Hisatsugu Yamada

Ruthenium Complexes with Hydrophobic Ligands That Are Key Factors for the Optical Imaging of Physiological Hypoxia

The phosphorescence emission of ruthenium complexes was applied to the optical imaging of physiological hypoxia. We prepared three complexes with hydrophobic substituents on the phenanthroline ligand and characterized their emission, which was quenched by molecular oxygen. Among the complexes synthesized in this study, a pyrene chromophore-linked ruthenium complex, Ru-Py, exhibited optimal properties for the imaging of hypoxia; the prolonged lifetime of the triplet excited state of the ruthenium chromophore, which was induced by efficient energy distribution and transfer from the pyrene unit, provided the highest sensitivity towards molecular oxygen. The introduction of hydrophobic pyrene increased the lipophilicity of the complex, leading to enhanced cellular uptake. Consequently, the bright phosphorescence of Ru-Py was seen in the cytoplasm of viable hypoxic cells, whereas the signal from aerobic cells was markedly weaker. Thus, we could clearly discriminate between hypoxic and aerobic cells by monitoring the phosphorescence emission. Furthermore, Ru-Py was applied to optical imaging in live mice. An intramuscular injection of Ru-Py successfully visualized ischemia-based hypoxia, which was constructed by leg banding.

Monitoring of biological one-electron reduction by 19F NMR using hypoxia selective activation of an 19F-labeled indolequinone derivative

Biological reduction of fluorine-labeled indolequinone derivative (IQ-F) was characterized by (19)F NMR for quantitative molecular understanding. The chemical shift change in (19)F NMR allowed monitoring of the enzymatic reduction of IQ-F. Upon hypoxic treatment of IQ-F with NADPH:cytochrome P450 reductase, IQ-F was activated via catalytic one-electron reduction to release nonafluoro-tert-butyl alcohol (F-OH), while the formation of F-OH was significantly suppressed under aerobic conditions. Similar hypoxia-selective reduction of IQ-F occurred within A549 cells, which expresses NADPH:cytochrome P450 reductase. The kinetic analysis was also performed to propose a reaction mechanism. The molecular oxygen slightly prevents the binding of IQ-F to reductase, while the rate of net reaction was decreased due to oxidation of a semiquinone anion radical intermediate generated by one-electron reduction of IQ-F. The disappearance of IQ-F and appearance of F-OH were imaged by (19)F fast spin echo, thus visualizing the hypoxia-selective reduction of IQ-F by means of MR imaging.

Radiation- and Photo-induced Activation of 5-Fluorouracil Prodrugs as a Strategy for the Selective Treatment of Solid Tumors

5-Fluorouracil (5-FU) is used widely as an anticancer drug to treat solid cancers, such as colon, breast, rectal, and pancreatic cancers, although its clinical application is limited because 5-FU has gastrointestinal and hematological toxicity. Many groups are searching for prodrugs with functions that are tumor selective in their delivery and can be activated to improve the clinical utility of 5-FU as an important cancer chemotherapeutic agent. UV and ionizing radiation can cause chemical reactions in a localized area of the body, and these have been applied in the development of site-specific drug activation and sensitization. In this review, we describe recent progress in the development of novel 5-FU prodrugs that are activated site specifically by UV light and ionizing radiation in the tumor microenvironment. We also discuss the chemical mechanisms underlying this activation.

DNA Hairpins Containing a Diaminostilbene Derivative as a Photoinduced Electron Donor for Probing the Effects of Single-Base Mismatches on Excess Electron Transfer in DNA

To investigate the effects of local structural disorder induced by a single-base mismatch on excess electron transfer (EET) in DNA, a novel hairpin DNA containing diaminostilbene (DAS) as a photoinducible electron donor has been developed. It was clearly demonstrated that EET efficiency depends on the electron injection modes from the electron donors and redox properties of the mismatched bases.

Magnetic Resonance Imaging of Tumor with a Self-Traceable Phosphorylcholine Polymer

Polymers are concentration-amplified with respect to the monomeric units. We show here that a phosphorylcholine polymer enriched with (13)C/(15)N at the methyl groups is self-traceable by multiple-resonance (heteronuclear-correlation) NMR in tumor-bearing mice inoculated with the mouse rectal cancer cell line (colon 26). Preliminary measurements indicated that the present polymeric nanoprobe was satisfactorily distinguished from lipids and detectable with far sub-micromolar spectroscopic and far sub-millimolar imaging sensitivities. Detailed ex vivo and in vivo studies for the tumor-bearing mice administered the probe with a mean molecular weight of 63,000 and a mean size of 13 nm, revealed the following: (1) this probe accumulates in the tumor highly selectively (besides renal excretion) and efficiently (up to 30% of the injected dose), (2) the tumor can thus be clearly in vivo imaged, the lowest clearly imageable dose of the probe being 100 mg/kg or 2.0 mg/20-g mouse, and (3) the competition between renal excretion and tumor accumulation is size-controlled; that is, the larger (higher molecular-weight) and smaller (lower molecular-weight) portions of the probe undergo tumor accumulation and renal excretion, respectively. The observed size dependence suggests that the efficient tumor-targeting of the present probe is stimulated primarily by the so-called enhanced permeability and retention (EPR) effect, that is, size-allowed invasion of the probe into the tumor tissue via defective vascular wall. Self-traceable polymers thus open an important area of magnetic resonance imaging (MRI) of tumors and may provide a highly potential tool to visualize various delivery/localization processes using synthetic polymers.

