1-(N,N-Dialkylcarbamoyl)-1,1-difluoromethanesulfonyl ester as a stable and effective precursor for a neopentyl labeling group with astatine-211.

Radiohalogens with a short half-life are useful radioisotopes for radiotheranostics. Astatine-211 is an α-emitting radiohalogen and is expected to be applicable to targeted α therapy. A neopentyl labeling group is an effective hydrophilic labeling unit for various radiohalogens, which includes 211At. In this study, a 1-(N,N-dialkylcarbamoyl)-1,1-difluoromethanesulfonyl (CDf) ester was developed as a stable precursor for labeling with 211At, 77Br and 125I through a neopentyl labeling group. The CDf ester remained stable in an acetonitrile solution at room temperature and enabled the successful syntheses of 211At-labeled compounds in a highly radiochemical conversion in the presence of K2CO3. 77Br- and 125I-labeled compounds can be prepared from the CDf ester without a base. The utility of the CDf ester was demonstrated in the synthesis of a benzylguanidine with a neopentyl 211At-labeling group. The developed method afforded a 32% radiochemical yield of 211At-labeled benzylguanidine. However, a partial deastatination was observed under acidic conditions during the removal of an N-Boc protecting group. Deprotecting these groups under milder acidic conditions may improve the radiochemical yield. In conclusion, the CDf ester facilitates the syntheses of 211At, 125I and 77Br-labeled compounds that use a neopentyl labeling group for radiotheranostic applications. Further optimization of protecting groups and reaction conditions should enhance the total radiochemical yield of the 211At-labeled compounds.

Radioactive isotope separation with 3D-printed flow-based device.

Radioactive isotope (RI) metals are a new type of tracer for positron emission tomography generated from the target metal by proton irradiation using a cyclotron. The generated metal RIs need to be separated from the target metal rapidly and effectively. In the present study, we developed a 3D-printed flow device to separate metal RIs from target metals. The separation was performed with selective formation of ethylenediaminetetraacetic acid (EDTA) complex based on the difference in formation constants. The RI metal selectively formed a EDTA complex, thus changing its ionic charge in solution. The solution was then introduced into a cation exchange column for selective adsorption of the target metal. The solution with added chelator and controlled pH was introduced into the developed system and automatically separated metal RI from target metals within 14 min. The separation method was applied to separate RI 67Ga from target metal Zn using a mixture of 107 pg L-1 67Ga in 250 mg L-1 Zn2+. The recoveries of 67Ga and Zn were 97% and 100%, respectively. Furthermore, an ultraviolet (UV) radiation reactor was integrated into the system to decompose the EDTA complex and recover the Ga3+ ion. Ga3+ recovery by UV radiation was effective, 87%. The developed system was also successfully applied to the separation of Zr and Y. Therefore, the method and system can be applied to separate other metal RIs from target metals.

Comparative evaluation of radionuclide therapy using 90Y and 177Lu.

OBJECTIVE: Both 90Y and 177Lu are attractive ß-emitters for radionuclide therapy and have been used in clinical practice. Nevertheless, comparative evaluation between 90Y- and 177Lu-labeled molecules has not been fully conducted. Thus, in this study, the features of 90Y and 177Lu for radionuclide therapy were assessed in tumor-bearing mice. METHODS: Two tumor cell lines with different growth rates were used. Biodistribution studies of 177Lu-labeled antibodies (177Lu-Abs) were conducted in each tumor-bearing mouse model. Subsequently, the therapeutic effect of 90Y- and 177Lu-Ab were assessed in tumor-bearing mice. The absorbed radiation dose for the tumor was estimated using the Monte Carlo simulation. RESULTS: 177Lu-Abs demonstrated high tumor accumulation in both tumor-xerograph. In the fast-growing tumor model, 90Y-Ab showed a better therapeutic effect than 177Lu-Ab, reflecting a higher absorbed radiation dose of 90Y-Ab than that of 177Lu-Ab. In the slow-growing tumor model, both 90Y- and 177Lu-Ab showed an excellent therapeutic effect; however, 177Lu-Ab had a longer efficacy period than 90Y-Ab, which could be attributed to the longer half-life and better dose uniformity of 177Lu than those of 90Y. CONCLUSIONS: To accomplish a maximum therapeutic effect, selecting 90Y or 177Lu, to depend on the growth rate of individual cancer, would be helpful.

