上原 知也

In this study we developed a neopentyl 211At‐labeled activated ester that incorporates a triazole spacer and applied it to the synthesis of an 211At‐labeled cetuximab. The activated ester was synthesized via the nucleophilic 211At‐astatination of a neopentyl sulfonate carrying two long alkyl chains that serve as a lipid tag, which was followed by the hydrolysis of an acetal. Additionally, we developed a novel Resin‐Assisted Purification and Deprotection (RAPD) protocol involving a solid‐phase extraction of the protected 211At‐labeled compound from the mixture of the labeling reaction, hydrolysis of the acetal on the resin, and finally an elution of the 211At‐labeled activator from the resin. This method allows the synthesis of an 211At‐labeled activated ester with high purity through a simplified procedure that circumvents the need for HPLC purification. Using this 211At‐labeled activated ester, we efficiently synthesized 211At‐labeled cetuximab in 27±1% radiochemical yield with 95% radiochemical purity. This 211At‐activated ester demonstrated high reactivity, and enabled the completion of the reaction with the antibody within 10 min. In comparative biodistribution studies between 211At‐labeled cetuximab and the corresponding 125I‐labeled cetuximab in normal mice, both the thyroid and stomach showed radioactivity levels that were less than 1.0% of the injected dose.

BACKGROUND: L-type amino acid transporter 1 (LAT1) is overexpressed in various cancers; therefore, radiohalogen-labeled amino acid derivatives targeting LAT1 have emerged as promising candidates for cancer radiotheranostics. However, 211At-labeled amino acid derivatives exhibit instability against deastatination in vivo, making it challenging to use 211At for radiotherapy. In this study, radiohalogen-labeled amino acid derivatives with high dehalogenation stability were developed. RESULTS: We designed and synthesized new radiohalogen-labeled amino acid derivatives ([211At]At-NpGT, [125I]I-NpGT, and [18F]F-NpGT) in which L-tyrosine was introduced into the neopentyl glycol (NpG) structure. The radiolabeled amino acid derivatives were recognized as substrates of LAT1 in the in vitro studies using C6 glioma cells. In a biodistribution study using C6 glioma-bearing mice, these agents exhibited high stability against in vivo dehalogenation and similar biodistributions. The similarity of [211At]At-NpGT and [18F]F-NpGT indicated that these pairs of radiolabeled compounds would be helpful in radiotheranostics. Moreover, [211At]At-NpGT exhibited a dose-dependent inhibitory effect on the growth of C6 glioma-bearing mice. CONCLUSIONS: [211At]At-NpGT exhibited a dose-dependent inhibitory effect on the tumor growth of glioma-bearing mice, and its biodistribution was similar to that of other radiohalogen-labeled amino acid derivatives. These findings suggest that radiotheranostics using [18F]F-NpGT and [123/131I]I-NpGT for diagnostic applications and [211At]At-NpGT and [131I]I-NpGT for therapeutic applications are promising.

放射性核種を含む薬剤を生体に投与し、体内から患部に放射線を照射する治療薬剤を核医学治療薬剤という。近年、神経内分泌腫瘍や褐色細胞腫に対する核医学治療薬剤が本邦で立て続けに認可されるなど徐々に広まってきている。更に、海外では前立腺がんに対する核医学治療薬剤の認可例もみられ、今後国内への導入も期待されている。本稿では、核医学治療薬剤の現状と開発に向けた最近の知見について概説する。

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.

Copper-64 (64Cu)-labeled antibody fragments such as Fab are useful for molecular imaging (immuno-PET) and radioimmunotherapy. However, these fragments cause high and persistent localization of radioactivity in the kidneys after injection. To solve this problem, this study assessed the applicability of a molecular design to 64Cu, which reduces renal radioactivity levels by liberating a urinary excretory radiometabolite from antibody fragments at the renal brush border membrane (BBM). Since 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) forms a stable complex with Cu, NOTA-conjugated Met-Val-Lys-maleimide (NOTA-MVK-Mal), which is a radio-gallium labeling agent for antibody fragments, was evaluated for applicability to 64Cu. The MVK linkage was recognized by the BBM enzymes to liberate [64Cu]Cu-NOTA-Met although the recognition of the MVK sequence for the [64Cu]Cu-NOTA-MVK derivative was reduced compared with that of its [67Ga]Ga-counterpart, probably due to the difference in the charge of the metal-NOTA complexes. When injected into mice, [64Cu]Cu-NOTA-MVK-Fab resulted in similar renal radioactivity levels to the 67Ga-labeled counterpart. In addition, [64Cu]Cu-NOTA-MVK-Fab resulted in lower renal radioactivity levels than those from 64Cu-labeled Fab using a conventional method, without a reduction in the tumor radioactivity levels. These findings indicate that our approach to reducing renal radioactivity levels by liberating a radiolabeled compound from antibody fragments at the renal BBM for urinary excretion is applicable to 64Cu-labeled antibody fragments and useful for immuno-PET and radioimmunotherapy.

