Mechanisms of Action
MDMA interacts with plasma monoamine transporters and storage vesicles to increase extracellular levels of 5-HT, dopamine, and norepinephrine (Cozzi et al. 1999; Fitzgerald and Reid 1990; Gudelsky and Nash 1996; Hiramatsu and Cho 1990; Kankaanpaa et al. 1998; Nash and Brodkin 1991; Rudnick and Wall 1992; Schuldiner et al. 1993). Direct MDMA stimulation of postsynaptic 5-HT2A receptors and !-2 adrenoceptors also contributes to MDMA's effects. For example, dopamine release is also indirectly increased by MDMA stimulation of 5-HT2A receptors on GABAergic striatonigral neurons (Gudelsky and Nash 1996; Koch and Galloway 1997; Palfreyman et al. 1993; Schmidt et al. 1992; Yamamoto et al. 1995).
Although the specific mechanisms of MDMA's therapeutic effects are not fully understood, several observations and hypotheses can be made. Increased extracellular levels of dopamine and norepinephrine are known to be important to the reinforcing effects of psychostimulants (Ritz and Kuhar 1993; Rothman et al. 2001; Wise and Bozarth 1985). These neurotransmitters likely play a similar role with MDMA, producing feelings of excitement, euphoria, and well-being. When the D2 receptor antagonist haloperidol was combined with MDMA, human volunteers reported less positive mood and greater anxiety (Liechti and Vollenweider 2000a), findings in keeping with these hypotheses. Central dopamine and norepinephrine are also thought to regulate readiness for action and arousal, with dopamine possibly mediating behavioral readiness, and locus coeruleus norepinephrine mediating conscious registration of external stimuli (Clark et al. 1987; Robbins and Everitt 2000). Increasing these neurotransmitters may therefore place the individual in a state of alertness that is ideal for recalling or even re-experiencing state-dependent memories of stressful events. This potentially aversive state may be modified by MDMA effects on both the serotonergic system and postsynaptic !-2 adrenoceptors.
MDMA effects on the serotonergic system are likely important for its therapeutic effects. MDMA induces 5-HT release and is a 5-HT2 agonist. Serotonergic dysfunction is associated with anxiety, aggression, and depression. Increasing 5-HT release is thought to have opposite effects. For example, stimulation of 5-HT1A and 5-HT1B receptors decreases anxiety and aggression in rodent behavioral studies (Brunner and Hen 1997; Graeff et al. 1996) and likely contributes to reduced defensiveness and increased self-confidence reported after MDMA. 5- HT1A receptors in the hippocampus have also been specifically hypothesized to enable disengaging from previously learned associations if they lead to adverse outcomes (Guimaraes et al. 1993). MDMA also has moderate 5-HT2A activity (Nash et al. 1994), which leads to modest alterations in perception of meaning (Liechti et al. 2000b), possibly facilitating new ways of thinking. Case reports suggest increasing extracellular 5-HT levels may facilitate recovery of remote memories (Robertson 1997), a phenomenon that has been reported by psychotherapists administering MDMA to patients (Downing 1985). Studies in humans suggest that serotonergic activity plays an important role in generating the subjective effects of MDMA, since co- administration of a serotonin uptake inhibitor reduces most subjective effects (Liechti et al. 2000a; Tancer and Johanson 2004). Thus, MDMA effects on the serotonergic system may decrease anxiety and aggression and create a state of mind that is conducive to psychotherapy. Direct MDMA stimulation of postsynaptic !-2 adrenoceptors may modify this state by altering the balance of !-1 and !-2 stimulation, allowing the individual to remain emotionally calm despite noradrenergic activation. MDMA is an !-2 agonist (Lavelle et al. 1999). Like other !-2 agonists, such as guanfacine and clonidine (Arnsten 1998), MDMA produces feelings of calmness and relaxation (Cami et al. 2000). It is worth noting that open label trials suggest that clonidine may be helpful for treating symptoms of PTSD (Harmon and Riggs 1996; Kinzie and Leung 1989), indicating that !-adrenergic action may possess anxiolytic effects in humans.
Drug Activity Related to Proposed Action
MDMA has a unique profile of psychopharmacological effects making it well suited to intensive psychotherapy. In the context of psychotherapy, MDMA has been noted to reduce defenses and fear of emotional injury while enhancing communication and capacity for introspection (Greer and Tolbert 1998; Grinspoon and Bakalar 1986). Placebo-controlled clinical trials have confirmed that MDMA produces an easily-controlled intoxication characterized by euphoria, increased well being, sociability, self-confidence, and extroversion (Cami et al. 2000; Harris et al. 2002; Hernandez-Lopez et al. 2002; Liechti et al. 2000a; Liechti et al. 2001a; Liechti et al. 2000b; Liechti and Vollenweider 2000a; Tancer and Johanson 2001; Tancer and Johanson 2003; Vollenweider et al. 1998). These effects make it likely that MDMA would be useful in psychotherapeutic treatment of many different complaints.
