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ABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION
Radiation Biology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland
Received February 6, 2004; accepted April 15, 2004.
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Abstract |
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; Ilangovan et al., 2002; Kuppusamy et al., 2002; Yamada et al., 2002; Kasazaki et al., 2003). When a nitroxyl spin probe is administered to a living animal, in vivo EPR signal intensities of the probe show characteristic signal intensity profiles as a function of time in the animal, depending on the organ investigated. Generally, in vivo EPR signal decay rates are obtained based on a suitable region of its decay curve to estimate redox status in the animal (Yamada et al., 2002).
Several nitroxyl radicals have been used in studies that exhibit EPR signal decay profiles, depending on the spin probe used. The decay constant of a spin probe depends on the route of administration (i.e., intravenous, intraperitoneal) and methodology of the analysis (i.e., one-compartment model, two-compartment model) (Kocherginsky and Swartz, 1995). Moreover, species, strains, and gender of the experimental animal was found to affect the EPR signal decay profiles (Kocherginsky and Swartz, 1995; Matsumoto et al., 2000).
The following general observations can be summarized from earlier studies. The in vivo EPR signal decay rates of the spin probes are enhanced by reactive oxygen species (Utsumi et al., 1993; Sano et al., 1998; Phumala et al., 1999; Han et al., 2001), such as hydroxyl radical and superoxide, which reduce nitroxyl radical in presence of H atom donor, such as NAD(P)H or glutathione (Samuni et al., 1988; Krishna et al., 1992; Samuni et al., 2002; Takeshita et al., 2002). In contrast, the decay rates are decreased due to reoxidation of the hydroxylamine to the nitroxyl radical under the oxidative atmosphere such as exposure to hyperoxia or generation of hydrogen peroxide (Quaresima et al., 1993; Matsumoto et al., 2000). Under reducing conditions as in hypoxic tumors, the decay rate constants of nitroxyl radicals were found to increase because reoxidation may be less efficient in such conditions (Minetti and Scorza, 1991; Ilangovan et al., 2002; Kuppusamy et al., 2002).
The signal decay rate also depends on several kinetic factors such as the distribution of the spin probe from the blood to the tissues and vice versa, urinary excretion through the kidney, fecal excretion through liver and bile, and entrapment into specific tissues/organs. To estimate redox information independent of clearance, the pharmacokinetic analysis of nitroxyl spin probes is therefore needed. Several groups have reported the comparison of pharmacokinetics of several nitroxyl spin probes in experimental animals (Komarov et al., 1994; Takechi et al., 1997; Hahn et al., 1998).
Blood and/or plasma concentration of a drug in the paramagnetic form is a most common index to estimate its in vivo pharmacokinetics. In rats, the time course of EPR signal intensity of the spin probes in the blood can be obtained in real time by the monitoring the circulating blood using X-band EPR (Takechi et al., 1997). However in mice, the small blood volume and difficulty of surgical procedures make it difficult to conduct repeat sampling and/or real-time measurement of the circulating blood. Therefore, in most cases of pharmacokinetic studies of nitroxyl radicals in mice, EPR measurements were carried out in mouse-tail instead of blood (Komarov et al., 1994). These results include pharmacokinetic information not only from the blood but also from tissue in the sampled region. In addition, it is not possible to determine the concentration of spin probes from in vivo EPR studies because the in vivo EPR signal intensity is not quantitative in most cases.
In our laboratory, the EPR-related oximetry, redox status estimation, and related functional imaging techniques are being investigated using mouse tumor models (Yamada et al., 2002; Matsumoto et al., 2003). Nitroxyl radicals are used in some of these studies to noninvasively assess tissue redox status. Additionally, nitroxyl radicals are used as in vivo radioprotectors. Therefore, the pharmacokinetic data of nitroxyl radicals in mouse blood will be useful to assess tumor redox status.
In this study, we evaluated the acute toxicity of nitroxyl spin probe after i.v. injection. We also describe a novel procedure to measure the time course of spin probe concentration in the blood of a mouse using X-band EPR spectroscopy. Using this technique, the pharmacokinetics of several nitroxyl spin probes was estimated and the pharmacokinetics results were also simulated. Suitable properties for in vivo redox probes and the radiation protection agents are discussed.
