A R Green

Effect of NXY‐059 on infarct volume after transient or permanent middle cerebral artery occlusion in the rat; studies on dose, plasma concentration and therapeutic time window

The efficacy of the free radical trapping agent NXY‐059 in reducing the infarct volume following both transient and permanent focal ischaemia has been examined in rats. In the transient ischaemia model, rats were subjected to a 2 h occlusion of the middle cerebral artery (MCA). Intravenous infusion of NXY‐059 (1, 10 and 30 mg kg−1 h) for 21.75 h starting 2.25 h after the occlusion, produced a dose‐dependent decrease in both neurological impairment and the histologically measured infarct volume (a mean 59% decrease at 10 mg kg−1 h). In the permanent ischaemia model, animals were injected (s.c.) with a loading dose of NXY‐059 of 32.5, 53.8 or 75.4 mg kg−1 and osmotic minipumps were implanted which had been primed to deliver respectively 30, 50 or 70 mg kg−1 h. When treatment was initiated 5 min after MCA occlusion there was a dose dependent protection of both cortical and sub‐cortical tissue (cortex: 63% at the mid‐range dose). Protection was related linearly to plasma concentration (plasma unbound NXY‐059 concentration at 1 h: 37±16 μmol l−1 at the mid‐range dose). When the mid range dose was administered between 5 min – 4 h after MCA occlusion, a marked and statistically significant protection was seen at all time points (44% protection in cortex at 4 h). These data demonstrate the substantial neuroprotective efficacy of NXY‐059 at plasma concentrations that can be achieved clinically and indicate that NXY‐059 also has a therapeutic window of opportunity that is clinically relevant.

The mechanisms involved in the long-lasting neuroprotective effect of fluoxetine against MDMA (‘ecstasy')-induced degeneration of 5-HT nerve endings in rat brain

It has been reported that co‐administration of fluoxetine with 3,4‐methylenedioxymethamphetamine (MDMA, ‘ecstasy’) prevents MDMA‐induced degeneration of 5‐HT nerve endings in rat brain. The mechanisms involved have now been investigated. MDMA (15 mg kg−1, i.p.) administration produced a neurotoxic loss of 5‐HT and 5‐hydroxyindoleacetic acid (5‐HIAA) in cortex, hippocampus and striatum and a reduction in cortical [3H]‐paroxetine binding 7 days later. Fluoxetine (10 mg kg−1, i.p., ×2, 60 min apart) administered concurrently with MDMA or given 2 and 4 days earlier provided complete protection, and significant protection when given 7 days earlier. Fluvoxamine (15 mg kg−1, i.p., ×2, 60 min apart) only produced neuroprotection when administered concurrently. Fluoxetine (10 mg kg−1, ×2) markedly increased the KD and reduced the Bmax of cortical [3H]‐paroxetine binding 2 and 4 days later. The Bmax was still decreased 7 days later, but the KD was unchanged. [3H]‐Paroxetine binding characteristics were unchanged 24 h after fluvoxamine (15 mg kg−1, ×2). A significant cerebral concentration of fluoxetine plus norfluoxetine was detected over the 7 days following fluoxetine administration. The fluvoxamine concentration had decreased markedly by 24 h. Pretreatment with fluoxetine (10 mg kg−1, ×2) failed to alter cerebral MDMA accumulation compared to saline pretreated controls. Neither fluoxetine or fluvoxamine altered MDMA‐induced acute hyperthermia. These data demonstrate that fluoxetine produces long‐lasting protection against MDMA‐induced neurodegeneration, an effect apparently related to the presence of the drug and its active metabolite inhibiting the 5‐HT transporter. Fluoxetine does not alter the metabolism of MDMA or its rate of cerebral accumulation.

Role of hyperthermia in the protective action of clomethiazole against MDMA ('ecstasy')-induced neurodegeneration, comparison with the novel NMDA channel blocker AR-R15896AR.