Copper-mediated radioiodination and radiobromination via aryl boronic precursor and its application to 125I/77Br-labeled prostate-specific membrane antigen imaging probes.

Prostate-specific membrane antigen (PSMA), expressed in prostate cancer cells, is being investigated extensively worldwide as a target for imaging and therapy of prostate cancer. Various radioiodinated PSMA imaging probes have been developed, and their structure has a peptidomimetic urea-based skeleton as a pharmacophore. For direct radioiodination of molecules containing these peptidomimetic structures, prior studies performed radioiododestannylation or electrophilic radioiodination of tyrosine residues. However, although these radiolabeling methods are frequently used, there are some issues with precursor toxicity and by-product production. Therefore, it is required to investigate a radiolabeling method that can be used for the radiosynthesis of radioiodinated PSMA imaging probes with urea-based peptidomimetic structures. We recently reported that copper-mediated radioiodination via a boronic precursor is an effective method for directly labeling a peptide. This radiohalogenation method was expected to be an effective method for radiosynthesis of PSMA imaging probes with a peptidomimetic structure. In this study, to confirm that this labeling method applies to the synthesis of the PSMA imaging probe, we synthesized PSMA imaging probes labeled with 125I and 77Br ([125I]mIB-PS and [77Br]mBrB-PS) using a copper-mediated radiohalogenation via common boronic precursors and investigated optimal boronic precursor and labeling conditions. As a result, the radiochemical yields of [125I]mIB-PS and [77Br]mBrB-PS were improved to > 93% at room temperature by optimizing the structure of the boronic precursor. We demonstrate that copper-mediated nucleophilic radiochemistry using a boronic precursor is a promising radiosynthetic method of PSMA imaging probes. Although we focused on the synthesis of PSMA imaging probes, the results in this study will also be useful for the synthesis of various radioiodine or radiobromine-labeled bioactive molecules.

Organic cation transporter 3 mediates the non-norepinephrine transporter driven uptake of meta-[211At]astato-benzylguanidine.

INTRODUCTION: Meta-[211At]astato-benzylguanidine ([211At]MABG) accumulates in pheochromocytoma via norepinephrine transporter (NET) and leads to a strong antitumor effect, but it also distributed in normal tissues non-specifically. Meta-[131I]iodo-benzylguanidine ([131I]MIBG), an iodine-labeled analog of [211At]MABG, is known to be transported by not only NET but also organic cation transporter (OCT). The involvement of OCT in [211At]MABG uptake is still largely unknown. We investigated the involvement of OCT in the non-NET-driven uptake of [211At]MABG both in vitro and in vivo. METHODS: [123I]MIBG and [211At]MABG uptake was investigated in PC-12 (rat pheochromocytoma cell line), NIH/3T3 (mouse fibroblasts cell line), ACHN (human renal cancer cell line), and BxPC-3 (human pancreatic cancer cell line). Herein, we used desipramine and dl-norepinephrine to inhibit NET, and we used steroids (hydrocortisone and prednisolone) to inhibit OCT3. The [211At]MABG uptake in OCT3-knockdown cells established with OCT3-selective siRNA was also investigated. We investigated the biodistribution of [211At]MABG in PC-12 tumor-bearing mice after a preloading of phosphate-buffered saline (PBS) or hydrocortisone solution. RESULTS: The uptake of both [123I]MIBG and [211At]MABG was significantly inhibited by desipramine in PC-12 cells but not the other cell lines. The expression of OCT3 was relatively higher than those of the other OCT subtypes in ACHN and BxPC-3 cells. The expression of OCTs was not observed in NIH/3T3 cells. The uptake of both [123I]MIBG and [211At]MABG in ACHN and BxPC-3 cells was significantly inhibited by the steroid treatments. The [211At]MABG uptake was also reduced in OCT3-knockdown cells (p < 0.001). The radioactivity of [211At]MABG was significantly reduced in normal tissues by the preloading of hydrocortisone. In contrast, there was an increasing trend of [211At]MABG uptake in the PC-12 tumors. The tumor-to-normal tissue ratio was significantly increased by the preloading of hydrocortisone compared to that of PBS. CONCLUSION: Our results suggest that OCT3 is involved in non-NET-driven [211At]MABG uptake. The preloading of hydrocortisone selectively reduced [211At]MABG accumulation in normal organs in vivo. OCT3 inhibition may therefore be beneficial for a reduction of the radiation risk in healthy organs in the treatment of malignant pheochromocytomas.

