A.J. Cross

Review of the pharmacology and clinical pharmacology of 3,4-methylenedioxymethamphetamine (MDMA or “Ecstasy”)

Abstract3,4-Methylenedioxymethamphetamine (MDMA or “Ecstasy”) was first synthesised 80 years ago, but has recently received prominence as an illegally synthesised recreational drug of abuse. There is a widely held belief among misusers that it is safe. In the last 2–3 years there have been a number of reports of the drug producing severe acute toxicity and death and there are concerns that it may cause long term toxic damage to 5-hydroxytryptamine (5-HT) nerve terminals. There is a considerable literature on the acute pharmacological effects of MDMA in experimental animals, and this is reviewed. The drug produces both hyperthermia and the “serotonin syndrome”, a series of behavioural changes which result from increased 5-HT function. Acute clinical toxicity problems following MDMA ingestion also include hyperthermia and the appearance of the serotonin syndrome. The hyperthermia appears to precipitate other severe clinical problems and the outcome can be fatal. In agreement with others, we suggest that the recent increase in the number of reports of MDMA toxicity probably results from the widespread use of the drug at all night dance parties or “raves”. The phenomenon of amphetamine aggregation toxicity in mice was reported 40 years ago. If applicable to MDMA-induced toxicity in humans, all the conditions necessary to induce or enhance toxicity are present at raves: crowded conditions (aggregation), high ambient temperature, loud noise and dehydrated subjects. Administration of MDMA to rodents and non-human primates results in a long term neurotoxic decrease in 5-HT content in several brain regions and there is clear biochemical and histological evidence that this reflects neurodegeneration of 5-HT terminals. Unequivocal data demonstrating that similar changes occur in human brain do not exist, but limited and indirect clinical evidence gives grounds for concern. There are also data suggesting that long term psychiatric changes can occur, although there are problems of interpretation and these are reviewed. Suggestions for the rational treatment of the acute toxicity are made on the basis of both pharmacological studies in animals and current clinical practice. Cases presenting clinically are usually emergencies and unlikely to allow carefully controlled studies. Proposals include decreasing body temperature (possibly with ice), the use of dantrolene and anticonvulsant and sedative medication, particularly benzodiazepines. The use of neuroleptics requires care because of the theoretical risk of producing the neuroleptic malignant syndrome and the possibility of precipitating seizures. In rats, chlormethiazole antagonises the hyperthermia produced by MDMA and has been shown clinically to block MDMA-induced convulsive activity.

Animal models of acute ischaemic stroke: can they predict clinically successful neuroprotective drugs?

Substantial efforts are being made to develop drugs which will protect the brain from the neurodegeneration that follows an acute ischaemic stroke. However, while there are already a significant number of animal models of stroke, there is currently no information as to whether activity of a compound in any of them will predict clinical efficacy. In this article, Jackie Hunter, Richard Green and Alan Cross review the major models of acute cerebral ischaemia and propose rational protocols for examining novel neuroprotective agents.

Neuroprotective activity of chlormethiazole following transient forebrain ischaemia in the gerbil

1 The effect of chlormethiazole, and other drugs which potentiate γ‐aminobutyric acid (GABA) function on delayed neuronal death in the hippocampus has been examined in the gerbil. 2 Chlormethiazole (100 mg kg−1, i.p.) and two other drugs previously reported to be neuroprotective (dizocilpine, 3 mg kg−1, i.p. and ifenprodil, 4 mg kg−1, i.p.) were all found to prevent neurodegeneration of CA1/CA2 neurones in the hippocampus when given 30 min before a 5 min episode of bilateral carotid artery occlusion. 3 Chlormethiazole (100 mg kg−1) was neuroprotective when given up to 3 h, after the ischaemic episode. 4 Given 1 h after the cartoid artery occlusion, chlormethiazole produced significant protection against hippocampal neurodegeneration at a dose of 50 mg kg−1, but not at 25 mg kg−1. 5 Phenobarbitone (100 mg kg−1, i.p.) and Saffan (alphaxalone, 45 mg kg−1plus alphadalone, 15 mg kg−1, i.p.) were not protective when given 1 h after the ischaemic episode while pentobarbitone (30 mg kg−1, i.p.) had a modest protective effect. 6 Evidence is presented to show that neither the operating procedure nor the chlormethiazole administration lowered rectal or cerebral temperature. 7 The data suggest that chlormethiazole may be a useful treatment in the prevention of neurodegeneration following stroke or cardiac arrest.

