Ken-ichiro Matsumoto

High-Resolution Mapping of Tumor Redox Status by Magnetic Resonance Imaging Using Nitroxides as Redox-Sensitive Contrast Agents

Purpose: There is considerable research directed toward the identification and development of functional contrast agents for medical imaging that superimpose tissue biochemical/molecular information with anatomical structures. Nitroxide radicals were identified as in vivo radioprotectors. Being paramagnetic, they can provide image contrast in magnetic resonance imaging (MRI) and electron paramagnetic resonance imaging (EPRI). The present study sought to determine the efficacy of nitroxide radioprotectors as functional image contrast agents. Experimental Design: Nitroxide radioprotectors, which act as contrast agents, were tested by EPRI and MRI to provide tissue redox status information noninvasively. Results: Phantom studies showed that the nitroxide, 3-carbamoyl-PROXYL (3CP), undergoes time-dependent reduction to the corresponding diamagnetic hydroxylamine only in the presence of reducing agents. The reduction rates of 3CP obtained by EPRI and MRI were in agreement suggesting the feasibility of using MRI to monitor nitroxide levels in tissues. The levels of 3CP were examined by EPRI and MRI for differences in reduction between muscle and tumor (squamous cell carcinoma) implanted in the hind leg of C3H mice simultaneously. In vivo experiments showed a T1-dependent image intensity enhancement afforded by 3CP which decreased in a time-dependent manner. Reduction of 3CP was found to be the dominant mechanism of contrast loss. The tumor regions exhibited a faster decay rate of the nitroxide compared to muscle (0.097 min-1 versus 0.067 min-1, respectively). Conclusions: This study shows that MRI can be successfully used to co-register tissue redox status along with anatomic images, thus providing potentially valuable biochemical information from the region of interest.

A new nitroxyl-probe with high retention in the brain and its application for brain imaging.

In order to estimate free radical reactions and image them in the brain of living animals, a nitroxyl spin-probe, carboxy-PROXYL acetoxymethyl ester (CxP-AM) was newly synthesized. CxP-AM was designed to be hydrolyzed by esterase, but not by lipase, so that it would pass through the blood-brain barrier and be retained in the cytosolic phase of parenchymal cells in the brain after intravenous injection. The pharmacokinetics of CxP-AM was compared with those of carboxy-PROXYL (CxP) and its methyl ester (CxP-M). Carboxyl esterase almost completely hydrolyzed CxP-AM within 3 min. After intravenous injection, the brain retained 1.8 times more CxP-AM than CxP-M, and retained it for more than 30 min. Electron spin resonance computed tomographic (ESR-CT) imaging of CxP-AM in the heads of mice produced marked contrast in the encephalon region, while CxP was distributed only in the extracranial region and CxP-M was distributed in both regions, confirming the pharmacokinetics of CxP-AM. The decay rate of CxP-AM determined with time-resolved ESR-CT imaging was different in the two brain regions, suggesting regional differences in the total reducing capability. CxP-AM should become a powerful probe for the investigation and diagnosis of free radical reactions and their imaging in the brain.

A mitochondrial superoxide theory for oxidative stress diseases and aging.

Fridovich identified CuZnSOD in 1969 and manganese superoxide dismutase (MnSOD) in 1973, and proposed ”the Superoxide Theory,” which postulates that superoxide (O2•−) is the origin of most reactive oxygen species (ROS) and that it undergoes a chain reaction in a cell, playing a central role in the ROS producing system. Increased oxidative stress on an organism causes damage to cells, the smallest constituent unit of an organism, which can lead to the onset of a variety of chronic diseases, such as Alzheimer’s, Parkinson’s, amyotrophic lateral sclerosis and other neurological diseases caused by abnormalities in biological defenses or increased intracellular reactive oxygen levels. Oxidative stress also plays a role in aging. Antioxidant systems, including non-enzyme low-molecular-weight antioxidants (such as, vitamins A, C and E, polyphenols, glutathione, and coenzyme Q10) and antioxidant enzymes, fight against oxidants in cells. Superoxide is considered to be a major factor in oxidant toxicity, and mitochondrial MnSOD enzymes constitute an essential defense against superoxide. Mitochondria are the major source of superoxide. The reaction of superoxide generated from mitochondria with nitric oxide is faster than SOD catalyzed reaction, and produces peroxynitrite. Thus, based on research conducted after Fridovich’s seminal studies, we now propose a modified superoxide theory; i.e., superoxide is the origin of reactive oxygen and nitrogen species (RONS) and, as such, causes various redox related diseases and aging.

