ZNT5-6 and ZNT7 play an integral role in protein N-glycosylation by supplying Zn2+ to Golgi α-mannosidase II.

The stepwise addition of monosaccharides to N-glycans attached to client proteins to generate a repertoire of mature proteins involves a concerted action of many glycosidases and glycosyltransferases. Here, we report that Golgi α-mannosidase II (GMII), a pivotal enzyme catalyzing the first step in the conversion of hybrid- to complex-type N-glycans, is activated by Zn2+ supplied by the early secretory compartment-resident ZNT5-ZNT6 heterodimers (ZNT5-6) and ZNT7 homodimers (ZNT7). Loss of ZNT5-6 and ZNT7 function results in marked accumulation of hybrid-type and complex/hybrid glycans with concomitant reduction of complex- and high-mannose-type glycans. In cells lacking the ZNT5-6 and ZNT7 functions, the GMII activity is substantially decreased. In contrast, the activity of its homolog, lysosomal mannosidase (LAMAN), is not decreased. Moreover, we show that the growth of pancreatic cancer MIA PaCa-2 cells lacking ZNT5-6 and ZNT7 is significantly decreased in a nude mouse xenograft model. Our results indicate the integral roles of ZNT5-6 and ZNT7 in N-glycosylation and highlight their potential as novel target proteins for cancer therapy.

Optimizing the pharmacokinetics of an 211At-labeled RGD peptide with an albumin-binding moiety via the administration of an albumin-binding inhibitor.

PURPOSE: A probe for targeted alpha therapy (TAT) using the RGD peptide (Ga-DOTA-K([211At]APBA)-c(RGDfK) ([211At]1)) with albumin-binding moiety (ABM) was recently developed. [211At]1 highly accumulated in tumors and significantly inhibited tumor growth in U-87 MG tumor-bearing mice. However, high [211At]1 retention in blood may cause critical adverse events, such as hematotoxicity. Therefore, we attempted to accelerate the blood clearance of [211At]1 by competitively inhibiting the binding of [211At]1 to albumin to modulate the pharmacokinetics of the former. METHODS: To evaluate the effects of albumin-binding inhibitors in normal mice, sodium 4-(4-iodophenyl)butanoate at 2, 5, or 10 molar equivalents of blood albumin was administered at 1-h postinjection of [211At]1. The biodistribution of [211At]1, SPECT/CT imaging of [67Ga]Ga-DOTA-K(IPBA)-c(RGDfK) ([67Ga]2), and the therapeutic effects of [211At]1 were compared with or without IPBA administration in U-87 MG tumor-bearing mice. RESULTS: Blood radioactivity of [211At]1 was decreased in a dose-dependent manner with IPBA in normal mice. In U-87 MG tumor-bearing mice, the blood radioactivity and accumulation in nontarget tissues of [211At]1 were decreased by IPBA. Meanwhile, tumor [211At]1 accumulation was not changed at 3-h postinjection of IPBA. In SPECT/CT imaging of [67Ga]2, IPBA administration dramatically decreased radioactivity in nontarget tissues, and only tumor tissue was visualized. In therapeutic experiments, [211At]1 with IPBA injected-group significantly inhibited tumor growth compared to the control group. CONCLUSION: IPBA administration (as an albumin-binding inhibitor) could modulate the pharmacokinetics and enhance the therapeutic effects of [211At]1.

Development of probes for radiotheranostics with albumin binding moiety to increase the therapeutic effects of astatine-211 (211At).

