酵素疎水場による金属イオン-基質結合促進に関するモデル研究

金沢大学薬学部研究課題/領域番号:62740342, 研究期間(年度):1987出典：研究課題「酵素疎水場による金属イオン-基質結合促進に関するモデル研究」課題番号62740342（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-62740342/）を加工して作

Stability and mode of coordination complexes formed in the silver(i)/nucleoside systems

Complexes of silver(i) with nucleosides (adenosine, cytidine, uridine and thymidine) have been studied using potentiometric, spectral and theoretical methods. Stability constants of the complexes as well as their coordination modes have been determined. Results of the equilibrium studies have provided evidence for the formation of only ML and ML(OH) type complexes. The basicity of nucleosides was found to substantially influence the stability constant of the ML type complexes. Spectral data have allowed us to identify the sites of silver attachment to the nucleoside. Additionally a new silver-adenosine complex of stoichiometry Ag(Ado)(OH) was prepared from aqueous solution at pH close to 6. Its characterization and comparison with complexes formed in solution are described. The structures of complexes formed in solution and in solid state were confirmed through computational calculations (DFT/B3LYP: lanL2DZ theoretical procedure). © 2011 The Royal Society of Chemistry and the Centre National de la Recherche Scientifique

A complex of rab3A, SNAP-25, VAMP/synaptobrevin-2 and syntaxins in brain presynaptic terminals

AbstractTwo monoclonal antibodies (SPM-1 and SPM-2) immunoprecipitate brain N-type calcium channels. On immunoaffinity chromatography of digitonin extracts of bovine brain membranes on SPM-1- and SPM-2-Sepharose, proteins of 36 (syntaxins A and B), 28 and 19 kDa are specifically retained by both columns. Here we show that the 19 and 28 kDa bands contain VAMP/synaptobrevin-2, and rab3A/smg25A and SNAP-25, respectively. Since SPM-1 and SPM-2 recognize only syntaxins and the 28 kDa band (rab3A/smg25A and SNAP-25), respectively, the results indicate that all these proteins form a complex. Our results suggest tight linkage between the components involved in neurotransmitter release

Evaluation of Chlorella as a Decorporation Agent to Enhance the Elimination of Radioactive Strontium from Body

Background Release of radionuclides, such as 137Cs and 90Sr, into the atmosphere and the ocean presents an important problem because internal exposure to 137Cs and 90Sr could be very harmful to humans. Chlorella has been reported to be effective in enhancing the excretion of heavy metals; thus, we hypothesized that Chlorella could also enhance the elimination of 137Cs or 90Sr from the body. We evaluated the potential of Chlorella as a decorporation agent in vitro and in vivo, using 85Sr instead of 90Sr. Methods In vitro experiments of adsorption of 137Cs and 85Sr to Chlorella were performed under wide pH conditions. The maximum sorption capacity of Chlorella to strontium was estimated using the Langmuir model. A 85Sr solution was orally administrated to mice pretreated with Chlorella. At 48 h after 85Sr administration, the biodistribution of radioactivity was determined. Results In the in vitro experiments, although 85Sr barely adsorbed to Chlorella at low pH, the 85Sr adsorption ratio to Chlorella increased with increasing pH. The maximum sorption capacity of Chlorella to strontium was 9.06 mg / g. 137Cs barely adsorbed to Chlorella under any pH conditions. In the biodistribution experiments, bone accumulation of radioactivity after 85Sr administration was significantly decreased in the Chlorella pretreatment group compared with the non-treatment control group. Conclusions In conclusion, these results indicated that Chlorella could inhibit the absorption of 90Sr into the blood and enhance the elimination of 90Sr from the body through adsorption in intestine. Further studies are required to elucidate the mechanism and the components of Chlorella needed for adsorption to strontium and could promote the development of more effective decorporation agents. © 2016 Ogawa et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Effectiveness of two novel anionic and cationic platinum complexes in the treatment of osteosarcoma

Aim: This study aimed to characterize the cellular basis of the platinum cytotoxicity of two novel platinum complexes, 3Pt and 1Pt, in comparison with that of cisplatin. 3Pt comprises anionic phosphate moieties, while 1Pt comprises neutral aromatic ligands. Methods: We compared the cytotoxic potency of 3Pt and 1Pt with that of cisplatin in osteosarcoma cell lines and an orthotopic mouse model. Results: The cytotoxic potency of 3Pt was markedly higher than that of cisplatin in all cell lines. Both novel platinum complexes showed a complete lack of cross resistance in cisplatin-resistant cells. Caffeine enhanced the cytotoxic potency of these novel platinum complexes, as observed for cisplatin. Apoptosis after drug administration was observed by DNA ladder formation and an annexin V/PI assay. DNA double-strand breaks were confirmed by phosphorylation of histone H2AX. In vivo, the antitumor activity of 3Pt and 1Pt was superior and similar, respectively, to that of cisplatin. Both novel platinum complexes exerted strong antitumor effects on osteosarcoma in vitro and in vivo. Conclusions: 3Pt may be an effective drug for the treatment of bone cancer because the PO3 moiety has a high affinity to bone, as exhibited by bisphosphonates, and is expected to decrease the incidence of side effects at extraskeletal sites and overcome drug resistance. Cationic 1Pt may also be an effective antitumor drug because of its unique chemical structure and properties. Further investigations to detail the antitumor effects of these ionic Pt complexes on osteosarcoma are warranted. © 2015 Bentham Science Publishers

