J. Martin Elliott

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.

A study of the mechanisms involved in the neurotoxic action of 3,4-methylenedioxymethamphetamine (MDMA, 'ecstasy') on dopamine neurones in mouse brain

Administration of 3,4‐methylenedioxymethamphetamine (MDMA, ‘ecstasy’) to mice produces acute hyperthermia and long‐term degeneration of striatal dopamine nerve terminals. Attenuation of the hyperthermia decreases the neurodegeneration. We have investigated the mechanisms involved in producing the neurotoxic loss of striatal dopamine. MDMA produced a dose‐dependent loss in striatal dopamine concentration 7 days later with 3 doses of 25 mg kg−1 (3 h apart) producing a 70% loss. Pretreatment 30 min before each MDMA dose with either of the N‐methyl‐D‐aspartate antagonists AR‐R15896AR (20, 5, 5 mg kg−1) or MK‐801 (0.5 mg kg−1×3) failed to provide neuroprotection. Pretreatment with clomethiazole (50 mg kg−1×3) was similarly ineffective in protecting against MDMA‐induced dopamine loss. The free radical trapping compound PBN (150 mg kg−1×3) was neuroprotective, but it proved impossible to separate neuroprotection from a hypothermic effect on body temperature. Pretreatment with the nitric oxide synthase (NOS) inhibitor 7‐NI (50 mg kg−1×3) produced neuroprotection, but also significant hypothermia. Two other NOS inhibitors, S‐methyl‐L‐thiocitrulline (10 mg kg−1×3) and AR‐R17477AR (5 mg kg−1×3), provided significant neuroprotection and had little effect on MDMA‐induced hyperthermia. MDMA (20 mg kg−1) increased 2,3‐dihydroxybenzoic acid formation from salicylic acid perfused through a microdialysis tube implanted in the striatum, indicating increased free radical formation. This increase was prevented by AR‐R17477AR administration. Since AR‐R17477AR was also found to have no radical trapping activity this result suggests that MDMA‐induced neurotoxicity results from MDMA or dopamine metabolites producing radicals that combine with NO to form tissue‐damaging peroxynitrites.

Studies on the effect of MDMA (‘ecstasy') on the body temperature of rats housed at different ambient room temperatures

3,4‐Methylenedioxymethamphetamine (MDMA, ‘ecstasy’) administration to rats produces hyperthermia if they are housed in normal or warm ambient room temperature (Ta) conditions (20°C), but hypothermia when in cool conditions (Ta17°C). We have now investigated some of the mechanisms involved. MDMA (5 mg kg−1 i.p.) produced a rapid decrease in rectal temperature in rats at Ta 15°C. This response was blocked by pretreatment with the dopamine D2 receptor antagonist remoxipride (10 mg kg−1 i.p.), but unaltered by pretreatment with the D1 antagonist SCH23390 (1.1 mg kg−1 i.p.). MDMA (5 mg kg−1) did not alter the tail temperature of rats at Ta 15°C, but decreased the tail temperature of rats at Ta 30°C. A neurotoxic dose of MDMA (three doses of 5 mg kg−1 given 3 h apart) decreased cortical and hippocampal 5‐HT content by approximately 30% 7 days later. This lesion did not influence the rise in tail temperature when rats were moved from Ta 20°C to 30°C compared to nonlesioned controls, but did result in a lower tail temperature than that of controls when they were returned to Ta 24°C. Acute administration of MDMA (5 mg kg−1) to MDMA‐lesioned rats produced a sustained decrease in tail temperature in rats housed at Ta 30°C compared to nonlesioned controls. These data suggest that the thermoregulatory problems previously observed in MDMA‐lesioned rats housed at Ta 30°C result, partially, from their inability to lose heat by vasodilation of the tail, a major heat‐loss organ in this species.

Effect of repeated ('binge') dosing of MDMA to rats housed at normal and high temperature on neurotoxic damage to cerebral 5-HT and dopamine neurones.

The technique of ‘binge’ dosing (several doses in one session) by recreational users of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) requires evaluation in terms of its consequences on the acute hyperthermic response and long-term neurotoxicity. We examined the neurotoxic effects of this dosing schedule on 5-HT and dopamineneurones in the rat brain. When repeated (three) doses of MDMA (2, 4 and 6 mg/kg i.p.) were given 3 h apart to rats housed at 19 °C, a dose-dependent acute hyperthermia and long-term loss of 5-HT was observed in several brain regions (hippocampus, cortex and striatum), with an approximate 50% loss following 3 × 4mg/kg and 65% decrease following 3 × 6mg/kg. No decrease in striatal dopamine content was detected. When MDMA (4 mg/kg i.p.) was given repeatedly to rats housed at 30 °C, a larger acute hyperthermic response than that observed in rats treated at 19 °C environment was seen (maximum response 2.6 ± 0.1 °C versus 1.3 ± 0.2 °C). A long-term cerebral 5-HT loss of approximately 65% was also detected in both the cortex and hippocampus, but no loss in striatal dopamine content occurred. These data emphasize the increased acute hyperthermic response and neurotoxicity which occurs when MDMA is administered in a hot room environment compared to normal room temperature conditions, and support the view that MDMA is a selective 5-HT neurotoxin, even when a binge dosing schedule is employed and the rats are present in a hot environment.