PURPOSE: We have developed probes for multiradionuclides radiotheranostics using RGD peptide ([67Ga]Ga-DOTA-c[RGDf(4-I)K] ([67Ga]1) and Ga-DOTA-[211At]c[RGDf(4-At)K] ([211At]2)) for clinical applications. The introduction of an albumin binding moiety (ABM), such as 4-(4-iodophenyl)-butyric acid (IPBA), that has high affinity with the blood albumin and prolongs the circulation half-life can improve the pharmacokinetics of drugs. To perform more effective targeted alpha therapy (TAT), we designed and synthesized Ga-DOTA-K([211At]APBA)-c(RGDfK) ([211At]5) with 4-(4-astatophenyl)-butyric acid (APBA), which has an astato group instead of an iodo group in IPBA. We evaluated whether APBA functions as ABM and [211At]5 is effective for TAT. In addition, we prepared 67Ga-labeled RGD peptide without ABM, [67Ga]Ga-DOTA-K-c(RGDfK) ([67Ga]3), and 125I-labeled RGD peptide with ABM, Ga-DOTA-K([125I]IPBA)-c(RGDfK) ([125I]4), to compare with [211At]5. METHODS: Biodistribution experiments of [67Ga]3 without ABM, [125I]4 and [211At]5 with ABM were conducted in normal mice and U-87 MG tumor-bearing mice. In addition, two doses of [211At]5 (370 or 925 kBq) were administered to U-87 MG tumor-bearing mice to confirm the therapeutic effects. RESULTS: The blood retention of [125I]4 and [211At]5 was remarkably increased compared to [67Ga]3. Also, [125I]4 and [211At]5 showed similar biodistribution and significantly greater tumor accumulation and retention compared to [67Ga]3. In addition, [211At]5 inhibited tumor growth in a dose-dependent manner. CONCLUSION: The functionality of APBA as ABM like IPBA, and the usefulness of [211At]5 as the radionuclide therapy agent for TAT was revealed.

Synthesis and Evaluation of Radiogallium Labeled Bone-Imaging Probes Using Oligo-γ-Carboxy Glutamic Acid Peptides as Carriers to Bone.

We investigated the importance of the carboxy group density in bone affinity during the development of peptide-based bone-seeking radiopharmaceuticals and carriers. Oligo-γ-carboxy glutamic acid peptides [(Gla)n] with higher carboxy group density than oligo-glutamic acid peptides [(Glu)n] and oligo-aspartic acid peptides [(Asp)n] were chosen. Using the radiogallium chelator N,N'-bis-[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid (HBED-CC), we synthesized [67Ga]Ga-HBED-CC-(Gla)n (n = 1, 2, 5, 8, 11, or 14) with high yields. Hydroxyapatite-binding assays, biodistribution, and SPECT imaging showed higher affinity and bone accumulation for [67Ga]Ga-HBED-CC-(Gla)n compared to [67Ga]Ga-HBED-CC-(Glu)n. Notably, [67Ga]Ga-HBED-CC-(Gla)8 and [67Ga]Ga-HBED-CC-(Gla)11 exhibited superior bone accumulation and rapid blood clearance. SPECT/CT imaging with [67Ga]Ga-HBED-CC-(Gla)8 exclusively visualized the bone tissue. These findings support the potential use of [67Ga]Ga-HBED-CC-(Gla)n as excellent bone-imaging PET probes, suggesting (Gla)n peptides are superior bone-seeking carriers.

Chemical design of radioiodinated probes with a metabolizable linkage for target-selective imaging of systemic amyloidosis.

INTRODUCTION: Systemic amyloidosis is a rare disease caused by the deposition of amyloid fibrils in various organs. Amyloid-targeted radiopharmaceuticals have been developed and applied to diagnose systemic amyloidosis peripherally; however, high-contrast imaging has not been achieved because of the high background signals in normal organs. To overcome this problem, we designed an amyloid-targeted radioiodinated probe 1 with a metabolizable linkage (ester bond) to release of radiolabeled metabolites (m-iodohippuric acid) in normal organs that could be rapidly excreted in the urine. METHODS: Compound 1 was synthesized by conjugating 2-(4-(methylamino)phenyl)benzo[d]thiazol-6-ol, an amyloid-targeting compound, with m-iodohippuric acid. [125I]1 was synthesized via iododestannylation using a tributyltin precursor. Mouse models of amyloid A (AA) amyloidosis, a type of systemic amyloidosis, were prepared by administering amyloid-enhancing factor to mice and used for in vitro autoradiography using organ sections and in vivo evaluation. RESULTS: [125I]1 was obtained with a radiochemical yield of 59% and radiochemical purity of over 95%. An in vitro autoradiographic study demonstrated that [125I]1 specifically binds to amyloid in the splenic tissue. Upon administration to normal mice, [125I]1 was distributed to organs throughout the body, followed by the rapid excretion of radioactivity in the urine as m-[125I]iodohippuric acid. Furthermore, ex vivo autoradiography showed that [125I]1 bound to the amyloid formed around the follicles in the spleens of AA amyloidosis model mice. CONCLUSION: These results suggest that the interposition of a metabolizable linkage between an amyloid-targeting moiety and a radiolabeled hippuric acid would be useful in the design of radiopharmaceuticals for high-contrast imaging of systemic amyloidosis.