The effects of chlormethiazole and nimodipine on cortical infarct area after focal cerebral ischaemia in the rat

Focal ischaemia in the rat cerebral cortex was produced by means of a photochemically induced thrombosis of cerebral arteries. This was achieved by intravenous infusion of the photosensitive dye Rose Bengal and illumination of the skull with focused green light. Initial experiments justified the use of tetrazolium staining as an index of infarct damage. Using this technique it was demonstrated that chlormethiazole (200 mg/kg, i.p.) given 5 min post ischaemia markedly reduced the area of infarcted cortical tissue. A second experiment replicated this observation and showed that, in contrast, nimodipine (0.5 mg/kg, i.p.) given 5 min post infarct was without effect on infarct size. The pattern of Evans Blue extravasation indicated that the infarct developed over a 24-h period with the major damage occurring in the first 4.5 h. The spread of the infarct beyond the initial core of damage was decreased by an estimated value of almost 50% by injection of chlormethiazole (200 mg/kg, i.p.) 5 min after the light exposure. These data indicate that chlormethiazole is an effective drug in protecting against the effects of focal ischaemia in the rat and, taken with earlier observations that chlormethiazole protects against the effects of global ischaemia in the gerbil, suggest that the drug may be an effective treatment against the ischaemic cell death that can occur following a stroke or cardiac arrest.

A comparative study in rats of the in vitro and in vivo pharmacology of the acetylcholinesterase inhibitors tacrine, donepezil and NXX-066

The in vitro and in vivo effects of the novel acetylcholinesterase inhibitors donepezil and NXX-066 have been compared to tacrine. Using purified acetylcholinesterase from electric eel both tacrine and donepezil were shown to be reversible mixed type inhibitors, binding to a similar site on the enzyme. In contrast, NXX-066 was an irreversible non-competitive inhibitor. All three compounds were potent inhibitors of rat brain acetylcholinesterase (IC50 [nM]; tacrine: 125 +/- 23; NXX-066: 148 +/- 15; donepezil: 33 +/- 12). Tacrine was also a potent butyrylcholinesterase inhibitor. Donepezil and tacrine displaced [3H]pirenzepine binding in rat brain homogenates (IC50 values [microM]; tacrine: 0.7; donepezil: 0.5) but NXX-066 was around 80 times less potent at this M1-muscarinic site. Studies of carbachol stimulated increases in [Ca2+]i in neuroblastoma cells demonstrated that both donepezil and tacrine were M1 antagonists. Ligand binding suggested little activity of likely pharmacological significance with any of the drugs at other neurotransmitter sites. Intraperitoneal administration of the compounds to rats produced dose dependent increases in salivation and tremor (ED50 [micromol/kg]; tacrine: 15, NXX-066: 35, donepezil: 6) with NXX-066 having the most sustained effect on tremor. Following oral administration, NXX-066 had the slowest onset but the greatest duration of action. The relative potency also changed, tacrine having low potency (ED50 [micromol/kg]; tacrine: 200, NXX-066: 30, donepezil: 50). Salivation was severe only in tacrine treated animals. Using in vivo microdialysis in cerebral cortex, both NXX-066 and tacrine were found to produce a marked (at least 30-fold) increase in extracellular acetylcholine which remained elevated for more than 2 h after tacrine and 4 h after NXX-066.