Highly Efficient Separation of Ultratrace Radioactive Copper Using a Flow Electrolysis Cell.

Preparing compounds containing the radioisotope 64Cu for use in positron emission tomography cancer diagnostics is an ongoing area of research. In this study, a highly efficient separation method to recover 64Cu generated by irradiating the target 64Ni with a proton beam was developed by employing a flow electrolysis cell (FE). This system consists of (1) applying a reduction potential for the selective adsorption of 64Cu from the target solution when dissolved in HCl and (2) recovering the 64Cu deposited onto the carbon working electrode by desorbing it from the FE during elution with 10 mmol/L HNO3, which applies an oxidation potential. The 64Cu was selectively eluted at approximately 30 min under a flow rate of 0.5 mL/min from the injection to recovery. The newly developed flow electrolysis system can separate the femtomolar level of ultratrace radioisotopes from the larger amount of target metals as an alternative to conventional column chromatography.

Nonclinical study and applicability of the absorbed dose conversion method with a single biodistribution measurement for targeted alpha-nuclide therapy.

BACKGROUND: We recently reported a new absorbed dose conversion method, RAP (RAtio of Pharmacokinetics), for 211At-meta-astatobenzylguanidine (211At-MABG) using a single biodistribution measurement, the percent injected dose/g. However, there were some mathematical ambiguities in determining the optimal timing of a single measurement of the percent injected dose/g. Thus, we aimed to mathematically reconstruct the RAP method and to examine the optimal timing of a single measurement. METHODS: We derived a new formalism of the RAP dose conversion method at time t. In addition, we acquired a formula to determine the optimal timing of a single measurement of the percent injected dose/g, assuming the one-compartment model for biological clearance. RESULTS: We investigated the new formalism's performance using a representative RAP coefficient with radioactive decay weighting. Dose conversions by representative RAP coefficients predicted the true [211At]MABG absorbed doses with an error of 10% or less. The inverses of the representative RAP coefficients plotted at 4 h post-injection, which was the optimal timing reported in the previous work, were very close to the new inverses of the RAP coefficients 4 h post-injection. Next, the behavior of the optimal timing was analyzed by radiolabeled compounds with physical half-lives of 7.2 h and 10 d on various biological clearance half-lives. Behavior maps of optimal timing showed a tendency to converge to a constant value as the biological clearance half-life of a target increased. The areas of optimal timing for both compounds within a 5% or 10% prediction error were distributed around the optimal timing when the biological clearance half-life of a target was equal to that of the reference. Finally, an example of RAP dose conversion was demonstrated for [211At]MABG. CONCLUSIONS: The RAP dose conversion method renovated by the new formalism was able to estimate the [211At]MABG absorbed dose using a similar pharmacokinetics, such as [131I]MIBG. The present formalism revealed optimizing imaging time points on absorbed dose conversion between two radiopharmaceuticals. Further analysis and clinical data will be needed to elucidate the validity of a behavior map of the optimal timing of a single measurement for targeted alpha-nuclide therapy.

Rapid Flow-Based System for Separation of Radioactive Metals by Selective Complex Formation.

Short-lived radioactive metals are important tracers in clinical diagnosis. Radioactive metals for clinical use are produced from suitable target metals in cyclotrons. The trace amount of radioactive metal produced is contained in a relatively large amount of target metal. A rapid and effective method is required to isolate the radioactive metal. In the present study, selective complex formation followed by cation-exchange adsorption was performed in a continuous flow-based system. Ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA) was selected as the ligand after simulation of the separation of radioactive Ga from the target (Zn). Selectively, the Ga-EDTA complex passed through the cation trap, while Zn2+ was trapped. This separation principle is opposite to that of typical solid-phase extraction, which captures the target ion. The proposed separation was performed in a flow-based system with a parallel, open-channel ion trap. The performance was optimized by altering the channel dimensions, channel-filling mesh, and flow rate. Finally, the target radioactive metal, Ga, was selectively and effectively (>99%) separated from a mixture of 50 fg Ga/L and 100 mg Zn/L. The concentration of Zn remaining in the Ga solution was 2.3 µg/L. The complexed Ga was converted to free Ga3+ by a simple UV irradiation method. The proposed method effectively and rapidly separates trace amounts of radioactive metals contained in larger amounts of target metals using a simple flow system that can be operated on site.