Preparation and evaluation of a radiogallium complex-conjugated bisphosphonate as a bone scintigraphy agent

金沢大学医薬保健研究域薬学系Introduction: 68Ga is a radionuclide of great interest as a positron emitter for positron emission tomography (PET). To develop a new bone-imaging agent with radiogallium, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was chosen as a chelating site and Ga-DOTA complex-conjugated bisphosphonate, which has a high affinity for bone, was prepared and evaluated. Although we are interested in developing 68Ga-labeled bone imaging agents for PET, in these initial studies 67Ga was used because of its longer half-life. Methods: DOTA-conjugated bisphosphonate (DOTA-Bn-SCN-HBP) was synthesized by conjugation of 2-(4-isothiocyanatebenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid to 4-amino-1-hydroxybutylidene-1,1-bisphosphonate (alendronate). 67Ga-DOTA-Bn-SCN-HBP was prepared by coordination with 67Ga, and its in vitro and in vivo evaluations were performed. Results: 67Ga-DOTA-Bn-SCN-HBP was prepared with a radiochemical purity of over 95% without purification. 67Ga-DOTA-Bn-SCN-HBP had great affinity for hydroxyapatite in binding assay. In biodistribution experiments, 67Ga-DOTA-Bn-SCN-HBP accumulated in bone rapidly but was hardly observed in tissues other than bone. Pretreatment of an excess amount of alendronate inhibited the bone accumulation of 67Ga-DOTA-Bn-SCN-HBP. Conclusions: 67Ga-DOTA-Bn-SCN-HBP showed ideal biodistribution characteristics as a bone-imaging agent. These findings should provide useful information on the drug design of bone imaging agents for PET with 68Ga. © 2010 Elsevier Inc. All rights reserved

Preparation and evaluation of 186/188Re-labeled antibody (A7) for radioimmunotherapy with rhenium(I) tricarbonyl core as a chelate site

Objective: Rhenium is one of the most valuable elements for internal radiotherapy because 186Re and 188Re have favorable physical characteristics. However, there are problems when proteins such as antibodies are used as carriers of 186/188Re. Labeling methods that use bifunctional chelating agents such as MAG3 require the conjugation of the 186/188Re complex to protein after radiolabeling with the bifunctional chelating agent. These processes are complicated. Therefore, we planned the preparation by a simple method and evaluation of a stable 186/188Re-labeled antibody. For this purpose, we selected 186/188Re(I) tricarbonyl complex as a chelating site. In this study, A7 (an IgG1 murine monoclonal antibody) was used as a model protein. 186/188Re-labeled A7 was prepared by directly reacting a 186/188Re(I) tricarbonyl precursor, [186/188Re(CO)3(H2O)3]+, with A7. We then compared the biodistribution of 186/188Re-labeled A7 in tumor-bearing mice with 125I-labeled A7. Methods: For labeling A7, [186/188Re(CO)3(H2O)3]+ was prepared according to a published procedure. 186/188Re-labeled A7 (186/188Re-(CO)3-A7) was prepared by reacting [186/188Re(CO)3(H2O)3]+ with A7 at 43°C for 2 h. Biodistribution experiments were performed by the intravenous administration of 186/188Re-(CO)3-A7 solution into tumor-bearing mice. Results: 186Re-(CO)3-A7 and 188Re-(CO)3-A7 were prepared with radiochemical yields of 23 and 28%, respectively. After purification with a PD-10 column, 186/188Re-(CO)3-A7 showed a radiochemical purity of over 95%. In biodistribution experiments, 13.1 and 13.2% of the injected dose/g of 186Re-(CO)3-A7 and 188Re-(CO)3-A7, respectively, accumulated in the tumor at 24-h postinjection, and the tumor-to-blood ratios were over 2.0 at the same time point. Meanwhile, uptake of 125I-A7 in the tumor was almost the same as that of 186/188Re-(CO)3-A7 at 24-h postinjection. Blood clearances of 186/188Re-(CO)3-A7 were faster than those of 125I-A7. Conclusion: 186/188Re-labeled A7 showed high uptakes in the tumor. However, further modification of the labeling method would be necessary to improve radiochemical yields and their biodistribution. © 2009 The Japanese Society of Nuclear Medicine

Evaluation of Ga-DOTA-(D-Asp)n as bone imaging agents: D-aspartic acid peptides as carriers to bone

