[show abstract][hide abstract] ABSTRACT: Cyclic nitroxides are a diverse group of stable free radicals that have unique antioxidant properties. Because of their ability to interact with free radicals, they have been used for many years as biophysical tools. During the past 15–20 years, however, many interesting biochemical interactions have been discovered and harnessed for therapeutic applications. Biologically relevant effects of nitroxides have been described, including their ability to degrade superoxide and peroxide, inhibit Fenton reactions, and undergo radical–radical recombination. Cellular studies defined the activity of nitroxides in vitro. By modifying oxidative stress and altering the redox status of tissues, nitroxides have been found to interact with and alter many metabolic processes. These interactions can be exploited for therapeutic and research use, including protection against ionizing radiation, as probes in functional magnetic resonance imaging, cancer prevention and treatment, control of hypertension and weight, and protection from damage resulting from ischemia/reperfusion injury. Although much remains to be done, many applications have been well studied and some are currently being tested in clinical trials. The therapeutic and research uses of nitroxide compounds are reviewed here with a focus on the progress from initial development to modern trials.
[show abstract][hide abstract] ABSTRACT: Electron spin resonance (ESR) studies were carried out for 2mM
3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine-1-oxyl (MC-PROXYL) and
3-carboxy-2,2,5,5,-tetramethyl-1-pyrrolidinyloxy (carboxy-PROXYL) in
pure water and various concentrations of liposomal solutions by using
300 MHz ESR spectrometer. The ESR parameters such as the line width,
hyperfine coupling constant, g-factor, rotational correlation time,
partition parameter and permeability were reported for the samples. The
permeable and impermeable nature of nitroxyl spin probes was
demonstrated. The increased rotational correlation time was observed for
MC-PROXYL, which indicates the permeable and less mobile nature of the
nitroxyl spin probe. The obtained partition parameter and permeability
values are used to quantify the permeable nature of the nitroxyl spin
probe. The peaks correspond to lipid phase were observed for MC-PROXYL
in liposomal solution, but that peak was not observed for
carboxy-PROXYL. These results indicate the permeable and the impermeable
nature of nitroxyl spin probes.
[show abstract][hide abstract] ABSTRACT: The recent development of a bi-modality magnetic resonance imaging/electron paramagnetic resonance imaging (MRI/EPRI) platform has enabled longitudinal monitoring of both tumor oxygenation and redox status in murine cancer models. The current study used this imaging platform to test the hypothesis that a more reducing tumor microenvironment accompanies the development of tumor hypoxia. To test this, the redox status of the tumor was measured using Tempol as a redox‑sensitive MRI contrast agent, and tumor hypoxia was measured with Oxo63, which is an oxygen-sensitive EPRI spin probe. Images were acquired every 1-2 days in mice bearing SCCVII tumors. The median pO2 decreased from 14 mmHg at 7 days after tumor implantation to 7 mmHg at 15 days after implantation. Additionally, the hypoxic fraction, defined as the percentage of the tumor that exhibited a pO2<10 mmHg, increased with tumor size (from 10% at 500 mm3 to 60% at 3,500 mm3). The rate of Tempol reduction increased as a function of tumor volume (0.4 min-1 at 500 mm3 to 1.7 min-1 at 3,500 mm3), suggesting that the tumor microenvironment became more reduced as the tumor grew. The results show that rapid Tempol reduction correlates with decreased tumor oxygenation, and that the Tempol decay rate constant may be a surrogate marker for tumor hypoxia.
International Journal of Oncology 09/2012; · 2.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: Electron spin resonance studies were carried out for 2mM 14N labeled 2H
(MC-PROXYL) in pure water and 100, 200, 300, 400 mM concentrations of
liposomal solution and 3-carboxy-2,2,5,5,-tetramethyl-1-pyrrolidinyloxy
(carboxy-PROXYL) in pure water and 400 mM concentration of liposomal
solution by using L-band ESR spectrometer. This study reveals that the
permeability of MC-PROXYL in liposomal solution and impermeability of
carboxy-PROXYL in 400 mM concentration of liposomal solution. This study
illustrates that the ESR can be used to differentiate between the
intra-and extra-membrane water by loading the liposome vesicles with a
lipid permeable nitroxyl spin probe. The lipid membrane permeability of
two different nitroxyl spin probes was demonstrated.