Preparation and Evaluation of Thermosensitive Liposomes Encapsulating I-125-Labeled Doxorubicin Derivatives for Auger Electron Therapy.

Auger electrons (AEs) are very low-energy electrons emitted by radionuclides such as I-125 (125I). This energy is deposited across a small distance (<0.5 µm), resulting in high linear energy transfer that is potent for causing lethal damage to cancer cells. Thus, AE-emitting radiotherapeutic agents have great potential for cancer treatment. In this study, thermosensitive liposomes (TSLs) encapsulating 125I-labeled doxorubicin (DOX) derivatives were developed for Auger electron therapy, targeting the DNA of cancer cells. A radioiodinated DOX derivative [125I]5 highly accumulated in the nuclei of cancer cells and showed potent cytotoxicity against Colon 26 cancer cells by AEs. Subsequently, [125I]5 was loaded into the TSLs with high encapsulation efficiency. Potent release of [125I]5 from TSLs was achieved with heating, whereas a decreased release was observed without heating. Furthermore, TSLs encapsulating [125I]5 showed a high uptake in the nuclei at 42 °C for 1 h. We supposed that [125I]5 was released by heating at 42 °C and accumulated in the nuclei in the cells. These results suggest that the combination of TSLs encapsulating [125I]5 and hyperthermia is an effective cancer therapy.

Borealin-Derived Peptides as Survivin-Targeting Cancer Imaging and Therapeutic Agents.

Survivin is overexpressed in most cancer cells but is rarely expressed in normal adult tissues. It is associated with poor prognosis and resistance to radiation therapy and chemotherapy. In this study, we designed and synthesized borealin-derived small peptides (Bor peptides) to function as survivin-targeting agents for the diagnosis and treatment of cancers. These peptides exhibited binding affinities for recombinant human survivin (Kd = 49.6-193 nM), with Bor65-75 showing the highest affinity (Kd = 49.6 nM). Fluorescence images of fluorescein isothiocyanate-labeled Bor65-75 showed its co-localization with survivin expression in the human pancreatic cancer cell line, MIA PaCa-2. In the WST-1 assay, cell penetrable nona-d-arginine-conjugated Bor65-75 (r9-Bor65-75) inhibited the growth of MIA PaCa-2 cells and MDA-MB-231 cells (89 and 88% inhibition at 10 µM, respectively), whereas it had almost no effect on the human mammary epithelial cell line, MCF-10A, that inherently does not have high survivin expression. Flow cytometry with annexin V and propidium iodide staining revealed that r9-Bor65-75 induced apoptosis in MIA PaCa-2 cells in a dose-dependent manner. An increase in cleaved poly ADP-ribose polymerase protein expression was observed in MIA PaCa-2 cells exposed to r9-Bor65-75 by western blotting, suggesting that r9-Bor65-75 inhibits cell proliferation by inducing apoptosis. In vivo, r9-Bor65-75 significantly suppressed tumor growth in MIA PaCa-2 xenograft mice, without any marked weight loss. Hence, Bor peptides are promising candidates for the development of cancer imaging and anticancer agents targeting survivin.

Enhanced Therapeutic Effect of Liposomal Doxorubicin via Bio-Orthogonal Chemical Reactions in Tumors.