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Materials and Methods |
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Animals. Female C3H mice were supplied by the Frederick Cancer Research Center, Animal Production (Frederick, MD). Animals, received at 6 weeks of age, were housed five per cage in climate-controlled circadian rhythm-adjusted rooms and were allowed food and water ad libitum. Experiments are carried out in compliance with the Guide for the Care and Use of Laboratory Animal Resources (1996), National Research Council, and approved by the National Cancer Institute Animal Care and Use Committee. Pharmacological experiments were performed within 2 weeks of their arrival at the facility.
Acute Toxicity by Intravenous Injection. Mice were anesthetized by isoflurane (1.5% in medical air; 700 ml/min). Several doses (1.50, 1.00, 0.75, 0.50, and 0.25 µmol/g b.wt.) were tried and acute toxicities were tested.
Measurement of Time Course of the Spin Probe Concentrations in Blood. Each mouse was anesthetized by isoflurane (1.5% in medical air; 700 ml/min). The tail vein was cannulated to inject spin probe solution. The jugular vein was cannulated by polyethylene-10 tubing. The jugular cannulation was through the X-band EPR cavity, and the end of the cannulation tube was connected to a syringe. The schematics of the experimental arrangement to monitor blood levels of the spin probes continuously using EPR spectroscopy are shown in Fig. 1. The cannulation line and cylinder were filled with heparinized saline. Spin probe solutions were injected via the cannula in the tail vein. Two different doses were tested for time-course study of each probe except for TEMPO. The dose of TEMPO was 0.25 µmol/g b.wt. based on the maximum tolerated dose (MTD). Similarly, the higher dose was determined as 0.50 µmol/g b.wt. for amino-TEMPO and CAT-1, 1.00 µmol/g b.wt. for TEMPONE, and 1.50 µmol/g b.wt. for other probes. The lower dose was one-half of the higher dose. Immediately after the injection of the spin probe, blood was pumped up to the X-band EPR cavity (E line; Varian, Palo Alto, CA) from the jugular vein cannulation, and the EPR signal was measured. The blood was pushed back into the jugular vein after the measurement. The X-band EPR measurements were repeated over 20 min (30 min for carbamoyl-PROXYL, carboxy-PROXYL, and CAT-1) after injection of a spin probe solution. After each measurement, 
10 to 30 µl of heparinized saline was additionally flushed. The EPR conditions were as follows; microwave frequency was 9.4 GHz, microwave power was 10 mW, magnetic field modulation frequency was 100 kHz, magnetic field modulation amplitude was 1.0 Gauss, and time constant was 0.064 s. The spin probe solutions were diluted by phosphate-buffered saline at several concentrations and measured with same system to obtain the standard curves.
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Data Analysis of the Time Course of the Spin Probe Concentrations in Blood. The estimation of the decay constant is affected by the selection of a time window, which may be subjective. To eliminate such empirical factors, we processed the data as follows. 1) Experimental endpoint was decided based on 0.05, 0.5, and 1.2% of predicted initial blood concentration C0 for TEMPO derivatives, carbamoyl-PROXYL, and carboxy-PROXYL. The prediction of initial blood concentration was calculated by the dose and the initial distribution volume. A blood volume of 1.95 ml for a 25-g mouse (Satoh, 1986) was used. The plasma volume was 1.07 ml for a 25-g mouse (55% of blood volume) (Rygaard and Povlsen, 1974; Kutscher and Schmalbach, 1975). The initial distribution volume was, at this time, double that of plasma volume. 2) The slow phase was estimated by the concentration region between the final concentration (at endpoint) and 5-fold of the final concentration and slow component k
was thus calculated. 3) The fast phase was estimated by the concentration region between 10-fold of the final concentration and 80% of predicted initial blood concentration. 4) After deducting the slow phase components from the fast phase, the fast component k
was calculated.
Simulation of Nitroxyl Decay Curve in Mouse Blood. A simulation of decay of spin probe in blood was carried out according with the following equations:
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t is increment of data time point (minutes); and kD is distribution rate (minutes-1). (Tt-1 
D/V: kD = kD0 or
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The values used for the simulation was shown in Table 1. For initial 15 s (0.25 min), Tt was assumed to increase linearly by injection, i.e., eqs. 1 and 3 have an additional term (+ Ti/0.25 x t). Ti is expected initial blood concentration of total probe, which is given as D/V0. V0 depends on the O/W partition coefficient, P. Expected initial blood concentration of nitroxyl radical Ni is same value as Ti when radical form was administered. Ni was set as 0 when hydroxylamine form was administered. Tt, Bt, and Nt at time t = 0 are T0 = B0 = N0 = 0.
