Hiroyuki Kimura

Epithelial cell adhesion molecule (EpCAM) is overexpressed in various epithelial malignancies and represents an attractive target for molecular imaging and radionuclide therapy. This study aimed to develop and evaluate a novel EpCAM-targeted monoclonal antibody for single-photon emission computed tomography (SPECT) imaging. To this end, Ep4-1, a high-affinity anti-EpCAM monoclonal antibody, was conjugated to the bifunctional chelator p-SCN-Bn-diethylenetriaminepentaacetic acid (DTPA) and radiolabeled with indium-111 (111In). In vitro EpCAM-binding affinity, plasma stability, and cellular internalization were examined using high-EpCAM-expressing DLD-1 cells. In vivo biodistribution, blocking studies, and SPECT/computed tomography (CT) imaging were performed in DLD-1 tumor-bearing mice. Results showed that [111In]In-DTPA-Ep4-1 retained high, EpCAM-specific binding after chelator conjugation and showed excellent plasma stability. The radiolabeled antibody exhibited pronounced cellular internalization; approximately 93% of cell-associated radioactivity was internalized in DLD-1 cells at 6 h, exceeding the reported internalization efficiency of previously described EpCAM-targeted antibodies. In vivo biodistribution indicated progressive and selective tumor uptake, reaching 15.1 ± 1.3% ID/g at 72 h after injection. Co-injection of excess unlabeled Ep4-1 reduced tumor accumulation, consistent with EpCAM-specific targeting. Uptake in the liver and spleen was consistent with the expected pharmacokinetics of intact IgG antibodies. SPECT/CT imaging enabled clear visualization of EpCAM-expressing tumors at 72 h after injection. In conclusion, Ep4-1 is a highly specific and strongly internalizing EpCAM-targeted monoclonal antibody that enables robust SPECT imaging of EpCAM-positive tumors. Given the favorable internalization properties of Ep4-1, it may serve as a promising imaging platform and a potential candidate for future EpCAM-directed radiotheranostics applications, including α-particle-based radionuclide therapy.

Current methods for diagnosing primary aldosteronism (PA) are limited by their invasiveness and diagnostic accuracy. This study aimed to develop nuclear medicine imaging tracers targeting CYP11B2, which is overexpressed in patients with PA. We successfully synthesized iodinated and fluoroethoxynated pyridinyldihydroquinolinone (PDHQ) derivatives, among which PDHQ-1 exhibited the highest selectivity for CYP11B2. Furthermore, [125I]PDHQ-1 accumulated in the adrenal gland soon after administration, reaching its highest saturation compared to that in other organs 5 min after administration; however, its radioactivity decreased over time. Autoradiographic analysis revealed that [125I]PDHQ-1 displayed a 4.4-fold higher accumulation in the CYP11B2 region of adrenal sections from human patients with aldosterone-producing adenomas than in the CYP11B1 region. In contrast, [125I]IMTO, which is a highly specific radiotracer for imaging adrenocortical tissue, displayed similar accumulation in the CYP11B2 and CYP11B1 regions. Collectively, our results suggest that [125I]PDHQ-1, featuring a pyridinyldihydroquinolinone scaffold, shows potential as an imaging tracer for PA.

A streamlined radiosynthetic methodology enables the precise sequential assembly of boronic ester precursors on diol resin, seamlessly integrating copper-mediated radioiododeboronation and azide-alkyne cycloaddition. This approach yields radioiodinated compounds with high radiochemical conversion (>90%) without requiring column purification until the final stage, thereby accelerating the efficient development of advanced radioiodinated molecular probes.

Recently, we demonstrated that the alopecia observed in vitamin D receptor gene-deficient (Vdr-KO) rats is not seen in rats with a mutant VDR(R270L/H301Q), which lacks ligand-binding ability, suggesting that the ligand-independent action of VDR plays a crucial role in maintaining the hair cycle. Since Vdr-KO rats also showed abnormalities in the skin, the relationship between alopecia and skin abnormalities was examined. To clarify the mechanism of actions of vitamin D and VDR in the skin, protein composition, and gene expression patterns in the skin were compared among Vdr-KO, Vdr-R270L/H301Q, and wild-type (WT) rats. While Vdr-R270L/H301Q rats exhibited normal skin formation similar to WT rats, Vdr-KO rats showed remarkable hyperkeratosis and trans-epidermal water loss in the skin. RNA sequencing and proteomic analysis revealed that the gene and protein expression patterns in Vdr-KO rats significantly differed from those in WT and Vdr-R270L/H301Q rats, with a marked decrease in the expression of factors involved in Shh, Wnt, and Bmp signaling pathways, a dramatic reduction in the expression of hair keratins, and a substantial increase in the expression of epidermal keratins. This study clearly demonstrated that non-liganded VDR is significantly involved in the differentiation, proliferation, and cell death of keratinocytes in hair follicles and the epidermis.