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: Electron paramagnetic resonance (EPR) oximetry at 700 MHz operating frequency employing a surface coil resonator is used to assess tissue partial pressure of oxygen (pO(2)) using paramagnetic media whose linewidth and decay constant are related to oxygen concentration. Differences in extracellular and intracellular pO(2) in squamous cell carcinoma (SCC) tumor tissue were tested using several types of water-soluble paramagnetic media, which localize extracellularly or permeate through the cell membrane. The nitroxide carboxy-PROXYL (CxP) can only be distributed in blood plasma and extracellular fluids whereas the nitroxides carbamoyl-PROXYL (CmP) and TEMPOL (TPL) can permeate cell membranes and localize intracellularly. EPR signal decay constant and the linewidth of the intravenously administered nitroxides in SCC tumor tissues implanted in mouse thigh and the contralateral normal muscle of healthy mice breathing gases with different pO(2) were compared. The pO(2) in the blood can depend on the oxygen content in the breathing gas while tissue pO(2) was not directly influenced by pO(2) in the breathing gas. The decay constants of CmP and TPL in tumor tissue were significantly larger than in the normal muscles, and lower linewidths of CmP and TPL in tumor tissue was observed. The SCC tumor showed intracellular hypoxia even though the extracellular pO(2) is similar to normal tissue in the peripheral region.
[show abstract][hide abstract] ABSTRACT: Nitroxyl contrast agents (nitroxyl radicals, also known as nitroxide) are paramagnetic species, which can react with reactive oxygen species (ROS) to lose paramagnetism to be diamagnetic species. The paramagnetic nitroxyl radical forms can be detected by using electron paramagnetic resonance imaging (EPRI), Overhauser MRI (OMRI), or MRI. The time course of in vivo image intensity induced by paramagnetic redox-sensitive contrast agent can give tissue redox information, which is the so-called redox imaging technique. The redox imaging technique employing a blood-brain barrier permeable nitroxyl contrast agent can be applied to analyze the pathophysiological functions in the brain. A brief theory of redox imaging techniques is described, and applications of redox imaging techniques to brain are introduced.
Methods in molecular biology (Clifton, N.J.) 01/2011; 711:397-419.
[show abstract][hide abstract] ABSTRACT: A sensitive and simultaneous analytical technique for visualizing multiple endogenous molecules is now strongly required in biological science. Here, we show the applicability of a matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) system for getting chemically diverse metabolite profiles on a single-mammalian cell. This ultrahighly sensitive MALDI-MS technique enabled a spatially resolved detection of a broad range of metabolites including nucleotides, cofactors, phosphorylated sugars, amino acids, lipids, and carboxylic acids in normal mouse brain tissue with their unique distributions. Furthermore, a combination of MS imaging and metabolic pathway analysis of a rat transient middle cerebral artery occlusion model visualized a spatiotemporal behavior of metabolites in the central metabolic pathway regulated by an ischemia reperfusion. These findings highlight potential applications of an in situ metabolomic imaging technique to visualize spatiotemporal dynamics of the tissue metabolome, which will facilitate biological discovery in both preclinical and clinical settings.
[show abstract][hide abstract] ABSTRACT: Geldanamycin (GM), a benzoquinone ansamycin antibiotic, is a natural product inhibitor of Hsp90 with potent and broad anti-cancer properties. Because of its adverse effects on liver, its less toxic derivatives 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) and 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG) are currently being evaluated for the treatment of cancer. Previously, it has been demonstrated that the redox cycling of GM by NADPH-cytochrome P450 reductase leads to the formation of the GM semiquinone and superoxide radicals, the latter being identified using spin-trapping. We hypothesized that the different hepatotoxicity induced by GM, 17-AAG and 17-DMAG reflects the redox active properties of the quinone moiety and possibly the extent of superoxide formation, which may stimulate cellular oxidative injury. Our data demonstrate that superoxide can be efficiently trapped during the reduction of GM, 17-AAG and 17-DMAG by NADPH-cytochrome P450 reductase, and that superoxide formation rate followed the order 17-DMAG > 17-AAG > GM. In the absence of superoxide scavengers, the rate of NADPH oxidation followed the order 17-DMAG > GM > 17-AAG. The half-wave one-electron reduction potentials (E(1/2)) of GM, 17-AAG and 17-DMAG in DMSO have been determined to be -0.37, -0.13 and -0.015V (vs. Ag/AgCl), respectively. If the same order of E(1/2) follows in neutral aqueous media, thermodynamic considerations imply that 17-DMAG is more readily reduced by the P450 reductase as well as by superoxide. The order of the drug cytotoxicity toward rat primary hepatocytes, as determined by their effect on cell viability and on intracellular oxidant level, was opposite to the order of E(1/2) of the respective quinone/semiquinone couples. These results suggest that hepatotoxicity exhibited by the Hsp90 inhibitors belonging to benzoquinone ansamycins could be attributed to superoxide. The apparent discrepancy between the order of toxicity and the orders of superoxide formation rate, which is correlated with E(1/2), is discussed.
