Esther O'Shea

The Pharmacology and Clinical Pharmacology of 3,4-Methylenedioxymethamphetamine (MDMA, “Ecstasy”)

The amphetamine derivative (±)-3,4-methylenedioxymethamphetamine (MDMA, ecstasy) is a popular recreational drug among young people, particularly those involved in the dance culture. MDMA produces an acute, rapid enhancement in the release of both serotonin (5-HT) and dopamine from nerve endings in the brains of experimental animals. It produces increased locomotor activity and the serotonin behavioral syndrome in rats. Crucially, it produces dose-dependent hyperthermia that is potentially fatal in rodents, primates, and humans. Some recovery of 5-HT stores can be seen within 24 h of MDMA administration. However, cerebral 5-HT concentrations then decline due to specific neurotoxic damage to 5-HT nerve endings in the forebrain. This neurodegeneration, which has been demonstrated both biochemically and histologically, lasts for months in rats and years in primates. In general, other neurotransmitters appear unaffected. In contrast, MDMA produces a selective long-term loss of dopamine nerve endings in mice. Studies on the mechanisms involved in the neurotoxicity in both rats and mice implicate the formation of tissue-damaging free radicals. Increased free radical formation may result from the further breakdown of MDMA metabolic products. Evidence for the occurrence of MDMA-induced neurotoxic damage in human users remains equivocal, although some biochemical and functional data suggest that damage may occur in the brains of heavy users. There is also some evidence for long-term physiological and psychological changes occurring in human recreational users. However, such evidence is complicated by the lack of knowledge of doses ingested and the fact that many subjects studied are or have been poly-drug users.

The pharmacology of the acute hyperthermic response that follows administration of 3,4-methylenedioxymethamphetamine (MDMA, 'ecstasy') to rats

The pharmacology of the acute hyperthermia that follows 3,4‐methylenedioxymethamphetamine (MDMA, ‘ecstasy’) administration to rats has been investigated. MDMA (12.5 mg kg−1 i.p.) produced acute hyperthermia (measured rectally). The tail skin temperature did not increase, suggesting that MDMA may impair heat dissipation. Pretreatment with the 5‐HT1/2 antagonist methysergide (10 mg kg−1), the 5‐HT2A antagonist MDL 100,907 (0.1 mg kg−1) or the 5‐HT2C antagonist SB 242084 (3 mg kg−1) failed to alter the hyperthermia. The 5‐HT2 antagonist ritanserin (1 mg kg−1) was without effect, but MDL 11,939 (5 mg kg−1) blocked the hyperthermia, possibly because of activity at non‐serotonergic receptors. The 5‐HT uptake inhibitor zimeldine (10 mg kg−1) had no effect on MDMA‐induced hyperthermia. The uptake inhibitor fluoxetine (10 mg kg−1) markedly attenuated the MDMA‐induced increase in hippocampal extracellular 5‐HT, also without altering hyperthermia. The dopamine D2 antagonist remoxipride (10 mg kg−1) did not alter MDMA‐induced hyperthermia, but the D1 antagonist SCH 23390 (0.3 – 2.0 mg kg−1) dose‐dependently antagonized it. The dopamine uptake inhibitor GBR 12909 (10 mg kg−1) did not alter the hyperthermic response and microdialysis demonstrated that it did not inhibit MDMA‐induced striatal dopamine release. These results demonstrate that in vivo MDMA‐induced 5‐HT release is inhibited by 5‐HT uptake inhibitors, but MDMA‐induced dopamine release may not be altered by a dopamine uptake inhibitor. It is suggested that MDMA‐induced hyperthermia results not from MDMA‐induced 5‐HT release, but rather from the increased release of dopamine that acts at D1 receptors. This has implications for the clinical treatment of MDMA‐induced hyperthermia.

In vivo evidence for free radical involvement in the degeneration of rat brain 5‐HT following administration of MDMA (‘ecstasy’) and p‐chloroamphetamine but not the degeneration following fenfluramine