P-cadherin is overexpressed in various cancers and can be a target for radioimmunotherapy. We investigated the preclinical pharmacokinetics and pharmacology of FF-21101, an 111In- or 90Y-conjugated monoclonal antibody against P-cadherin, to evaluate its clinical applications. Methods: The radiochemical purity, binding affinity, and in vitro serum stability of 111In or 90Y-labeled FF-21101 were evaluated. The pharmacokinetics of 111In or 90Y-FF-21101 were compared in normal mice. Tumor accumulation after 111In-FF-21101 administration was investigated in mice bearing subcutaneous tumors with high (NCI-H1373), moderate (EBC-1), or no (A549) P-cadherin expression. The tumor suppression effect after a single intravenous injection of 90Y-FF-21101 was assessed in NCI-H1373 and EBC-1 mouse xenograft models. The relationship between antibody dose and tumor accumulation was investigated in the NCI-H1373 mouse xenograft model. The absorbed radiation dose in humans after injection of 90Y-FF-21101 was estimated using γ-camera images of cynomolgus monkeys. Results: The radiochemical purities of 111In- and 90Y-FF-21101 were 98.2% ± 2.5% (n = 9) and 99.3% ± 0.6% (n = 5), respectively. The dissociation constants were 1.083 nM for 111In-FF-21101 and 1.367 nM for 90Y-FF-21101. Both 111In- and 90Y-FF-21101 were stable in human serum after 96 h of incubation and exhibited similar pharmacokinetics in normal mice. The tumor accumulation of 111In-FF-21101 was closely related to the intensity of P-cadherin expression in the cells. 90Y-FF-21101 showed significant tumor growth inhibition, indicating that NCI-H1373 and EBC-1 recurrence was not observed after intravenous administration of 3.7 and 7.4 MBq, respectively of 90Y-FF-21101 per animal. Tumor uptake in the mouse xenograft model and estimated absorbed radiation doses in the spleen of monkeys decreased with increasing antibody doses of 111In-FF-21101. Conversely, the estimated absorbed radiation dose in the red marrow increased with increasing antibody dose. An antibody dose of 4.8 mg/m2 was considered appropriate for humans, on the basis of efficacy and safety. The maximum tolerated administered activity of 90Y-FF-21101 was estimated to be 2,886 MBq/human. Conclusion: FF-21101 radioimmunotherapy exhibited high antitumor affinity and antitumor efficacy in mouse xenograft models. Extrapolation of the pharmacokinetics in monkeys to humans suggests the potential for clinical application of FF-21101 for treating P-cadherin-expressing tumor.

This survey was performed in order to investigate the incidence of adverse reactions to radiopharmaceuticals in FY2019 in Japan. It was based on responses to questionnaires sent to nuclear medicine institutions. Replies were obtained from 1,003 institutions out of 1,213 to which the questionnaire had been sent. A total of 1,037,568 radiopharmaceutical administrations were reported. Eighteen cases of adverse reactions were reported. The incidence of adverse reactions per 100,000 cases was 1.7. No case of defective products was reported.

The high and persistent renal radioactivity levels after injection of radiolabeled low-molecular-weight polypeptides constitute a significant problem for their diagnostic and therapeutic applications, especially when they are labeled with metallic radionuclides. To improve the renal radioactivity levels of technetium-99m (99mTc)-labeled Fab fragments, a mercaptoacetyltriglycine (MAG3)-based new bifunctional chelating agent with a cleavable glycyl-phenylalanyl-lysine (GFK) linkage, MAG3-GFK-suc-TFP, was designed, synthesized, and evaluated. 99mTc-labeled Fab was obtained by reacting MAG3-GFK-Fab conjugate with 99mTc-glucarate. The GFK linkage remained stable in plasma but was cleaved by enzymes on the renal brush border membrane. The comparative biodistribution studies with indium-111 (111In)-labeled Fab using SCN-CHX-A″-DTPA showed that while both radiolabeled Fabs exhibited similar elimination rates from the blood, [99mTc]Tc-MAG3-GFK-Fab registered much lower renal radioactivity levels from 30 min post-injection onward due to the release and subsequent urinary excretion of [99mTc]Tc-MAG3-Gly. However, [99mTc]Tc-MAG3-GFK-Fab showed an increase in the intestinal radioactivity levels with the time that was not observed with 111In-labeled Fab. The analysis of the intestinal contents suggested the redistribution of [99mTc]Tc-MAG3-Gly to the intestine. The retrospective comparison of [99mTc]Tc-MAG3-GFK-Fab with the radiolabeled Fabs so far prepared under the identical concept suggested that some portion of [99mTc]Tc-MAG3-Gly was generated after the coated vesicle formation and they were excreted into the blood, and subsequently redistributed in the intestine via hepatobiliary excretion. In conclusion, MAG3-GFK-suc-TFP provided 99mTc-labeled Fabs that exhibit low renal radioactivity shortly after injection by the post-labeling procedure. The present study indicated that, contrary to our earlier proposal, the generation of the radiometabolites would proceed not only during the internalization process of the parental antibody fragments but also after coated vesicle formation. This study also showed that the intracellular behaviors of radiometabolites played crucial roles in the elimination rates and the routes of the radioactivity from the kidney.