Therapists conducting MDMA-assisted psychotherapy sometimes administered a smaller dose of MDMA approximately two to three hours after the first administration to prolong the session (Greer and Tolbert 1986; Stolaroff 1988). The supplemental dose of MDMA was usually smaller than the initial dose, and use of the supplemental dose was tolerated. Controlled, double-blind studies of repeated dosing with MDMA have used two equally large doses, either spaced four hours apart (Pacifici et al. 2001) or one day apart (Farre et al. 2004; Pacifici et al. 2001). To date, only immunological findings have been reported for the study with the schedule of repeated doses that most closely resembles the proposed study (Pacifici et al. 2001).
Past reports of psychotherapeutic use of MDMA in people with terminal illnesses suggested that MDMA in combination with psychotherapy might assist in reduction of cancer diagnosis anxiety in people with advanced stage cancer. Therapists made use of the reduced anxiety in relation to emotionally upsetting thoughts or memories, the greater accessibility of deeply emotional material and the increased acceptance of self and others in helping people deal with anxiety and impending death (Holland 2001; Greer and Tolbert 1998; Stevens 1997; Stevens 1999; Stevens 2000). Narrative accounts of MDMA-assisted psychotherapy also point to increased analgesia, strengthened communication with loved ones, and less guilt or distress on noting deteriorating health.
People with advanced stage cancer may face a reduction in quality of life arising from anxiety and distress stemming from the diagnosis, lack of control over life events and increasing need to rely on others. Anxiety may be amenable to treatment with conventional anxiolytics, and people may decide to avoid anxiolytics because of their side effects. MDMA-assisted psychotherapy offers another means of reducing anxiety and increasing quality of life to people with advanced stage cancer by administering MDMA in a setting that enhances and augments subjective effects assumed to be therapeutic, such as increased recall of deeply emotional material and reduced anxiety. These effects are probably mediated partially or wholly through release and inhibition of serotonin, norepinephrine and dopamine uptake, and through direct or indirect action on monoamine neurotransmitters. It is also possible that changes in neurohormone release, such as increased release of arginine vasopressin (Forsling et al. 2001) may be involved in producing some of these therapeutic effects.
The psychotherapeutic effects of MDMA are accompanied by dose-dependent physiological effects including vasoconstriction and increased heart rate and blood pressure (Lester et al. 2000; Liechti et al. 2001; Mas et al. 1999; Tancer and Johanson 2001, and see pp 44-48 of IND #63,384). Physiological effects of MDMA reach their maximum within 1 and 2 hrs after oral MDMA administration and have largely subsided within 6 hrs of drug administration (Gamma et al. 2000; Mas et al. 1999; Tancer and Johanson 2003; Vollenweider et al. 1998, see also Baggott et al. 2001). Data on maximum changes in heart rate and blood pressure collected from human studies published or in preparation in mid-2001 are summarized in Table 3.1 in the Investigators' Brochure. Data from more recent reports (e.g. Farre et al. 2004; Hernandez-Lopez et al. 2002; Lamers et al. 2003; Tancer and Johanson 2003) are similar to data collected in previous reports. Pre-treatment a serotonin uptake inhibitor attenuated or prevented elevations in blood pressure and heart rate (Liechti and Vollenweider 2000), and the 5HT2A receptor antagonist ketanserin reduced elevated diastolic pressure (Liechti et al. 2000a), while the D2 antagonist haloperidol failed to attenuate any of the cardiovascular effects of MDMA (Liechti et al. 2000b). These findings suggest that cardiovascular effects are at least partially due to serotonergic activity. When given in controlled settings, MDMA produced only slight increases in body temperature (Harris et al. 2002; Lester et al. 2000; Liechti et al. 2000b; Tancer and Johanson 2003), with the increase undetected in a number of studies (Farre et al. 2004; de la Torre et al. 2000; Liechti et al. 2000a).