Liposomes are highly biocompatible drug carriers in drug delivery systems (DDSs). Preferential accumulation of liposomes and acceleration of drug release at target tumor sites are essential for effective cancer therapy using liposomal formulations; however, conventional liposomes are unsuitable for on-demand drug release. We have previously reported that drug release can be accelerated via a bio-orthogonal inverse electron demand Diels-Alder (IEDDA) reaction between amphiphilic tetrazine (Tz)-containing liposomes and norbornene (NB) derivatives in vitro. In this study, we prepared HSTz-liposomes composed of hydrogenated soybean phosphatidylcholine (HSPC) and Tz compound (2-hexadecyl-N-(6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)octadecanamide) with particle sizes of 60-80 nm and ζ-potentials of -5 to 0 mV. Similar to our previous report, the addition of 5-norbornene-2-carboxylic acid (NBCOOH) to HSTz-liposomes accelerated drug release from the liposomes in vitro. In the biodistribution study using colon26 tumor-bearing mice, the radiolabeled HSTz-liposomes were accumulated and retained in the tumor at 6-48 h post-injection, whereas the radioactivity in the blood almost disappeared at 48 h. Therefore, the timing of the injection of NBCOOH was selected to be 48 h after the injection of the HSTz-liposome to avoid the IEDDA reaction in the bloodstream. We investigated the in vivo drug release by evaluating the intratumoral localization of doxorubicin (DOX) encapsulated in HSTz-liposomes labeled with fluorescent lipids. In the tumors treated with HSTz-liposomes and NBCOOH, DOX was more widely dispersed in the tumor compared with fluorescent lipid, suggesting that the release of encapsulated drugs (DOX) from HSTz-liposomes was enhanced in the tumor tissue via the bio-orthogonal IEDDA reaction. Furthermore, the combination of DOX-encapsulated HSTz-liposomes with NBCOOH significantly suppressed tumor growth compared to conventional DOX-encapsulated liposomes. In conclusion, the bio-orthogonal IEDDA reactions in the liposomal membrane enabled the acceleration of drug release from HSTz-liposomes in vivo, suggesting a promising strategy for effective cancer therapy.

Substituent effects of the phenyl ring at different positions from the α-carbon of TEMPO-type nitroxide.

Nitroxides are known to undergo oxidation, reduction, and radical scavenging reactions due to their stable radicals. Nitroxides have a wide range of applications due to their reactivities, including radical detecting probes and catalysts. Because nitroxides are easily reduced by ascorbate, a reducing agent, in biological applications, it is critical to control their reactivity to use them as a probe to trace the target reaction. On the other hand, the phenyl group, which is present in many functional organic molecules, is useful for controlling the electronic and steric effects. However, there has been few systematic studies on the substituent effects of TEMPO-type nitroxides with phenyl rings in the vicinity of a radical (α-position). In this study, we synthesized three nitroxides with a phenyl group at the α-position of a TEMPO-type nitroxide and tested their redox properties. The results showed that the reduction reactivity and redox potential differed depending on the position of the phenyl group, implying that the phenyl group one carbon away from the α-carbon of the N-O moiety increases the degree of steric hindrance. This finding is expected to be the basis for the development of functional nitroxides.

Development of a radioiodinated thioflavin-T-Congo-red hybrid probe for diagnosis of systemic amyloidosis.

INTRODUCTION: Systemic amyloidosis is a group of diseases characterized by the deposition of amyloid protein in multiple organs throughout the body and causing their dysfunction. As amyloid deposition is observed at an early phase and is highly specific to systemic amyloidosis, noninvasive detection of amyloid is considered useful for the early diagnosis of systemic amyloidosis. In this study, we designed and synthesized a novel radiolabeled amyloid imaging probe, sodium (E)-4-amino-3-((4-(6-iodobenzothiazol-2-yl)phenyl)diazenyl)naphthalene-1-sulfonate (1), which combines two amyloid-binding compounds, thioflavin-T and Congo-red, and evaluated its effectiveness in diagnosing amyloidosis. METHODS: A tributyltin precursor was synthesized through a 5-step reaction from 2-amino-6-bromobenzothiazole, and [125I]1 was synthesized by an iododestannylation reaction with a tributyltin precursor. Mouse models of amyloid A (AA) amyloidosis, a type of systemic amyloidosis, were prepared by intraperitoneal injection of amyloid-enhancing factor into mice. An in vitro autoradiographic study was performed using spleen sections from normal mice and AA amyloidosis mice. Furthermore, [125I]1 was intravenously injected into mice, and its distribution was evaluated. Finally, an ex vivo autoradiographic study was performed using AA amyloidosis mice. RESULTS: [125I]1 was obtained with a radiochemical yield of 66% and a radiochemical purity of over 95%. In vitro autoradiography revealed specific binding of [125I]1 to thioflavin-S-stained regions in the spleen. Normal mice showed relatively rapid clearance of [125I]1 from the organs, whereas radioactivity was retained in the spleen, where amyloid deposition was observed in model mice. Furthermore, ex vivo autoradiography showed a heterogeneous distribution of [125I]1, which was co-localized with thioflavin-S-stained regions in the spleen of model mice. CONCLUSION: These results indicate the potential of radioiodinated 1 as a nuclear imaging probe for diagnosing AA amyloidosis.