The role of 5-HT in the impairment of thermoregulation observed in rats administered MDMA ('ecstasy') when housed at high ambient temperature.

RationaleAdministration to rats of a neurotoxic dose of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) produces an impairment in thermoregulation which is reflected in a prolonged hyperthermic response to a subsequent dose of MDMA given to rats housed at high ambient temperature.ObjectiveWe wished to examine whether the impaired thermoregulation was associated with decreased cerebral 5-HT content produced by the prior neurotoxic dose of MDMA.MethodsRats were injected with drugs decreasing 5-HT function [the tryptophan hydroxlase inhibitor p-chlorophenylalanine (PCPA), and 5-HT receptor antagonists] and rectal temperature was measured after administering MDMA to rats housed at 30°C.ResultsPCPA pretreatment decreased 5-HT and 5-HIAA concentrations in cortex, hippocampus and striatum by >80% and prolonged the hyperthermia induced in rats housed at 30°C by administering MDMA (5 mg/kg i.p.). A similar prolongation of the hyperthermic response to MDMA was seen when rats were pretreated with methysergide (10 mg/kg i.p.) or the 5-HT1A antagonist WAY100635 (0.5 mg/kg s.c.).ConclusionsDecreasing 5-HT function in diverse ways enhanced the hyperthermic response to MDMA given to rats housed at high ambient temperature. This suggests that loss of 5-HT acting on 5-HT1A receptors leads to impaired thermoregulation in rats and suggests that the impairment seen in MDMA pretreated rats housed at high ambient temperature is due to a loss in 5-HT function. These data could have implications for recreational users of MDMA, who may have damaged serotoninergic neurons because of prior heavy or frequent use of the drug, when taking further doses of MDMA in hot environments such as dance clubs.

Hyperthermic and neurotoxic effect of 3,4-methylenedioxymethamphetamine (MDMA) in guinea pigs

The report (Ricaurte et al. 2002) that frequent repeat dosing of primates with MDMA produced damage to dopaminergic neurones, in addition to the expected damage to serotonin nerve endings, has recently been withdrawn as a drug supply error had resulted in the animals being dosed with methamphetamine by mistake (Ricaurte et al. 2003). Consequently, the view that MDMA is a selective serotonin neurotoxin has been reinforced. Essentially all studies of the neurotoxic effect of MDMA in rats have failed to detect damage to dopamine neurones (see Green et al. 2003) and all studies on primates (both monkeys and humans) have similarly failed to demonstrate neurotoxic loss of dopamine or dopaminergic markers (see Green et al. 2003). However, administration of MDMA to mice does result in neurotoxic damage to dopamine nerve endings (O’Shea et al. 2001), with little evidence for serotonin loss except in selenium deficient animals (Sanchez et al. 2003). It still, therefore, appears possible that MDMA might be capable of producing dopaminergic damage in other species, which would weaken the notion that it is primarily a selective neurotoxin of serotoninergic nerve endings. We have now briefly examined whether MDMA induces long-term dopamine loss in another small experimental animal, the guinea pig. There appear to have only been two studies performed on the effect of MDMA in guinea pigs. Commins et al. (1987) gave two doses of MDMA (20 mg/kg) daily for 4 days and reported a major loss in striatal 5-HT content (71%) 14 days later and, crucially, a statistically significant loss (24%) of dopamine content. Battaglia et al (1988) confirmed the long-term MDMA-induced striatal 5-HT loss, but did not measure the dopamine concentration. Neither study examined whether there was an acute hyperthermic response following MDMA administration to guinea pigs. However, in rats there is general agreement that the induction of hyperthermia is a major, but not necessarily essential, factor in the degree of subsequent neurotoxic damage (Broening et al. 1995; Malberg and Seiden 1998; O’Shea et al. 1998). We have now examined in guinea pigs both the acute hyperthermic response following MDMA administration and the striatal 5-HT and dopamine concentration 1 week following two different dosing regimes of MDMA. Male guinea pigs (Hall, Burton on Trent, UK), weighing 350–425 g at the start of the experiment, were injected IP with either saline or MDMA using two different dose schedules (group I: 20 mg/kg 2 during 1 day; group II: 20 mg/kg twice daily for 3 days). Injections were given at 10.00 and 16.00 hours. Rectal temperature was measured in lightly restrained animals by use of a rectal probe with digital readout over 8 h on the first day (all groups) and for 4 h following the fifth injection (day 3) in saline-injected and group II. Seven days following the final injection, all groups were killed and striatal 5-HT and dopamine were measured by HPLC with electrochemical detection (see O’Shea et al. 2001). The first injection of MDMA produced an acute hyperthermic response (Fig. 1), but the second dose failed to produce an increase in rectal temperature and there was no significant increase in rectal temperature following the fifth dose (Fig. 1, insert). One week after MDMA administration there was a major (72%) loss in striatal 5-HT and 5-HIAA concentration, and this loss was similar whether the rats were given two or six doses of MDMA. Neither dose regime resulted in a neurotoxic loss of striatal dopamine content, K. S. Saadat · A. R. Green Neuropharmacology Research Group, School of Pharmacy, De Montfort University, The Gateway, Leicester, LE1 9BH, UK