金沢大学新学術創成研究機構67Ga-DOTA-(L-Asp)11 and 67Ga-DOTA-(L-Asp)14, which have been developed as bone imaging agents, showed a high accumulation in bone and a rapid blood clearance in mice. However, peptides composed of D-amino acids are more stable in vivo than those composed of their L-equivalents. In this study, 67Ga-DOTA-(D-Asp)n (n = 2, 5, 8, 11, or 14) were synthesized using the Fmoc-based solid-phase methodology and evaluated. In hydroxyapatite binding assay, binding of 67Ga-DOTA-(D-Asp)n tended to increase with increasing length of the amino acid chain. 67Ga-DOTA-(D-Asp)11 and 67Ga-DOTA-(D-Asp)14 caused a high accumulation of radioactivity in the bones of the mice. However, the results for 67Ga-DOTA-(D-Asp)n and 67Ga-DOTA-(L-Asp)n were comparable. In urine analyses, the proportion of intact complex after injection of 67Ga-DOTA-(D-Asp)14 was significantly higher than that of 67Ga-DOTA-(L-Asp)14. Although 67Ga-DOTA-(D-Asp)14 was more stable than 67Ga-DOTA-(L-Asp)14, the properties of 67Ga-DOTA-(D-Asp)n and 67Ga-DOTA-(L-Asp)n as bone imaging agents may be comparable. © 2017 The Author(s)

Development of novel radiogallium-labeled bone imaging agents using oligo-aspartic acid peptides as carriers

金沢大学疾患モデル総合研究センター68Ga (T1/2 = 68 min, a generator-produced nuclide) has great potential as a radionuclide for clinical positron emission tomography (PET). Because poly-glutamic and poly-aspartic acids have high affinity for hydroxyapatite, to develop new bone targeting 68Ga-labeled bone imaging agents for PET, we used 1,4,7,10-tetraazacyclododecane-1,4,7,10- tetraacetic acid (DOTA) as a chelating site and conjugated aspartic acid peptides of varying lengths. Subsequently, we compared Ga complexes, Ga-DOTA-(Asp)n (n = 2, 5, 8, 11, or 14) with easy-to-handle 67Ga, with the previously described 67Ga-DOTA complex conjugated bisphosphonate, 67Ga-DOTA-Bn-SCN-HBP. After synthesizing DOTA-(Asp)n by a Fmoc-based solid-phase method, complexes were formed with 67Ga, resulting in 67Ga-DOTA-(Asp)n with a radiochemical purity of over 95% after HPLC purification. In hydroxyapatite binding assays, the binding rate of 67Ga-DOTA-(Asp)n increased with the increase in the length of the conjugated aspartate peptide. Moreover, in biodistribution experiments, 67Ga-DOTA-(Asp) 8, 67Ga-DOTA-(Asp)11, and 67Ga-DOTA- (Asp)14 showed high accumulation in bone (10.5±1.5, 15.1±2.6, and 12.8±1.7% ID/g, respectively) but were barely observed in other tissues at 60 min after injection. Although bone accumulation of 67Ga-DOTA-(Asp)n was lower than that of 67Ga-DOTA-Bn-SCN-HBP, blood clearance of 67Ga-DOTA-(Asp)n was more rapid. Accordingly, the bone/blood ratios of 67Ga-DOTA-(Asp) 11 and 67Ga-DOTA-(Asp)14 were comparable with those of 67Ga-DOTA-Bn-SCN-HBP. In conclusion, these data provide useful insights into the drug design of 68Ga-PET tracers for the diagnosis of bone disorders, such as bone metastases. © 2013 Ogawa et al.CC-BY 4.

Radiobrominated benzimidazole-quinoline derivatives as Platelet-derived growth factor receptor beta (PDGFRβ) imaging probes

金沢大学疾患モデル総合研究センターPlatelet-derived growth factor receptor beta (PDGFRβ) affects in numerous human cancers and has been recognized as a promising molecular target for cancer therapies. The overexpression of PDGFRβ could be a biomarker for cancer diagnosis. Radiolabeled ligands having high affinity for the molecular target could be useful tools for the imaging of overexpressed receptors in tumors. In this study, we aimed to develop radiobrominated PDGFRβ ligands and evaluate their effectiveness as PDGFRβ imaging probes. The radiolabeled ligands were designed by modification of 1-{2-[5-(2-methoxyethoxy)-1H- benzo[d]imidazol-1-yl]quinolin-8-yl}piperidin-4-amine (1), which shows selective inhibition profile toward PDGFRβ. The bromine atom was introduced directly into C-5 of the quinoline group of 1, or indirectly by the conjugation of 1 with the 3-bromo benzoyl group. [77Br]1-{5-Bromo-2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]quinoline-8-yl}piperidin-4-amine ([77Br]2) and [77Br]-N-3-bromobenzoyl-1-{2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]quinolin-8-yl}-piperidin-4-amine ([77Br]3) were prepared using a bromodestannylation reaction. In a cellular uptake study, [77Br]2 and [77Br]3 more highly accumulatd in BxPC3-luc cells (PDGFRβ-positive) than in MCF7 cells (PDGFRβ-negative), and their accumulation was significantly reduced by pretreatment with inhibitors. In biodistribution experiments, [77Br]2 accumulation was higher than [77Br]3 accumulation at 1 h postinjection. These findings suggest that [76Br]2 is more promising for positron emission tomography (PET) imaging of PDGFRβ than [76Br]3. © 2018 The Author(s).CC-BY 4.