Administration of 3,4‐methylenedioxymethamphetamine (MDMA or ‘ecstasy’) to several species results in a long lasting neurotoxic degeneration of 5‐hydroxytryptaminergic neurones in several regions of the brain. We have now investigated whether this degeneration is likely to be the result of free radical‐induced damage. Free radical formation can be assessed by measuring the formation of 2,3‐ and 2,5‐dihydroxybenzoic acid (2,3‐DHBA and 2,5‐DHBA) from salicylic acid. An existing method involving implantation of a probe into the hippocampus and in vivo microdialysis was modified and validated. Administration of MDMA (15 mg kg−1, i.p.) to Dark Agouti (DA) rats increased the formation of 2,3‐DHBA (but not 2,5‐DHBA) for at least 6 h. Seven days after this dose of MDMA, the concentration of 5‐hydroxytryptamine (5‐HT) and 5‐hydroxyindoleacetic acid (5‐HIAA) was reduced by over 50% in hippocampus, cortex and striatum, reflecting neurotoxic damage. There was no change in the concentration of dopamine or 3,4‐dihydroxyphenylacetic acid (DOPAC) in the striatum. p‐Chloroamphetamine (PCA), another compound which produces a neurotoxic loss of cerebral 5‐HT content, when given at a dose of 5 mg kg−1 also significantly increased the formation of 2,3‐DHBA (but not 2,5‐DHBA) in the dialysate for over 4.5 h. post‐injection starting 2 h after treatment. In contrast, fenfluramine administration (15 mg kg−1, i.p.) failed to increase the 2,3‐DHBA or 2,5‐DHBA concentration in the dialysate. A single fenfluramine injection nevertheless also markedly decreased the concentration of 5‐HT and 5‐HIAA in the hippocampus, cortex and striatum seven days later. When rats pretreated with fenfluramine (15 mg kg−1, i.p.) seven days earlier were given MDMA (15 mg kg−1, i.p.) no increase in 2,3‐DHBA was seen in the dialysate from the hippocampal probe. This indicates that the increase in free radical formation following MDMA is occurring in 5‐HT neurones which have been damaged by the prior fenfluramine injection. Administration of the free radical scavenging agent α‐phenyl‐N‐tert‐butyl nitrone (PBN; 120 mg kg−1, i.p.) 10 min before and 120 min after an MDMA (15 mg kg−1, i.p.) injection prevented the acute rise in the 2,3‐DHBA concentration in the dialysate and attenuated by 30% the long term damage to hippocampal 5‐HT neurones (as indicated by a smaller MDMA‐induced decrease in both the concentration of 5‐HT and 5‐HIAA and also the binding of [3H]‐paroxetine). These data indicate that a major mechanism by which MDMA and PCA induce damage to 5‐hydroxytryptaminergic neurones in rat brain is by increasing the formation of free radicals. These probably result from the degradation of catechol and quinone metabolites of these substituted amphetamines. In contrast, fenfluramine induces damage by another mechanism not involving free radicals; a proposal supported by some of our earlier indirect studies. We suggest that these different modes of action render untenable the recent suggestion that MDMA will not be neurotoxic in humans because fenfluramine appears safe at clinical doses.

A study of the neurotoxic effect of MDMA ('ecstasy') on 5-HT neurones in the brains of mothers and neonates following administration of the drug during pregnancy

It is well established that 3,4‐methylenedioxymethamphetamine (MDMA or ‘ecstasy’) is neurotoxic and produces long term degeneration of cerebral 5‐hydroxytryptamine (5‐HT) nerve terminals in many species. Since MDMA is used extensively as a recreational drug by young people, it is being ingested by many women of child bearing age. We have therefore examined the effect of administering high doses of MDMA to rats during pregnancy on the cerebral content of both the dams and the neonates. MDMA (20 mg kg−1, s.c.) was injected twice daily on days 14–17 of the gestation period. The initial dose produced a marked hyperthermic response in the dam which was progressively attenuated in both peak height and area under the curve following further doses of the drug. The body weight of the dams decreased during the period of treatment. There was a modest decrease in litter size (−20%) of the MDMA‐treated dams. The concentration of 5‐HT and its metabolite 5‐HIAA was decreased by over 65% in the hippocampus and striatum and 40% in the cortex of the dams 1 week after parturition. In contrast, the content of 5‐HT and 5‐HIAA in the dorsal telencephalon of the pups of the MDMA‐treated dams was the same as that seen in tissue from pups born to control animals. Administration of MDMA (40 mg kg−1, s.c.) to adult rats increased thiobarbituric acid reacting substances (TBARS) in cortical tissue 3 h and 6 h later, indicating increased lipid peroxidation. No increase in TBARS was seen in the cortical tissue of 7–10 day neonates injected with this dose of MDMA 3 h or 6 h earlier. The data suggest that exposure to MDMA in utero during the maturation phase does not produce damage to 5‐HT nerve terminals in the foetal rat brain, in contrast to the damage seen in the brains of the mothers. This may be due to MDMA being metabolized to free radical producing entities in the adult brain but not in the immature brain or, alternatively, to more effective or more active free radical scavenging mechanisms being present in the immature brain.