Substrate/Product-targeted NMR monitoring of pyrimidine catabolism and its inhibition by a clinical drug.

We report the application of one-dimensional triple-resonance NMR to metabolic analysis and thereon-based evaluation of drug activity. Doubly (13)C/(15)N-labeled uracil ([(15)N1,(13)C6]-uracil) was prepared. Its catabolic (degradative) conversion to [(13)C3,(15)N4]-β-alanine and inhibition thereof by gimeracil, a clinical co-drug used with the antitumor agent 5-fluorouracil, in mouse liver lysates were monitored specifically using one-dimensional triple-resonance ((1)H-{(13)C-(15)N}) NMR, but not double-resonance ((1)H-{(13)C}) NMR, in a ratiometric manner. The administration of labeled uracil to a mouse resulted in its non-selective distribution in various organs, with efficient catabolism to labeled β-alanine exclusively in the liver. The co-administration of gimeracil inhibited the catabolic conversion of uracil in the liver. In marked contrast to in vitro results, however, gimeracil had practically no effect on the level of uracil in the liver. The potentiality of triple-resonance NMR in the analysis of in vivo pharmaceutical activity of drugs targeting particular metabolic reactions is discussed.

In situ analysis of [8-13C-7-15N]-double-labelled theophylline by a triple resonance NMR technique

[8-13C-7-15N]-labelled theophylline was synthesized and applied for in situ analysis using a 1H–{13C–15N} triple resonance NMR technique. The triple resonance analysis enabled selective detection of labelled theophylline in a mixture containing excess amino acids or crude mouse liver extract, where conventional 1H NMR analysis suffers from difficulty because of background signals.

Pharmacokinetics of Chiral Dendrimer-Triamine-Coordinated Gd-MRI Contrast Agents Evaluated by in Vivo MRI and Estimated by in Vitro QCM.

Recently, we developed novel chiral dendrimer-triamine-coordinated Gd-MRI contrast agents (Gd-MRI CAs), which showed longitudinal relaxivity (r1) values about four times higher than that of clinically used Gd-DTPA (Magnevist®, Bayer). In our continuing study of pharmacokinetic differences derived from both the chirality and generation of Gd-MRI CAs, we found that the ability of chiral dendrimer Gd-MRI CAs to circulate within the body can be directly evaluated by in vitro MRI (7 T). In this study, the association constants (Ka) of chiral dendrimer Gd-MRI CAs to bovine serum albumin (BSA), measured and calculated with a quartz crystal microbalance (QCM) in vitro, were found to be an extremely easy means for evaluating the body-circulation ability of chiral dendrimer Gd-MRI CAs. The Ka values of S-isomeric dendrimer Gd-MRI CAs were generally greater than those of R-isomeric dendrimer Gd-MRI CAs, which is consistent with the results of our previous MRI study in vivo.

13C/15N‐Enriched l‐Dopa as a Triple‐Resonance NMR Probe to Monitor Neurotransmitter Dopamine in the Brain and Liver Extracts of Mice

Abstract In an attempt to monitor μm‐level trace constituents, we applied here 1H‐{13C‐15N} triple‐resonance nuclear magnetic resonance (NMR) to 13C/15N‐enriched l‐Dopa as the inevitable precursor of the neurotransmitter dopamine in the brain. The perfect selectivity (to render endogenous components silent) and μm‐level sensitivity (700 MHz spectrometer equipped with a cryogenic probe) of triple‐resonance allowed the unambiguous and quantitative metabolic and pharmacokinetic analyses of administered l‐Dopa/dopamine in the brain and liver of mice. The level of dopamine generated in the brain (within the range 7–76 μm, which covers the typical stimulated level of ∼30 μm) could be clearly monitored ex vivo, but was slightly short of the detection limit of a 7 T MR machine for small animals. This work suggests that μm‐level trace constituents are potential targets of ex vivo monitoring as long as they contain N atom(s) and their appropriate 13C/15N‐enrichment is synthetically accessible.

Magnetic resonance imaging of tumor with a self-traceable polymer conjugated with an antibody fragment

A (13)C-enriched phosphorylcholine polymer ((13)C-PMPC) as a self-traceable MR (magnetic resonance) tag was conjugated with a fragment (scFv) of Herceptin, a clinical antibody against antigen Her2. When injected in model mice bearing Her2(+) (gastric) and Her2(-) (pancreatic) tumors, the antibody-tag conjugate (13)C-PMPC-scFv selectively accumulated in the Her2(+) tumor with a rapid build-up/decay (accumulation/clearance) profile and, with the use of the (1)H-(13)C double-resonance (heteronuclear correlation) technique, the Her2(+) gastric tumor was clearly MR imaged.