pharmacokinetics of 131I-MIBG with some limitations. The RAP method would be applicable to a large number of subjects for targeted nuclide therapy.

The 211At-labeled compound, 4-[211At]astato-L-phenylalanine, is one of the most promising amino acid derivatives for use in targeted alpha therapy (TAT) for various cancers. Electrophilic demetallation of a stannyl precursor is the most widely used approach for labeling biomolecules with 211At. However, the low acid-resistance of the stannyl precursor necessitates the use of an N- and C- terminus-protected precursor, which results in a low overall radiochemical yield (RCY) due to the multiple synthetic steps involved. In this study, a deprotected organosilyl compound, 4-triethylsilyl-L-phenylalanine, was employed for the direct synthesis of astatinated phenylalanines. 211At was separately recovered from the irradiated 209Bi target using chloroform (CHCl3) and N-chlorosuccinimide-methanol (NCS-MeOH) solution. The RCYs of 4-[211At]astato-L-phenylalanine obtained from the triethylsilyl precursor with the use of 211At, isolated in CHCl3 and NCS-MeOH solution, were 75% and 64% respectively. In both cases, the retention time of the 4-[211At]astato-L-phenylalanine was found to be about 20 min, which showed reasonable correlation with the retention time of non-radioactive 4-halo-L-phenylalanines (4-chloro-, 4-bromo-, and 4-iodo-L-phenylalanine). The one-step reaction examined in this study involved mild reaction conditions (70 °C) and a short time (10 min) compared to the other currently reported procedures for astatination. Electrophilic desilylation was found to be very effective for the labeling of aromatic amino acids with 211At.

全身に転移したがんの治療法として、アルファ線を放出する放射性同位元素（RI）を組み込んだ薬剤あるいはRIそのものを体内に投与し、がん細胞に送り届ける、アルファ線放出RI内用療法が注目されている。我々は、アルファ線がん治療薬211At-MABGを用いた非臨床試験を進め、優れた抗腫瘍効果を明らかにしてきた。現在、211At-MABGの殺細胞メカニズムの解明に取り組んでいる。本発表では、RNAシーケンス解析により明らかとなった211At-MABGの殺細胞メカニズムと211At-MABG特異的な応答について報告する。, QST高崎サイエンスフェスタ2019

Targeted α-particle therapy is a promising option for patients with malignant pheochromocytoma. Recent observations of α-emitting meta-[At-211]-astato-benzylguanidine ([At-211]-MABG) in a pheochromocytoma mouse model showed a strong anti-tumor effect [1], though the molecular mechanism remained elusive. Here, we show the comprehensive RNA-sequencing (RNA-seq) data for rat pheochromocytoma cell line PC12 cells based on in vitro [At-211]-MABG administration experiments. Enrichment analysis of differentially expressed genes (DEGs) and analysis of the gene expression profiles of cell cycle checkpoints revealed the mode of cell death via the p53-p21 signaling pathway after [At-211]-MABG treatment. Only p53-targeted genes were ranked among representative DEGs for decreased survival at all time points. Also, we estimated the low fluence rate of α-particles in in vitro [At-211]-MABG administration, approximately “one α-particle emission per one cell in one hour” for 10% survival. Long-lasting p53-induced signaling may break the wall of the “low fluence rate” in PC12 tumor cell death to exert anti-tumor effects. We recently published these findings on RNA-seq of in vitro PC12 transcriptome, suggesting that p53-p21 signaling pathway was an important cell death mechanism in anti-tumor effect of [At-211]-MABG [2]. References: [1] Y. Ohshima et al., Eur J Nucl Med Mol Imaging, 2018. [2] Y. Ohshima et al., Theranostics, 2019, 日本放射線影響学会第62回大会

The astatine-211 (At-211) labeled compound, 4-[211At]astato-L-phenylalanine, is one of the most promising amino acid derivatives for use in targeted alpha therapy (TAT) for various cancers. Electrophilic demetallation of stannyl precursor is the most widely used approach for labeling biomolecules with At-211. However, the low acid-resistance of the stannyl precursor necessitates the use of a N- and C- terminus protected precursor, which causes low overall radiochemical yield (RCY) due to the multiple synthetic steps involved. A deprotected organosilyl compound, 4-triethylsilylphenylalanine, was employed for direct synthesis of the astatinated phenylalanine in this study., 11th International Symposium on Targeted-Alpha-Therapy (TAT11)

Sequencing empowers clinical diagnostics and therapy, but targeted alpha therapy has not yet fully received it. Here, we used RNA-sequencing to identify genes relevant to induced anti-tumor alpha-particle radiation response compared with 60Co γ-rays. The gene expression changes of PC12 cells were observed at 3, 6 and 12 h after 211At-MABG treatment and 60Co γ-rays irradiation, controlling dose of irradiation at survival levels of 10% and 80%. Minimum of 7 million reads were sequenced for each condition. Overall transcriptome profiles were strikingly distinct, but important gene expression changes of 10% survival for cell-cycle checkpoints finally reached the same level with a time delay. We now explore possible therapy or molecular imaging target genes for malignant pheochromocytoma., 第5回セラノスティクス国際会議(Theranostics2019)への出席