Differential Radiation Protection of Salivary Glands versus Tumor by Tempol with Accompanying Tissue Assessment of Tempol by Magnetic Resonance Imaging

Purpose: The nitroxide free radical, Tempol, was evaluated for potential differential radiation protection of salivary glands and tumor using fractionated radiation. Mechanistic information was explored by monitoring the presence and bioreduction of Tempol in both tissues noninvasively by magnetic resonance imaging (MRI). Experimental Design: Female C3H mice were immobilized using custom-made Lucite jigs for localized irradiation (five daily fractions) either to the oral cavity or tumor-bearing leg. Tempol (275 mg/kg) was administered (i.p.) 10 min before each radiation fraction. Salivary gland damage was assessed 8 weeks after radiation by measuring pilocarpine-mediated saliva output. Tumor growth was assessed by standard radiation regrowth methods. Dynamic T1-weighted magnetic resonance scans were acquired before and after Tempol injection using a 4.7T animal MRI instrument. Results: Tempol treatment was found to protect salivary glands significantly against radiation damage (∼60% improvement); whereas no tumor protection was observed. Intracellular reduction of Tempol to the nonradioprotective hydroxylamine as assessed by MRI was 2-fold faster in tumor compared with salivary glands or muscle. Conclusions: Tempol provided salivary gland radioprotection and did not protect tumor, consistent with the hypothesis that differential radioprotection by Tempol resides in faster reduction to the nonradioprotective hydroxylamine in tumor compared with normal tissues. The unique paramagnetic properties of Tempol afforded noninvasive MRI monitoring of dynamic changes of Tempol levels in tissue to support the finding. These data support further development and consideration of Tempol for human clinical trials as a selective protector against radiation-induced salivary gland damage.

Absolute oxygen tension (pO2) in murine fatty and muscle tissue as determined by EPR

The absolute partial pressure of oxygen (pO2) in the mammary gland pad and femoral muscle of female mice was measured using EPR oximetry at 700 MHz. A small quantity of lithium phthalocyanine (LiPc) crystals was implanted in both mammary and femoral muscle tissue of female C3H mice. Subsequent EPR measurements were carried out 1–30 days after implantation with or without control of core body temperature. The pO2 values in the tissue became stable 2 weeks after implantation of LiPc crystals. The pO2 level was found to be higher in the femoral muscle than in the mammary tissue. However, the pO2 values showed a strong dependence on the core body temperature of the mice. The pO2 values were responsive to carbogen (95% O2, 5% CO2) breathing even 44–58 days after the implantation of LiPc. The LiPc linewidth was also sensitive to changes in the blood supply even 60 days after implantation of the crystals. This study further validates the use of LiPc crystals and EPR oximetry for long‐term non‐invasive assessment of pO2 levels in tissues, underscores the importance of maintaining normal body core temperature during the measurements, and demonstrates that mammary tissue functions at a lower pO2 level than muscle in female C3H mice. Magn Reson Med, 2005. Published 2005 Wiley‐Liss, Inc.

Electron paramagnetic resonance imaging of tumor hypoxia : Enhanced spatial and temporal resolution for in vivo pO2 determination

The time‐domain (TD) mode of electron paramagnetic resonance (EPR) data collection offers a means of estimating the concentration of a paramagnetic probe and the oxygen‐dependent linewidth (LW) to generate pO2 maps with minimal errors. A methodology for noninvasive pO2 imaging based on the application of TD‐EPR using oxygen‐induced LW broadening of a triarylmethyl (TAM)‐based radical is presented. The decay of pixel intensities in an image is used to estimate T  2* , which is inversely proportional to pO2. Factors affecting T  2* in each pixel are critically analyzed to extract the contribution of dissolved oxygen to EPR line‐broadening. Suitable experimental and image‐processing parameters were obtained to produce pO2 maps with minimal artifacts. Image artifacts were also minimized with the use of a novel data collection strategy using multiple gradients. Results from a phantom and in vivo imaging of tumor‐bearing mice validated this novel method of noninvasive oximetry. The current imaging protocols achieve a spatial resolution of ∼1.0 mm and a temporal resolution of ∼9 s for 2D pO2 mapping, with a reliable oxygen resolution of ∼1 mmHg (0.12% oxygen in gas phase). This work demonstrates that in vivo oximetry can be performed with good sensitivity, accuracy, and high spatial and temporal resolution. Magn Reson Med, 2006. Published 2006 Wiley‐Liss, Inc.

