鈴木 博元

<jats:title>Abstract</jats:title><jats:sec> <jats:title>Background</jats:title> <jats:p>Prostate cancer is a common cancer among men worldwide that has a very poor prognosis, especially when it progresses to metastatic castration-resistant prostate cancer (mCRPC). Therefore, novel therapeutic agents for mCRPC are urgently required. Because prostate-specific membrane antigen (PSMA) is overexpressed in mCRPC, targeted alpha therapy (TAT) for PSMA is a promising treatment for mCRPC. Astatine-211 (<jats:sup>211</jats:sup>At) is a versatile α-emitting radionuclide that can be produced using a cyclotron. Therefore, <jats:sup>211</jats:sup>At-labeled PSMA compounds could be useful for TAT; however, <jats:sup>211</jats:sup>At-labeled compounds are unstable against deastatination in vivo. In this study, to develop in vivo stable <jats:sup>211</jats:sup>At-labeled PSMA derivatives, we designed and synthesized <jats:sup>211</jats:sup>At-labeled PSMA derivatives using a neopentyl glycol (NpG) structure that can stably retain <jats:sup>211</jats:sup>At in vivo. We also evaluated their biodistribution in normal and tumor-bearing mice.</jats:p> </jats:sec><jats:sec> <jats:title>Results</jats:title> <jats:p>We designed and synthesized <jats:sup>211</jats:sup>At-labeled PSMA derivatives containing two glutamic acid (Glu) linkers between the NpG structure and asymmetric urea (NpG-L-PSMA ((L-Glu)<jats:sub>2</jats:sub> linker used) and NpG-D-PSMA ((D-Glu)<jats:sub>2</jats:sub> linker used)). First, we evaluated the characteristics of <jats:sup>125</jats:sup>I-labeled NpG derivatives because <jats:sup>125</jats:sup>I was readily available. [<jats:sup>125</jats:sup>I]I-NpG-L-PSMA and [<jats:sup>125</jats:sup>I]I-NpG-D-PSMA showed low accumulation in the stomach and thyroid, indicating their high in vivo stability against deiodination. [<jats:sup>125</jats:sup>I]I-NpG-L-PSMA was excreted in urine as hydrophilic radiometabolites in addition to the intact form. Meanwhile, [<jats:sup>125</jats:sup>I]I-NpG-D-PSMA was excreted in urine in an intact form. In both cases, no radioactivity was observed in the free iodine fraction. [<jats:sup>125</jats:sup>I]I-NpG-D-PSMA showed higher tumor accumulation than [<jats:sup>125</jats:sup>I]I-NpG-L-PSMA. We then developed <jats:sup>211</jats:sup>At-labeled PSMA using the NpG-D-PSMA structure. [<jats:sup>211</jats:sup>At]At-NpG-D-PSMA showed low accumulation in the stomach and thyroid in normal mice, indicating its high stability against deastatination in vivo. Moreover, [<jats:sup>211</jats:sup>At]At-NpG-D-PSMA showed high accumulation in tumor similar to that of [<jats:sup>125</jats:sup>I]I-NpG-D-PSMA.</jats:p> </jats:sec><jats:sec> <jats:title>Conclusions</jats:title> <jats:p>[<jats:sup>211</jats:sup>At]At-NpG-D-PSMA showed high in vivo stability against deastatination and high tumor accumulation. [<jats:sup>211</jats:sup>At]At-NpG-D-PSMA should be considered as a potential new TAT for mCRPC.</jats:p> </jats:sec>

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.

Abstract 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Purpose: This study was undertaken to evaluate the renal radioactivity levels of a newly designed 67Ga-labeled antibody fragment with a linkage cleaved by enzymes present on the brush border membrane (BBM) lining the lumen of the renal tubule.Experimental Design:67Ga-labeled S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (SCN-Bn-NOTA) was conjugated with an antibody Fab fragment through a Met-Val-Lys linkage (67Ga-NOTA-MVK-Fab) considering that a Met-Val sequence is a substrate of enzymes on the renal BBM and 67Ga-NOTA-Met is excreted from the kidney into the urine. The enzymatic recognition of the linkage was evaluated with a low-molecular-weight 67Ga-NOTA-Met-Val-Lys derivative. Biodistribution of radioactivity after injection of 67Ga-NOTA-MVK-Fab into mice was compared with 67Ga-NOTA-conjugated Fab fragments through a Met-Ile linkage that liberates 67Ga-NOTA-Met (67Ga-NOTA-MI-Fab) or a conventional thiourea linkage (67Ga-NOTA-Fab).Results: The MVK linkage remained stable in plasma and was recognized by enzymes on renal BBM to liberate 67Ga-NOTA-Met. When injected into mice, all three 67Ga-labeled Fab exhibited similar blood clearance rates and tumor accumulation. Significant differences were observed in the kidney where 67Ga-NOTA-MVK-Fab registered the lowest renal radioactivity levels from early postinjection time (P < 0.05), followed by 67Ga-NOTA-MI-Fab, which was well reflected in the SPECT/CT images.Conclusions: These findings indicated that our proposal of liberating a radiolabeled compound to urinary excretion from antibody fragments at the renal BBM to reduce the renal radioactivity levels was applicable to 67/68Ga-labeled antibody fragments. Because antibody fragments and constructs share similar metabolic fates in the kidney, the present labeling procedure would also apply to a variety of antibody fragments and constructs of interest. Clin Cancer Res; 24(14); 3309-16. ©2018 AACR.