Development and evaluation of a theranostic probe with RGD peptide introduced platinum complex to enable tumor-specific accumulation.

Cisplatin (CDDP) has been widely used for chemotherapy. However, it has several unfavorable side effects due to its low tumor selectivity. In this study, we designed, synthesized, and evaluated Pt(IV)-[c(RGDyK)]2 (9), in which two molecules of an RGD peptide are introduced as a carrier molecule to cancer into oxoplatin, a Pt(IV) prodrug of CDDP, to enhance cancer selectivity. Furthermore, we prepared and evaluated Pt(IV)-[c(RGDyK)]{[125I]c[RGDy(3-I)K]} ([125I]10) for a preliminary step of nuclear medicine imaging and theranostics. Compound 9 inhibited cell growth in the cell viability assay and, [125I]10 was highly accumulated in tumor tissues (1 h: 3.53 ± 0.53 %ID/g) in the biodistribution study. These results indicate that implementing RGD peptides into oxoplatin enabled tumor-specific accumulation, and combining [123/124I]10 and 9 for diagnostic imaging and therapy could be useful for cancer theranostics.

A Radiolabeled Self-assembled Nanoparticle Probe for Diagnosis of Lung-Metastatic Melanoma.

Melanoma is a highly malignant skin cancer that frequently metastasizes to the lung, bone, and brain at an early phase. Therefore, noninvasive detection of metastasized melanoma could be beneficial to determine suitable therapeutic strategies. We previously reported a biocompatible ternary anionic complex composed of plasmid DNA (pDNA), polyethyleneimine (PEI), and γ-polyglutamic acid (γ-PGA) based on an electrostatic interaction, which was highly taken up by melanoma cells (B16-F10), even if it was negatively charged. Here, we developed a radiolabeled γ-PGA complex by using indium-111 (111In)-labeled polyamidoamine dendrimer (4th generation; G4) instead of pDNA and iodine-125 (125I)-labeled PEI instead of native PEI, and evaluated its effectiveness as a melanoma-targeted imaging probe. This ternary complex was synthesized at a theoretical charge ratio; carboxyl groups of 111In-diethylenetriaminepentaacetic acid (DTPA)-G4 : amino groups of 125I-PEI : carboxyl groups of γ-PGA was 1 : 8 : 16, and the size and zeta potential were approximately 29 nm and -33 mV, respectively. This complex was taken up by B16-F10 cells with time. Furthermore, a biodistribution study, using normal mice, demonstrated its accumulation in the liver, spleen, and lung, where macrophage cells are abundant. Almost the same level of radioactivity derived from both 111In and 125I was observed in these organs at an early phase after probe injection. Compared with the normal mice, significantly higher lung-to-blood ratios of radioactivity were observed in the B16-F10-lung metastatic cancer model. In conclusion, the radiolabeled γ-PGA complex would hold potentialities for nuclear medical imaging of lung metastatic melanoma.

Inverse Electron Demand Diels-Alder Reactions in the Liposomal Membrane Accelerates Release of the Encapsulated Drugs.