The neurotoxic effects of methamphetamine on 5-hydroxytryptamine and dopamine in brain: Evidence for the protective effect of chlormethiazole

Studies were undertaken in mice and rats on the neurotoxic effects of methamphetamine on dopaminergic and 5-hydroxytryptaminergic neurones in the brain and the neuroprotective action of chlormethiazole. In initial studies, mice were injected with methamphetamine (5 mg/kg, i.p.) at 2 hr intervals, to a total of 4 times. This procedure produced a 66% loss of striatal dopamine and a 50% loss of tyrosine hydroxylase activity 3 days later. Chlormethiazole (50 mg/kg, i.p.), given 15 min before each dose of methamphetamine, totally prevented the methamphetamine-induced loss of tyrosine hydroxylase activity and partly prevented the loss of dopamine. Phencyclidine (20 mg/kg, i.p.), given in place of chlormethiazole, also prevented the loss of tyrosine hydroxylase. Administration to rats of 4 doses of methamphetamine (15 mg/kg, i.p.) at 3 hr intervals resulted in a 75% loss of striatal dopamine 3 days later and a similar loss of 5-HT and 5-HIAA in cortex and hippocampus. Chlormethiazole (50 mg/kg, i.p.), given 15 min before each injection of methamphetamine, protected against the loss of dopamine and indoleamine content, in the respective regions. Pentobarbital (25 mg/kg, i.p.) also provided substantial protection but diazepam (2.5 mg/kg, i.p.) was without effect. Confirming earlier studies, dizocilpine (1 mg/kg) also provided substantial protection against the methamphetamine-induced neurotoxicity. Preliminary data indicated that chlormethiazole was not neuroprotective because of a hypothermic action. These data therefore demonstrate that chlormethiazole is an effective neuroprotective agent against methamphetamine-induced neurotoxicity and extend the evidence for the possible value of this drug in preventing neurodegeneration.

The cholinergic pharmacology of tetrahydroaminoacridine in vivo and in vitro

1 The effect of tetrahydroaminoacridine (THA) on cholinergically mediated behaviour in the rat and mouse has been investigated. In addition the actions of this compound on cholinesterase activity and on muscarinic and nicotinic receptors has also been examined. 2 Administration of THA (5–20 mg kg−1, i.p.) produced a dose‐dependent increase in tremor, hypothermia and salivation in both rats and mice. A similar profile of activity was seen following physostigmine (0.1‐0.6 mg kg−1) administration. 3 THA was approximately fifty fold less potent than physostigmine in inducing behavioural change but its effects persisted for over twice as long as those of physostigmine. For example THA‐induced hypothermia was still present at 4 h in the mouse and 8 h in the rat. 4 In vitro THA was a potent non‐competitive inhibitor of rat brain cholinesterase (IC50: 57 + 6 nM) and bovine erythrocyte acetylcholinesterase (IC50: 50 + 10 nM) but was a more potent inhibitor of horse serum butyrylcholinesterase (IC50: 7.2 + 1.4 nM). 5 Radioligand binding studies indicated that THA binds non‐selectively but with moderate potency to both M1 (Ki: 600 nM) and M2 (Ki: 880 nM) muscarinic receptors. THA also interacted with the allosteric site present on cardiac M2 receptors. 6 It is concluded that THA is a reversible non‐competitive inhibitor of cholinesterase with a long half life (compared with physostigmine). It also may antagonize muscarinic receptors at high doses. The long half life may account for its reported efficacy in the treatment of Alzheimers disease.