The immediate effect of administration of 3,4‐methylenedioxymethamphetamine (MDMA or ‘ecstasy’) on rectal temperature and the effect of putative neuroprotective agents on this change has been examined in rats. The influence of the temperature changes on the long term MDMA‐induced neurodegeneration of cerebral 5‐hydroxytryptamine (5‐HT) nerve terminals was also examined. The novel low affinity N‐methyl‐D‐aspartate (NMDA) receptor channel blocker AR‐R15896AR (20 mg kg−1, i.p.) given 5 min before and 55 min after MDMA (15 mg kg−1, i.p.) did not prevent the MDMA‐induced hyperthermia and did not alter either the MDMA‐induced neurodegenerative loss of 5‐HT and 5‐hydroxyindoleacetic acid (5‐HIAA) in cortex, striatum and hippocampus or loss of [3H]‐paroxetine binding in cortex 7 days later. The neuroprotective agent clomethiazole (50 mg kg−1, i.p.) given 5 min before and 55 min after MDMA (15 mg kg−1) abolished the MDMA‐induced hyperthermic response and markedly attenuated the loss of 5‐HT, 5‐HIAA and [3H]‐paroxetine binding in the brain regions examined 7 days later. When rats treated with MDMA plus clomethiazole were kept at high ambient temperature for 5 h post‐MDMA, thereby keeping their body temperature elevated to near that seen in rats given MDMA alone, the MDMA‐induced loss of 5‐HT, 5‐HIAA and [3H]‐paroxetine was still attenuated. However, the protection (39%) afforded by the clomethiazole administration was less than seen in rats kept at normal ambient temperature (75%). These data support the proposals of others that NMDA receptor antagonists are neuroprotective against MDMA‐induced degeneration only if they induce hypothermia and further suggest that increased glutamate activity may not be involved in the neurotoxic action of MDMA. These data further demonstrate that a proportion of the neuroprotective action of clomethiazole is due to an effect on body temperature but that, in addition, the compound protects against MDMA‐induced damage by an unrelated mechanism.

Clomethiazole is neuroprotective in models of global and focal cerebral ischemia when infused at doses producing clinically relevant plasma concentrations

We investigated the neuroprotective effect of infusing various doses of clomethiazole in models of global and focal cerebral ischemia. In a model of global ischemia, gerbils were infused with clomethiazole (intravenous), attaining steady state plasma concentrations of between 1 and 13 microM for 24 h. In a transient middle cerebral artery occlusion model in rats, clomethiazole was administered subcutaneously over 22.75 h using osmotic minipumps producing steady state plasma concentrations of between 1 and 13 microM. Clomethiazole was protective in these models at plasma concentrations of respectively 6.1 microM and above and 3.5 microM and above. Clomethiazole is thus neuroprotective in both global and focal ischemia at plasma concentrations known to be well tolerated in stroke patients.

The use of the Chandler loop to examine the interaction potential of NXY-059 on the thrombolytic properties of rtPA on human thrombi in vitro

Recombinant tissue‐type plasminogen activator (rtPA) is the only globally approved treatment for acute ischaemic stroke. Other potential treatments might be administered with rtPA, making it important to discover whether compounds interfere with rtPA‐induced lysis. We evaluated methods for examining the effect of the neuroprotectant NXY‐059 on the lytic property of rtPA.

Neuroprotective efficacy of AR-A008055, a clomethiazole analogue, in a global model of acute ischaemic stroke and its effect on ischaemia-induced glutamate and GABA efflux in vitro