Enhancing the Therapeutic Effect of 2-211At-astato-α-methyl-L-phenylalanine with Probenecid Loading.

L-type amino acid transporter 1 (LAT1) might be a useful target for tumor therapy since it is highly expressed in various types of cancers. We previously developed an astatine-211 (211At)-labeled amino acid derivative, 2-211At-astato-α-methyl-L-phenylalanine (2-211At-AAMP), and demonstrated its therapeutic potential for LAT1-positive cancers. However, the therapeutic effect of 2-211At-AAMP was insufficient, probably due to its low tumor retention. The preloading of probenecid, an organic anion transporter inhibitor, can delay the clearance of some amino acid tracers from the blood and consequently increase their accumulation in tumors. In this study, we evaluated the effect of probenecid preloading on the biodistribution and therapeutic effect of 2-211At-AAMP in mice. In biodistribution studies, the blood radioactivity of 2-211At-AAMP significantly increased with probenecid preloading. Consequently, the accumulation of 2-211At-AAMP in tumors was significantly higher with probenecid than without probenecid loading. In a therapeutic study, tumor growth was suppressed by 2-211At-AAMP with probenecid, and the tumor volume was significantly lower in the treatment group than in the untreated control group from day 2 to day 30 (end of the follow-up period) after treatment. These results indicate that probenecid loading could improve the therapeutic effect of 2-211At-AAMP by increasing its accumulation in tumors.

Neopentyl Glycol as a Scaffold to Provide Radiohalogenated Theranostic Pairs of High In Vivo Stability.

The high in vivo stability of 2,2-dihydroxymethyl-3-[18F]fluoropropyl-2-nitroimidazole ([18F]DiFA) prompted us to evaluate neopentyl as a scaffold to prepare a radiotheranostic system with radioiodine and astatine. Three DiFA analogues with one, two, or without a hydroxyl group were synthesized. While all 125I-labeled compounds remained stable against nucleophilic substitution, only a 125I-labeled neopentyl glycol was stable against cytochrome P450 (CYP)-mediated metabolism and showed high stability against in vivo deiodination. 211At-labeled neopentyl glycol also remained stable against both nucleophilic substitution and CYP-mediated metabolism. 211At-labeled neopentyl glycol showed the biodistribution profiles similar to those of its radioiodinated counterpart in contrast to the 125I/211At-labeled benzoate pair. The urine analyses confirmed that 211At-labeled neopentyl glycol was excreted in the urine as a glucuronide conjugate with the absence of free [211At]At-. These findings indicate that neopentyl glycol would constitute a promising scaffold to prepare a radiotheranostic system with radioiodine and 211At.

Re-Evaluations of Zr-DFO Complex Coordination Chemistry for the Estimation of Radiochemical Yields and Chelator-to-Antibody Ratios of 89Zr Immune-PET Tracers.

(1) Background: Deferoxamine B (DFO) is the most widely used chelator for labeling of zirconium-89 (89Zr) to monoclonal antibody (mAb). Despite the remarkable developments of the clinical 89Zr-immuno-PET, chemical species and stability constants of the Zr-DFO complexes remain controversial. The aim of this study was to re-evaluate their stability constants by identifying species of Zr-DFO complexes and demonstrate that the stability constants can estimate radiochemical yield (RCY) and chelator-to-antibody ratio (CAR). (2) Methods: Zr-DFO species were determined by UV and ESI-MS spectroscopy. Stability constants and speciation of the Zr-DFO complex were redetermined by potentiometric titration. Complexation inhibition of Zr-DFO by residual impurities was investigated by competition titration. (3) Results: Unknown species, ZrHqDFO2, were successfully detected by nano-ESI-Q-MS analysis. We revealed that a dominant specie under radiolabeling condition (pH 7) was ZrHDFO, and its stability constant (logß111) was 49.1 ± 0.3. Competition titration revealed that residual oxalate inhibits Zr-DFO complex formation. RCYs in different oxalate concentration (0.1 and 0.04 mol/L) were estimated to be 86% and >99%, which was in good agreement with reported results (87%, 97%). (4) Conclusion: This study succeeded in obtaining accurate stability constants of Zr-DFO complexes and estimating RCY and CAR from accurate stability constants established in this study.