The accumulation of 99mTc-labeled probes targeting saturable systems of the body is hindered by the presence of a large excess of unlabeled ligands needed to ensure high radiochemical yields in a short reaction time. To address the issue, we recently reported a novel concept of a metal-coordination-mediated synthesis of a bivalent 99mTc-labeled probe from a monovalent ligand using d-penicillamine (Pen) as a chelating molecule and c(RGDfK) as a model targeting device. The Pen-conjugated c(RGDfK) via a hexanoate linkage (Pen-Hx-c(RGDfK)) provided a bivalent [99mTc]Tc-[(Pen-Hx-c(RGDfK))2 that possessed much higher integrin αvβ3 binding affinity than Pen-Hx-c(RGDfK) and visualized a murine tumor without purification. However, high radioactivity levels were observed in the abdominal regions, which necessitated improved pharmacokinetics of the probes for practical applications. In this study, a pharmacokinetic (PK) modifier was introduced to manipulate the pharmacokinetics of the 99mTc-Pen2-based bivalent probe. The Hx linkage in Pen-Hx-c(RGDfK) was replaced with acetyl-d-serine-d-serine-glycine (Ac-ssG) or hexanoyl-d-serine-d-serine-d-serine (Hx-sss) to prepare Pen-Ac-ssG-c(RGDfK) or Pen-Hx-sss-c(RGDfK). Pen-Ac-ssG-c(RGDfK) impaired the complexation ability of Pen-Hx-c(RGDfK), and a monovalent 99mTc-labeled compound was generated at low ligand concentration. However, Pen-Hx-sss-c(RGDfK) provided the objective bivalent 99mTc-labeled probe in high radiochemical yields at a concentration similar to that of Pen-Hx-c(RGDfK). [99mTc]Tc-[Pen-Hx-sss-c(RGDfK)]2 also possessed stability and integrin αvβ3 binding affinity similar to those of [99mTc]Tc-[Pen-Hx-c(RGDfK)]2. As a result, [99mTc]Tc-[Pen-Hx-sss-c(RGDfK)]2 exhibited tumor and abdominal radioactivity levels similar to and significantly lower than those of [99mTc]Tc-[Pen-Hx-c(RGDfK)]2. These findings indicate the incorporation of a tripeptide PK modifier to Pen-Hx-c(RGDfK) preserved the complexation ability and improved the pharmacokinetics of the resulting 99mTc-labeled bivalent probe without impairing the targeting ability. Thus, the [Pen-Hx-(PK modifier)-(targeting device)] would constitute a basic formulation for preparing the 99mTc-Pen2-based bivalent probes for imaging saturable targets of the body.

OBJECTIVE: This pharmacovigilance-based survey was aimed at determining the prevalence of, and association between, radiopharmaceuticals and adverse reactions to radiopharmaceuticals from 1975 to 2017 in Japan. METHODS: The Subcommittee for Safety Issues of Radiopharmaceuticals of the Japan Radioisotope Association's Medical Science and Pharmaceutical Committee mailed a form for reporting adverse reactions to radiopharmaceuticals to all institutes performing nuclear medicine examinations in Japan. This investigation included adverse reactions to diagnostic radiopharmaceuticals labeled with both single-photon- and positron-emitting radionuclides and therapeutic sodium iodide labeled with 131I. Each institute returned the reporting form to the subcommittee each time an adverse reaction occurred. RESULTS: Replies were obtained from 75% of the institutions. In total, 1099 adverse reactions were reported from 46,645,580 radiopharmaceutical administrations, giving a prevalence of 2.4 adverse reactions per 100,000 administrations (95% confidence interval 2.2-2.5). Adverse reactions were most frequently observed for 131I-iodomethylnorcholesterol (230.1/105 administrations), followed by 131I-HSA (76.3/105 administrations), 131I-sodium iodohippurate (31.2/105 administrations), and 99mTc-DTPA (12.0/105 administrations). Comparison of adverse reactions between before and after 1997 revealed that prevalence dropped from 3.7/105 administrations (95% confidence interval 3.5-4.1) to 1.5/105 administrations (95% confidence interval 1.4-1.6). During the study period, vasovagal reactions accounted for 50.3% of adverse reactions, fever for 7.5%, allergic reactions for 25.7%, and other for 16.5%; 3.7% of all adverse reactions were considered severe but none were lethal. A definite, probable, possible, less likely, and uncertain causal relationship with radiopharmaceuticals was observed in 13.1%, 33.7%, 39.9%, 6.2%, and 7.1% of adverse reactions, respectively. CONCLUSIONS: These results suggest that nuclear medicine staff must be aware of the possibility of adverse reactions from radiopharmaceuticals, despite their rarity.

BACKGROUND: Recently, antiprogrammed cell death protein 1 (aPD-1) and antiprogrammed death-ligand 1 (aPD-L1) monoclonal antibodies (mAbs) have been approved. Even though aPD-1 and aPD-L1 mAbs target the same PD-1/PD-L1 axis, it is still unclear whether both mAbs exert equivalent pharmacological activity in patients who are sensitive to PD-1/PD-L1 blockade therapy, as there is no direct comparison of their pharmacokinetics (PK) and antitumor effects. Therefore, we evaluated the differences between both mAbs in PK and therapeutic effects in PD-1/PD-L1 blockade-sensitive mouse models. METHODS: Herein, murine breast MM48 and colon MC38 xenografts were used to analyze the pharmacological activity of aPD-1 and aPD-L1 mAbs. The PK of the mAbs in the tumor-bearing mice was investigated at low and high doses using two radioisotopes (Indium-111 and Iodine-125) to evaluate the accumulation and degradation of the mAbs. RESULTS: aPD-1 mAb showed antitumor effect in a dose-dependent manner, indicating that the tumor model was sensitive to PD-1/PD-L1 blockade therapy, whereas aPD-L1 mAb failed to suppress tumor growth. The PK study showed that aPD-L1 mAb was accumulated largely in normal organs such as the spleen, liver, and kidney, resulting in low blood concentration and low distributions to tumors at a low dose, even though the tumors expressed PD-L1. Sufficient accumulation of aPD-L1 mAb in tumors was achieved by administration at a high dose owing to the saturation of target-mediated binding in healthy organs. However, degradation of aPD-L1 mAb in tumors was greater than that of aPD-1 mAb, which resulted in poor outcome presumably due to less inhibition of PD-L1 by aPD-L1 mAb than that of PD-1 by aPD-1 mAb. CONCLUSION: According to the PK studies, aPD-1 mAb showed linear PK, whereas aPD-L1 mAb showed non-linear PK between low and high doses. Collectively, the poor PK characteristics of aPD-L1 mAb caused lower antitumor activity than of aPD-1 mAb. These results clearly indicated that aPD-L1 mAb required higher doses than aPD-1 mAb in clinical setting. Thus, targeting of PD-1 would be more advantageous than PD-L1 in terms of PK.

