Tatsuya Naganuma

Whole-body kinetic image of a redox probe in mice using Overhauser-enhanced MRI.

Overhauser-enhanced MRI (OMRI) enables visualization of free radicals in animals based on dynamic nuclear polarization. Real-time data of tissue redox status gathered from kinetic images of redox-sensitive nitroxyl radical probes using OMRI provided both anatomic and physiological information. Phantom experiments demonstrated the linear correlation between the enhancement factor and the concentration of a membrane-impermeable probe, carboxy-PROXYL (3-carboxy-2,2,5,5-tetramethyl- pyrrolidine-1-oxyl). Whole-body OMRI images illustrated the in vivo kinetics of carboxy-PROXYL for 25 min. Initial distribution was observed in lung, heart, liver, and kidney, but not brain, corresponding to its minimal lipophilicity. Based on these images (pixel size, 1.33 × 1.33 mm; slice thickness, 50mm), a time-concentration curve with low coefficient of variance (<0.21) was created to assess pharmacokinetic behaviors. A biexponential curve showed a distribution phase from 1 to 10 min and an elimination phase from 15 to 25 min. The α rate constant was greater than the β rate constant in ROIs, confirming that its pharmacokinetics obeyed a two-compartment model. As a noninvasive technique, combining OMRI imaging with redox probes to monitor tissue redox status may be useful in acquiring valuable information regarding organ function for preclinical and clinical studies of oxidative diseases.

Dynamic nuclear polarization studies of redox-sensitive nitroxyl spin probes in liposomal solution

Overhauser-enhanced magnetic resonance imaging (OMRI) studies of a membrane-permeable nitroxyl spin probe, (2)H-enriched 3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine-1-oxyl (MC-PROXYL), used in simultaneous molecular imaging is reported. Phantom imaging was performed with liposomal solutions of MC-PROXYL at varying spin probe and liposome concentrations using a field-cycle mode, custom-built OMRI scanner. Dynamic nuclear polarization (DNP) spectra of the liposomal solution of the spin probe, measured at 14.529mT using a 5mT sweep of the electron paramagnetic resonance (EPR) irradiation field showed splitting of the low and high filed hyperfine lines. Spectral measurements using D(2)O and a spin broadening agent, K(3)Fe(CN)(6) confirmed that these peaks originated from water molecules in two different environments, compartmentalized with liposomes. The nuclear Overhauser enhancement measured at different EPR irradiation times and power levels showed reduction in water nuclear magnetic resonance (NMR) signal enhancement in liposomal membrane due to the reduction in the coupling constant, rho. This study illustrates that OMRI can be used to differentiate between the intra- and extra- membrane water by loading the liposome vesicles with a lipid-permeable nitroxyl spin probe.

Overhauser-enhanced magnetic resonance imaging characterization of mitochondria functional changes in the 6-hydroxydopamine rat model

Oxidative stress may be involved in the dopaminergic neurodegenerations seen in 6-OHDA-lesioned rats through its production of free radicals and through mitochondrial dysfunction. In this study, we noninvasively demonstrate brain redox alterations in 6-OHDA-lesioned rats using Overhauser-enhanced magnetic resonance imaging (OMRI). The reduction rate of 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-l-oxyl (methoxycarbonyl-PROXYL), a redox-sensitive contrast agent, was used as an index of the redox status in vivo. The methoxycarbonyl-PROXYL reduction rate, calculated from continuous images, decreased significantly in lesioned hemispheres compared to their corresponding contralateral hemispheres. The reduction rates in cellular fractions obtained from the striatum were estimated by X-band electron spin resonance (ESR) and calculated by assuming first-order kinetics for their time-dependent decreases. When methoxycarbonyl-PROXYL was mixed with cytoplasm fractions, the reduction rates were the same in both hemispheres. However, the ESR signal of methoxycarbonyl-PROXYL in the mitochondrial fraction of the lesioned hemispheres decayed more slowly than that of the corresponding contralateral hemispheres. Concordantly, biochemical assays showed that the activity of mitochondrial complex I also decreased more slowly in lesioned hemispheres. Thus, this method of noninvasively imaging brain redox alterations faithfully reflects changes in mitochondrial complex I activity in 6-OHDA-lesioned rats.