Absorbed dose simulation of meta-211At-astato-benzylguanidine using pharmacokinetics of 131I-MIBG and a novel dose conversion method, RAP.

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. In addition, 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.

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

INTRODUCTION: Targeted α-radionuclide therapy has attracted attention as a promising therapy for refractory cancers. However, the application is limited to certain types of cancer. Since L-type amino acid transporter 1 (LAT1) is highly expressed in various human cancers, we prepared an LAT1-selective α-radionuclide-labeled amino acid analog, 2-[211At]astato-α-methyl-L-phenylalanine (2-[211At]AAMP), and evaluated its potential as a therapeutic agent. METHODS: 2-[211At]AAMP was prepared from the stannyl precursor. Stability of 2-[211At]AAMP was evaluated both in vitro and in vivo. In vitro studies using an LAT1-expressing human ovarian cancer cell line, SKOV3, were performed to evaluate cellular uptake and cytotoxicity of 2-[211At]AAMP. Biodistribution and therapeutic studies in SKOV3-bearing mice were performed after intravenous injection of 2-[211At]AAMP. RESULTS: 2-[211At]AAMP was stable in murine plasma in vitro and excreted intact into urine. Cellular uptake of 2-[211At]AAMP was inhibited by treatment with an LAT1-selective inhibitor. After 24 h incubation, 2-[211At]AAMP suppressed clonogenic growth at 10 kBq/ml, and induced cell death and DNA double-strand breaks at 25 kBq/ml. When injected into mice, 2-[211At]AAMP exhibited peak accumulation in the tumor at 30 min postinjection, and radioactivity levels in the tumor were retained up to 60 min. The majority of the radioactivity was rapidly eliminated from the body into urine in an intact form immediately after injection. 2-[211At]AAMP significantly improved the survival of mice (P < 0.05) without serious side effects. CONCLUSION: 2-[211At]AAMP showed α-radiation-dependent cellular growth inhibition after it was taken up via LAT1. In addition, 2-[211At]AAMP had a beneficial effect on survival in vivo. These findings suggest that 2-[211At]AAMP would be useful for the treatment of LAT1-positive cancer. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: This is the first report of an LAT1-targeting radiopharmaceutical for α-radionuclide therapy; this agent would be applicable for the treatment of various types of cancer.

Electrodialytic Handling of Radioactive Metal Ions for Preparation of Tracer Reagents.

Radioactive metals are applied in biochemistry, medical diagnosis such as positron emission tomography (PET), and cancer therapy. However, the activity of radioisotopes exponentially decreases with time; therefore, rapid and reliable probe preparation methods are strongly recommended. In the present study, electrodialytic radioactive metal ion handling is studied for counter ion conversion and in-line probe synthesis. Presently, counter ion conversion and probe synthesis are achieved by evaporative dryness and solution mixing, respectively. Evaporative dryness is time-consuming and is a possible process that can lead to loss of radioactive metal ions. Mixing of solutions for synthesis makes dilution and undesirable effects of counter ion on the synthesis. An optimized electrodialytic flow device can transfer a radioisotope, 64Cu2+, with high recovery from HCl matrices to HNO3 (∼100%). Matrices can also be transferred into acetic acid and citric acid, even though the concentration of the metal ion is at the picomolar level. The ion transfer can also be achieved with simultaneous counter ion conversion, complex synthesis, and enrichment. When the ligand was dissolved in an acceptor solution, the transferred metal ions from the donor were well mixed and formed a complex with the ligand in-line. The efficiency of the synthesis was ∼100% for 1.0 pM 64Cu. A relatively larger donor-to-acceptor flow rate can enrich the metal ion in the acceptor solution continuously. The flow rate ratio of 10 (donor/acceptor) can achieve 10 times enrichment. The present electrodialytic ion handling method can treat ultra-trace radioisotopes in a closed system. With this method, rapid, effective, and safe radioisotope treatments were achieved.

