Takeshi Fuchigami

SVS-1, a cationic amphiphilic peptide (CAP), adopts a β-hairpin conformation only upon interacting with the surface of cancer cells, thereby disrupting their membrane. Previously, we developed [67Ga]Ga-NOTA-KV6, an SVS-1 derivative for tumor imaging, which demonstrated favorable early tumor uptake but poor retention. To address this limitation, we designed and evaluated a novel d-amino acid–based SVS-1 analogue, [67Ga]Ga-NOTA-d-KV6, to enhance metabolic stability and improve pharmacokinetic properties. Fluorescein isothiocyanate (FITC)-labeled d-KV6 exhibited high accumulation in cancer cells, similar to that of FITC-KV6. In addition, uptake of [67Ga]Ga-NOTA-d-KV6 was significantly greater in cancer cells than in normal cells. Similar to [67Ga]Ga-NOTA-KV6, cellular uptake of [67Ga]Ga-NOTA-d-KV6 in cancer cells was significantly reduced at low temperature (4 °C) and in the presence of several different types of endocytic inhibitors, indicating internalization via multiple endocytosis pathways. Metabolic stability studies revealed that [67Ga]Ga-NOTA-d-KV6 was significantly more stable than [67Ga]Ga-NOTA-KV6. An in vivo biodistribution study of [67Ga]Ga-NOTA-d-KV6 revealed low blood retention, high accumulation in the liver and kidneys, and minimal uptake by other organs. Tumor accumulation of [67Ga]Ga-NOTA-d-KV6 was moderate, but tumor retention was improved compared with that of [67Ga]Ga-NOTA-KV6, leading to a tumor-to-blood ratio of 17.3 at 240 min postinjection. These findings demonstrate that d-amino acid substitution improved the plasma stability and tumor retention of the CAP derivative while maintaining preferential uptake in cancer cells. Although further optimization is required to reduce liver and kidney accumulation and improve tumor-to-background contrast, mirror peptide formation represents a promising strategy for the development of CAP-based cancer theranostic agents.

Macropa has formed more stable complexes with 225Ac, although DOTA has been extensively used as a chelator for 225Ac. This study aimed to design and synthesize two novel 225Ac-labeled RGD peptides, [225Ac]Ac-Macropa-c(RGDyK) ([225Ac]1) and [225Ac]Ac-Macropa-[c(RGDyK)]2 ([225Ac]2), and to evaluate their potential for targeted alpha therapy (TAT) of glioblastoma. Further, whether combination therapy with homoarginine (hArg), a modulator of lysosomal function, could further improve therapeutic efficacy was investigated. In Colon-26 cells, [225Ac]2 demonstrated significantly higher cellular uptake and cytotoxicity than [225Ac]1. In Colon-26 tumor-bearing mice, [225Ac]2 exhibited higher tumor accumulation and significant tumor growth inhibition. In GL261 glioblastoma cells, [225Ac]2 showed comparable cellular uptake to that in Colon-26 cells, and combination with hArg in vitro improved its cytotoxicity. However, no significant difference in therapeutic efficacy was observed between the hArg-treated and control groups in the orthotopic GL261 glioblastoma mouse model. The results of this study indicate the potential of 225Ac-labeled RGD peptides incorporating Macropa as a chelator as promising agents for TAT. However, the further optimization of combination strategies and pharmacokinetics is required.

In Auger electron therapy, the effective delivery of Auger electron-emitting radionuclides to the nuclei of cancer cells is essential for achieving high therapeutic efficacy. In this study, we developed a novel strategy that combined thermosensitive liposomes (TSLs) encapsulating 125I-labeled DNA-targeted compounds. This approach enabled enhanced tumor accumulation via TSL-mediated delivery and efficient nuclear translocation of radioactivity by releasing an 125I-labeled doxorubicin derivative ([125I]I-DOX) within the tumor microenvironment. [125I]I-DOX exhibited high cellular and nuclear uptake, producing potent cytotoxicity through Auger electrons emitted from 125I. [125I]I-DOX-loaded TSLs efficiently released [125I]I-DOX at 43 °C, enhancing its cellular uptake by Colon-26 cells. Tumor accumulation of radioactivity in the heating group was much higher than in the nonheating group, and the accumulation was maintained for up to 48 h postinjection. Intravenous injection of [125I]I-DOX-loaded TSLs (5.0 MBq) and localized tumor heating significantly inhibited tumor growth without body weight loss. The combination of [125I]I-DOX-loaded TSLs and hyperthermia represents a promising strategy for Auger electron therapy targeting the nuclei of cancer cells.

