Lubeluzole protects hippocampal neurons from excitotoxicity in vitro and reduces brain damage caused by ischemia

Abstract

Previously reported effects of lubeluzole, such as inhibition of glutamate release, inhibition of nitric oxide (NO) synthesis and blockage of voltage-gated Na⁺- and Ca²⁺-ion channels, suggest a neuroprotective action of this drug. Here we report about the effects of lubeluzole and its R-isomer on glutamate-induced neuronal cell death in mixed hippocampal cultures. In addition, we studied the effect of lubeluzole in focal cerebral ischemia models in mice and rats. In hippocampal cultures exposed to 500 nM glutamate for 1 h, lubeluzole (0.1–100 nM), but not the R-isomer (1–100 nM), reduced the percentage of damaged neurons from 42±8% to 18±7% (P<0.01). In mice and rats, lubeluzole reduced ischemic brain damage, when administered immediately after middle cerebral artery occlusion. Interestingly, the protective effect (reduction of the infarct volume in rats to 77% of control; P<0.01) was also found when the lubeluzole treatment (2.5 mg/kg) was started 3 h after ischemia. Especially this latter effect suggests that lubeluzole will be a useful drug for stroke therapy.

Introduction

Various biochemical events that occur as a result of a dramatically reduced supply of oxygen and glucose have been proposed to play a role in the development of cerebral infarction after ischemic insults (for references see Krieglstein and Oberpichler-Schwenk, 1994; Krieglstein, 1996). Many experimental studies in vitro and in vivo have demonstrated the neuroprotective potential of agents that interact with Ca²⁺-channels, inhibit the release of excitatory amino acids or block their receptors or reduce the generation of free radicals or the synthesis of NO (O'Neill et al., 1995; Krieglstein, 1996). However, only a few of the experimentally investigated neuroprotective compounds were proven to be beneficial in clinical stroke trials and many of the effective compounds had severe side effects (Marshall and Mohr, 1993; Krieglstein, 1996; Lees and Anzal, 1996).

Lubeluzole, the S-isomer of a novel benzothiazole derivative, was investigated in clinical trials for the treatment of ischemic stroke. Previously reported effects of lubeluzole, such as inhibition of glutamate release, inhibition of glutamate-activated NO synthesis and blockage of voltage-gated Na⁺- and Ca²⁺-ion channels, suggest that it has neuroprotective potential (De Ryck et al., 1994, De Ryck et al., 1995, De Ryck et al., 1996; Scheller et al., 1995; Lesage et al., 1996). For the R-isomer of lubeluzole or the racemate the described effects were either moderate or not evident. The compound improved the neurological outcome in a model of photochemically induced thrombotic cerebral infarction in rats when administered within 6 h after the onset of ischemia, whereas the R-isomer of lubeluzole was inactive (De Ryck et al., 1994, De Ryck et al., 1996). However, to our knowledge there is no evidence, so far, that this improvement in neurological outcome by lubeluzole administered 3–6 h after the onset of the ischemic insult corresponds to a reduction in damage to brain tissue. In contrast, in a rat model of transient focal ischemia, lubeluzole reduced the infarct volume only when treatment was started immediately after the onset of ischemia (Aronowski et al., 1996a, Aronowski et al., 1996b). A recent phase II clinical trial involving 232 patients with acute cerebral infarction showed that mortality could be reduced by intravenous infusion of lubeluzole (10 mg/d), whereas the higher dose (20 mg/d) was found to increase mortality although neurological variables were improved (Diener et al., 1996). Thus, it is of interest to further study acute and delayed administration of lubeluzole in experimental models of ischemia to provide support for the promising results obtained with lubeluzole in clinical stroke trials.

The aim of this study was to investigate the neuroprotective potency of lubeluzole and its R-isomer against l-glutamate-induced excitotoxicity in primary mixed hippocampal cultures containing astrocytes and neurons. Furthermore, we tested whether lubeluzole could protect brain tissue from ischemic damage when administered immediately and 3–4 h after permanent middle cerebral artery occlusion.