Nitroxides prevent exacerbation of indomethacin-induced gastric damage in adjuvant arthritis rats

Nonsteroidal anti-inflammatory drugs are the drugs of choice in the treatment of rheumatoid arthritis (RA) because of their rapid analgesic effect. However, they induce severe gastric damage in RA patients and animals by a process mediated by reactive oxygen species (ROS). Nitroxides (nitroxyl radicals) are widely used as imaging agents and antioxidants to explore the role of ROS generation in the pathogenesis of disease. In this study, the effectiveness of the newly synthesized nitroxides 8-aza-7,7,9,9-tetramethyl-1,4-dioxaspiro[4.5]undecan-8-oxyl (compound 1) and 4-oxo-2,2,6,6-tetraethylpiperidine-1-oxyl (compound 2) in the prevention of gastric ulcers in adjuvant arthritis rats treated with indomethacin was evaluated by monitoring the reaction of reactive oxygen species in gastric tissue with Overhauser-enhanced magnetic resonance imaging (OMRI). Pretreatment with all tested nitroxides suppressed the ulcers induced by indomethacin treatment in arthritic rats. OMRI using compounds 1 and 2 as well as 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) demonstrated a redox imbalance in the stomach of these rats. Lipid peroxide and interleukin (IL)-1β levels in the gastric mucosa were significantly suppressed by compound 1 and TEMPOL, whereas CINC/gro, a member of the IL-8 family, was significantly suppressed by compound 1 only. These results suggest that the preventive effects of nitroxides on gastric ulcers may operate by different mechanisms.

Mouse lactate dehydrogenase X: A promising magnetic resonance reporter protein using hyperpolarized pyruvic acid derivative Y

We developed a promising candidate for a hypersensitive magnetic resonance (MR) reporter protein. The reporter protein is comprised of mouse lactate dehydrogenase-X (mLDH-X) and a hyperpolarized pyruvic acid derivative, substrate Y that gives no false-positive signals by endogenous human lactate dehydrogenases (hLDHs). The X–Y pair shows high MR sensitivity and sufficient reaction kinetics, thus enabling direct detection of reporter protein X in crude biological mixtures by monitoring the 13C NMR signal of hyperpolarized product Yr.

Nitroxyl radicals-modified dendritic poly(l-lysine) as a contrast agent for Overhauser-enhanced MRI

Overhauser-enhanced magnetic resonance imaging (OMRI), which is a double resonance technique, creates images of free radical distribution in animals by enhancing the water proton signal intensity by the overhauser effect. In this study, we constructed a contrast agent by combining PROXYL groups that have nitroxyl radicals with PEG-modified dendritic poly(l-lysine) that accumulates in the tumor by enhanced permeability and retention (EPR) effect. Addition of the PROXYL groups at the PEG chains’ termini on KG6 was advantageous in OMRI, because the ESR signal of the nitroxyl radical was maintained without decay caused by mobility restriction, even if the PROXYL groups were attached at 25 mol% on one molecule. After intramuscular injection of the molecule modified at 25 mol%, that is, PR25-PEG-KG6, a significant OMRI signal was observed at the injected site. However, no signal was detected in the tumor after intravenous injection of PR25-PEG-KG6 to a tumor-bearing mouse, although PR25-PEG-KG6 itself accumulated in the tumor. The reason was that the nitroxyl radicals were immediately reduced in the blood after the injection, suggesting that use of stable nitroxyl radicals will enable detection of tumors by OMRI after intravenous injection.

Development of a new redox molecular imaging method

For indirect tissue observation, electron-spin, Overhauser-enhanced, dynamic nuclear polarization magnetic resonance imaging (DNP-MRI) is a useful technique. However, its sensitivity and resolution are low compared with the clinical MRI apparatus. By switching to electron spin resonance (ESR) excitation, the magnetic field of the NMR detection, field cycle technique, which aims to improve resolution, was proposed. However, the effect of eddy currents or current value was altered unsatisfactorily. A team at Kyushu University proposed a new DNP-MRI technique capable of improving NMR detection field by preparing in advance a magnetic field, which was connected by the sample transport system. By developing a mobile MRI method that can be used while moving, and fastening the sample in a disk that rotates at a constant speed, they have developed a circular transport DNP-MRI method that greatly reduces the load on the sample. The circular transport DNP-MRI system comprises a circular sample transport system, detection of an MRI magnetic field of 1.5 T, and ESR excitation magnetic field of 20 mT. The developed DNP-MRI had a clear glass tube phantom and resolution of 0.15 mm, and was successful in imaging multiple radical resonant points. It has been commercialized by Japan Redox Limited. In the process of equipment commercialization, a new digital spectrometer has been developed, which expanded the MRI apparatus.

Effect of ionic interaction between a hyperpolarized magnetic resonance chemical probe and a gadolinium contrast agent for the hyperpolarized lifetime after dissolution

In hyperpolarization of (13)C-enriched magnetic resonance chemical probes in the solid-state, a trace amount of gadolinium (Gd) contrast agent can be used to maximize polarization of the (13)C nuclear spins. Here, we report systematic measurement of the spin-lattice relaxation time (T1) and enhancement level of (13)C-enriched chemical probes in the presence of various Gd contrast agents in the liquid-state after dissolution. Using two different (13)C probes having opposite electric charges at neutral pH, we clearly show the T1 of hyperpolarized (13)C was barely affected by the use of a Gd complex that displays repulsive interaction with the (13)C probe in solution, whilst T1 was drastically shortened when there was ionic attraction between probe and complex.