Recently, we demonstrated that the alopecia observed in vitamin D receptor gene deficient (VDR-KO) rats is not seen in rats with a mutant VDR (R270L/H301Q), which lacks ligand-binding ability, suggesting that the ligand-independent action of VDR plays a crucial role in maintaining the hair cycle. Since VDR-KO rats also showed abnormalities in the skin, the relationship between alopecia and skin abnormalities was examined. To clarify the mechanism of actions of vitamin D and VDR in the skin, protein composition, and gene expression patterns in the skin were compared among VDR-KO, VDR (R270L/H301Q), and wild-type (WT) rats. While R270L/H301Q rats exhibited normal skin formation similarly to WT rats, VDR-KO rats showed a remarkable hyperkeratosis and trans-epidermal water loss in the skin. RNA sequencing and proteomic analysis revealed that the gene and protein expression patterns in VDR-KO rats significantly differed from those in WT and VDR (R270L/H301Q) rats, with a marked decrease in the expression of factors involved in Shh, Wnt, and Bmp signaling pathways, a dramatic reduction in the expression of hair keratins, and a substantial increase in the expression of epidermal keratins. This study clearly demonstrated that non-liganded VDR is significantly involved in the differentiation, proliferation, and cell death of keratinocytes in hair follicles and the epidermis.

The copper‐catalyzed Chan–Evans–Lam (CEL) coupling reaction advances carbon–heteroatom cross‐coupling and has facilitated the development of radiohalogenation methodologies in radiochemistry. This study investigated the mechanisms and side reactions of CEL iodination under conditions relevant to radiosynthesis and typical organic synthesis, focusing on the effects of sodium iodide. The concentrations of copper and iodide, as well as the copper‐to‐iodide ratio, were identified as significant factors for successful copper‐mediated CEL iodination, influencing the reaction mechanisms and side reactions. Excess iodide relative to the copper salt led to the formation of poorly soluble iodinated copper(I) complexes that competed with that of the desired aryl iodide. Additionally, the predominant copper complex involved in the catalytic cycle differed between the early and late stages of the reaction, depending on the copper‐to‐iodide ratio. The results of this study indicate that the specialized radiosynthesis conditions meet the requirements for efficient CEL iodination. In particular, an extremely low concentration of iodide is optimal for CEL iodination. These in‐depth mechanistic insights not only provide a detailed comparison of CEL iodination across radiochemistry and synthetic organic chemistry but can also inspire the development of novel (radio)iodination methods.

We designed and synthesized a novel platinum complex conjugated with 2-fluorinated 2-deoxyglucoside, named FGC-Pt, to capitalize on the Warburg effect and metabolic trapping properties of [¹⁸F]2-deoxy-2-fluoro-d-glucose ([¹⁸F]FDG). Then, we conducted comprehensive in vitro and in vivo studies to evaluate the effects of FGC-Pt. In vitro cytotoxicity assays using HeLa cells revealed that FGC-Pt exhibited concentration-dependent cytotoxicity, even though its cytotoxic effect was less pronounced than that of cisplatin. In the evaluation of in vivo biodistribution in mice, platinum concentration in tumors and major organs (muscle, bone, blood, liver, and kidney) and the ratio of platinum concentration in tumors to major organs following the tail vein injection of FGC-Pt and cisplatin suggest that FGC-Pt is more retained in tumors than in other organs and tends to accumulate in tumors more than cisplatin. Furthermore, an in vivo assessment of the antitumor effect conducted in A549 cell-bearing mice demonstrated that FGC-Pt possesses substantial potential as an antitumor agent. It exhibited a tumor growth-inhibitory effect comparable to that of cisplatin while inducing lower toxicity, as evidenced by lower weight loss after administration. Herein, we successfully produced a novel compound with a tumor-growth-inhibitory effect comparable to that of cisplatin and low toxicity.

Copper-mediated one-pot two-step radioiodination proceeds under mild conditions. Furthermore, this methodology operates with continuous incorporation of reagents into the reaction system and a single-step purification.

Immunogenic responses by protein therapeutics often lead to reduced therapeutic effects and/or adverse effects via the generation of neutralizing antibodies and/or antidrug antibodies (ADA). Mirror-image proteins of the variable domain of the heavy chain of the heavy chain antibody (VHH) are potential novel protein therapeutics with high-affinity binding to target proteins and reduced immunogenicity because these mirror-image VHHs (d-VHHs) are less susceptible to proteolytic degradation in antigen-presenting cells (APCs). In this study, we investigated the preparation protocols of d-VHHs and their biological properties, including stereoselective target binding and immunogenicity. Initially, we established a facile synthetic process of two model VHHs [anti-GFP VHH and PMP12A2h1 (monomeric VHH of caplacizumab)] and their mirror-image proteins by three-step native chemical ligations (NCLs) from four peptide segments. The folded synthetic VHHs (L-anti-GFP VHH and L-PMP12A2h1) bound to the target proteins (EGFP and vWF-A1 domain, respectively), while their mirror-image proteins (d-anti-GFP VHH and d-PMP12A2h1) showed no binding to the native proteins. For biodistribution studies, L-VHH and d-VHH with single radioactive indium diethylenetriamine-pentaacid (In-111-DTPA) labeling at the C-terminus were designed and synthesized by the established protocol. The distribution profiles were essentially similar between L-VHH and d-VHH, in which the probes accumulated in the kidney within 15 min after intravenous administration in mice, because of the small molecular size of VHHs. Comparative assessment of the immunogenicity responses revealed that d-VHH-induced levels of ADA generation were significantly lower than those of native VHH, regardless of the peptide sequences and administration routes. The resulting scaffold investigated should be applicable in the design of d-VHHs with various C-terminal CDR3 sequences, which can be identified by screening using display technologies.