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). The MTD of TEMPO was below 0.25 µmol/g b.wt., which was approximately one-sixth that of TEMPOL. The MTD of TEMPONE should be between 1.00 and 1.50 µmol/g b.wt. which is lower than TEMPOL. A previous report (Hahn et al., 1998) also determined the MTD of TEMPONE by i.p. injection to be 225 mg/kg b.wt. = 1.32 µmol/g b.wt., which was in the same range in the present study. The MTD of amino-TEMPO should be between 0.50 and 0.75 µmol/g b.wt., which is lower than TEMPOL and TEMPONE. However, a previous report (Hahn et al., 1998) showed the MTD of amino-TEMPO by i.p. injection to be 250 mg/kg b.wt. = 1.46 µmol/g b.wt. The intraperitoneal injection route used in that study may have reduced the toxicity of amino-TEMPO. Octanol/water partition coefficients for the piperidine nitroxyl radicals have been reported in the following order: TEMPO >> TEMPOL > TEMPONE > amino-TEMPO > carboxy-TEMPO (Takechi et al., 1997). Hydrophilic amino-TEMPO injected i.p. may be retained in ascites for relatively long period rather than being transported into blood circulation through the cell membrane and might be prevented to reach target tissues/organs of toxicity. The MTD of CAT-1 should be between 0.50 and 0.75 µmol/g b.wt., which is in the same range as amino-TEMPO. Therefore, acute toxicity in mice by i.v. injection followed the order TEMPO > CAT-1 
amino-TEMPO > TEMPONE > TEMPOL. No acute deaths of mice were observed for other spin probes at the doses used in this study.
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Time Course of the Spin Probe Concentrations in Blood. Figure 2 shows a logarithmic plot of decay curves of several spin probes in mouse blood measured by EPR. Decay curves showed biphasic profiles except for TEMPO and CAT-1. Because the lipophilic TEMPO molecule adsorbed and remained in the polyethylene-10 tube, quantification in the blood was inaccurate below 0.03 mM. Although data points could not be obtained for sufficient time periods because of high background to reliably characterize it, the decay curve of TEMPO (Fig. 2C) seemed to be biphasic as shown by simulation (Fig. 3C). In addition, TEMPO showed relatively low initial blood concentrations than the expected value from dose administered. CAT-1, which is an extracellular probe, showed a triphasic behavior, consisting of a short first phase, followed by slow second phase, and then a final linear decrease.
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A similar triphasic decay, as observed for CAT-1, was shown for another membrane-impermeable molecule, tri[8-carboxy-2,2,6,6-tetrakis(2-hydroxymethyl)benzo[1,2-d:4,5-d']bis(1,3)dithio-4-yl]methyl radical (Oxo63), which is a triarylmethyl radical used as EPR oximetry probe (manuscript submitted for publication). The slow second phase was due to the saturation of urinary excretion. Oxo63 exhibited saturated excretion at a relatively high dose (1.50 µmol/g b.wt. bolus i.v. injection), whereas CAT-1 exhibited evidence of saturation at 0.25 µmol/g b.wt. bolus i.v. injection. Oxo63 has a diuretic effect, whereas no diuretic effect for any nitroxyl spin probes has been reported to our knowledge. The third phase of Oxo63 started when the plasma concentration fell below 2.7 mM, whereas the third phases of CAT-1 started below 0.15 mM as shown in Fig. 2H. Total CAT-1 concentration, i.e., the total of reduced and oxidized forms, should be higher than the concentration indicated in Fig. 2H, because CAT-1 undergoes reduction in the mouse.