Effect of GBR 12909 and fluoxetine on the acute and long term changes induced by MDMA ('ecstasy') on the 5-HT and dopamine concentrations in mouse brain

We examined the long term effect of 3,4 methylenedioxymethamphetamine (MDMA, 10, 20 and 30 mg/kg, i.p.) on the cerebral 5-hydroxytryptamine (5-HT) and dopamine content in Swiss Webster mice. Three injections of MDMA (20 or 30 mg/kg, i.p.) given 3 h apart produced a marked depletion in the striatal content of dopamine and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) 7 days later. None of the doses administered altered the concentration of 5-HT or its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in several brain areas. Pre-treatment with the dopamine uptake inhibitor GBR 12909 (10 mg/kg, i.p.), 30 min before each of the three MDMA (30 mg/kg, i.p.) injections, completely prevented the long term loss in the striatal catechol concentrations. However, GBR 12909 (10 mg/kg, i.p.) not only failed to prevent the acute effects induced by MDMA (30 mg/kg x 3, i.p.) on dopamine metabolism 30 min later, but in fact potentiated them. The 5-HT uptake inhibitor, fluoxetine (10 mg/kg, i. p.) failed to prevent both the acute and long term dopaminergic deficits. MDMA (30 mg/kg x 3) altered the body temperature of the mice biphasically, producing a rapid hyperthermia followed by prolonged hypothermia. In contrast, MDMA (20 mg/kg x 3) produced an initial hypothermia followed by hyperthermia. The present experiments therefore appear to rule out any direct relationship between the neurotoxic effects of MDMA and its acute effects on body temperature in mice. Fluoxetine administered 30 min before each MDMA (30 mg/kg) injection prevented these temperature changes, while GBR 12909 was without effect. This suggests that the neuroprotective effect of GBR 12909 against MDMA-induced neurotoxicity is not directly related to its ability to inhibit the MDMA-induced acute effects on dopamine metabolism or alter the MDMA-induced temperature change. The data illustrate major differences in the neurotoxic profile of MDMA in mice and rats.

Studies on the role of dopamine in the degeneration of 5‐HT nerve endings in the brain of Dark Agouti rats following 3,4‐methylenedioxymethamphetamine (MDMA or ‘ecstasy’) administration

We investigated whether dopamine plays a role in the neurodegeneration of 5‐hydroxytryptamine (5‐HT) nerve endings occurring in Dark Agouti rat brain after 3,4‐methylenedioxymethamphetamine (MDMA or ‘ecstasy’) administration. Haloperidol (2 mg kg−1 i.p.) injected 5 min prior and 55 min post MDMA (15 mg kg−1 i.p.) abolished the acute MDMA‐induced hyperthermia and attenuated the neurotoxic loss of 5‐HT 7 days later. When the rectal temperature of MDMA+haloperidol treated rats was kept elevated, this protective effect was marginal. MDMA (15 mg kg−1) increased the dopamine concentration in the dialysate from a striatal microdialysis probe by 800%. L‐DOPA (25 mg kg−1 i.p., plus benserazide, 6.25 mg kg−1 i.p.) injected 2 h after MDMA (15 mg kg−1) enhanced the increase in dopamine in the dialysate, but subsequent neurodegeneration was unaltered. L‐DOPA (25 mg kg−1) injected before a sub‐toxic dose of MDMA (5 mg kg−1) failed to induce neurodegeneration. The MDMA‐induced increase in free radical formation in the hippocampus (indicated by increased 2,3‐ and 2,5‐dihydroxybenzoic acid in a microdialysis probe perfused with salicylic acid) was unaltered by L‐DOPA. The neuroprotective drug clomethiazole (50 mg kg−1 i.p.) did not influence the MDMA‐induced increase in extracellular dopamine. These data suggest that previous observations on the protective effect of haloperidol and potentiating effect of L‐DOPA on MDMA‐induced neurodegeneration may have resulted from effects on MDMA‐induced hyperthermia. The increased extracellular dopamine concentration following MDMA may result from effects of MDMA on dopamine re‐uptake, monoamine oxidase and 5‐HT release rather than an ‘amphetamine‐like’ action on dopamine release, thus explaining why the drug does not induce degeneration of dopamine nerve endings.

