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Radionuclide imaging and therapy are promising methods for controlling systemic cancers; however, their clinical application has been limited by excessive radionuclide accumulation in healthy tissues. To minimize radionuclide accumulation in non-cancerous tissues while ensuring sufficient build up in tumors, we aimed to develop a method that controlled the in vivo dynamics of radionuclides post-administration. To this end, we describe a novel strategy that combines liposomes, a potent carrier system for drug delivery, with unique radionuclide-ligand complexes based on 111 In-ethylenedicysteine. Conventional 111 In-ligand-complexes-carrying liposomes delivered substantial amounts of radionuclides to tumors; however, they also accumulated in the liver and spleen. In contrast, 111 In-ethylenedicysteine-carrying liposomes greatly reduced non-specific accumulation, while being retained selectively at high doses within tumors. Liposomes were rapidly broken down in the liver, releasing encapsulated 111 In-ligand complexes. Among the chelates used, only 111 In-ethylenedicysteine could escape from the liver and be excreted in the urine. Instead, most liposomes remained intact in tumors, retaining the radionuclide-ligand complexes within them. Therefore, high tumor accumulation was obtained regardless of the type of 111 In-ligand complexes in the liposomes. In vivo single photon emission computed tomography/computed tomography imaging with 111 In-ethylenedicysteine-carrying liposomes accurately revealed tumor-selective radionuclide retention with little background. Hence, our new strategy could greatly enhance tumor-to-healthy tissue ratios, improve diagnostic imaging, boost therapeutic efficacy, reduce toxicity to healthy tissues, and facilitate radionuclide imaging and therapy., Competing Interests: Declaration of Competing Interest The authors have no relevant financial or non-financial interests to disclose., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The interposition of a cleavable linkage by enzymes on the renal brush border membrane constitutes a promising approach for reducing the renal radioactivity levels of radiolabeled low-molecular-weight antibody fragments and constructs (LMW Abs). Herein, we applied the molecular design to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-based reagents for radiotheranostic applications with trivalent radiometals. DOTA or a derivative thereof was conjugated to a Fab through an FGK linkage ([ 111 In]In-DO3A i Bu-Bn-FGK-Fab or [ 111 In]In-DOTA-Bn-FGK-Fab). When injected into mice, both generated radiometabolites, [ 111 In]In-DO3A i Bu-Bn-F and [ 111 In]In-DOTA-Bn-F, by the angiotensin-converting enzyme at similar rates. Both exhibited significantly lower renal radioactivity levels than a 111 In-labeled Fab prepared by the conventional procedure ([ 111 In]In-DOTA-Bn-SCN-Fab). The different elimination rates of each radiometabolite from the kidney significantly affected the renal radioactivity levels. [ 111 In]In-DO3A i Bu-Bn-FGK-Fab preferentially reduced the renal localization without impairing tumor accumulation. These findings would pave the way for developing a DOTA-based radiotheranostic platform for LMW Abs bearing cleavable linkers for renal brush border enzymes.

Chemotherapy for peritoneal dissemination is poorly effective owing to limited drug transfer from the blood to the intraperitoneal (i.p.) compartment after intravenous (i.v.) administration. i.p. chemotherapy has been investigated to improve drug delivery to tumors; however, the efficacy continues to be debated. As anticancer drugs have low molecular weight and are rapidly excreted through the peritoneal blood vessels, maintaining the i.p. concentration as high as expected is a challenge. In this study, we examined whether i.p. administration is an efficient route of administration of high-molecular-weight immune checkpoint inhibitors (ICIs) for the treatment of peritoneal dissemination using a model of peritoneal disseminated carcinoma. After i.p. administration, the amount of anti-PD-L1 antibody transferred into i.p. tumors increased by approximately eight folds compared to that after i.v. administration. Intratumoral distribution analysis revealed that anti-PD-L1 antibodies were delivered directly from the i.p. space to the surface of tumor tissue, and that they deeply penetrated the tumor tissues after i.p. administration; in contrast, after i.v. administration, anti-PD-L1 antibodies were only distributed around blood vessels in tumor tissues via the enhanced permeability and retention (EPR) effect. Owing to the enhanced delivery, the therapeutic efficacy of anti-PD-L1 antibody in the peritoneal dissemination models was also improved after i.p. administration compared to that after i.v. administration. This is the first study to clearly demonstrate an EPR-independent delivery of ICIs to i.p. tumors by which ICIs were delivered in a massive amount to the tumor tissue via direct penetration after i.p. administration., Competing Interests: Declaration of Competing Interest Authors declare that they have no competing interests in this research., (Copyright © 2022 Elsevier B.V. All rights reserved.)