On the basis of data from human studies of physiological effects, an initial dose of 25 mg is expected to have a minimal impact on blood pressure, heart rate, or body temperature, and effects are also expected to be minimal after a total dose of 37.5 mg MDMA, though findings from at least one study suggest that this dose might produce detectable increases in tension and relaxation (Harris et al. 2002). Both the initial dose of 83.3 mg and 125 mg, which will serve as the total dose during the first session and the initial dose on the second session, are similar or identical to the dose used in the study of MDMA-assisted therapy in people with PTSD (IND #63,384). These doses are expected to produce significant increases in blood pressure and heart rate, but are not expected to produce sustained increases in heart rate or blood pressure above 170/100 mm Hg. There is no data from controlled studies on the effects of 187.5 mg MDMA, the total dose for the second experimental session. It is expected that elevation in blood pressure and heart rate may be greater than the elevation seen after 125 mg, but with the increase in blood pressure and heart rate not greatly exceeding the elevation reported after 2.5 mg/kg MDMA. The physiological effects of a second dose of MDMA that is half the original dose and given two and a half hours after the first dose are not yet known. Administering a second dose of 100 mg MDMA a day after an initial 100 mg dose increased systolic blood pressure, diastolic blood pressure and heart rate to levels greater than seen after the initial dose, but not significantly greater.
MDMA dose-dependently and acutely increases cortisol, prolactin, and adrenocortictropic hormone concentrations (Farre et al. 2004; Grob et al. 1996; Grob et al. Unpublished; Harris et al. 2002; Mas et al. 1999; Pacifici et al. 2004; Tancer and Johanson 2003), while growth hormone is unchanged by up to 125 mg MDMA (Mas et al. 1999). Increases in cortisol and prolactin peak at about 2 hours after MDMA administration. A second dose of 100 mg MDMA given four hours after an initial dose of 100 mg produced a second increase in cortisol during an interval when cortisol levels were declining (Pacifici et al. 2001), and a dose of 100 mg MDMA given 24 hours after an initial dose stimulated a greater release of cortisol, but not prolactin (Farre et al. 2004). In a study of the effects of 0.5 and 1.5 mg/kg MDMA in eight people, there was a trend for increased levels of the hormone dehydroepiandrosterone (DHEA) after 0.5 mg/kg MDMA, and a significant increase after 1.5 mg/kg MDMA (Harris et al. 2002), with DHEA levels peaking 2 to 3 h post-drug. Harris and colleagues failed to detect any changes in luteinizing hormone (LH), estradiol, progesterone or follicle stimulating hormone (FSH) in women participants. 40 mg MDMA acutely increased circulating levels of antidiuretic hormone (arginine vasopressin) in eight male volunteers (Forsling et al. 2001; Henry et al. 1998). Antidiuretic hormone reached maximum levels between 1 to 2 hours after MDMA administration. Increased retention of fluid is unlikely to be of any consequences in a clinical setting. Nonetheless, precautions will be taken to avoid dilutional hyponatremia, including providing electrolyte-containing beverages and restrictions on fluid consumption. Studies conducted in Spain suggest that MDMA acutely affects the immune system (Pacifici et al. 1999;; Pacifici et al. 2000; Pacifici et al. 2001; Pacifici et al. 2002; Pacifici et al. 2004). These acute changes in immunologic function include reduced CD4 T-cell count, increased NK cell count, and decreased phytohaemoagglutin A-induced lymphocyte proliferation. MDMA decreased levels of the immune system stimulating and proinflammatory cytokine interleukin 2 (IL-2) and increased levels of the immunosuppressive and anti-inflammatory cytokine interleukin 10 (IL-10) (Pacifici et al. 2004; Pacifici et al. 2001). Generally, MDMA appears to decrease the concentration of Th1 cytokines and increase the amount of Th2 cytokines measured in blood. Immunological changes seen after an initial dose of MDMA are enhanced by a second dose of identical size given four hours after the first dose (Pacifici et al. 2001; Pacifici et al. 2002), and a second dose of identical size given 24 hours after the first dose produced the same immunological effects over the same time course, but with greater intensity than after the first dose (Pacifici et al. 2002). Given this data, it is possible that administering a smaller supplemental dose 2.5 h after the first dose will slightly enhance the immunological effects set in motion by the first dose. These acute changes are unlikely to be of consequence in healthy individuals and are of a similar magnitude to changes produced by other pharmacological agents. For example, the CD4 T-cell count decrease was similar in magnitude to that produced by 0.8 g/kg oral ethanol (the equivalent of 4-5 drinks) in the same report (Pacifici et al. 2001). The mechanism of this MDMA-induced immunomodulation is unclear but may involve MDMA- induced glucocorticoid release or sympathomimetic activity, and activity at alpha adrenergic receptors (Connor et al. 2004). Serotonin release probably plays a role in these changes, since paroxetine pretreatment attenuates and in some cases completely eliminated the immunological effects of MDMA (Pacifici et al. 2004) while only partially reducing elevated cortisol levels after MDMA. Acute alterations in immune functioning after MDMA administration have also been noted in mice (House et al. 1995) and rats (Connor et al. 1998; Connor et al. 2000a; Connor et al. 2000b; Connor et al. 2004). This immunomodulation is an acute effect of MDMA and is not likely to persist for more than 48 hours after MDMA administration.