The modulation by chlormethiazole of the GABAA‐receptor complex in rat brain

1 The interactions of chlormethiazole with γ‐aminobutyric acid (GABA) synthesis and release, and with ligand binding to sites associated with the GABAA‐receptor complex and the GABAB‐receptor have been studied in the rat. The GABAA‐receptor was studied using [3H]‐muscimol, [3H]‐flunitrazepam was used to label the benzodiazepine modulatory site, and [35S]‐butylbicyclophosphorothionate ([35S]‐TBPS) to label the chloride channel. 2 Chlormethiazole had no effect on GABA synthesis in the cortex, hippocampus and striatum or on GABA release from cortical slices in vitro. Chlormethiazole did not displace [3H]‐baclofen binding to the GABAB‐receptor. 3 Chlormethiazole (IC50 = 140 μm) and pentobarbitone (IC50 = 95 μm) both inhibited [35S]‐TBPS binding by increasing the rate of [35S]‐TBPS dissociation. In addition, chlormethiazole caused an apparent decrease in the affinity of [35S]‐TBPS binding. 4 Chlormethiazole enhanced the binding of [3H]‐muscimol but had no effect on [3H]‐flunitrazepam binding. In contrast, the sedative barbiturate pentobarbitone enhanced both [3H]‐muscimol and [3H]‐flunitrazepam binding. 5 It is concluded that the sedative and anticonvulsant effects of chlormethiazole are probably mediated through an action at the GABAA‐receptor. However, chlormethiazole does not interact with the GABAA‐receptor complex in an identical manner to the sedative barbiturate pentobarbitone.

The effect of chlormethiazole on neuronal damage in a model of transient focal ischaemia

1 The effect of chlormethiazole has been studied in a transient middle cerebral artery (MCA) occlusion model of cerebral ischaemia in the rat. The MCA was occluded for 1 h by use of an intraluminal suture technique, with reperfusion for 24 h following removal of the occluding filament. Neuronal damage was determined by measurement of the area of necrosis following Cresyl Violet staining of sections taken through the ischaemic region. 2 In the initial experiment, occlusion of the MCA produced a large volume of ischaemic damage in both cortex and striatum, characterized by necrosis and pyknosis (total volume of damage, 287 ± 13 mm3; n = 9). Rats injected with chlormethiazole (1000 μmol kg−1, i.p.) 60 min before occlusion had a reduced volume of damage in both regions (104 ± 11 mm3; n = 9; P < 0.001). 3 In a subsequent study systemic physiological parameters (heart rate, blood pressure, blood pH, blood gases and rectal temperature) were measured throughout the ischaemic period. 4 Chlormethiazole (10000 μmol kg−1) pretreatment produced little change in systemic physiology and the neuroprotective effect of the drug when given 60 min prior to the MCA occlusion was confirmed. Chlormethiazole was also neuroprotective when given 10 min following the start of reperfusion (control group: 244 ± 52 mm3, n = 10; chlormethiazole pretreatment group: 102 ± 23 mm3, n − 10; P < 0.001; chlormethiazole post‐ischaemia group: 122 ± 16mm3; P<0.001, n= 10). 5 It is concluded that chlormethiazole is an effective neuroprotective agent in this model of transient focal ischaemia. The observation that chlormethiazole is protective when given after reperfusion indicates that the effect of the drug is unlikely to be due to an alteration of intra‐ischaemic cerebral blood flow, but is more probably a direct effect on the development of ischaemic damage.

Effect of chlormethiazole, dizocilpine and pentobarbital on harmaline-induced increase of cerebellar cyclic GMP and tremor

Administration to mice of harmaline (100 mg/kg SC) resulted in a greater than two-fold increase in cyclic GMP in the cerebellum 15 min later. This response was inhibited by pretreatment 5 min before the harmaline with pentobarbital (ED50 6.5 mg/kg), chlormethiazole (ED50 10.4 mg/kg) and dizocilpine (ED50 0.5 mg/kg). Harmaline-induced tremor was inhibited by pentobarbital (ED50 30 mg/kg) and chlormethiazole (ED50 50 mg/kg) but not dizocilpine. The data demonstrate that the harmaline-induced tremor and cerebellar cyclic GMP rise are probably not associated. They also demonstrate that chlormethiazole is able to inhibit a biochemical response (the increase in cerebellar cyclic GMP) which results from increased glutamate function.