1,4,7,10-Tetraazacyclododecane-1,4,7-triacetic acid (DO3A) has been used to prepare 68 Ga-labeled probes for the diagnostic counterpart of radiotheranostic applications. While DO3A provides stable complexes with therapeutic radionuclides such as 90 Y, 177 Lu, and 225 Ac, further improvement of the in vivo stability of the Ga-DO3A complex is required. Considering the high stability of an intact Ga-DOTA complex, the stability of Ga complexes of DOTA and DO3A derivatives, including benzyl-DOTA (Bn-DOTA), was evaluated to gain fundamental knowledge for developing the next-generation radiotheranostic probes using 68 Ga as a diagnostic counterpart. Following the complexation reaction to prepare 67 Ga-labeled DOTA and DO3A derivatives, the stability of the resulting 67 Ga-labeled compounds was evaluated in murine plasma and apo-transferrin challenge. [ 67 Ga]Ga-Bn-DOTA produced two isomers, and one of the isomers exhibited the highest stability among the tested complexes. The X-ray crystallography showed that the less stable isomer of Ga-Bn-DOTA suggested an N 3 O 3 coordination geometry, while Ga-DOTA and Ga-Bn-DO3A show N 4 O 2 coordination. To further evaluate the stability, a synthetic somatostatin analogue, [Tyr 3 ]octreotide (TOC), was used as a model peptide, and p -COOH-Bn-DOTA and DO3A were conjugated with TOC to prepare DOTA-Bn-TOC and DOTATOC. [ 67 Ga]Ga-DOTA-Bn-TOC also yielded two isomers with varying stability, and one isomer exhibited significantly higher stability than [ 67 Ga]Ga-DOTATOC both in vitro and in vivo . These findings indicate that para -substituted Bn-DOTA would constitute a suitable chelating agent for developing next-generation radiotheranostic probes, although high-performance liquid chromatography purification is needed. Thus, further chemical modification on the Bn-DOTA molecule is also needed to avoid the formation of a Ga complex with the N 3 O 3 configuration., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

Introduction: Radiolabeled peptides and low-molecular-weight (LMW) polypeptides show high and persistent radioactivity levels in the kidney. To develop a DOTA-based bifunctional chelating agent that provides a radiometabolite with a rapid elimination rate from the kidney, a para-carboxyl Bn-DOTA (p-COOH-Bn-DOTA) was designed, synthesized, and evaluated., Methods: A precursor compound, p-COOH-Bn-DOTA( t Bu) 4 , was synthesized in 9 steps using N-Boc-p-iodo-L-phenylalanine as the starting material. A synthetic somatostatin analog (TOC) was used as a representative peptide metabolized in the renal lysosomes. p-COOH-Bn-DOTA-conjugated TOC (DOTA-Bn-TOC) was synthesized by the conventional solid-phase peptide synthesis using p-COOH-Bn-DOTA( t Bu) 4 . DOTA-tris( t Bu ester) was also conjugated with TOC to prepare DOTATOC. 111 In-labeling of the peptides was conducted under similar conditions. The radiochemical conversions, stability against apo-transferrin (apoTf), and in vivo behaviors were compared., Results: [ 111 In]In-DOTA-Bn-TOC was obtained with higher radiochemical conversions than [ 111 In]In-DOTATOC. Both 111 In-labeled TOC derivatives remained stable against apoTf. In biodistribution studies, [ 111 In]In-DOTA-Bn-TOC exhibited higher initial uptake in the kidney, followed by a faster elimination rate of radioactivity into the urine than [ 111 In]In-DOTATOC. The metabolic studies showed that the shorter residence time of the radiometabolite from [ 111 In]In-DOTA-Bn-TOC was responsible for the renal radioactivity decline., Conclusion: p-COOH-Bn-DOTA provides stable 111 In-labeled peptides in high yields at low peptide concentrations. p-COOH-Bn-DOTA also provides a radiometabolite with a short residence time in the kidney. Such characteristics would render p-COOH-Bn-DOTA useful to the future application to radiolabeled LMW polypeptides with low renal radioactivity levels., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Prostate cancer frequently metastasizes to the bone. Because patients with bone metastases suffer from skeletal-related events, the diagnosis and treatment of bone