[¹¹¹In]In-diethylenetriaminepentaacetic acid-5,10,15,20-tetraphenylporphyrin ([¹¹¹In]In-DTPA-TPP) nanodroplets were developed for cancer theranostics, featuring ultrasound-sensitive properties. The designed nanodroplets that encapsulate the low-boiling-point liquid perfluorocarbon and IR-780 iodide, a near-infrared fluorescent dye, with surface conjugation of ¹¹¹In-labeled porphyrin derivative, were synthesized and evaluated by in vitro and in vivo experiments. The cellular uptake of [¹¹¹In]In-DTPA-TPP nanodroplets was significantly higher than that of control nanodroplets without TPP. Biodistribution experiments revealed greater tumor accumulation in mice injected with [¹¹¹In]In-DTPA-TPP nanodroplets than in those injected with control nanodroplets lacking TPP. Additionally, the accumulation of [¹¹¹In]In-DTPA-TPP nanodroplets in the tumor was visualized by single-photon emission computed tomography. Sonodynamic therapeutic experiments revealed that DTPA-TPP nanodroplets at 10 µmol total lipids/kg weight with a single ultrasound irradiation onto the tumor area significantly inhibited tumor growth. These results indicate that [¹¹¹In]In-DTPA-TPP nanodroplets would be promising cancer theranostic agents.

Legumain (LGMN), a lysosomal cysteine protease, is crucial for tumor progression, invasion, and metastasis, making it a promising target for cancer imaging and therapy. This study developed novel ⁶⁷Ga-labeled 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA)-conjugated LGMN-cleavable peptide probes ([⁶⁷Ga]Ga-NOTA-LCPs) composed of polyarginine and polyglutamic acid sequences linked by LGMN-cleavable sites for nuclear medicine imaging of LGMN activity. The probes were synthesized via fluorenylmethoxycarbonyl solid-phase peptide synthesis and radiolabeled in high radiochemical yields. In vitro assays with HCT116 cells showed significantly higher uptake of [⁶⁷Ga]Ga-NOTA-LCPs compared to non-cleavable controls, confirming efficient cleavage and cellular uptake. In vivo studies in tumor-bearing mice revealed rapid renal clearance, low non-specific binding, and favorable tumor-to-blood ratios, particularly for [⁶⁷Ga]Ga-NOTA-LCP-1. These results demonstrate the potential of [⁶⁷Ga]Ga-NOTA-LCPs as effective LGMN-responsive imaging agents, with further optimization needed to improve tumor specificity and reduce off-target accumulation.

Bone-seeking radiopharmaceuticals are essential for the early detection of bone metastases. In this study, we developed three Technetium-99m (99mTc)-labeled oligopeptides composed of acidic amino acids and evaluated their bone-targeting ability. Hydrazinonicotinamide (HYNIC)-conjugated oligopeptides with five residues of aspartic acid (Asp), glutamic acid (Glu), or γ-carboxyglutamic acid (Gla) were synthesized and radiolabeled with 99mTc using tricine and 3-acetylpyridine as co-ligands. Their hydroxyapatite binding, in vitro stability, biodistribution, and single-photon emission computed tomography (SPECT)/CT imaging in normal mice were evaluated. Among the three tracers, [99mTc]Tc-HYNIC-(tricine)(AcP)-(dl-Gla)5 ([99mTc]3) showed the highest hydroxyapatite binding and bone uptake, with clear visualization in SPECT/CT. All compounds exhibited high radiochemical purity and in vivo stability. Oligopeptides containing Gla residues exhibited superior bone affinity and imaging performance, suggesting that Gla-based oligopeptides are promising scaffolds for the development of 99mTc-labeled bone imaging agents.