Neurological Effects
In clinical studies, doses of MDMA similar to that currently proposed (125 mg) have led to acute neurological changes such as impaired gait, tremor, or nystagmus in a minority of volunteers. The incidence of these effects in clinical MDMA studies is summarized in Tables 2.2 to 2.4 in the Investigators' Brochure. Studies published subsequent to the Investigator's Brochure found similar effects, as reviewed in the first and second updates to the Investigator's Brochure. These effects resolve within several hours. Lasting neurological effects have not been noted.
MDMA appears to produce modest acute changes in neurocognitive performance during peak drug effects. The acute effects of MDMA, generally at doses of either 125 mg or 1.7 mg/kg, have been assessed using the digit symbol substitution task (Cami et al. 2000), a simple reaction time task (Cami et al. 2000; Hernandez-Lopez et al. 2002), a continuous performance attention task (Gamma et al. 2000), the Stroop task (Vollenweider et al. 1998), and a prepulse inhibition measure of sensorimotor gating (Liechti et al. 2001b; Vollenweider et al. 1999b). Of these tasks, only the digit symbol substitution task and the prepulse inhibition task have detected MDMA-induced performance alterations. A study employing the slightly lower dose of 75 mg assessed skills potentially used in driving motor vehicles (Lamers et al. 2003), including visual tracking, divided attention, Object Estimation Under Divided Attention (OMEDA), the Tower of London, and verbal fluency (word generation). Seventy-five mg MDMA did not affect performance on most of the tasks listed above except for the estimation of time needed for a temporarily hidden object to move from one place to another.
Participation in clinical MDMA studies has not been associated with chronic alterations in neurocognitive performance. Data collected by Grob and associates (described in "Previous Human Experience" below) and by Vollenweider and colleagues (Ludewig et al. 2003; Vollenweider et al, 2000, see also pp. 189-190 for IND #63,384) indicate that performance on tests of neurocognitive function is not altered after receiving one or two doses of MDMA in a clinical setting. In contrast, studies of illicit ecstasy users have suggested that repeated MDMA use may be associated with lowered neurocognitive performance, specifically in the areas of memory and executive function (planning and decision making). While a majority of studies have detected these differences (see the Investigator's Brochure and subsequent updates (Baggott et al. 2001; Baggott and Jerome 2003; Jerome 2004 for a detailed discussion), not all studies have detected lower cognitive performance in ecstasy users. A number of studies employing more appropriately matched controls (Halpern et al. 2004; Thomasius et al. 2003) have tended to find fewer differences in cognitive function, with Halpern and colleagues failing to find impaired verbal memory even in ecstasy users reporting use on 50 or more occasions, indicating that differences detected in earlier studies were at least partially due to use of other drugs, or factors associated with polysubstance use. Subtle but detectable impairments in cognitive function may also appear in people reporting heavy use of ecstasy (Back-Madruga et al. 2004; Bolla et al. 1998; Gouzoulis-Mayfrank et al. 2003; Halpern et al. 2004). In a retrospective study finding impairment in very high dose recreational users of ecstasy, there was no effect seen among those who had taken up to an estimated 440 mg of "ecstasy" per month for a year or longer and had used it a minimum of 25 times (unpublished table from published study, Bolla et al. 1998). A recent study employing samples of ecstasy users and non-ecstasy users well-matched for moderate use of other substances detected impaired information processing and executive function in people who reported taking ecstasy on 50 or more occasions, but not in people who reported taking ecstasy on fewer than 50 occasions (Halpern et al. 2004). Another study detected impaired memory in ecstasy users who had consumed at least 80 ecstasy tablets over a lifetime, but failed to detect memory impairment in ecstasy users who had taken fewer than 80 tablets (Gouzoulis-Mayfrank et al. 2003).
An examination of the literature relating to ecstasy use and signs and symptoms of anxiety, depression, and other psychiatric symptoms found inconclusive support for increased psychopathology or psychological difficulties in ecstasy users. A number of recent investigations failed to support claims that ecstasy use is uniquely associated with increases in psychological problems. Increased rates of psychiatric symptoms or psychological difficulties in ecstasy users appear to be more strongly associated with polysubstance use or with pre-existing conditions associated with drug use (see for example Dafters et al. 2004; Daumann et al. 2004; Daumann et al. 2001; Lieb et al. 2002; Thomasius et al. 2003). Given the tenuous link between repeated ecstasy use and psychiatric symptoms, it is not expected that two doses of MDMA will have any effects upon subsequent psychological well-being.