The decay constants of various spin probes were calculated from those plots based on a two-compartment model analysis except for TEMPO and CAT-1 as shown in Table 3. No marked differences were obtained between the high dose and the low dose except for TEMPOL and carboxy-TEMPO. In this experiment, the k values followed the order TEM-PONE > amino-TEMPO > TEMPOL > carboxy-TEMPO > carboxy-PROXYL > carbamoyl-PROXYL. This finding is in agreement with previous reports (Komarov et al., 1994; Takechi et al., 1997), except that several results reported for the order of TEMPO and amino-TEMPO was dependent on the experimental design and/or analysis. However, the ka showed a different order. For low doses, k of the probes followed the order amino-TEMPO > TEMPONE > TEM-POL > carboxy-TEMPO carbamoyl-PROXYL > carboxy-TEMPO. For high doses, the k of TEMPONE and TEMPOL were similar.
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Simulation of Nitroxyl Decay Curve in Mouse Blood. The urinary excretion rate kU was determined to be 0.035 min-1 based on the pharmacokinetics data of the paramagnetic spin probe Oxo63. The pharmacokinetics of Oxo63 should be influenced by distribution, excretion, and less in vivo reduction. Therefore, kU was obtained from the very late time region (30–60 min after injection) of the Oxo63 decay curve. The kU should be a significantly smaller value, i.e., being saturated when the Tt-1 was higher than 2.7 mM, which is the value based on the pharmacokinetics of Oxo63. The distribution rate kD might be larger than 0.5 min-1, whereas it was difficult to estimate because it probably changes with time. For nitroxyl spin probe, the distribution rate kD may be dependent on the distribution volume, i.e., O/W partition coefficient and/or membrane permeability. The kD may be faster for probes with low membrane permeability and their distribution may occur by penetration through cell interstitial spaces. In contrast, high membrane-permeable probes should have a low kD because of the distribution through cell membrane. The kD gradually slows down when the Tt-1 goes below D/Vf. The final distribution volume Vf should be estimated considering membrane permeability. V0 is the initial distribution volume, which consists of the plasma volume and volumes associated with tissue cell interstices, cell membranes, and intracellular regions. Therefore, the V0 is also dependent on membrane permeability. Lipophilicity may increase V0, whereas extremely high lipophilicity may tend to decrease V0 because lipophilic probes may be within membranes. The kB should also be dependent based on the membrane permeability. The reabsorption rate, kA, was set to equal kB, and the fecal excretion rate, kF, was set at 1/10 of the kB. The reoxidation rate, kO, may depend on the kR and also on the membrane permeability. Finally, reduction rate, kR, was chosen depending on the shape of decay profile. The values used in simulation were selected considering the order of the redox potential and the partition coefficient shown in previous reports (Krishna et al., 1992; Takechi et al., 1997). The values used for simulations are shown in Table 1.
Simulated decay curves are shown in Fig. 3. Simulated decay profiles showed similar patterns as obtained in animal experiments. The desired values kR followed the order of TEMPO > TEMPOL > amino-TEMPO > TEMPONE >> carboxy-TEMPO > CAT-1 > carboxy-PROXYL >> carbamoyl-PROXYL. This order is dependent on the oxidation/reduction potentials within a class of probes, i.e., TEMPO or PROXYL derivatives.
Figure 4 shows the simulation of nitroxyl radical concentration after the administration of 1.5 µmol/g b.wt. hydroxylamine for TEMPOL and carbamoyl-PROXYL. This simulation was carried out using the same equation by setting the expected initial concentration of radical form Ni = 0. The administration of the hydroxylamine form of carbamoyl-PROXYL may not give enough concentration of radical form (Fig. 4A). However, the administration of TEMPOL-H, the hydroxylamine form of TEMPOL, can reach the same radical concentration as TEMPOL administered 3 min after administration (Fig. 4B).
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). TEMPONE was, however, less toxic than amino-TEMPO in mice. In a living mouse, TEMPONE may be metabolized to the less toxic TEMPOL at a relatively fast rate (Kroll and Borchert, 1999). After injection of TEM-PONE, a small portion of TEMPOL signal occurred and gradually increased. TEMPONE almost completely disappeared within 10 min, after which time, only the TEMPOL signal was observed. When potassium ferricyanide was added to collected blood 25 min after injection of TEMPONE, recovered signal was only TEMPOL (data not shown).