Novel 18F-Labeled α-Methyl-Phenylalanine Derivative with High Tumor Accumulation and Ideal Pharmacokinetics for Tumor-Specific Imaging.

Positron emission tomography (PET) imaging with 18F-labeled α-methyl-substituted amino acids exerts significant influence on differential diagnosis of malignant tumors and tumor-like lesions. Exclusive uptake via L-type amino acid transporter 1 (LAT1), a tumor-specific transporter, accounts for their excellent tumor specificity and low background accumulation. However, further refinement and optimization in their tumor accumulation and pharmacokinetics are sorely needed. To address these issues, we newly designed 18F-labeled α-methyl-phenylalanine (18F-FAMP) regioisomers (2-, 3-, or 4-18F-FAMP) and stereoisomers (L- or D-form), and we comprehensively evaluated their potential as tumor-imaging agents. 18F-FAMPs were prepared from α-methyl phenylalanine by electrophilic radiofluorination and purified by reversed-phase HPLC. In biodistribution studies on normal mice, L-2-18F-FAMP and the three D-18F-FAMPs showed faster blood clearance and lower renal accumulation than L-3-18F-FAMP or L-4-18F-FAMP. In LS180 human colorectal cancer cell line xenograft mice, L-2-18F-FAMP exhibited significantly higher tumor accumulation than the D-18F-FAMPs or a clinically relevant tracer, L-3-18F-α-methyl-tyrosine (18F-FAMT) (p < 0.05). The renal accumulation levels of L-2-18F-FAMP were significantly lower than that of 18F-FAMT (p < 0.01). LAT-1 specificity of L-2-18F-FAMP was validated in the cellular uptake studies. The PET imaging with L-2-18F-FAMP clearly visualized the tumor as early as 1 h after injection, and the high tumor accumulation level was retained for 3 h. These findings suggest that L-2-18F-FAMP constitutes a potential PET tracer for tumor-specific imaging.

Anti-tumor effects and potential therapeutic response biomarkers in α-emitting meta-211At-astato-benzylguanidine therapy for malignant pheochromocytoma explored by RNA-sequencing.

Targeted α-particle therapy is a promising option for patients with malignant pheochromocytoma. Recent observations regarding meta-211At-astato-benzylguanidine (211At-MABG) in a pheochromocytoma mouse model showed a strong anti-tumor effect, though the molecular mechanism remains elusive. Here, we present the first comprehensive RNA-sequencing (RNA-seq) data for pheochromocytoma cells based on in vitro211At-MABG administration experiments. Key genes and pathways in the tumor α-particle radiation response are also examined to obtain potential response biomarkers. Methods: We evaluated genome-wide transcriptional alterations in the rat pheochromocytoma cell line PC12 at 3, 6, and 12 h after 211At-MABG treatment; a control experiment using 60Co γ-ray irradiation was carried out to highlight 211At-MABG-specific gene expression. For comparisons, 10% and 80% iso-survival doses (0.8 and 0.1 kBq/mL for 211At-MABG and 10 and 1 Gy for 60Co γ-rays) were used. Results: Enrichment analysis of differentially expressed genes (DEGs) and analysis of the gene expression profiles of cell cycle checkpoints revealed similar modes of cell death via the p53-p21 signaling pathway after 211At-MABG treatment and γ-ray irradiation. The top list of ranked DEGs demonstrated the expression of key genes on the decrease in the survival following 211At-MABG exposure, and four potential genes (Mien1, Otub1, Vdac1 and Vegfa genes) of 211At-MABG therapy. Western blot analysis indicated increased expression of TSPO in 211At-MABG-treated cells, suggesting its potential as a PET imaging probe. Conclusion: Comprehensive RNA-seq revealed contrasting cellular responses to γ-ray and α-particle therapy, leading to the identification of four potential candidate genes that may serve as molecular imaging and 211At-MABG therapy targets.

Preclinical Evaluation of the Acute Radiotoxicity of the α-Emitting Molecular-Targeted Therapeutic Agent 211At-MABG for the Treatment of Malignant Pheochromocytoma in Normal Mice.