Clinical studies have investigated the effects of MDMA on cerebral blood flow. MDMA acutely alters regional cerebral blood flow (rCBF) and may decrease rCBF for several weeks after drug exposure. Gamma et al. (2000a) used [H2 15O]-Positron Emission Tomography (PET) to measure rCBF at 75 min after 1.7 mg/kg MDMA in 16 volunteers. They detected increases in prefrontal, inferior temporal, and cerebellar cortex rCBF. Decreased rCBF was detected in limbic, paralimbic, central frontal, and temporal areas. These acute effects of MDMA on rCBF may be followed by decreases in rCBF (Chang et al. 2000), as found in a study where SPECT was performed upon eight volunteers 10 to 21 days after receiving the second of two doses of MDMA administered in a clinical setting. These decreases appeared to be time-limited. Two additional volunteers assessed at 41 and 80 days after last MDMA exposure did not show decreases. Similarly, Gamma et al. did not detect differences in cerebral blood flow between ecstasy users and nonusers during a vigilance task (Gamma et al. 2001). Finally, in the study of acute changes in rCBF (Gamma et al. 2000), the eight volunteers who received 1.7 mg/kg MDMA in their first session did not have altered cerebral blood flow in their second session, which was conducted at least two weeks later (Vollenweider 2001, letter of support, pp. 189-190, Mithoefer and Wagner 2001; IND #63,384).
Cardiovascular Effects
The acute cardiovascular effects of MDMA were investigated by Lester et al. (2000). 8 volunteers were administered placebo, 0.5 mg/kg, and 1.5 mg/kg (approximately 105 mg) MDMA in a three session placebo-controlled, double blind study. Two-dimensional Doppler echocardiograms were performed one hour after MDMA administration. MDMA was well tolerated and produced hemodynamic effects similar in magnitude to the !-agonist dobutamine, 40 ug/kg per minute intravenously. As discussed above, the dose-dependent effects of up to 2.5 mg/kg (approximately 175 mg) MDMA on heart rate and blood pressure have been characterized by five different research groups, including three in the United States.
In vitro studies of human heart cells demonstrate that MDMA activates 5-HT2B receptors, which stimulate heart valve cell growth (Setola et al. 2003). 5-HT2B receptor agonism is associated with increased incidence of heart valve disease associated with the serotonin releaser fenfluramine (Rothman and Baumann 2002). However, only fenfluramine and its metabolite dexfenfluramine produced statistically significant increases in heart valve cell growth. It is also important to note that valvular heart disease is associated with daily use of fenfluramine, whereas MDMA will not be administered on a daily basis in this study.
MDMA is classified as a Schedule I compound with a high potential for abuse, primarily because of its use in settings such as "rave" dance parties. It should be noted that instead of experiencing euphoria, individuals undergoing MDMA-assisted psychotherapy are likely to experience deeply emotional thoughts, feelings, and memories, including thoughts associated with grief, rage, and fear. As a result, it seems unlikely that people undergoing this emotionally challenging psychotherapy will find the experience pleasurable or safe enough to pursue MDMA use in unsupervised and uncontrolled settings.
There is no evidence that MDMA-naive healthy volunteers exposed to MDMA in previous Phase 1 clinical studies with MDMA have been motivated to seek out and use MDMA in non-medical settings. Liechti et al. (2001) reviewed the effects of MDMA in 54 male and 20 female volunteers who had participated in clinical studies and stated "none of the participants expressed any interest in taking MDMA as a recreational drug" after participation in an MDMA study.
In the currently proposed study, diversion is not an issue because MDMA will only be administered under supervision of a research psychiatrist and no take-home doses will be permitted. As discussed elsewhere, MDMA will be stored and handled in compliance with Federal and local regulations for Schedule I compounds. The issue of abuse liability is discussed in more detail in this application under "Additional Information."