[show abstract][hide abstract] ABSTRACT: Radiation is used in the treatment of a broad range of malignancies. Exposure of normal tissue to radiation may result in both acute and chronic toxicities that can result in an inability to deliver the intended therapy, a range of symptoms, and a decrease in quality of life. Radioprotectors are compounds that are designed to reduce the damage in normal tissues caused by radiation. These compounds are often antioxidants and must be present before or at the time of radiation for effectiveness. Other agents, termed mitigators, may be used to minimize toxicity even after radiation has been delivered. Herein, we review agents in clinical use or in development as radioprotectors and mitigators of radiation-induced normal tissue injury. Few agents are approved for clinical use, but many new compounds show promising results in preclinical testing.
The Oncologist 01/2010; 15(4):360-71. · 4.10 Impact Factor
[show abstract][hide abstract] ABSTRACT: The partial pressure of oxygen (pO(2)) plays a determining role in the energy metabolism of aerobic cells. However, low pO(2) level induces pathophysiological conditions such as tumor hypoxia, ischemia or reperfusion injury, and delayed/altered wound healing. Especially, pO(2) level in the tumor is known to be related to tumor progression and effectiveness of radiotherapy. To monitor the pO(2) levels in vivo, continuous wave (CW) and time-domain (TD) electron paramagnetic resonance (EPR) spectroscopy method was used, in which surface coil resonator and Lithium phthalocyanine (LiPc) as oxygen sensor were crucial. Once LiPc particles are embedded in a desired location of organ/tissue, the pO(2) level can be monitored repeatedly and non-invasively. This method is based on the effect of oxygen concentration on the EPR spectra of LiPc which offers several advantages as follows: (1) high sensitivity, (2) minimum invasiveness, (3) repeated measurements, (4) absence of toxicity (non-toxic), and (5) measurement in a local region of the tissue with embedded LiPc. Therefore, in this chapter, we describe the method using CW and TD EPR spectroscopy with oxygen-sensitive particle, LiPc, for in vivo monitoring of oxygen.
Methods in molecular biology (Clifton, N.J.) 01/2010; 610:29-39.
[show abstract][hide abstract] ABSTRACT: The partial pressure of oxygen (pO2) plays a determining role in the energy metabolism of aerobic cells. However, low pO2 level induces pathophysiological conditions such as tumor hypoxia, ischemia or reperfusion injury, and delayed/altered wound
healing. Especially, pO2 level in the tumor is known to be related to tumor progression and effectiveness of radiotherapy. To monitor the pO2 levels in vivo, continuous wave (CW) and time-domain (TD) electron paramagnetic resonance (EPR) spectroscopy method was used,
in which surface coil resonator and Lithium phthalocyanine (LiPc) as oxygen sensor were crucial. Once LiPc particles are embedded
in a desired location of organ/tissue, the pO2 level can be monitored repeatedly and non-invasively. This method is based on the effect of oxygen concentration on the EPR
spectra of LiPc which offers several advantages as follows: (1) high sensitivity, (2) minimum invasiveness, (3) repeated measurements,
(4) absence of toxicity (non-toxic), and (5) measurement in a local region of the tissue with embedded LiPc. Therefore, in
this chapter, we describe the method using CW and TD EPR spectroscopy with oxygen-sensitive particle, LiPc, for in vivo monitoring
Key wordsLithium phthalocyanine (LiPc)-oxymetry-in vivo tissue oxygen-tumor-EPR
[show abstract][hide abstract] ABSTRACT: Recognition of importance of angiogenesis to tumor growth, metastasis, and treatment outcome has led to efforts to develop non-invasive methods for longitudinal monitoring of tumor microvasculature. We describe a steady-state MRI technique to determine absolute blood volume (BV) as a marker of microvascular density with improved spatial and temporal resolution using an ultra small super paramagnetic iron oxide (USPIO). A noise reduction scheme for BV imaging was also proposed based on weighting factors derived by pre-contrast signal level as an adjustable additive constant. Gradient echo sequence was used for BV imaging with MRI at 7T. Optimal imaging conditions (USPIO dose and echo time) were determined by USPIO dose-dependent studies ex vivo and in vivo. Improved analysis strategies were at first applied for cerebral BV estimation in mice, which were found in good agreement with the literature values. These methods were then used to determine tumor BV in mice. The optimal concentration of USPIO for BV estimates was found to range from 3.6 to 4.48 mM (estimated as Fe concentration) in ex vivo experiments corresponding to an in vivo dosage of 215-287 micromol/kg body weight, whereas a USPIO dose of 287 micromol/kg leads to higher cerebral BV estimate in vivo than the reported values. Application of the BV imaging method to evaluation of anti-angiogenic effect of Sunitinib in squamous cell carcinoma (SCC) tumor bearing mice revealed approximately 46% reduction in tumor BV 4 days after start of Sunitinib treatment. The results show that the MRI approach using USPIO yields high-resolution absolute BV images and the method can be conveniently applied to monitor longitudinal tumor microvessel density changes as a function of growth or in response to treatment.