Persistent MDMA-induced dopaminergic neurotoxicity in the striatum and substantia nigra of mice

Acute administration of repeated doses of 3,4‐methylenedioxymethamphetamine (MDMA, ecstasy) dramatically reduces striatal dopamine (DA) content, tyrosine hydroxylase (TH), and DA transporter‐immunoreactivity in mice. In this study, we show for the first time the spatiotemporal pattern of dopaminergic damage and related molecular events produced by MDMA administration in mice. Our results include the novel finding that MDMA produces a significant decrease in the number of TH‐immunoreactive neurons in the substantia nigra (SN). This decrease appears 1 day after injection, remains stable for at least 30 days, and is accompanied by a dose‐dependent long‐lasting decrease in TH‐ and DA transporter‐immunoreactivity in the striatum, which peaked 1 day after treatment and persisted for at least 30 days, however, some recovery was evident from day 3 onwards, evidencing sprouting of TH fibers. No change is observed in the NAc indicating that MDMA causes selective destruction of DA‐containing neurons in the nigrostriatal pathway, sparing the mesolimbic pathway. The expression of Mac‐1 increased 1 day after MDMA treatment and glial fibrillary acidic protein increased 3 days post‐treatment in the striatum and SN but not in the NAc, in strict anatomical correlation with dopaminergic damage. These data provide the first evidence that MDMA causes persistent loss of dopaminergic cell bodies in the SN.

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.

Studies, using in vivo microdialysis, on the effect of the dopamine uptake inhibitor GBR 12909 on 3,4-methylenedioxymethamphetamine ('ecstasy')-induced dopamine release and free radical formation in the mouse striatum

The present study examined the mechanisms by which 3,4‐methylenedioxymethamphetamine (MDMA) produces long‐term neurotoxicity of striatal dopamine neurones in mice and the protective action of the dopamine uptake inhibitor GBR 12909. MDMA (30 mg/kg, i.p.), given three times at 3‐h intervals, produced a rapid increase in striatal dopamine release measured by in vivo microdialysis (maximum increase to 380 ± 64% of baseline). This increase was enhanced to 576 ± 109% of baseline by GBR 12909 (10 mg/kg, i.p.) administered 30 min before each dose of MDMA, supporting the contention that MDMA enters the terminal by diffusion and not via the dopamine uptake site. This, in addition to the fact that perfusion of the probe with a low Ca2+ medium inhibited the MDMA‐induced increase in extracellular dopamine, indicates that the neurotransmitter may be released by a Ca2+‐dependent mechanism not related to the dopamine transporter. MDMA (30 mg/kg × 3) increased the formation of 2,3‐dihydroxybenzoic acid (2,3‐DHBA) from salicylic acid perfused through a probe implanted in the striatum, indicating that MDMA increased free radical formation. GBR 12909 pre‐treatment attenuated the MDMA‐induced increase in 2,3‐DHBA formation by approximately 50%, but had no significant intrinsic radical trapping activity. MDMA administration increased lipid peroxidation in striatal synaptosomes, an effect reduced by approximately 60% by GBR 12909 pre‐treatment. GBR 12909 did not modify the MDMA‐induced changes in body temperature. These data suggest that MDMA‐induced toxicity of dopamine neurones in mice results from free radical formation which in turn induces an oxidative stress process. The data also indicate that the free radical formation is probably not associated with the MDMA‐induced dopamine release and that MDMA does not induce dopamine release via an action at the dopamine transporter.

3,4‐Methylenedioxymethamphetamine increases interleukin‐1β levels and activates microglia in rat brain: studies on the relationship with acute hyperthermia and 5‐HT depletion

3,4‐Methylenedioxymethamphetamine (MDMA) administration to rats produces acute hyperthermia and 5‐HT release. Interleukin‐1β (IL‐1β) is a pro‐inflammatory pyrogen produced by activated microglia in the brain. We examined the effect of a neurotoxic dose of MDMA on IL‐1β concentration and glial activation and their relationship with acute hyperthermia and 5‐HT depletion. MDMA, given to rats housed at 22°C, increased IL‐1β levels in hypothalamus and cortex from 1 to 6 h and [3H]‐(1‐(2‐chlorophenyl)‐N‐methyl‐N‐(1‐methylpropyl)3‐isoquinolinecarboxamide) binding between 3 and 48 h. Increased immunoreactivity to OX‐42 was also detected. Rats became hyperthermic immediately after MDMA and up to at least 12 h later. The IL‐1 receptor antagonist did not modify MDMA‐induced hyperthermia indicating that IL‐1β release is a consequence, not the cause, of the rise in body temperature. When MDMA was given to rats housed at 4°C, hyperthermia was abolished and the IL‐1β increase significantly reduced. The MDMA‐induced acute 5‐HT depletion was prevented by fluoxetine coadministration but the IL‐1β increase and hyperthermia were unaffected. Therefore, the rise in IL‐1β is not related to the acute 5‐HT release but is linked to the hyperthermia. Contrary to IL‐1β levels, microglial activation is not significantly modified when hyperthermia is prevented, suggesting that it might be a process not dependent on the hyperthermic response induced by MDMA.