Original contribution
Effects of metalloporphyrin catalytic antioxidants in experimental brain ischemia

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Abstract

Reactive oxygen species play a role in the response of brain to ischemia. The effects of metalloporphyrin catalytic antioxidants (AEOL 10113 and AEOL 10150) were examined after murine middle cerebral artery occlusion (MCAO). Ninety minutes after reperfusion from 90 min MCAO in the rat, AEOL 10113, AEOL 10150, or vehicle were given intracerebroventricularly. AEOL 10113 and AEOL 10150 similarly reduced infarct size (35%) and neurologic deficit. AEOL 10113 caused behavioral side effects at twice the neuroprotective dose while AEOL 10150 required a 15-fold increase from the neuroprotective dose to cause behavioral changes. AEOL 10150, given 6 h after 90 min MCAO, reduced total infarct size by 43% without temperature effects. Brain AEOL 10150 elimination t1/2 was 10 h. In the mouse, intravenous AEOL 10150 infusion post-MCAO reduced both infarct size (25%) and neurologic deficit. Brain AEOL 10150 uptake, greater in the ischemic hemisphere, was dose- and time-dependent. AEOL 10150 had direct effects on proteomic events and ameliorated changes caused by ischemia. In primary mixed neuronal/glial cultures exposed to 2 h of O2/glucose deprivation, AEOL 10150 reduced lactate dehydrogenase release dose-dependently and selectively preserved aconitase activity in concentrations consistent with neuroprotection in vivo. AEOL 10150 is an effective neuroprotective compound offering a wide therapeutic window with a large margin of safety against adverse behavioral side effects.

Introduction

Reactive oxygen and nitrogen species play critical roles in the response of brain to an ischemic insult [1]. Renewed interest in therapeutic application of antioxidants has been derived from three independent sources of information. First, hydroxyl radical production persists for at least several hours after onset of ischemia in rodent models, suggesting the potential for a clinically relevant therapeutic window [2], [3]. This is consistent with the observation that superoxide (radical dotO2) production is sustained after reperfusion from middle cerebral artery occlusion (MCAO) [4], [5]. Second, investigations with transgenic mice have demonstrated consistent improvement of histologic/neurologic outcome from either focal or global ischemia when overexpression of either copper/zinc (CuZn-SOD), manganese (Mn-SOD) [6] or extracellular (EC-SOD) superoxide dismutase is achieved [7], [8], [9]. Conversely, targeted 2deletion of the Mn-SOD [10], CuZn-SOD [11], or EC-SOD isozymes [12] worsens ischemic outcome. Third, pharmacologic advances in synthesis of catalytic antioxidants have led to evaluation of such molecules in a variety of stroke models [13], [14], [15], [16]. Efficacy in reducing infarct size and neurologic deficit, despite a substantial delay in administration after onset of acute brain injury, has raised hope that compounds possessing this mechanism of action may find clinical utility.

A metalloporphyrin catalytic antioxidant has recently been shown to be effective in the modification of temporary focal ischemia in rodent models [16]. This metalloporphyrin, AEOL 10113 [17], [18], is limited by the fact that it is composed of several stereoisomers that make a pharmaceutically acceptable formulation difficult and severely limit the ability to assess tissue levels and carry out pharmacokinetic studies. AEOL 10150 is a structurally different metalloporphyrin catalytic antioxidant than AEOL 10113, which possesses imidizole side chain substitutions (Fig. 1). AEOL 10150 provides a nearly 2-fold increase in SOD potency compared to AEOL 10113 and reduces the IC50 for in vitro lipid peroxidation by 50% (Table 1). In contrast to AEOL 10113, AEOL 10150 does not exist as a stereoisomer, is relatively easy to synthesize, and can be accurately and simply analyzed in tissues [19]. We hypothesized that AEOL 10150 would yield neuroprotection equivalent to AEOL 10113 at doses markedly less than those required to produce neurotoxicity in a rat model of focal cerebral ischemia. The work was extended to define intravenous (i.v.) AEOL 10150 efficacy in a mouse MCAO model and to perform pharmacokinetic assessment of the transfer of AEOL 10150 from blood to brain under conditions of ischemia/reperfusion. We then sought to define interactions between AEOL 10150 and the proteomic response of brain to focal ischemia and probe mechanisms for the protection observed in primary mixed neuronal/glial cultures exposed to oxygen/glucose deprivation (OGD). We hypothesized that AEOL 10150 would reduce changes in protein expression attributable to ischemia, reduce neuronal cell death induced by oxidative stress in vitro, and preserve aconitase activity as a marker of superoxide mediated cell injury.

Section snippets

Materials and methods

The following studies were approved by the Duke University Animal Care and Use Committee.

Experiment one (relative behavioral neurotoxicity of AEOL 10113 vs. AEOL 10150)

For AEOL 10113, an i.c.v. dose of 600 ng was found to cause just noticeable behavioral side effects. For AEOL 10150, 9 μg was found to produce just noticeable behavioral effects. Thus, toxicity from AEOL 10150 was 1/15th that of AEOL 10113. We, therefore, studied an AEOL 10150 dose of 300 ng to provide direct comparison to AEOL 10113 and an AEOL 10150 dose of 4500 ng as a maximal dose clearly devoid of adverse behavioral effects.

Experiment two (rat MCAO/i.c.v. AEOL 10113 vs. AEOL 10150)

Physiologic values are given in Table 2. There were no

Discussion

The principal findings of this series of studies were as follows. Intracerebroventricular AEOL 10150 had 1/15th the potency of AEOL 10113 for inducing adverse neurobehavioral side effects in the rat. AEOL 10150 and AEOL 10113 had similar potency to reduce cerebral infarct size and neurologic deficit when given i.c.v. 90 min after reperfusion from MCAO. When given 6 h after reperfusion, AEOL 10150 reduced cerebral infarct volume by 43%. AEOL 10150 also reduced infarct size and neurologic deficit

Acknowledgements

This work was supported by U.S. Public Health Service grants R01 NS38944-03, U10 HL 63397, PO1 HL 31992, RO1 NS39587 and Incara Pharmaceuticals Corp., Research Triangle Park, NC, USA. Drs. Crapo, Day, and Warner hold an equity position in and serve as consultants for Incara, Inc. The authors are grateful to Ann D. Brinkhous for expert technical assistance.

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