The diaminedithiol (N2S2) tetradentate ligand constitutes a useful chelating molecule for preparing 99mTc-labeled compounds of high in vivo stability in high radiochemical yields. However, since the thiol groups in the N2S2 ligand are easy to be oxidized to disulfide bonds, they need to be protected with an appropriate protecting group, which hinders the broad applications of the N2S2 ligand for radiopharmaceuticals. In this study, a Zn chelate of N2S2 was evaluated as a precursor for purification-free 99mTc-labeled N2S2 under the mild and simple procedure. Zn-N2S2 was prepared by reacting Zn acetate with N2S2, and the Zn-N2S2 remained stable under aerobic conditions at room temperature. 99mTc-N2S2 was obtained over 90% radiochemical yields at room temperature by a one-pot reaction, consisting of Zn-N2S2 (10-5 M), 99mTcO4-, ethylenediaminetetraacetic acid (EDTA), and a reducing agent (Sn2+) at pH = 5.5 to 7.5. 99mTc-N2S2 was also obtained over 90% radiochemical yields when the reaction was conducted in the presence of an equimolar amount of IgG antibody. These findings indicate the Zn complex of N2S2 ligand constitutes a stable and useful precursor to prepare 99mTc-labeled N2S2 compounds in high yields under the mild and simple procedure.

本調査は、2018年度に投与された放射性医薬品に関連して発生した副作用事例の発生頻度とその内容を調べる目的で実施された。調査は、調査票を核医学診療施設に送付して回答を求めるアンケート方式で実施した。調査対象1,230施設のうち、999施設より回答が得られた。副作用事例は14件報告された。回答を得た999施設における放射性医薬品の投与件数は1,017,702件であった。副作用発生率は100,000件あたり1.4件であった。不良品事例の報告はなかった。(著者抄録)

This survey was performed in order to investigate the incidence of adverse reactions to radiopharmaceuticals in FY2018 in Japan. It was based on the responses to questionnaires sent to nuclear medicine institutions. Replies were obtained from 999 institutions among 1,230 to which the questionnaire had been sent. Fourteen cases of adverse reactions were reported. A total of 1,017,702 radiopharmaceutical administrations were reported. The incidence of adverse reactions per 100,000 cases was 1.4. No case of deficient products was reported.

Inflammation after myocardial infarction (MI) may be a major factor influencing ventricular remodeling, leading to congestive heart failure and arrhythmia. Therefore, inflammation in the heart needs to be monitored. Tenascin-C (TNC) is an extracellular matrix molecule not normally expressed, but it is strongly upregulated when associated with active inflammation. Based on this characteristic, we successfully imaged in vivo inflammatory lesions in rat models using 111Indium (111In)-labeled anti-TNC antibodies. The aim of the present study was to further assess the applicability of this molecular imaging probe to detect inflammatory activity in primate hearts.We generated an MI model of cynomolgus monkeys (Macaca fascicularis) by coronary artery ligation and performed dual-isotope single-photon emission computed tomography (SPECT) imaging with an 111In-labeled anti-TNC antibody Fab' fragment (111In-TNC Fab') and 99mtechnetium methoxy-isobutyl isonitrile (99mTc-MIBI). Dual autoradiography was used to compare the uptake of 111In-TNC Fab' with histology and immunostaining for TNC. Dual-isotope SPECT showed the regional myocardial uptake of 111In-TNC Fab' complementary to a defect in the perfusion image by 99mTc-MIBI. The high radioactivity of 111In-TNC Fab' by autoradiography corresponded to immunostaining for TNC, which was observed in inflammatory lesions at the border zone between the infarcted and non-infarcted areas of the left ventricle and at the epi/pericarditis lesions of the right ventricle. These results demonstrate the potential of 111In-TNC-Fab' imaging to monitor myocardial injury and inflammation and suggest the feasibility of the non-invasive detection of cardiac inflammation following acute MI in a preclinical stage before testing in humans.

The presence of excess unlabeled ligands in the injectate hinders the target uptake of 99mTc-labeled targeting vectors. To address the issue, we previously developed a chemical design which provides a 99mTc-labeled trivalent RGD probe upon CN-βAla-Gly-Gly-c(RGDfK) (Lβ) coordination to [99mTc][Tc(CO)3]+ core at pH 6.0. In this study, we extended our coordination mediated synthesis of the trivalent RGD probe to that of a tetravalent one. Our initial attempts reacting Lβ with [99mTc][Tc(CO)3]+ core at pH 8.0 failed to provide [99mTc][Tc(CO)2(Lβ)4]+ due to the formation of multiple side products. A γ-aminobutylic acid (GABA) based isonitrile ligand CN-GABA-Gly-Gly-c(RGDfK) (LG), on the other hand, avoided the side reaction and selectively provided [99mTc][Tc(CO)2(LG)4]+ (99mTc-[LG]4) at pH 8.0. 99mTc-[LG]4 exhibited higher binding affinity to integrin αvβ3 than its unlabeled ligand, and visualized U87MG tumor without tedious post-labeling purification. These results indicate that the metal coordination-mediated syntheses of 99mTc-labeled multivalent probes have been successfully applied to a tetravalent one, which would allow a wider range of choices for designing novel 99mTc-labeled multivalent probes of high in vivo target uptake.