Pharmacokinetics/Toxicokinetics
Summary of Pharmacokinetic Parameters
Table 4. MDMA Pharmacokinetics
| MDMA Dose | N | Cmax γg/l |
Tmax H |
AUC 0-24 γg*h/l |
AUC/dose γg*h/(l*mg) |
Reference |
| 50 | 2 | 19.8 and 82.8 | 2 and 3 | 100.1 and 813.9 | 2 and 16.3 | de la Torre et al. 2000a |
| 75 | 8 | 130.9 ± 38.6 | 1.8 ± 0.38 | 1331.5 ± 646.03 | 17.8 ± 8 .6 | Mas et al. 1999 |
| 75 | 12 | 178 (no SD) | 3 | Not reported | NA | Lamers et al. 2003 |
| 100 | 8 | 222.5 ± 26.06 | 2.3 ± 1.1 | 2431.38 ± 766.52 | 24.31 ± 7.7 | de la Torre et al. 2000b |
| 100 | 9 | 180 ± 33 | 2 ± 0.26 | 1452 ± 771 | 14.52 ± 7.7 | Farre et al. 2004 |
| 100 | 7 | 208.7 ± 17.1 | 16 ± 0.4 | Not reported | NA | Pizarro et al. 2004 |
| 125 | 8 | 236.4 ± 57.97 | 2.4 ± 0.98 | 2623.7 ± 572.9 | 21 ± 4.6 | Mas et al. 1999 |
| 150 | 2 | 441.9 and 486.9 | 1.5 and 2 | 5132.8 and 5232 | 34.2 and 34.9 | de la Torre et al. 2000a |
| 50 | 2 | Na | na | 2.7 and 5.1 | Na | de la Torre et al. 2000b |
| 75 | 8 | 2.3835 ± 2.1362 | 0.1171 ± 0.0818 | 7.86 ± 3.58 | 0.42 ± 0.2 | Mas et al. 1999 |
| 100 | 8 | 2.7 ± 1.53 | 0.081 ± 0.018 | 8.96 ± 2.27 | 1.31 ± 0.55 | De la Torre et al. 2000b |
| 100 | 7 | na | 0.07 ± 0.03 | 11.8 ± 4.4 | na | Pizarro et al. 2004 |
| 125 | 8 | 2.1253 ± 1.1001 | 0.0923 ± 0.0428 | 8.73 ± 3.29 | 0.41 ± 0.22 | Mas et al. 1999 |
| 150 | 2 | Na | na | 6.9 and 7.2 | Na | De la Torre et al. 2000a |
The pharmacokinetics of MDMA, summarized above in Table 4, have been primarily characterized by a group of Spanish researchers, with the exception of one publication from a team of researchers in the Netherlands that was not primarily concerned with pharmacokinetics. Additional pharmacokinetic parameters for MDMA and metabolites are given in the papers cited in Table 4. For example, after 125 mg MDMA, total clearance for MDMA was 51.1 14.1 per hr, while renal clearance was 13.0 5.4 per hr (de la Torre et al. 2000a). The findings of the Spanish researchers are consistent with other investigations using limited doses (Fallon et al. 1999; Hensley and Cody 1999) or illicit users (Crifasi and Long 1996; Moore et al. 1996; Ramcharan et al. 1998).
As can be seen in Table 4, MDMA kinetics are dose dependent within the range of commonly administered doses (de la Torre et al. 2000b). These dose-dependent kinetics appear to be due to dose-dependent metabolism rather than changes in absorption or excretion. Mas et al. (1999) reported that 75 mg and 125 mg doses of MDMA had similar absorption constants and absorption half-lives. On the other hand, non-renal clearance for 125 mg MDMA was approximately half that of 75 mg MDMA. The dose-dependent metabolism of MDMA is at least partially due to inhibition of CYP2D6, as discussed below. It has also been established that the fraction of MDMA bound to dog plasma proteins is approximately 0.4 and is concentration-independent over a wide range of concentrations (Garrett et al. 1991). Therefore, changes in plasma partitioning are not likely to be significant.
Farre and colleagues reported the pharmacokinetics of a second dose of 100 mg MDMA given 24 hours after an initial 100 mg dose in nine men (Farre et al. 2004). Cmax was 232. 39 /L, AUC(24-48) was 2564 762 g/*h/L, Tmax(24-48) was 25.5 0.33 h, and AUC/dose was 25.64 7.6 g/*h/1*mg. Maximal MDMA concentration after the second dose was similar to maximal concentration after the slightly higher dose of 125 mg (see Table 4 above), and probably results from non-linear pharmacokinetics. Based on these findings, metabolism of an initial dose will also be affected by a supplemental dose. However, since the size and timing of this dose are different from the dosing regimen employed by Farre and colleagues, it is not clear whether the supplemental dose will produce slightly higher maximal values than expected after the supplemental dose only or the combined dose, or whether it will instead lengthen Tmax.
Absorption/Distribution/Metabolism/Excretion
The pharmacokinetics of MDMA in humans have been characterized in blood and urine samples using oral doses of up to 150 mg MDMA. Metabolites of MDMA which have been identified in humans include 3,4-methylenedioxyamphetamine (MDA), 4-hydroxy- 3-methoxy-methamphetamine (HMMA), 4-hydroxy-3-methoxyamphetamine (HMA), 3,4-dihydroxyamphetamine (DHA, also called alpha-methyldopamine), 3,4- dihydroxymethamphetamine (DHMA, also called HHMA), 3,4-methylenedioxyphenylacetone, and N-hydroxy-3,4-methylenedioxyamphetamine (de Boer et al. 1997; Helmlin et al. 1996; Helmlin and Brenneisen 1992; Lanz et al. 1997; Ortuno et al. 1999; Pizarro et al. 2002; Segura et al. 2001). Thus far, human plasma levels of MDMA and the metabolites HMMA, HMA, and MDA have been published (de la Torre et al. 2000; Pizarro et al. 2002; Pizarro et al. 2003; Pizarro et al. 2004). HMMA appears to be the main metabolite in humans (Pizarro et al. 2004). Metabolites are primarily excreted as glucuronide and sulfate conjugates (Helmlin et al. 1996).
