Hideo Saji

Zinc is an essential trace element involved in many biological activities; however, its functions are not fully understood. To elucidate the role of endogenous labile Zn²⁺, we developed a novel ratiometric fluorescence probe, 5-(4-methoxyphenyl)-4-(methylsulfanyl)-[2,2′-bipyridin]-6-amine (6 (rBpyZ)) based on the 6-amino-2,2′-bipyridine scaffold, which acts as both the chelating agent for Zn²⁺ and the fluorescent moiety. The methoxy group acted as an electron donor, enabling the intramolecular charge transfer state of 6 (rBpyZ), and a ratiometric fluorescence response consisting of a decrease at the emission wavelength of 438 nm and a corresponding increase at the emission wavelength of 465 nm was observed. The ratiometric probe 6 (rBpyZ) exhibited a nanomolar-level dissociation constant (K_d = 0.77 nM), a large Stokes shift (139 nm), and an excellent detection limit (0.10 nM) under physiological conditions. Moreover, fluorescence imaging using A549 human lung adenocarcinoma cells revealed that 6 (rBpyZ) had good cell membrane permeability and could clearly visualize endogenous labile Zn²⁺. These results suggest that the ratiometric fluorescence probe 6 (rBpyZ) has considerable potential as a valuable tool for understanding the role of Zn²⁺ in living systems.

Hyperlipidemia causes systemic lipid disorder, which leads to hepatic steatosis and atherosclerosis. Thus, it is necessary to detect these syndromes early and precisely to improve prognosis. In the affected regions, abnormal formation and growth of lipid droplets is observed; therefore, lipid droplets may be a suitable target for the diagnosis of hyperlipidemia-related syndromes. In this study, we designed and synthesized [^99mTc]Tc-BOD and [^99mTc]Tc-MBOD composed of one technetium-99m and two BODIPY scaffolds with hydroxamamide (Ham) or N-methylated hydroxamamide (MHam) in radiochemical yields of 54 and 35%, respectively, with a radiochemical purity of over 95%. [^99mTc]Tc-BOD showed significantly higher accumulation levels in foam cells than in non-foam cells (foam cells: 213.8 ± 64.8, non-foam cell: 126.2 ± 26.9 %dose/mg protein, p < 0.05) 2 h after incubation. In contrast, [^99mTc]Tc-MBOD showed similar accumulation levels in foam cells and non-foam cells (foam cells: 92.2 ± 23.3, non-foam cell: 83.8 ± 19.8 %dose/mg protein). In normal mice, [^99mTc]Tc-BOD exhibited gradual blood clearance (0.5 h: 4.98 ± 0.35, 6 h: 1.94 ± 0.12 %ID/g) and relatively high accumulation in the liver 6 h after administration (15.22 ± 1.72 %ID/g). Therefore, [^99mTc]Tc-BOD may have potential as an imaging probe for detecting lipid droplets in disease lesions of hyperlipidemia.

A small extent of endogenous labile zinc is involved in many vital physiological roles in living systems. However, its detailed functions have not been fully elucidated. In this study, we developed a novel biheteroaryl-based low molecular weight fluorescent sensor, 3-(phenylsulfonyl)-pyrazine–pyridone (5b), and applied it for the detection of endogenous labile zinc ions from lung cancer cells during apoptosis. The electron-withdrawing property of the sulfonyl group between the phenyl ring as an electron donor and the pyridone ring as a fluorophore inhibited the intramolecular charge transfer state, and the background fluorescence of the sensor was decreased in aqueous media. From the structure–fluorescence relationship analysis of the substituent effects with/without Zn²⁺, compound 5b acting as a sensor possessed favorable properties, including a longer emission wavelength, a large Stokes shift (over 100 nm), a large fluorescence enhancement in response to Zn²⁺ under physical conditions, and good cell membrane permeability in living cells. Fluorescence imaging studies of human lung adenocarcinoma cells (A549) undergoing apoptosis revealed that compound 5b could detect endogenous labile zinc ions. These experiments suggested that the low molecular weight compound 5b is a potential fluorescence sensor for Zn²⁺ toward understanding its functions in living systems.

Carbonic anhydrase IX (CA-IX) is regarded as a favorable target for in vivo imaging because of its specific expression in hypoxic regions of tumors. Hypoxia assists tumor propagation and growth and is resistant to chemotherapy and radiotherapy. Here, we designed and synthesized [^99mTc]hydroxamamide ([^99mTc]Ham) and [^99mTc]methyl-substituted-hydroxamamide ([^99mTc]MHam) complexes including a bivalent CA-IX ligand, sulfonamide (SA), and ureidosulfonamide (UR). In a cell binding assay, [^99mTc]Ham complexes with bivalent SA ([^99mTc]SAB2A and [^99mTc]SAB2B) and UR ([^99mTc]URB2A and [^99mTc]URB2B) showed significantly greater uptake into CA-IX high-expressing (HT-29) cells than that into CA-IX low-expressing cells. Since the binding affinity of [^99mTc]URB2A and [^99mTc]URB2B for CA-IX was significantly higher than that of [^99mTc]SAB2A and [^99mTc]SAB2B, we additionally synthesized [^99mTc]MURB2 (a [^99mTc]MHam complex with bivalent UR) and evaluated the CA-IX-specific binding affinity of [^99mTc]URB2A, [^99mTc]URB2B, and [^99mTc]MURB2. Their uptake into HT-29 cells was reduced by the addition of a CA inhibitor, acetazolamide, suggesting their CA-IX-specific binding affinity. A biodistribution study in HT-29 tumor-bearing mice was carried out using [^99mTc]URB2A and [^99mTc]MURB2 with the highest specificity for HT-29 cells. [^99mTc]URB2A showed moderate tumor uptake and reduction by coinjection with acetazolamide; however, the tumor/blood ratio was insufficient for in vivo imaging. These results provided key information for the design of novel Ham-based imaging probes targeting CA-IX.

Deposition of islet amyloid consisting of amylin constitutes one of pathological hallmarks of type 2 diabetes mellitus (T2DM), and it may be involved in the development and progression of T2DM. However, the details about the relationship between the deposition of islet amyloid and the pathology of T2DM remain unclear, since no useful imaging tracer enabling the visualization of pancreatic amylin is available. In the present study, we synthesized and evaluated six novel ¹⁸F-labeled phenoxymethylpyridine (PMP) derivatives as amylin imaging probes. All ¹⁸F-labeled PMP derivatives showed not only affinity for islet amyloid in the post-mortem T2DM pancreatic sections but also excellent pharmacokinetics in normal mice. Furthermore, ex vivo autoradiographic studies demonstrated that [¹⁸F]FPMP-5 showed intense labeling of islet amyloids in the diabetes model mouse pancreas in vivo. The preclinical studies suggested that [¹⁸F]FPMP-5 may have potential as an imaging probe that targets amylin aggregates in the T2DM pancreas.