Generally, doses of commonly used pharmaceuticals are administered at concentrations less than 10 nmol/g in humans. The dose of spin probes used in this experiment was between 10- and 100-fold higher than this value. Saturation of excretion processes could occur at such a high dose. From the pharmacokinetic results of Oxo63, the urinary excretion rate was not so high (0.035 min-1) (manuscript submitted for publication). When the blood concentration of the probe was higher than a certain extent, urinary excretion may be saturated. The time period of the saturated excretion may be shortened with decreasing dose, below which the decay curves of all probes become biphasic. The saturation period can also be shortened by membrane permeability of the probe, i.e., increasing distribution volume and fecal excretion through bile. However, total concentrations of membrane-permeable probes were kept in blood and/or tissues for relatively long time periods as shown in a previous report (Hahn et al., 1998) due to enterohepatic circulation.
The decay curve profiles can be influenced by several rate factors, such as distribution, excretion, reduction, and reoxidation rates. The distribution rate should stabilize and should decrease when the probes distribute in the body. The urinary excretion could be saturated when the probe concentration in the blood is high. The urinary excretion rate will recover when the blood concentration of total probe decreases. The fecal excretion can also be expected for membrane-permeable probes. The fecal excretion is comprised of three processes, i.e., excretion into bile, reabsorption from bile, and actual excretion into feces. The distribution rate and total excretion rate may change depending on blood and/or tissue concentration of the probe. Therefore, distribution and excretion may not exhibit first order decay kinetics and may make the biphasic decay indistinguishable under conditions when saturation of excretion has not occurred. This process should influence the total probe kinetics. In addition to this, the different types of reduction rates should be considered, but mainly a reversible one-electron reduction and an irreversible reduction. Moreover, reoxidation of hydroxylamine to nitroxyl radical should be considered. When the reduction rate predominates, the excretion phase can be neglected. However, in most of cases, the oxidation phase will make the profile pseudobiphasic, because reduction and oxidation will reach equilibrium, after which the decay rate depends primarily on the excretion rate. When the reduction rate was slow enough and the blood concentration of the probe was high enough, the saturation of total probe excretion was obvious on the decay curve of reduced form as a triphasic profile with or without a slight shoulder (Fig. 2H).
Based on the factors discussed above, a simulation of the decay of nitroxyl radicals in blood was proposed as described under Materials and Methods. Although this simulation was not comprehensive, some observations related to the mechanism of in vivo nitroxyl decay can be noted. Several simulations of the decay kinetics showed that a combination of distribution and excretion is responsible for the basic biphasic decay under nonsaturated conditions of excretion. When the redox contribution to the signal loss was neglected, the slope of the fast phases was dependent mainly on the distribution volume and bile excretion rate, i.e., membrane permeability. The change in reduction and reoxidation rates, i.e., redox status, could influence the slope of the fast phase and the time taken to transform from the fast phase to the slow phase, and then cancel each other, after which the decay curve will depend primarily on distribution and/or excretion. Therefore, the slow phase, especially at the later time regions after the administration of the probe, depends only on excretion rate. To estimate in vivo redox information from the decay constant of nitroxyl radicals in blood circulation, probes with fast reduction rates, such as the TEMPO, are sensitive because the reduction rates could predominate in the fast phase.
Most reported studies use nitroxyl radicals as redox probes where the EPR signal decay in a particular tissue and/or organ or the part of the body is followed. Different mechanisms for in vivo nitroxyl decay rate described in a previous report (Kocherginsky and Swartz, 1995) may be due to the ambiguity of measured volume and variation of treated phase. For the particular tissue and/or organ of interest, an additional compartment, which includes uptake phase and reduction phase, should be considered. Then, the decay curve will exhibit a peak followed by a biphasic profile with relatively slower initial fast phase or linear profile lacking fast phase. The reduction rate presumably predominates in EPR measurements of whole body or particular tissue and/or organ in the absence of gradients (Komarov et al., 1994; Kamatari et al., 2002). Therefore, redox status estimation using nitroxyls as probes will be more effective when implemented in combination with EPR imaging techniques.