The oxidation of the methylenedioxy group can take place via enzymes such as cytochrome p450 (Hiramatsu et al. 1990; Kumagai et al. 1991; Lim and Foltz 1988; Tucker et al. 1994) or by a non-enzymatic process involving the hydroxyl radical (Lin et al. 1992). The enzymes catalyzing this reaction have been examined in the rabbit (Kumagai et al. 1991), rat (Gollamudi et al. 1989; Hiramatsu and Cho 1990; Hiramatsu et al. 1990; Hiratsuka et al. 1995) and human (de la Torre et al. 2000; Kraemer and Maurer 2002; Kreth et al. 2000; Lin et al. 1997; Maurer et al. 2000; Tucker et al. 1994; Wu et al. 1997). In human liver microsomes, Michaelis-Menten kinetics for formation of dihydroxylated metabolites are biphasic (Kreth et al. 2000). The low Km component for demethylenation is CYP2D6 as it is selectively inhibited by quinidine. At higher concentrations of MDMA, other enzymes with higher Km also contribute to MDMA demethylenation, including CY1A2 and CYP3A4.
Although it was hypothesized that genetic variations in CYP2D6 activity might influence risk of MDMA toxicity, this is no longer a concern. Several in vitro studies have shown that MDMA is not just a substrate for CYP2D6 but also binds to it, forming an inhibitory complex (Brady et al. 1986; Delaforge et al. 1999; Heydari et al. 2004; Wu et al. 1997). Compelling in vivo evidence of enzyme inhibition was provided by de la Torre et al. (de la Torre et al. 2000a) who showed that plasma levels and 24-hour urinary recovery of HMMA are dose-independent. This is likely the result of inhibition of CYP2D6-mediated DHMA formation. The fact that CYP2D6 is apparently easily saturated makes this possible source of individual sensitivity appear less significant. In fact, there currently seems to be no evidence that the poor metabolizer genotype is by itself a major risk factor for acute MDMA toxicity. Kreth et al. (2000) reported that the poor metabolizer trait did not lead to significant alteration in maximal drug plasma concentrations in an individual participating in a clinical study of the MDMA analogue, MDE. At least one poor metabolizer has received MDMA as a participant in a study conducted by the Spanish team (Pacifici et al. 2002, see also Pacifici et al. 2004) without any adverse events occurring. The individual had 60% greater MDMA AUC after a first and a second dose, but the only other reported difference for this participant was a statistically significant increase in amount of NK cells. Issues involved in MDMA metabolism is addressed in a review by de la Torre and colleagues (De la Torre et al. 2004). Evidence from in vitro and in vivo studies and the cases described above provide further evidence that the role of CYP2D6 in MDMA metabolism is sufficiently limited that it is not a major risk factor for immunocompetent individuals participating in clinical research with MDMA.
Enzymes involved in the formation of MDA from MDMA in human liver microsomes have been investigated by two groups (Kreth et al. 2000; Maurer et al. 2000). Maurer et al. reported that formation of MDA was predominantly catalyzed by CYP1A2 (and to a lesser extent by CYP2D6), but did not present detailed results of their experiments. Kreth et al., in a publication focusing on MDE metabolism, reported high correlations between MDMA and MDE N-dealkylation and MDE N-dealkylation and human liver microsome CYP2B6 content. MDE N-dealkylation and CYP1A2 levels were also significantly correlated. This indicates that CYP2B6 and CYP1A2 participate in the formation of MDA. The role of CYP2B6 in human MDMA metabolism is consistent with rodent research (Gollamudi et al. 1989).