Monoamine reuptake inhibition and mood-enhancing potential of a specified oregano extract

A healthy, balanced diet is essential for both physical and mental well-being. Such a diet must include an adequate intake of micronutrients, essential fatty acids, amino acids and antioxidants. The monoamine neurotransmitters, serotonin, dopamine and noradrenaline, are derived from dietary amino acids and are involved in the modulation of mood, anxiety, cognition, sleep regulation and appetite. The capacity of nutritional interventions to elevate brain monoamine concentrations and, as a consequence, with the potential for mood enhancement, has not been extensively evaluated. The present study investigated an extract from oregano leaves, with a specified range of active constituents, identified via an unbiased, high-throughput screening programme. The oregano extract was demonstrated to inhibit the reuptake and degradation of the monoamine neurotransmitters in a dose-dependent manner, and microdialysis experiments in rats revealed an elevation of extracellular serotonin levels in the brain. Furthermore, following administration of oregano extract, behavioural responses were observed in mice that parallel the beneficial effects exhibited by monoamine-enhancing compounds when used in human subjects. In conclusion, these data show that an extract prepared from leaves of oregano, a major constituent of the Mediterranean diet, is brain-active, with moderate triple reuptake inhibitory activity, and exhibits positive behavioural effects in animal models. We postulate that such an extract may be effective in enhancing mental well-being in humans.

Parallel changes in serotonin levels in brain and blood following acute administration of MDMA

Recent studies have demonstrated a similar acute effect of 3,4- methylenedioxymethamphetamine (MDMA) in blood platelets and brain tissue via action on the serotonin transporter. To investigate the validity of blood serotonin as a peripheral marker for central serotonin in this regard, we administered MDMA (20 mg/kg i.p.) to rats and observed a parallel decrease in serotonin levels in the frontal cortex and blood at 2 h (63% and 46% respectively) with some recovery evident at 8 h (42% and 38%) and more so at 18 h (19% and 24% below control levels). Administration of a tryptophan supplement (82.5 mg/kg p.o.) to naïve rats produced parallel increases in serotonin levels 2 h later in the frontal cortex (39%) and blood (26%). Following MDMA administration, the same dose of tryptophan caused a smaller (26%) rise in brain serotonin whereas in blood it had no effect. We conclude that blood serotonin is a useful marker for brain serotonin levels in the rat following acute administration of MDMA and this finding highlights the possible use of platelet serotonin as a marker for brain serotonin in human studies involving MDMA.

Paradigmatic identification of MMP-2 and MT1-MMP activation systems in cardiac fibroblasts cultured as a monolayer.

Activations of MMP‐2 and membrane type 1‐matrix metalloproteinase (MT1‐MMP) have been correlated with cell migration, a key cellular event in the wound healing and tissue remodeling. We have previously demonstrated furin‐dependent MMP‐2 and MT1‐MMP activations induced by type I collagen in cardiac fibroblasts. To understand mechanistic aspects of the regulation of MMP‐2 and MT1‐MMP activations by potential non‐matrix factor(s) in cardiac fibroblasts, in the present study, we examined the effects of various agents including concanavalin A (ConA), a proteolytic phenotype‐producing agent. We showed that treatment of cells with ConA activated pro‐MMP‐2, and that this activation concurred with elevated levels of cellular MT1‐MMP and TIMP‐2. The presence of active MT1‐MMP and 43 and 36 kDa processed forms of MT1‐MMP in a fraction of intracellular proteins prepared from ConA‐treated cells suggests the possible internalization of differential forms of MT1‐MMP. The appearance of 36 kDa processed form of MT1‐MMP in conditioned media prepared from ConA‐treated cells indicates the possible extracellular release of the further processed MT1‐MMP fragment. Inhibition of furin in ConA‐treated cells attenuated pro‐MT1‐MMP processing and the cellular TIMP‐2 level, plus it reduced cell‐released active MMP‐2 in a time‐dependent manner. These results suggest the involvement of furin in the ConA‐induced activations of MT1‐MMP and MMP‐2. Furthermore, the existence of furin inhibitor‐insensitive pro‐ and active MMP‐2 species associated with ConA‐treated cells implies that a mechanism independent of furin may perhaps account for the binding of the MMP‐2 species to the cells. Supplementary material for this article can be found at http://www.mrw.interscience.wiley.com/suppmat/0730‐2312/suppmat/94/suppmat_guo.tif.