We have investigated the neuroprotective properties of AR-A008055 [(+/-)-1-(4-methyl-5-thiazolyl-1-phenyl-methylamine], a novel compound structurally related to clomethiazole. Administration (i.p.) of (+/-)-AR-A008055 60 min after 5 min of global cerebral ischaemia in gerbils produced a dose-dependent protection of the hippocampus from damage. Both enantiomers [(R)-(+)-AR-A008055 and (S)-(-)- AR-A008055] at 600 micromol/kg produced similar protection to that following clomethiazole (600& micromol/kg) and both produced similar and sustained neuroprotection, at 4, 7 and 21 days post-insult. When infused intravenously over a 2-h period, both enantiomers produced concentration-dependent neuroprotection, with the enantiomers providing similar protection at every plasma concentration (50-200 nmol/ml). The efficacy of (S)-(-)-AR-A008055 was similar to clomethiazole, but it was slightly less potent. Ischaemia-induced glutamate efflux from rat brain cortical prisms in vitro was inhibited by both isomers (100 microM). The inhibitory effect of (R)-(+)-AR-A008055 was blocked by bicuculline (10 microM) and picrotoxin (100 microM), while the effect of (S)-(-)-AR-A008055 was only antagonised by picrotoxin. This indicated that (S)-(-)-AR-A008055, like clomethiazole, is able to open the GABA(A)-chloride channel in the absence of endogenous GABA. (R)-(+)-AR-A008055 was more potent than (S)-(-)-AR-A008055 in enhancing the concentration of GABA in the medium following 30 min exposure of tissue to the ischaemic conditions, suggesting that it is an effective GABA uptake inhibitor. This action may explain both its effect on glutamate efflux in vitro and its neuroprotective effect in vivo.

The metabolism of clomethiazole in gerbils and the neuroprotective and sedative activity of the metabolites.

A single dose of clomethiazole (600 μmol kg−1 i.p.) has previously been shown to be neuroprotective in the gerbil model of global ischaemia. In gerbils, clomethiazole (600 μmol kg−1) injection produced a rapid appearance (peak within 5 min) of drug in plasma and brain and similar clearance (plasma t1/2: 40 min) from both tissues. The peak brain concentration (226±56 nmol g−1) was 40% higher than plasma. One major metabolite, 5‐(1‐hydroxyethyl‐2‐chloro)‐4‐methylthiazole (NLA‐715) and two minor metabolites 5‐(1‐hydroxyethyl)‐4‐methylthiazole (NLA‐272) and 5‐acetyl‐4‐methylthiazole (NLA‐511) were detected in plasma and brain. Evidence suggested that clomethiazole is metabolized directly to both NLA‐715 and NLA‐272. Injection of NLA‐715, NLA‐272 or NLA‐511 (each at 600 μmol kg−1) produced brain concentrations respectively 2.2, 38 and 92 times greater than seen after clomethiazole (600 μmol kg−1). Clomethiazole (600 μmol kg−1) injected 60 min after a 5 min bilateral carotid artery occlusion in gerbils attenuated the ischaemia‐induced degeneration of the hippocampus by approximately 70%. The metabolites were not neuroprotective at this dose. In mice, clomethiazole (600 μmol kg−1) produced peak plasma and brain concentrations approximately 100% higher than in gerbils, drug concentrations in several brain regions were similar but 35% higher than plasma. Clomethiazole (ED50: 180 μmol kg−1) and NLA‐715 (ED50: 240 μmol kg−1) inhibited spontaneous locomotor activity. The other metabolites were not sedative (ED50 >600 μmol kg−1). These data suggest that the neuroprotective action of clomethiazole results from an action of the parent compound and that NLA‐715 contributes to the sedative activity of the drug.

Nociceptin/orphanin FQ inhibits ischaemia-induced glutamate efflux from rat cerebrocortical slices

Nociceptin/orphanin FQ (NC), the endogenous ligand for the G-protein coupled nociceptin receptor (NCR), has a modulatory role in various physiological processes including neuro-transmitter release. We have examined the effects of NC, the analogues NC(1–13)NH2 and [F/G]NC(1–13)NH2 and the competitive antagonist [Nphe1]NC(1–13)NH2 (Nphe1) on glutamate efflux during an acute simulated ischaemic challenge in rat cerebrocortical slices. The increase in glutamate efflux seen with ischaemia was inhibited by NC (EC50 250 nM). At micromolar concentrations, the analogues were found to have a similar effect on glutamate efflux compared to NC. In all cases, inhibition of glutamate efflux was abolished by Nphe1 (30 μM). These results suggest a neuroprotective action for NC.