International Journal of Oncology 11/2009; 35(4):797-804. · 2.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: Nitroxides, unlike trityl radicals, have shorter T(2)s which until now were not detectable in vivo by a time-domain pulsed Electron Paramagnetic Resonance (EPR) spectrometer at 300 MHz since their phase memory times were shorter than the spectrometer recovery times. In the current version of the time-domain EPR spectrometer with improved spectrometer recovery times, the feasibility of detecting signals from nitroxide radicals was tested. Among the nitroxides evaluated, deuterated (15)N-Tempone ((15)N-PDT) was found to have the longest T(2). The signal intensity profile as a function of concentration of these agents was evaluated and a biphasic behavior was observed; beyond a nitroxide concentration of 1.5mM, signal intensity was found to decrease as a result of self-broadening. Imaging experiments were carried out with (15)N-PDT in solutions equilibrated with 0%, 5%, 10%, and 21% oxygen using the single point imaging (SPI) modality in EPR. The image intensity in these tubes was found to depend on the oxygen concentration which in turn influences the T(2) of (15)N-PDT. In vivo experiments were demonstrated with (15)N-PDT in anesthetized mice where the distribution and metabolism of (15)N-PDT could be monitored. This study, for the first time shows the capability to image a cell-permeable nitroxide in mice using pulsed EPR in the SPI modality.
Journal of Magnetic Resonance 01/2009; 197(2):181-5. · 2.30 Impact Factor
[show abstract][hide abstract] ABSTRACT: Radiation, a primary mode of cancer therapy, acutely damages cellular macromolecules and DNA and elicits stress responses that lead to cell death. The known cytoprotective activity of nitric oxide (NO) is blocked by thrombospondin-1, a potent antagonist of NO/cGMP signaling in ischemic soft tissues, suggesting that thrombospondin-1 signaling via its receptor CD47 could correspondingly increase radiosensitivity. We show here that soft tissues in thrombospondin-1-null mice are remarkably resistant to radiation injury. Twelve hours after 25-Gy hindlimb irradiation, thrombospondin-1-null mice showed significantly less cell death in both muscle and bone marrow. Two months after irradiation, skin and muscle units in null mice showed minimal histological evidence of radiation injury and near full retention of mitochondrial function. Additionally, both tissue perfusion and acute vascular responses to NO were preserved in irradiated thrombospondin-1-null hindlimbs. The role of thrombospondin-1 in radiosensitization is specific because thrombospondin-2-null mice were not protected. However, mice lacking CD47 showed radioresistance similar to thrombospondin-1-null mice. Both thrombospondin-1- and CD47-dependent radiosensitization is cell autonomous because vascular cells isolated from the respective null mice showed dramatically increased survival and improved proliferative capacity after irradiation in vitro. Therefore, thrombospondin-1/CD47 antagonists may have selective radioprotective activity for normal tissues.
American Journal Of Pathology 10/2008; 173(4):1100-12. · 4.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: Nitric oxide (NO) plays important physiological roles in the vasculature to regulate angiogenesis, blood flow, and hemostasis. In solid tumors, NO is generally acknowledged to mediate angiogenic responses to several growth factors. This contrasts with conflicting evidence that NO can acutely increase tumor perfusion through local vasodilation or diminish perfusion by preferential relaxation of peripheral vascular beds outside the tumor. Because thrombospondin 1 (TSP1) is an important physiological antagonist of NO in vascular cells, we examined whether, in addition to inhibiting tumor angiogenesis, TSP1 can acutely regulate tumor blood flow. We assessed this activity of TSP1 in the context of perfusion responses to NO as a vasodilator and epinephrine as a vasoconstrictor. Nitric oxide treatment of wild type and TSP1 null mice decreased perfusion of a syngeneic melanoma, whereas epinephrine transiently increased tumor perfusion. Acute vasoactive responses were also independent of the level of tumor-expressed TSP1 in a melanoma xenograft, but recovery of basal perfusion was modulated by TSP1 expression. In contrast, overexpression of truncated TSP1 lacking part of its CD47 binding domain lacked this modulating activity. These data indicate that TSP1 primarily regulates long-term vascular responses in tumors, in part, because the tumor vasculature has a limited capacity to acutely respond to vasoactive agents.
Neoplasia (New York, N.Y.) 09/2008; 10(8):886-96. · 5.48 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Journal of Pharmacy and Pharmacology 08/2008; 60(8):1049-60. · 2.03 Impact Factor