Bio-orthogonal inverse electron demand Diels-Alder (IEDDA) reactions between liposomes containing a tetrazine-based (Tz) compound and 2-norbornene (2-NB) could be a novel trigger for accelerating drug release from the liposomes via temporary membrane destabilization, as shown in our previous report. Herein, we evaluated the in vitro drug release using NB derivatives with carboxyl groups [5-norbornene-2-carboxylic acid (NBCOOH) and 5-norbornene-2,3-dicarboxylic acid (NB(COOH)2)] to investigate the effects of substituents at the NB backbone on the drug release rate. First, POTz-liposome composed of a Tz compound (2-hexadecyl-N-(6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)octadecanamide) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) were prepared. The mass spectrometry analysis revealed the binding of NB derivatives to the Tz compound via the IEDDA reaction after the POTz-liposome reacted with the NB derivatives. Indium-111-labeled diethylenetriaminepentaacetic acid (111In-DTPA) was encapsulated inside the liposomes, and the drug release rate was quantified by measuring radioactivity. At 24 h after incubation with 2-NB, NBCOOH, and NB(COOH)2, the release rates of 111In-DTPA from POTz-liposome were 21.0, 80.8, and 23.3%, respectively, which were significantly higher than those of POTz-liposome that was not treated with NB derivatives (4.2%), indicating the involvement of the IEDDA reaction for prompting drug release. Additionally, a thermodynamic evaluation using Langmuir monolayers was conducted to explore the mechanism of the accelerated drug release. An increase in membrane fluidity and a reduction in intermolecular repulsion between POPC and the Tz compound were observed after the reaction with NB derivatives, especially for NBCOOH. Thus, the IEDDA reaction in the liposomal membrane could be a potent trigger for accelerating the release of encapsulated drugs by regulating membrane fluidity and intermolecular repulsion in the liposomal membrane.

Synthesis of an amphiphilic tetrazine derivative and its application as a liposomal component to accelerate release of encapsulated drugs.

Tetrazine irreversibly reacts with dienophiles, and its derivatives find wide applications in the fields of biochemistry and biophysics. We have synthesized an amphiphilic tetrazine derivative (2-hexadecyl-N-(6-(6-(pyridin-2-yl)-1,2,4,5-tetrazine-3-yl)pyridin-3-yl)octadecanamide; 1), which has a hydrophilic tetrazine structure and hydrophobic alkyl chains. Liposomes composed of compound 1 and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) (PTz-liposome) were prepared. In search of a new drug delivery system (DDS), we investigated the viability of inverse electron-demand Diels-Alder, a reaction between tetrazine and 2-norbornene, on the surface of the liposomes to change membrane fluidity and promote spatial and temporal controlled release of the encapsulated drugs. Compound 1 was synthesized with a yield of 71%. MS analysis after incubation of 2-norbornene with PTz-liposome revealed the binding of 2-norbornene to tetrazine. Indium-111-labeled diethylenetriaminepentaacetic acid (111In-DTPA) was encapsulated inside PTz-liposome to evaluate the leakage of free 111In-DTPA from the liposomes quantitatively. After 24 h of adding 2-norbornene, the release percentage for PTz-liposome was significantly higher than that for the control liposome (without tetrazine structure). Furthermore, the membrane fluidity of the PTz-liposome was increased by adding 2-norbornene. These results suggested that the combination of dienophile and liposome containing a newly synthesized tetrazine derivative can be used as a controlled release DDS carrier.

Investigation of Biodistribution and Speciation Changes of Orally Administered Dual Radiolabeled Complex, Bis(5-chloro-7-[131I]iodo-8-quinolinolato)[65Zn]zinc.