The α-emitter 211At-labeled meta-astatobenzylguanidine (211At-MABG) has a strong antitumor effect on pheochromocytoma xenograft tumors and holds great promise as a new therapeutic option for malignant pheochromocytoma. To evaluate the acute radiation-related toxicity of 211At-MABG, we conducted biodistribution and dosimetry studies of 211At-MABG in ICR mice to estimate the doses absorbed by organs. We determined the maximum tolerated doses (MTD) of 211At-MABG on the basis of body weight loss and assessed the acute radiation-related toxicity induced by MTD administration on the basis of organ weights, histologic features, hematologic indices, and biochemical indices. The biodistribution and dosimetry studies of α-emitting 211At-MABG revealed high doses absorbed by most organs except the brain in ICR mice. The administration of 1.1, 2.2, and 3.3 MBq of 211At-MABG induced transient body weight loss, and 4.4 MBq of 211At-MABG induced unrecoverable body weight loss; thus, the MTD was 3.3 MBq for ICR mice. Although by day 5 the administration of 3.3 MBq had induced some radiation-related toxicity symptoms-such as body weight loss and leucopenia, which are generally observed in radiation therapy including ß--emitting radiopharmaceuticals-the mice had recovered by day 28. We observed no unexpected severe toxicity in ICR mice despite the high absorbed doses in most organs, especially the thyroid, heart, stomach, and adrenal glands. Our findings suggest that therapeutic treatments with appropriate doses of 211At-MABG estimated by dosimetry in each patient could be tolerated, although lower doses may initially be necessary to ensure patient safety in the first-in-human study.

A convenient and reproducible method for the synthesis of astatinated 4-[211At]astato-l-phenylalanine via electrophilic desilylation.

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.

Astatine-211 imaging by a Compton camera for targeted radiotherapy.

Astatine-211 is a promising radionuclide for targeted radiotherapy. It is required to image the distribution of targeted radiotherapeutic agents in a patient's body for optimization of treatment strategies. We proposed to image 211At with high-energy photons to overcome some problems in conventional planar or single-photon emission computed tomography imaging. We performed an imaging experiment of a point-like 211At source using a Compton camera, and demonstrated the capability of imaging 211At with the high-energy photons for the first time.

Antitumor effects of radionuclide treatment using α-emitting meta-211At-astato-benzylguanidine in a PC12 pheochromocytoma model.

PURPOSE: Therapeutic options for patients with malignant pheochromocytoma are currently limited, and therefore new treatment approaches are being sought. Targeted radionuclide therapy provides tumor-specific systemic treatments. The ß-emitting radiopharmaceutical meta-131I-iodo-benzylguanidine (131I-MIBG) provides limited survival benefits and has adverse effects. A new generation of radionuclides for therapy using α-particles including meta-211At-astato-benzylguanidine (211At-MABG) are expected to have strong therapeutic effects with minimal side effects. However, this possibility has not been evaluated in an animal model of pheochromocytoma. We aimed to evaluate the therapeutic effects of the α-emitter 211At-MABG in a pheochromocytoma model. METHODS: We evaluated tumor volume-reducing effects of 211At-MABG using rat pheochromocytoma cell line PC12 tumor-bearing mice. PC12 tumor-bearing mice received intravenous injections of 211At-MABG (0.28, 0.56, 1.11, 1.85, 3.70 and 5.55 MBq; five mice per group). Tumor volumes were evaluated for 8 weeks after 211At-MABG administration. The control group of ten mice received phosphate-buffered saline. RESULTS: The 211At-MABG-treated mice showed significantly lower relative tumor growth during the first 38 days than the control mice. The relative tumor volumes on day 21 were 509.2% ± 169.1% in the control mice and 9.6% ± 5.5% in the mice receiving 0.56 MBq (p < 0.01). In addition, the mice treated with 0.28, 0.56 and 1.11 MBq of 211At-MABG showed only a temporary weight reduction, with recovery in weight by day 10. CONCLUSION: 211At-MABG exhibited a strong tumor volume-reducing effect in a mouse model of pheochromocytoma without weight reduction. Therefore, 211At-MABG might be an effective therapeutic agent for the treatment of malignant pheochromocytoma.