Synthesis and imaging of blood‐brain‐barrier permeable nitroxyl‐probes for free radical reactions in brain of living mice

Three different lipophilic nitroxyl‐probes having capability to pass the blood‐brain barrier were, for the ftrst time, synthesized to estimate free radical reactions in brain of living animals. Two of the three were designed to be hydrolyzed by esterase and remain in cell. All 3 probes had high n‐octanol/buffer partition coefficients and gave 2 signal components in in vivo ESR spectra at head of living mice after intravenous injection. The ESR parameters of 2 components agreed with those of probes dissolved in water and lipidic phases. The ESR‐CT imaging on the nilroxyl‐proves after intravenous injection revealed that all probes presented in both encephalon and extracranial region of head. Tissue distribution of the nitroxyl‐probes demonstrated that the newly synthesized lipophilic nitroxyl‐probes had capability to pass the blood‐brain barrier and accumulated in brain than that of hydrophilic probe.

MR assessment of changes of tumor in response to hyperbaric oxygen treatment

Enhancement of image intensity, using the T1‐weighted spoiled gradient‐echo (SPGR) sequence, was measured in SCC tumor implanted in the flank of C3H mice while they were subjected to several types of oxygenation challenges inside a hyperbaric chamber designed and constructed to fit in an MRI resonator. The central portions of the tumor gave a positive enhancement, while the periphery showed signal reduction during both normobaric (NBO) and hyperbaric (HBO) oxygen challenges. In the contralateral normal leg, nearly 70% of the region showed a decrease in intensity, and the rest showed a positive enhancement. The positive signal enhancement was markedly greater under HBO compared to NBO. Calculated R1, R2, and M0 maps from multivariate fitting of images acquired by a multislice multiecho (MSME) sequence with variable TR before, during, and after HBO treatment confirm that the source of SPGR signal enhancement in the tumor is associated with shortening of T1. Magn Reson Med, 2006. Published 2006 Wiley‐Liss, Inc.

Assessment of Tissue Redox Status Using Metabolic Responsive Contrast Agents and Magnetic Resonance Imaging

Regulation of tissue redox status is important to maintain normal physiological conditions in the living body. Disruption of redox homoeostasis may lead to oxidative stress and can induce many pathological conditions such as cancer, neurological disorders and ageing. Therefore, imaging of tissue redox status could have clinical applications. Redox imaging employing magnetic resonance imaging (MRI) with nitroxides as cell‐permeable redox‐sensitive contrast agents has been used for non‐invasive monitoring of tissue redox status in animal models. The redox imaging applications of nitroxide electron paramagnetic resonance imaging (EPRI) and MRI are reviewed here, with a focus on application of tumour redox status monitoring. While particular emphasis has been placed on differences in the redox status in tumours compared to selected normal tissues, the technique possesses the potential to have broad applications to the study of other disease states, inflammatory processes and other circumstances where oxidative stress is implicated.

Nitroxyl radicals for labeling of conventional therapeutics and noninvasive magnetic resonance imaging of their permeability for blood-brain barrier: relationship between structure, blood clearance, and mri signal dynamic in the brain

The present study describes a novel nonradioactive methodology for in vivo noninvasive, real-time imaging of blood-brain barrier (BBB) permeability for conventional drugs, using nitroxyl radicals as spin-labels and magnetic resonance imaging (MRI). Two TEMPO-labeled analogues (SLENU and SLCNUgly) of the anticancer drug lomustine [1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea] were synthesized, using a substitution of the cyclohexyl part with nitroxyl radical. Nonmodified nitroxyl radical TEMPOL was used for comparison. The nitroxyl derivatives were injected intravenously in healthy mice via the tail vein, and MR imaging of the brain was performed on a 7.0 T MRI. The MRI signal dynamic of SLENU and SLCNUgly followed the same kinetics as nonmodified TEMPO radical. SLENU and SLCNUgly were rapidly transported and randomly distributed in the brain tissue, which indicated that the exchange of cyclohexyl part of lomustine with TEMPO radical did not suppress the permeability of the anticancer drug for BBB. The selected nitroxyl derivatives possessed different hydrophobicity, cell permeabilization ability, and blood clearance. Based on these differences, we investigated the relationship betweenthe structure of nitroxyl derivatives, their half-life in the circulation, and their MRI signal dynamic in the brain. This information was important for estimation of the merits and demerits of the described methodology and finding pathways for overcoming the restrictions.