Many zinc (Zn) complexes have been developed as promising oral antidiabetic agents. In vitro assays using adipocytes have demonstrated that the coordination structures of Zn complexes affect the uptake of Zn into cells and have insulinomimetic activities, for which moderate stability of Zn complexes is vital. The complexation of Zn plays a major role improving its bioavailability. However, investigation of the speciation changes of Zn complexes after oral administration is lacking. A dual radiolabeling approach was applied in order to investigate the speciation of bis(5-chloro-7-iodo-8-quinolinolato)zinc complex [Zn(Cq)2], which exhibits the antidiabetic activity in diabetic mice. In the present study, 65Zn- and 131I-labeled [Zn(Cq)2] were synthesized, and their biodistribution were analyzed after an oral administration using both invasive conventional assays and noninvasive gamma-ray emission imaging (GREI), a novel nuclear medicine imaging modality that enables analysis of multiple radionuclides simultaneously. The GREI experiments visualized the behavior of 65Zn and [131I]Cq from the stomach to large intestine and through the small intestine; most of the administered Zn was transported together with clioquinol (5-chloro-7-iodo-8-quinolinol) (Cq). Higher accumulation of 65Zn for [Zn(Cq)2] than ZnCl2 suggests that the Zn associated with Cq was highly absorbed by the intestinal tract. In particular, the molar ratio of administered iodine to Zn decreased during the distribution processes, indicating the dissociation of most [Zn(Cq)2] complexes. In conclusion, the present study successfully evaluated the speciation changes of orally administered [Zn(Cq)2] using the dual radiolabeling method.

Recent advances in the development of 225Ac- and 211At-labeled radioligands for radiotheranostics.

Radiotheranostics utilizes a set of radioligands incorporating diagnostic or therapeutic radionuclides to achieve both diagnosis and therapy. Imaging probes using diagnostic radionuclides have been used for systemic cancer imaging. Integration of therapeutic radionuclides into the imaging probes serves as potent agents for radionuclide therapy. Among them, targeted alpha therapy (TAT) is a promising next-generation cancer therapy. The α-particles emitted by the radioligands used in TAT result in a high linear energy transfer over a short range, inducing substantial damage to nearby cells surrounding the binding site. Therefore, the key to successful cancer treatment with minimal side effects by TAT depends on the selective delivery of radioligands to their targets. Recently, TAT agents targeting biomolecules highly expressed in various cancer cells, such as sodium/iodide symporter, norepinephrine transporter, somatostatin receptor, αvß3 integrin, prostate-specific membrane antigen, fibroblast-activation protein, and human epidermal growth factor receptor 2 have been developed and have made remarkable progress toward clinical application. In this review, we focus on two radionuclides, 225Ac and 211At, which are expected to have a wide range of applications in TAT. We also introduce recent fundamental and clinical studies of radiopharmaceuticals labeled with these radionuclides.

Astatine-211-labeled aza-vesamicol derivatives as sigma receptor ligands for targeted alpha therapy.

INTRODUCTION: As sigma receptors are abundantly expressed on different types of cancer cells, several radiolabeled sigma receptor ligands have been developed for cancer imaging and therapy. Previously, we synthesized and evaluated radioiodinated aza-vesamicol derivatives, [125I]pIC3NV, [125I]mIC2N5V, and [125I]mIC3N5V. They accumulated in tumors, and [125I]mIC2N5V and [125I]mIC3N5V showed higher tumor to non-target tissue ratios than [125I]pIC3NV. Therefore, we synthesized and evaluated the corresponding 211At-labeled compounds, [211At]mAtC2N5V and [211At]mAtC3N5V, for targeted alpha therapy (TAT). METHODS: [211At]mAtC2N5V and [211At]mAtC3N5V were prepared by the standard method of electrophilic astatodestannylation of the corresponding trimethylstannyl precursors. Cellular uptake experiments, and biodistribution experiments and therapeutic experiments in tumor-bearing mice were performed. RESULTS: The radiochemical yields of [211At]mAtC2N5V and [211At]mAtC3N5V were 45.5 ± 14.4% and 56.9 ± 13.8%, respectively. After HPLC purification, their radiochemical purities were over 95%. [211At]mAtC2N5V and [211At]mAtC3N5V showed high uptake in DU-145 cells. They demonstrated high accumulation in tumors (6.9 ± 1.4%injected dose/g and 5.1 ± 1.4%injected dose/g at 1 h, respectively) and similar biodistribution tendencies compared with the corresponding 125I-labeled compounds. A single injection of [211At]mAtC2N5V (0.48 MBq) or [211At]mAtC3N5V (0.48 MBq) significantly inhibited tumor growth. CONCLUSION: These results indicated that [211At]mAtC2N5V and [211At]mAtC3N5V could be potential candidates for TAT.