metastases in the early stage are important. In this study, to improve the sensitivity of detecting bone metastases in patients with prostate cancer, we designed, synthesized, and evaluated a multifunctional radiotracer, [ 67 Ga]Ga-D 11 -PSMA-617 ([ 67 Ga]3), with an undeca-aspartic acid as a bone-seeking moiety between [ 67 Ga]Ga-DOTA and a prostate-specific membrane antigen (PSMA) ligand based on the lysine-urea-glutamate motif. [ 67 Ga]3 showed a high affinity for hydroxyapatite and high uptake in PSMA-positive LNCaP cells. Moreover, in biodistribution experiments using tumor-bearing mice, [ 67 Ga]3 exhibited high accumulation in the bone and PSMA-positive tumor although the accumulation of [ 67 Ga]3 in the PSMA-positive tumor was lower than that of [ 67 Ga]Ga-PSMA-617. This study provides valuable information for developing radiotheranostic probes combining multiple carriers with different mechanisms., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Neuroinflammation and oxidative stress are hallmarks of neurodegenerative diseases. Microglia, the major important regulators of neuroinflammation, are activated in response to excessive generation of reactive oxygen species (ROS) from damaged cells and resulting in elevated and sustained damages. However, the relationship between microglia and ROS-regulatory system in the early stages of neuroinflammation prior to the appearance of neuronal damages have not been elucidated in detail. In this study, we analyzed the time-dependent changes in ROS generation during acute neuroinflammation in rats that were given an intrastriatal injection of lipopolysaccharide (LPS). We evaluated the effects of minocycline, an anti-inflammatory antibiotic, and N,N'-dimethylthiourea (DMTU), a radical scavenger, to understand the correlation between activated microglia and ROS generation. Ex vivo fluorescence imaging using dihydroethidium (DHE) clearly demonstrated an increased ROS level in the infused side of striatum in the rats treated with LPS. The level of ROS was changed in time-dependent manner, and the highest level of ROS was observed on day 3 after the infusion of LPS. Immunohistochemical studies revealed that time-dependent changes in ROS generation were well correlated to the presence of activated microglia. The inhibition of microglial activation by minocycline remarkably reduced ROS levels in the LPS-injected striatum, which indicated that the increased ROS generation caused by LPS was induced by activated microglia. DMTU decreased ROS generation and resulted in remarkable inhibitory effect on microglial activation. This study demonstrated that ROS generation during acute neuroinflammation induced by LPS was considerably associated with microglial activation, in an intact rat brain. The results provides a basis for understanding the interaction of ROS-regulatory system and activated microglia during neuroinflammation underlying neurodegenerative diseases., Competing Interests: Declaration of competing interest The authors declare that they have no competing of interest., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The high in vivo stability of 2,2-dihydroxymethyl-3-[ 18 F]fluoropropyl-2-nitroimidazole ([ 18 F]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 125 I-labeled compounds remained stable against nucleophilic substitution, only a 125 I-labeled neopentyl glycol was stable against cytochrome P450 (CYP)-mediated metabolism and showed high stability against in vivo deiodination. 211 At-labeled neopentyl glycol also remained stable against both nucleophilic substitution and CYP-mediated metabolism. 211 At-labeled neopentyl glycol showed the biodistribution profiles similar to those of its radioiodinated counterpart in contrast to the 125 I/ 211 At-labeled benzoate pair. The urine analyses confirmed that 211 At-labeled neopentyl glycol was excreted in the urine as a glucuronide conjugate with the absence of free [ 211 At]At - . These findings indicate that neopentyl glycol would constitute a promising scaffold to prepare a radiotheranostic system with radioiodine and 211 At.