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.

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.

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-tBu 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 67GaCl3, both diastereomers provided &gt 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 67Ga-labeled RGD-NOTGA remained stable during the apo-transferrin challenge, exhibiting similar affinity for integrin αvβ3 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- tBu prochelator. © 2012 American Chemical Society.

A 12-membered polyazamacrocycle, 1-oxa-4,7,10-triazacyclododecane-N, N′,N″-triacetic acid (ODTA), has been reported to provide an indium chelate of net neutral charge with thermodynamic stability higher than 1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid (DOTA). However, neither synthetic procedure for a C-functionalized ODTA (C-ODTA) nor its chelating ability with a trace amount of radioactive indium-111 ( 111In) has been elucidated. We herein present a facile synthetic procedure for C-ODTA, and estimated its ability as a chelating agent for radiolabeling peptides and proteins with 111In. The synthetic procedure involves the synthesis of a linear precursor using a para-substituted phenylalanine derivative as a starting material. The following intramolecular cyclization reaction was best performed (&gt 73% yield) when Boc-protected linear compound and the condensation reagent, HATU, were simultaneously added to the reaction vessel at the same flow rate. The cyclic compound was then reduced with BH 3 and alkylated with tert-butyl bromoacetate. The synthetic procedure was straightforward and some optimization would be required. However, most of the intermediate compounds were obtained easily in good yields, suggesting that the present synthetic procedure would be useful to synthesize C-ODTA derivatives. The intramolecular cyclization reaction might also be applicable to synthesize polyazamacrocycles of different ring sizes and cyclic peptides. In 111In radiolabeling reactions, C-ODTA provided 111In chelates in higher radiochemical yields at low ligand concentrations when compared with C-DOTA. The 111In-labeled C-ODTA remained unchanged in the presence of apo-transferrin. The biodistribution studies also showed that the 111In-labeled compound was mainly excreted into urine as intact. These findings indicate that C-ODTA would be useful to prepare 111In-labeled peptides of high specific activities in high radiochemical yields. © 2011 Elsevier Ltd. All rights reserved.

Poly(amidoamine) (PAMAM) dendrimers are highly branched spherical polymers that have a unique surface of primary amine groups and provide a versatile design for targeted delivery of pharmaceuticals and imaging agents. Acetylation or succinylation of surface amine groups of PAMAM dendrimer derivatives is frequently performed to reduce nonspecific uptake. However, since targeting molecules, drugs/imaging agents, and acylating reagents react with the amine groups on dendrimer, such modification may limit the number of targeting molecules and/or drugs or may result in insufficient charge reduction. In this study, a γ-glutamyl PAMAM dendrimer was designed and synthesized as a new precursor for targeting device. The relationship between surface electrical properties of the PAMAM dendrimer derivatives and pharmacokinetics was also determined. A PAMAM dendrimer (generation 4.0) was modified with a small number of Bolton-Hunter reagent to prepare Phe-P (pI 9.2). The amine residues of Phe-P were ã-glutamylated to prepare Glu-P (pI 7.1). The α-amine residues of Glu-P were then acetylated or succinylated to prepare Ac-Glu-P (pI 5.3) or Suc-Glu-P (pI 3.6). For comparison, Phe-P was acetylated or succinylated to prepare Ac-P (pI 6.0) or Suc-P (pI 5.1). All the PAMAM dendrimer derivatives exhibited similar molecular size (7.2 to 7.8 nm) except for Ac-P (5.1 nm). The biodistribution studies were performed after radioiodination of each PAMAM dendrimer derivative with Na[125I]I. When injected intravenously to mice, both [125I]Ac-P and [125I]Suc-P exhibited prolonged radioactivity levels in the blood and significantly lower hepatic and renal radioactivity levels than those of [125I]Phe-P. Both [ 125I]Glu-P and [125I]Ac-Glu-P showed residence times in the blood similar to those of [125I]Ac-P and [125I]Suc-P. However, [125I]Glu-P also registered higher radioactivity levels in the kidney. High hepatic and renal radioactivity levels were observed with highly anionic [125I]Suc-Glu-P. These results indicate that, while the manipulation of pI between 5 to 6 would be appropriate to enhance blood retention and reduce renal and hepatic uptake, the amount of primary amine residues on dendrimer surface may also play a crucial role in their renal uptake. The findings in this study show that γ-glutamyl PAMAM dendrimers would constitute versatile precursors to prepare PAMAM dendrimer-based targeting devices due to their neutral molecular charge (pI 7.1) and the presence of a large number of α-amine residues available for conjugation of targeting molecules and drugs/imaging agents. © 2010 American Chemical Society.