Effect of relative configuration of TEMPO-type nitroxides on ascorbate reduction.

2,2,6,6-Tetramethylpiperidin-N-oxyl (TEMPO)-type nitroxides are susceptible to bioreduction, leading to a loss of radical properties. Although it has been reported that the steric and electronic environments around the N-O moiety of nitroxides affect the reduction, how the relative configuration of nitroxide derivatives alters it is unclear. In this study, we investigated the effect of diastereomers on the radical properties of C2- and C4-disubstituted TEMPO-type nitroxides. We succeeded in isolating the diastereomers of the studied nitroxides for the first time. In addition, we compared the reactivities of nitroxide derivatives with different substituents at the C2 and C4 positions toward ascorbate reduction. We found that the bulky substituents at both C2 and C4 and the electronic effect of C4 affected the reduction of the isomers. C2- and C4-disubstituted nitroxides were administered to mice for electron spin resonance imaging to assess bioreduction in the brain. Similar to the reactivity to reduction in vitro, a difference in the bioreduction of diastereomers was observed in brain tissues. Our research strongly indicates that bioreduction can be controlled by changing the relative configuration, which can be used in the design of nitroxide derivatives for biological applications.

Preparation and evaluation of 111In-labeled liposomes containing phosphatidylglycerol for detection of macrophages in atherosclerotic plaques.

Macrophage infiltration is a characteristic feature of atherosclerotic plaque progression. Since liposomes containing 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG) are efficiently phagocytosed by macrophages, we deduced that radiolabeled liposomes containing DSPG could potentially be used for nuclear imaging of vulnerable atherosclerotic plaques. Indium-111 (111In)-labeled liposomes containing different ratios of DSPG were developed with a high labeling efficiency. 111In-labeled liposomes with higher DSPG content showed higher uptake by macrophage-like RAW264 cells. A biodistribution study demonstrated rapid blood clearance and selective accumulation in the liver and spleen, especially in normal mice injected with 111In-labeled liposomes with higher DSPG content. Accumulation in atherosclerotic plaques was evaluated using 111In-labeled DSPG liposomes, which had the highest DSPG content among the studied liposomes. 111In-labeled DSPG liposomes accumulated in the plaques and the radioactive regions were mostly consistent with the distribution of macrophages. The target-to-non-target ratio of 111In-labeled DSPG liposomes was higher than that of 111In-labeled control liposomes without DSPG. These results suggest that 111In-labeled liposomes containing DSPG are useful for nuclear medical diagnosis of atherosclerotic plaques.

RGD Peptide-Conjugated Dodecaborate with the Ga-DOTA Complex: A Preliminary Study for the Development of Theranostic Agents for Boron Neutron Capture Therapy and Its Companion Diagnostics.

A boron neutron capture therapy (BNCT) system, using boron-10-introduced agents coupled with companion diagnostics, is anticipated as a promising cancer theranostic. Thus, this study aimed to synthesize and evaluate a probe closo-dodecaborate-(Ga-DOTA)-c(RGDfK) (16) [Ga = gallium, DOTA =1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, and c(RGDfK) = cyclo(arginine-glycine-aspartate-d-phenylalanine-lysine] containing closo-dodecaborate ([B12H12]2-) as a boron cluster, a [67Ga]Ga-DOTA derivative for nuclear medicine imaging, and an RGD peptide for tumor targeting. Moreover, we prepared a radioiodinated probe [125I]17 in which I-125 is introduced into a closo-dodecaborate moiety of 16. [67Ga]16 and [125I]17 showed high stability and high uptake in cancer cells in vitro. Biodistribution experiments in tumor-bearing mice revealed similar biodistribution patterns between [67Ga]16 and [125I]17, such as a high uptake in the tumor and a low uptake in other non-target tissues. Meanwhile, [125I]17 exhibited higher accumulation in most tissues, including the tumor, than [67Ga]16, probably because of higher albumin binding. The higher the [125I]17 accumulation in the tumor, the more desirable it is for BNCT, with the possibility that the iodo-closo-dodecaborate site may work as an albumin binder.