本調査は、平成29年度に投与された放射性医薬品に関連して発生した副作用事例の発生頻度とその内容を調べる目的で実施された。調査は、調査票を核医学診療施設に送付して回答を求めるアンケート方式で実施した。調査対象1,231施設のうち、981施設より回答が得られた。副作用事例は12件報告された。回答を得た981施設における放射性医薬品の投与件数は1,028,886件であった。副作用発生率は100,000件あたり1.2件であった。不良品事例の報告はなかった。(著者抄録)

Macrophage mannose receptor (MMR/CD206) is a promising target for the detection and identification of sentinel lymph node (SLN). MMR-targeting probes have been developed using mannosylated dextran, however, impairment of efficient targeting of SLN was often caused because of retention of injection site in which macrophages and dendritic cells exist. In this study, we prepared new MMR-targeting probes from yeast mannan (85 kDa), and its bioditribution was investigated. In-vivo evaluation showed that 11.9% of injected dose of 99mTc-labeled mannan-S-cysteines (99mTc-MSCs) was accumulated in popliteal lymph node (the SLN in this model), however, significant level of radioactivity (approximately 80%) was remained in injection site. Interestingly, 99mTc-labeled low molecular weight mannan-S-cysteine mannan (99mTc-LSC) prepared from 50 and 25 kDa mannan showed a decreased specific accumulation of 99mTc-LSC in the popliteal lymph node, while the radioactivity at the injection site remained unchanged. These results suggest that the molecular size, or nature/shape of the sugar chain is important for the specific accumulation of 99mTc-MSC in popliteal lymph node.

本調査は、平成28年度に投与された放射性医薬品に関連して発生した副作用事例の発生頻度とその内容を調べる目的で実施された。調査は、調査票を核医学診療施設に送付して回答を求めるアンケート方式で実施した。調査対象1,235施設のうち、977施設より回答が得られた。副作用事例は9件報告された。回答を得た977施設における放射性医薬品の投与件数は1,052,650件であった。副作用発生率は100,000件あたり0.9件であった。不良品事例の報告はなかった。(著者抄録)

A large excess of unlabeled ligands over gallium-67 (67Ga) provides 67Ga-labeled probes with high radiochemical yields in a short reaction time. However, the unlabeled ligands hinder target accumulation of radiolabeled probes by competing for target molecules. To circumvent the problem, we investigated the way to prepare 67Ga-labeled multivalent probes from monovalent ligands. The reaction of a bi- or tridentate ligand with [67Ga]Ga-citrate resulted in 67Ga-labeled probes of insufficient stability. However, the reaction of [67Ga]Ga-citrate with a mixture of RGD-conjugated salicylaldehyde and triamine provided a 67Ga-labeled trivalent probe with stability sufficient for in vivo applications. Since the free Schiff base ligand decomposed rapidly upon injection, the 67Ga-labeled trivalent probe visualized the murine tumor without postlabeling purification, which was not achieved with a 67Ga-labeled trivalent probe from a trivalent ligand. These findings indicate the availability of Schiff base ligands to prepare 67Ga-labeled trivalent probes by a simple radiolabeling procedure.

The obstructive renal radioactivity after injection of antibody fragments/constructs labeled with metallic radionuclides would be improved by liberating a radiometal chelate of urinary excretion from the antibody molecules by enzymes on the renal brush border membrane (BBM). A tripeptide GFK sequence was newly evaluated as an enzyme-cleavable linkage and conjugated to a 99mTc chelate of an isonicotinic acid derivative of 2-picolylglycine (99mTc-IPG). 99mTc-IPG-glycine was liberated from 99mTc-IPG-GFK by the enzymes, while 99mTc-IPG-GK (where the tripeptide GFK was substituted with a dipeptide GK) did not. When injected into mice, 99mTc-IPG-GFK-conjugated Fab exhibited lower renal radioactivity levels than directly radioiodinated Fab shortly after injection without reducing the tumor radioactivity levels, due to a release and excretion of 99mTc-IPG-glycine by enzymes present on the renal BBM. These findings would provide insights to develop antibody fragments/constructs labeled with metallic radionuclides of the clinical relevance for improved renal radioactivity levels.

In the synthesis of technetium-99m (99mTc) labeled target-specific ligands, the presence of a large excess of unlabeled ligands over 99mTc in the injectate hinders target accumulation of 99mTc-labeled ligands by competing for target molecules. To circumvent the problem, we recently developed a concept of the metal coordination-mediated multivalency, and proved the concept with a 99mTc-labeled trivalent compound [99mTc(CO)3(CN-RGD)3]+. In this study, D-penicillamine (Pen) was selected as a chelating molecule and a cyclic RGDfK peptide was conjugated to Pen via a hexanoic linkage (Pen-Ahx-c(RGDfK)). 99mTc complexation reaction, and the stability, integrin αvβ3 binding affinity, and biodistribution of the 99mTc-labeled probe were investigated to evaluate the applicability of the concept to bivalent probes. 99mTc-[Pen-Ahx-c(RGDfK)]2 was obtained over 95% radiochemical yields under low Pen-Ahx-c(RGDfK) concentration (50 μM). 99mTc-[Pen-Ahx-c(RGDfK)]2 showed approximately 10-times higher integrin αvβ3 binding affinity than the monovalent compounds, Pen-Ahx-c(RGDfK) and c(RGDyV). In biodistribution studies, the tumor accumulation of 99mTc-[Pen-Ahx-c(RGDfK)]2 was decreased to 77% and 43% of HPLC-purified (Pen-Ahx-c(RGDfK)-free) 99mTc-[Pen-Ahx-c(RGDfK)]2 by the presence of 5 nmol of unlabeled Pen-Ahx-c(RGDfK) and Re-[Pen-Ahx-c(RGDfK)]2, respectively. 99mTc-[Pen-Ahx-c(RGDfK)]2 provided tumor image without removing unlabeled ligand, while a 99mTc-labeled monovalent probe prepared from a monovalent ligand could not. These findings indicate the availability of the design concept to prepare 99mTc-labeled bivalent probes with a variety of 99mTc core and other metallic radionuclides of clinical relevance.

