Original Contributions
Evaluation of the hydroxylamine tempol-H as an in vivo radioprotector

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Abstract

Nitroxides are stable free radical compounds that protect against the toxicity of reactive oxygen species in vitro and in vivo. Tempol (Aldrich, Milwaukee, WI, USA) is a cell-permeable hydrophilic nitroxide and has been shown to be an in vitro and in vivo radioprotector. The limitations of Tempol as a systemic radioprotector are that it causes substantial reductions in arterial blood pressure when administered intravenously and is associated with seizure activity. Furthermore, Tempol is rapidly reduced to its hydroxylamine form, Tempol-H, which limits the period of time the active form of the nitroxide is available for radioprotection. Based on initial pharmacological and blood pressure experiments performed in mice, we hypothesized that the systemic administration of Tempol-H in vivo would lead to an equilibration between Tempol and Tempol-H that would limit the toxicity of the nitroxide and provide in vivo radioprotection. Tempol-H was administered in increasing doses via an intraperitoneal route to C3H mice. The maximally tolerated dose was found to be 325 mg/kg. The whole-blood pharmacology of Tempol-H was investigated with electron paramagnetic resonance spectroscopy. These studies demonstrated the appearance of Tempol in whole blood immediately after intraperitoneal injection, suggesting that rapid oxidation of Tempol-H to Tempol takes place in vivo. Although the peak concentration of Tempol in whole blood after administration of Tempol-H did not reach the same levels as those observed when Tempol is administered, the whole-blood levels of Tempol were similar by 10 min after injection. Tempol-H provided protection against the lethality of whole-body radiation in C3H mice at 30 d with a dose modification factor of 1.3, which is similar to the results obtained with Tempol. Hemodynamic measurements in C3H mice after intravenous injection showed that Tempol-H produced little effect on blood pressure or pulse compared with Tempol. Tempol-H is a systemic in vivo radioprotector of C3H mice and is associated with less hemodynamic toxicity than Tempol.

Introduction

The nitroxides are stable free radical compounds that have been used as spin labels for biophysical studies and have been investigated as contrast agents in magnetic resonance imaging [1], [2]. These compounds also possess antioxidant activity, protecting V79 cells against the cytotoxicity of hydrogen peroxide [3] and tert-butyl hydroperoxide [4]. Several mechanisms may account for the antioxidant activity of nitroxides, including the oxidation of reduced transition metals [3], superoxide dismutase–like activity [5], and the scavenging of other free radicals [6], [7], [8]. The nitroxides also have been shown to provide in vitro [9], [10] and in vivo radioprotection [11], [12]. One water-soluble nitroxide, Tempol, protected V79 cells against radiation in a concentration-dependent fashion. It was also shown that the reduced form of Tempol, Tempol-H (the hydroxylamine), did not provide aerobic radioprotection [9]. Other nitroxides have also been shown to provide in vitro radioprotection [10].

It has been demonstrated that Tempol protects C3H mice against the lethality of whole-body radiation at 30 d [11]. In this study, the maximally tolerated dose of Tempol was administered intraperitoneally (ip) to mice 5 to 10 min before whole-body radiation. The LD50/30 dose (the dose of radiation that causes 50% lethality 30 d after radiation) was 9.97 Gy in the Tempol-treated mice and 7.84 Gy in the saline-treated mice. The dose modification factor (DMF) (i.e., the LD50/30 dose in the Tempol-treated group divided by the LD50/30 dose in the control group) was found to be 1.3. Subsequent experiments have identified other in vivo nitroxide radioprotectors [12].

The importance of nitroxide in vivo radioprotection was enhanced by studies that evaluated the differential protection of normal tissue (bone marrow) compared to that of tumor tissue [13]. Using growth delay in the radiation-induced fibrosarcoma (RIF-1) tumor, no significant difference in tumor control with radiation was observed between Tempol-treated and saline-treated mice [13]. Full x-ray dose-response experiments showed a TCD50/30 value of 37 Gy for Tempol-treated mice and 41 Gy for saline-treated mice [13]. The mechanism of differential radioprotection seems to be the differential bioreduction of Tempol to the hydroxylamine, which occurs to a greater extent in tumor than in bonemarrow [13], [14].

One obstacle to the development of the nitroxides as systemic radioprotectors is their toxicity when administered at high concentrations. Tempol and other nitroxides produce restlessness, agitation, and seizure activity when administered at levels that approach the maximally tolerated dose [11], [12]. Although some of the nitroxides may have direct neurotoxic effects [15], the toxicity seems to temporally correlate with the induction of hypotension and reflex tachycardia in mice [12]. The hemodynamic effects of the nitroxides may reflect their individual catalytic rate constants for superoxide dismutation, because some compounds such as Tempol produce substantial hypotension, although others have minimal effects. The dismutation of superoxide may alter the balance between superoxide and nitric oxide, leading to vasodilation [16].

The investigation of the hemodynamic effects of the nitroxides [12] led to an evaluation of Tempol-H. In this report, we describe the maximally tolerated dose, pharmacology, whole-body radioprotection, and hemodynamic effects of Tempol-H in C3H mice.

Section snippets

Chemicals

Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) was purchased from Aldrich (Milwaukee, WI, USA) and prepared in phosphate-buffered saline (PBS), pH 7.4 (Biofluids, Rockville, MD, USA). Tempol-H was prepared by hydrogenation in the presence of a platinum catalyst (yield > 90%) characterized by nuclear magnetic resonance, mass spectrum, and electron paramagnetic resonance (EPR). The product was found to contain less than 0.2% of the starting material, Tempol, as an impurity. Stock

Toxicity and determination of the maximally tolerated dose of Tempol-H

Tempol-H was administered ip to C3H mice. At doses up to 325 mg/kg, no lethality was observed (Fig. 1). Approximately 70% survival was observed at the 350 mg/kg dose level. Toxicity similar to that described with Tempol [11], including restlessness and seizure activity, was observed at the higher doses of Tempol-H. Based on these studies, the maximally tolerated dose of Tempol-H was determined to be 325 mg/kg and was used for subsequent whole-body radiation studies.

Whole-blood pharmacology of Tempol-H

Samples of whole blood from

Discussion

The development of radioprotectors is dependent on several factors. First, preclinical studies must show that compounds under development are in vivo radioprotectors. Second, the compounds must be associated with acceptable toxicity in preclinical studies at the doses that are required for radioprotection. Third, some therapeutic gain must be associated with the administration of the compound. In an ideal situation, this means that normal tissue protection can be achieved under conditions where

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