Astatine-211 (At-211) labeled phenylalanine were directly synthesized from an organosilylprecursor, (p-trimethylsilyl)phenylalanine, as a feasible study for the targeted alpha therapy (TAT) using amino acid derivatives. At-211 was isolated by dry distillation and then eluted with chloroform (CHCl3) or methanol-N-chlorosuccinimide solution (MeOH-NCS). After evaporating At-211 eluent, the mixture of the organosilylprecursor, NCS, and At-211 was heated at 70 oC in trifluoroacetic acid for 10 min. HPLC analysis demonstrated that a single radioactive peak was observed at retention time identical to that of the corresponding radioiodinated phenylalanine. Radiochemical yield (RCY) of the astatinated phenylalanine was 75% (CHCl3) and 64% (MeOH-NCS), respectively. These results clearly showed that the desired compound was successfully synthesized from an organosilylprecursor., 第58回日本核医学会学術総会

Sequencing empowers clinical diagnostics and therapy, but targeted alpha therapy has not yet fully received it. Recently, we observed strong anti-tumor effect of 211At-MABG in a pheochromocytoma mouse model. Here, we used RNA-sequencing to identify genes relevant to induced anti-tumor alpha-particle radiation response compared with 60Co γ-rays. The gene expression changes of PC12 cells were observed at 3, 6 and 12 h after 211At-MABG treatment and 60Co γ-rays irradiation, controlling dose of irradiation at survival levels of 10% and 80%. Minimum of 7 million reads were sequenced for each condition. Overall transcriptome profiles were strikingly distinct, but important gene expression changes of 10% survival for cell-cycle checkpoints finally reached the same level with a time delay. We now explore possible therapy or molecular imaging target genes for malignant pheochromocytoma., 第58回日本核医学会学術総会

Embryonic Stem Cells (ESC) possesses two distinct features; self-renewal and the potential to differentiate. Here we show the differentiation potential and growth rate of ESC correlates positively with the expression level of the gene encoding chromodomain helicase DNA binding protein 7 (CHD7). When ESCs are maintained in feeder-free conditions and single cell seeding, ESC KhES-1 having 4520 copies or more of CHD7 in 5 ng total RNA show differentiation potential, but this is lost when the CHD7 copy number is reduced in KhES-1 to less than 696 by alternative culture conditions. Introduction of siCHD7 reduced differentiation potential and growth rate of KhES-1. Interestingly, KhES-1 underwent spontaneous differentiation when mCHD7 was introduced and we could not obtain CHD7-overexpressing ESC in culture. These data suggest that CHD7 drives differentiation, and there is a lower limit for CHD7 to initiate differentiation and an upper limit for CHD7 if maintained in undifferentiated state, and such upper limit varies depending on culture condition. As CHD7 drives cell growth, ESC with the highest permissible CHD7 level in the given culture become dominant in a couple of passages. Thus, we can select differentiation resistance-free cell clones by optimizing the culture system using CHD7 as an index.

Objectives: Therapeutic options for patients with malignant pheochromocytoma are currently limited, and therefore new treatment approaches are being sought. Alpha-emitting radiopharmaceutical meta-211At-astatobenzylguanidine (211At-MABG) has potential as a malignant pheochromocytoma treatment. We previously reported the effects of using 30 µCi of 211At-MABG on tumor growth suppression in a pheochromocytoma mouse model. However, the histological impacts of 211At-MABG on tumor cells have not been evaluated. Therefore, the purpose of this study was to evaluate the histological changes associated with and therapeutic efficacy of various doses of 211At-MABG in the mouse pheochromocytoma model. Methods: Rat pheochromocytoma (PC-12) cells were subcutaneously inoculated into female nude mice. When tumor volumes reached approximately 50 mm3, mice bearing PC-12 tumors received 0 (saline), 15, 30, 50, 100 and 150 µCi of 211At-MABG (n = 5 each dose). The tumor volume and body weight were measured twice weekly for 8 weeks. Tumors and organs of interest were resected 8 weeks after 211At-MABG administration or when the mice were humanely euthanized in cases of excessive weight reduction. The tumor volume, tumor volume rate and body weight data were analyzed by ANOVA with the Student's t-test. For histological analysis of temporal change, tumors and organs of another group treated with 30 µCi of 211At-MABG were resected on days 1, 3 and 7. The histological sections of tumors and organs were stained with hematoxylin and eosin. Results: The control group injected with saline (0 µCi) had significantly increased tumor volume after 3 weeks (48.9 ± 7.7 mm3 to 296.1 ± 56.2 mm3, P < 0.01). In contrast, the 211At-MABG treatment group had significantly reduced tumor volumes 3 weeks after 211At-MABG administration (15 µCi: 48.9 ± 7.8 mm3 to 4.5 ± 2.3 mm3, P, SNMMI2017

Lens-free inline Holographic Microscopy (LHM) holds great promise for biomedical and industrial applications thanks to its conceptual simplicity. However, the challenge lies in achieving an image quality comparable to conventional microscopes. We demonstrate a high-throughput LHM system that is able to resolve 1.23μm-thin lines on a standard USAF 1951 test target with 1.67μm pixels at the full field-of-view (>29mm 2 ). The system is based on a unique multiwavelength iterative-phase-retrieval method, using customized hardware and real-time post-processing software. We have evaluated our system in experiments ranging from single-cell inspection to in-vitro imaging of stem-cell colonies.