本調査は、平成27年度に投与された放射性医薬品に関連して発生した副作用事例の発生頻度とその内容を調べる目的で実施された。調査は、調査票を核医学診療施設に送付して回答を求めるアンケート方式で実施した。調査対象1,274施設のうち、981施設より回答が得られた。副作用事例は15件報告された。回答を得た981施設における放射性医薬品の投与件数は1,056,828件であった。副作用発生率は100,000件あたり1.4件であった。不良品事例の報告はなかった。(著者抄録)

L-[methyl-11C]Methionine (11C-Met) is useful for estimating the therapeutic efficacy of particle radiotherapy at early stages of the treatment. Given the short half-life of 11C, the development of longer-lived 18F- and 123I-labeled probes that afford diagnostic information similar to 11C-Met, are being sought. Tumor uptake of 11C-Met is involved in many cellular functions such as amino acid transport System-L, protein synthesis, and transmethylation. Among these processes, since the energy-dependent intracellular functions involved with 11C-Met are more reflective of the radiotherapeutic effects, we evaluated the activity of the amino acid transport System-A as an another energy-dependent cellular function in order to estimate radiotherapeutic effects. In this study, using a carbon-ion beam as the radiation source, the activity of System-A was evaluated by a specific System-A substrate, alpha-[1-14C]-methyl-aminoisobutyric acid (14C-MeAIB). Cellular growth and the accumulation of 14C-MeAIB or 14C-Met were evaluated over time in vitro in cultured human salivary gland (HSG) tumor cells (3-Gy) or in vivo in murine xenografts of HSG tumors (6- or 25-Gy) before and after irradiation with the carbon-ion beam. Post 3-Gy irradiation, in vitro accumulation of 14C-Met and 14C-MeAIB decreased over a 5-day period. In xenografts of HSG tumors in mice, tumor re-growth was observed in vivo on day-10 after a 6-Gy irradiation dose, but no re-growth was detected after the 25-Gy irradiation dose. Consistent with the growth results, the in vivo tumor accumulation of 14C-MeAIB did not decrease after the 6-Gy irradiation dose, whereas a significant decrease was observed after the 25-Gy irradiation dose. These results indicate that the activity of energy dependent System-A transporter may reflect the therapeutic efficacy of carbon-ion radiotherapy and suggests that longer half-life radionuclide-labeled probes for System-A may also provide widely available probes to evaluate the effects of particle radiotherapy on tumors at early stage of the treatment.

This survey was performed to investigate the incidence of adverse reactions to radiopharmaceuticals in FY2015 in Japan. It was based on the responses to questionnaires sent to nuclear medicine institutions. The reply was obtained from 981 institutions among 1,274 to which the questionnaire had been sent. Fifteen cases of adverse reactions were reported. A total of 1,056,828 radiopharmaceutical administrations were reported. The incidence of adverse reactions per 100,000 cases was 1.4. No case of deficient products was reported.

UNLABELLED: Radiolabeled amino acids are superior PET tracers for the imaging of malignant tumors, and amino acids labeled with (76)Br, an attractive positron emitter because of its relatively long half-life (16.2 h), could potentially be a widely usable tumor imaging tracer. In this study, in consideration of its stability and tumor specificity, we designed two (76)Br-labeled amino acid derivatives, 2-(76)Br-bromo-α-methyl-l-phenylalanine (2-(76)Br-BAMP) and 4-(76)Br-bromo-α-methyl-l-phenylalanine (4-(76)Br-BAMP), and investigated their potential as tumor imaging agents. METHODS: Both (76)Br- and (77)Br-labeled amino acid derivatives were prepared. We performed in vitro and in vivo stability studies and cellular uptake studies using the LS180 colon adenocarcinoma cell line. Biodistribution studies in normal mice and in LS180 tumor-bearing mice were performed, and the tumors were imaged with a small-animal PET scanner. RESULTS: Both (77)Br-BAMPs were stable in the plasma and in the murine body. Although both (77)Br-BAMPs were taken up by LS180 cells and the uptake was inhibited by L-type amino acid transporter 1 inhibitors, 2-(77)Br-BAMP exhibited higher uptake than 4-(77)Br-BAMP. In the biodistribution studies, 2-(77)Br-BAMP showed more rapid blood clearance and lower renal accumulation than 4-(77)Br-BAMP. More than 90% of the injected radioactivity was excreted in the urine by 6 h after the injection of 2-(77)Br-BAMP. High tumor accumulation of 2-(77)Br-BAMP was observed in tumor-bearing mice, and PET imaging with 2-(76)Br-BAMP enabled clear visualization of the tumors. CONCLUSION: 2-(77)Br-BAMP exhibited preferred pharmacokinetics and high LS180 tumor accumulation, and 2-(76)Br-BAMP enabled clear visualization of the tumors by PET imaging. These findings suggest that 2-(76)Br-BAMP could constitute a potential new PET tracer for tumor imaging and may eventually enable the wider use of amino acid tracers.