One of the factors determining the suitability of nitroxyl radicals as redox probes is that the molecules should be able to go into the cell. However, while estimating the decay rate as an index of redox status, any traces of saturated excretion on the decay curve may be confounding. Optimally, the fast phase and the time period needed to shift to the slow phase should be as long as possible. For in vivo EPR imaging, the administered dose should be as high as possible. The saturation of excretion might be negligible for membrane-permeable probes, even at relatively high doses used. According to this hypothesis, TEMPOL and amino-TEMPO should be better redox probes among the compounds tested. Whereas TEMPOL and amino-TEMPO may be more sensitive for redox status, carbamoyl-PROXYL is better for EPR imaging. Although the simulation of decay of carbamoyl-PROXYL showed almost no difference between total probe concentration and radical concentration, it is expected that the reduction rate will be more responsive to the decay rate in tissues and/or organs.
Reoxidation of the hydroxylamine to the corresponding nitroxyl radical is a feasible approach for radiation protection in vivo, because radiation protection is shown only for the radical form not for the hydroxylamine in vitro (Mitchell et al., 1991; Sasaki et al., 1998). Administration of the hydroxylamine to living mice yields radioprotection because it is reoxidized to nitroxyl radical (Hahn et al., 2000). Reoxidization may occur more efficiently in normal tissues compared with the hypoxic conditions expected in the tumor. Therefore, the nitroxyl radical shows radiation protection only for normal cells. To obtain selective protection, it is important to keep a constant concentration of the hydroxylamine form in blood and/or tissues rather than the radical form. Therefore, administration in the hydroxylamine form should be better, because of lower toxicity (Zhdanov and Komarov, 1990; Hahn et al., 2000). Of the probes studied in this investigation, optimal choices may be carbamoyl-PROXYL and TEMPOL. However, treatment with radical form of carbamoyl-PROXYL may result in a higher concentration of the radical, which protects not only normal tissues but also cancer and/or tumor tissues. Therefore, TEMPOL and/or TEMPOL-H may be most suitable for in vivo radiation protection.
Using a novel X-band EPR technique, the pharmacokinetics of several nitroxyl spin probes were estimated and the suitability of these agents to probe in vivo redox status and as radiation protectors were described. EPR signal decay mechanisms of spin probes in the blood of living mice are analyzed by experiments and simulations. The one- or two-compartment model analysis is not sufficient in such a case, because the decay mechanisms are complicated. Because in vivo reduction rates predicted by the simulation reflect the oxidation/reduction potential of the spin probe, comparison of in vivo decay rates between different spin probes is difficult by one- or two-compartment model analysis. Considering simulation results, combination of distribution and excretion yields basic biphasic decay under nonsaturated urinary excretion conditions. Redox information could be reflected in the initial phase. The slow phase, especially at later period, depends only on excretion rate, because the reduction and the oxidation are at equilibrium. Based on sensitivity, MTD, and membrane permeability conditions, TEMPOL or amino-TEMPO are better as redox probes in blood, whereas carbamoyl-PROXYL may be more suitable spin probe to investigate tissue redox status by means of EPR imaging. To expect the selective protection against radiation for normal tissues rather than cancer/tumor, reoxidation of the probe in normal tissue is preferable. The hydroxylamine form of TEM-POL is probably a best radioprotector for in vivo use rather than TEMPOL.
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Footnotes |
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ABBREVIATIONS: EPR, electron paramagnetic resonance; TEMPO, 2,2,6,6-tetramethylpiperidine-N-oxyl; TEMPOL, 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl; TEMPONE, 4-oxo-2,2,6,6-tetramethylpiperidine-N-oxyl; amino-TEMPO, 4-amino-2,2,6,6-tetramethylpiperidine-N-oxyl; carboxy-TEMPO, 4-carboxy-2,2,6,6-tetramethylpiperidine-N-oxyl; CAT-1, 4-trimethylammonium-2,2,6,6-tetramethylpiperidine-N-oxyl iodide; carbamoyl-PROXYL, 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl; carboxy-PROXYL, 3-carboxy-2,2,5,5-tetramethylpyrrolidine-N-oxyl; MTD, maximum tolerated dose; O/W, octanol/water; Oxo63, tri[8-carboxy-2,2,6,6-tetrakis(2-hydroxymethyl)benzo[1,2-d:4,5-d']bis(1,3)dithio-4-yl]methyl radical.
Address correspondence to: Dr. Murali C. Krishna, Bldg. 10, Rm B3B69, National Institutes of Health, Bethesda, MD 20892-1002. E-mail: murali{at}helix.nih.gov
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