In recent years, radiotheranostics, a theranostic approach utilizing radioisotopes, has been gaining significant attention, primarily in the field of oncology. The success of this technology relies on the design of probes that specifically target desired sites, making the development of effective diagnostic and therapeutic agents a crucial area of research. This review provides an overview of the fundamental concepts and recent advancements in probe design for radiotheranostics, with a particular focus on pharmacokinetics and subcellular localization. Key topics discussed include: (1) multimerization, (2) introduction of the albumin-binding moiety, (3) charge modification, (4) glycosylation, (5) conjugation of cell-penetrating peptides, (6) introduction of the covalent binding moiety, (7) targeting the nucleus, and (8) utilizing drug release properties to control pharmacokinetics and intracellular localization. Through these strategies, we review the optimization and novel design possibilities for probes in radiotheranostics.

Boron neutron capture therapy (BNCT) is one of the promising cancer treatment methods with ¹⁰B-labeled compounds and neutron irradiation. The ¹⁰B(n,α)⁷Li reaction produces a ⁷Li nucleus and an α-particle with high linear energy transfer, which are responsible for the therapeutic effects. We hypothesized that BNCT could effectively treat bone metastases by selectively accumulating ¹⁰B at metastatic lesion sites. In this study, we designed, synthesized, and evaluated [^nat/67Ga]Ga-DOTA-K(ε-closo-dodecaborate)D₁₁ ([^nat/67Ga]6), a compound integrating closo-dodecaborate for BNCT, a [^nat/67Ga]Ga-DOTA derivative for nuclear imaging, and D₁₁, an aspartic acid peptide, for bone targeting. [⁶⁷Ga]6 and its precursor 5, which lacks gallium coordination, showed comparable biodistribution in normal mice, with high bone uptake and minimal off-target accumulation. These results support the potential of [^nat/67/68Ga]6 as an effective theranostic agent for bone metastases.

Purpose: Recently, an ²¹¹ At-labeled RGD peptide with an albumin-binding moiety, Ga-DOTA-K([ ²¹¹ At]APBA)-c(RGDfK) ([ ²¹¹ At] 1 ), was developed for targeted alpha therapy and radiotheranostics. [ ²¹¹ At] 1 showed high tumor accumulation and inhibited tumor growth in a dose-dependent manner. This study aimed to investigate whether [ ²¹¹ At] 1 can induce an antitumor immune response and whether the combination of [ ²¹¹ At] 1 and immune checkpoint blockade can enhance therapeutic efficacy. Methods: Biodistribution experiments of [ ²¹¹ At] 1 and therapeutic experiments of [ ²¹¹ At] 1 with or without anti-PD-1 or anti-CTLA-4 antibody were conducted in Colon-26 tumor-bearing BALB/c mice. Additionally, therapeutic experiments of [ ²¹¹ At] 1 were conducted in Colon-26 tumor-bearing BALB/c nu/nu mice to confirm the difference in the therapeutic effects derived from antitumor immune responses of [ ²¹¹ At] 1 . Infiltration of antitumor immune activity in Colon-26 tumors was confirmed by flow cytometry and immunofluorescence staining. Results: [ ²¹¹ At] 1 showed high tumor accumulation and inhibited tumor growth in a dose-dependent manner in Colon-26 tumor-bearing BALB/c mice. Additionally, mice treated with [ ²¹¹ At] 1 (675 kBq) and anti-CTLA-4 antibody showed superior tumor growth inhibition compared with mice treated with only [ ²¹¹ At] 1 (675 kBq). Tumor growth inhibition was weaker in BALB/c nu/nu mice than in BALB/c mice. Infiltration of CD4 ⁺ and CD8 ⁺ T cells was observed following treatment with [ ²¹¹ At] 1 (675 kBq) alone or in combination with anti-CTLA-4 antibody. Conclusion: [ ²¹¹ At] 1 induced an antitumor immune response and enhanced the therapeutic efficacy. The combination of [ ²¹¹ At] 1 and immune checkpoint blockade agents could be promising for cancer therapy.