Intrinsic drug resistance occurs in many renal carcinomas and is associated with increased expression of multidrug resistant proteins, which inhibits intracellular drug accumulation. Multidrug resistant protein 1, also known as P-glycoprotein, is a membrane drug efflux pump belonging to the ATP-binding cassette (ABC) transporter superfamily. ABC Sub-family B Member 2 (ABCG2) is widely distributed and is involved in the multidrug resistant phenotype. Sunitinib is a tyrosine kinase inhibitor used to treat kidney cancer that disrupts signaling pathways responsible for abnormal cancer cell proliferation and tumor angiogenesis. Multiple drug resistance is important in tyrosine kinase inhibitor-induced resistance. We hypothesized that inhibition of multidrug resistant transporters by elacridar (dual inhibitor of P-glycoprotein and ABCG 2) might overcome sunitinib resistance in experimental renal cell carcinoma. Human renal carcinoma cell lines 786-O, ACHN, and Caki-1 were treated with sunitinib or elacridar alone, or in combination. We showed that elacridar significantly enhanced sunitinib cytotoxicity in 786-O cells. P-glycoprotein activity, confirmed by P-glycoprotein function assay, was found to be inhibited by elacridar. ABCG2 expression was low in all renal carcinoma cell lines, and was suppressed only by combination treatment in 786-O cells. ABCG2 function was inhibited by sunitinib alone or combination with elacridar but not elacridar alone. These findings suggest that sunitinib resistance involves multidrug resistance transporters, and in combination with elacridar, can be reversed in renal carcinoma cells by P-glycoprotein inhibition.

The renal localization of gallium-67 or gallium-68 (Ga-67/68)-labeled low molecular weight (LMW) probes such as peptides and antibody fragments constitutes a problem in targeted imaging. Wu et al. previously showed that Ga-67-labeled S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (SCN-Bz-NOTA)-conjugated methionine (Ga-67-NOTA-Met) was rapidly excreted from the kidney in urine following lysosomal proteolysis of the parental Ga-67-NOTA-Bz-SCN-disulfide-stabilized Fv fragment (Bioconjugate Chem., (1997) 8, 365-369). In the present study, a new Ga-67/88-labeling reagent for LMW probes that liberates Ga-67/68-NOTA-Met was designed, synthesized, and evaluated using longer-lived Ga-67 in order to reduce renal radioactivity levels. We employed a methionine-isoleucine (MI) dipeptide bond as the cleavable linkage. The amine residue of MI was coupled with SCN-Bz-NOTA for Ga-67-labeling, while the carboxylic acid residue of MI was derivatized to maleimide for antibody conjugation in order to synthesize NOTA-MI-Mal. A Fab fragment of the anti-Her2 antibody was thiolated with iminothiolane, and NOTA-MI-Mal was conjugated with the antibody fragment by maleimide-thiol chemistry. The Fab fragment was also conjugated with SCN-Bz-NOTA (NOTA-Fab) for comparison. Ga-67-NOTA-MI-Fab was obtained at radiochemical yields of over 95% and was stable in murine serum for 24 h. In the biodistribution study using normal mice, Ga-67-NOTA-MI-Fab registered significantly lower renal radioactivity levels from 1 to 6 h postinjection than those of Ga-67-NOTA-Fab. An analysis of urine samples obtained 6 h after the injection of Ga-67-NOTA-MI-Fab showed that the majority of radioactivity was excreted as Ga-67-NOTA-Met. In the biodistribution study using tumor-bearing mice, the tumor to kidney ratios of Ga-67-NOTA-MI-Fab were 4 times higher (6 h postinjection) than those of Ga-67-NOTA-Fab. Although further studies including the structure of radiometabolites and/or cleavable linkages are required, the results of the present study indicate that the current chemical design is applicable to the development of Ga-67-labeled Fabs for low renal radioactivity levels.

Radionuclide bone imaging using polynuclear (99m)Tc complexes of bisphosphonates is the most common clinical practice in nuclear medicine. However, the improvement in the contrast between normal and osteogenic bone regions has been required. Herein we reported a new (99m)Tc-labeled compound considering the increased vascular permeability of osteogenic region. We selected penta-d-Asp as both a targeting motif to hydroxyapatite (HA) and a molecular size modifier, and two penta-d-Asp molecules were conjugated with the two carboxylate residues of ethylene dicysteine (EC) selected as the (99m)Tc chelating moiety to prepare EC-[(d-Asp)5]2. The molecular size, HA binding, and pharmacokinetics of (99m)Tc-EC-[(d-Asp)5]2 in normal mice and model rats bearing osteogenic tumor were compared to those of (99m)Tc-MDP and (99m)Tc-EC with one (d-Asp)5 motif, (99m)Tc-EC-(d-Asp)5. The molecular size of (99m)Tc-EC-[(d-Asp)5]2 was higher than that of (99m)Tc-MDP and (99m)Tc-EC-(d-Asp)5 when determined by permeability of the (99m)Tc-compounds through a membrane filter (10 kDa). The HA binding of (99m)Tc-EC-[(d-Asp)5]2 was higher than and similar to that of (99m)Tc-EC-(d-Asp)5 and (99m)Tc-MDP. (99m)Tc-EC-[(d-Asp)5]2 exhibited significantly lower accumulation in normal bone of mice than did (99m)Tc-MDP. In osteogenic tumor bearing model rats, (99m)Tc-EC-[(d-Asp)5]2 accumulated in the osteogenic and normal bone region similar to and lower than (99m)Tc-MDP, respectively. Although further studies including the chain length of d-Asp are required, these findings indicated that the present chemical design of (99m)Tc-labeled probe would be applicable to develop (99m)Tc-labeled probes for selective imaging of osteogenic bone region as well as develop therapeutic agents using therapeutic radionuclides such as (90)Y, (177)Lu, (186)Re, or (188)Re and cytotoxic agents to osteogenic bone tumor region.

Almost all small animal SPECT is performed with pinhole collimators (PH), including single-PH (SPH) and multi-PH (MPH). In the clinical study, not only PH but also parallel-hole collimator (PAH) is often used in planar and SPECT imaging. However, there have been no comparative studies on image quality with various collimators on the small animal imaging. This study compared the basic characteristics of PH and PAH in small animal imaging. Performance of planar and SPECT images was evaluated using (TcO4)-Tc-99m (-) and SPH, MPH and PAH with low energy and high resolution on the SPECT/CT scanner FX3200. We measured sensitivity, resolution, concentration linearity and uniformity. Planar imaging of mice with Tc-99m-labeled mercaptoacetyltriglycine (Tc-99m-MAG(3)) was performed using SPH and PAH. SPECT imaging with Tc-99m-methylene diphosphonate (Tc-99m-MDP) was performed using all collimators. With SPH, MPH and PAH, sensitivity was 43.5, 211.2 and 926.5 cps/MBq, respectively, and spatial resolution was 0.60/0.56, non/0.96, 5.20/5.34 mm full-width half maximum (planar/SPECT), respectively. There were marked correlations between the radioactivity counts on images and radioactivity with all collimators. Values of % standard deviation on planar imaging showed small differences between the SPH and PAH, while the values were the smallest on SPECT imaging with MPH. On imaging of mice, SPH yielded high-quality Tc-99m-MAG(3)-planar images when compared with PAH. MPH yielded sharper Tc-99m-MDP-SPECT images than SPH and PAH. The characteristics of PH and PAH differed on small animal imaging. Although sensitivity was higher with PAH, PH showed higher resolution. Among the PH collimators, SPH was more appropriate for planar imaging, and MPH was more suitable for SPECT imaging in a small animal imaging scanner with CZT detector.

We previously demonstrated that Fab fragments labeled with 3'-[(131)I]iodohippuryl N(ε)-maleoyl-l-lysine ([(131)I]HML) showed low renal radioactivity from early postinjection time, due to a liberation of m-[(131)I]iodohippuric acid by the action of renal brush border enzymes. Since there are lots of enzymes on renal brush border membrane, peptide linkages other than the glycyl-l-lysine were evaluated as the cleavable linkages to explore the chemical design. In this study, we evaluated four peptide linkages with a general formula of m-iodobenzoyl-glycyl-X (X: l-tyosine O-methyl, l-asparagine, l-glutamine, and N(ε)-Boc-l-lysine). In vitro studies using renal brush border membrane vesicles (BBMVs) demonstrated that 3'-[(125)I]iodohippuryl O-methyl-l-tyrosine (2c) liberated the highest amount of m-[(125)I]iodohippuric acid among the four substrates and the change in the linkage structure altered enzyme species responsible for the hydrolysis reaction. To further assess the applicability of the linkage, a radioiodination reagent containing a glycyl-tyrosine linkage, 3'-[(125)I]iodohippuryl O-((2-maleimidoethyl)carbamoyl)methyl-l-tyrosine (HMT, 12c), was designed, synthesized, and subsequently conjugated to an Fab fragment. [(125)I]HMT-Fab exhibited renal radioactivity levels similar to and significantly lower than [(125)I]HML-Fab and directly radioiodinated Fab, while the blood clearance rates of the three were similar. The analyses of urine for 24 h postinjection of [(125)I]HMT-Fab showed that m-[(125)I]iodohippuric acid was excreted as the major radiometabolite. The findings indicated that glycyl-tyrosine linkage is also available to reduce renal radioactivity levels of radioiodinated Fab fragments, due to liberation of m-iodohippuric acid by the action of enzymes present on renal brush border membrane. These findings suggest that an appropriate selection of peptide linkages would allow the liberation of a designed radiolabeled compound from covalently conjugated polypeptides to prepare radiolabeled polypeptides of low renal radioactivity levels. For the selection of the most appropriate peptide linkage, the in vitro system using BBMVs would be useful to narrow the candidates to just a few.

In the conventional synthesis of 1,4,7-tris-(glutaric acid)-1,4,7-triazacyclononane (NOTGA), four isomeric species are usually generated by the alkylation of 1,4,7-triazacyclononane with α-bromoglutaric acid diester. To estimate their biological efficacies as well as their stability and radiochemistry, the RRR/SSS and RRS/SSR NOTGA-(t)Bu prochelators were isolated and the corresponding cyclic RGDfK (RGD) conjugates with triethylene glycol linkages were prepared. The RRR/SSS and RRS/SSR diastereomers were obtained in 69% and 17% yields, respectively. In the complexation reaction with (67)GaCl(3), both diastereomers provided >98% radiochemical yields at pH 5 within 10 min when the reaction was conducted at room temperature. However, the RRR/SSS diastereomer exhibited more pH-sensitive radiochemical yields between pH 3.5 to 4.5. Despite their diasteromeric nature, both (67)Ga-labeled RGD-NOTGA remained stable during the apo-transferrin challenge, exhibiting similar affinity for integrin α(v)β(3) and biodistribution with predominant renal excretion. Similar tumor uptake was also observed in mice bearing U87MG tumor xenograft, which resulted in impressively high contrast SPECT/CT images. These findings indicate that the RGD-NOTGA conjugates of both diastereomers presented here possess equivalent biological efficacies and their combined usage would be feasible. It is worth noting that specific properties of a given biomolecule, cell expression levels of the corresponding target molecule, and presence or absence of a pharmacokinetic modifier would affect the structural differences between diastereomers on the ligand-receptor interactions and pharmacokinetics. Thus, the preparation of corresponding conjugates and evaluation of their chemical and biological performances still remains important for applying NOTGA to other biomolecules of interest using the diastereomerically pure NOTGA-(t)Bu prochelator.