MDMA is a chiral compound and has been almost exclusively administered as a racemate. Studies in human volunteers (Fallon et al. 1999; Hensley and Cody 1999; Pizarro et al. 2003; Pizarro et al. 2004) and rodents (Cho et al. 1990; Fitzgerald et al. 1990; Matsushima et al. 1998) indicate that the disposition of MDMA is stereoselective, with the S-enantiomer having a shorter elimination half-life and greater excretion that the R-enantiomer. For example, Fallon et al. (1999) reported that the area under the curve (AUC) of plasma concentrations was two to four times higher for the R-enantiomer than the S-enantiomer after 40 mg, p.o., in human volunteers. Moore et al. (1996) found greater levels of R-(-)-MDMA in blood, liver, vitreous and bile samples from an individual who died shortly after illicit MDMA use. Stereoselective analysis of biosamples in both an MDMA overdose and a traffic fatality had similar findings (Ramcharan et al., 1998; Crifasi and Long, 1996). The stereoselective pharmacokinetics of MDMA are reflected in formation of MDA and DHMA enantiomers (Fallon et al. 1999; Pizarro et al. 2004; Pizarro et al. 2003). In the first 24 hours after MDMA administration, greater plasma and urine concentrations of S-(+)-MDA than its R- enantiomer occur (Fallon et al. 1999; Moore et al. 1996). By contrast, R/S ratios of HMMA are more similar to those for MDA (greater amounts of R-(-)-HMMA than S-(+)- HMMA during the first 24 hours), or there is no difference between concentrations of the two enantiomers of HMMA (Pizarro et al. 2003; Pizarro et al. 2004).
Table 5. Urinary Recovery for MDMA and Metabolites (de la Torre et al. 2000a)
| Urinary Recovery (mol) | ||||||
| MDMA Dose mg (mol) |
N | MDMA | MDA | HMMA | HMA | Dose Excreted (%) |
| 50 (259) | 2 | 20.7 and 40.9 | 1.4 and 1.0 | 152.0 and 89.2 | 4.7 and 4.2 | 69.1 and 38.3 |
| 75 (358) | 8 | 71.2 ± 13.7 | 3.5 ± 0.9 | 128.3 ± 21.8 | 5.4 ± 0.4 | 53.7 ± 11.4 |
| 100 (518) | 2 | 232.6 and 74.7 | 1.4 and 5.6 | 59.8 and 124.0 | 2.9 and 6.8 | 57.3 and 40.7 |
| 125 (647) | 8 | 169.6 ± 69.5 | 6.4 ± 2.7 | 148.3 ± 102.8 | 6.2 ± 6.2 | 51.0 ± 16.2 |
| 150 (776) | 2 | 160.3 and 333.3 | 2.6 and 4.7 | 122.2 and 82.4 | 4.1 and 3.7 | 37.3 and 54.7 |
The urinary excretion of MDMA and its metabolites was first characterized by de la Torre and colleagues, with data from that study presented in Table 5 above. Metabolites are primarily excreted as glucuronide and sulfate conjugates (Helmlin et al. 1996). Subsequent studies examining metabolism after 100 mg MDMA reported excretion values similar to those reported by de la Torre and associates (Farre et al. 2004; Segura et al. 2001; Pizarro et al. 2004; Pizarro et al. 2003). Urinary excretion of the MDMA metabolite HHMA reported after the administration of 100 mg MDMA to four men are 91.8 23.8 mol and 17.7% recovery (Segura et al. 2001). As was the case for maximal plasma values, urinary recoveries for MDMA and MDA were higher after a second dose of 100 mg MDMA than after an initial dose of 100 mg MDMA (Farre et al. 2004).
The toxicity of MDMA has been investigated in numerous animal and in vitro studies published in peer-reviewed journals. In addition, hundreds of published case reports describe adverse events in illicit ecstasy users. Finally, 28-day toxicity studies in canines and rodents have been performed and are included in the MDMA Drug Master File (DMF #6293). Thus, the toxicity of MDMA is well characterized.
Serious MDMA toxicity is rare in uncontrolled settings, considering the millions of users taking "ecstasy" of unknown identity, potency, and purity (Baggott 2002; Gore 1999; Henry and Rella 2001). Under these conditions, the most common serious adverse event involves hyperthermia, which often appears to be influenced by prolonged physical exertion (dancing) in an area with a high ambient temperature. Reports of toxicity in illicit ecstasy users are summarized in the Investigator's Brochure (Baggott et al. 2001), and a brief review of more recent reports are covered in the latest update of the Investigator's Brochure (Jerome 2004). In addition to hyperthermic syndromes, other rare adverse events include dysphoric responses, hepatotoxicity, and hyponatremia. In the proposed clinical study, volunteers will be carefully monitored for signs and symptoms of these unlikely events, as discussed in "Monitoring for Toxicity," above. As described in "Previous Human Experience" below, exposure to MDMA in a controlled clinical setting has not been associated with toxicity. As well, improvement in quality of life occurring after MDMA-assisted psychotherapy should be weighed out against any concerns of MDMA toxicity.
Published animal and in vitro studies have specifically investigated the possibility of hyperthermia, hepatotoxicity and neurotoxicity after MDMA exposure. These types of toxicity appear to be dose-dependent and all available evidence indicates that the risks in these areas are minimal in the currently proposed study. These areas of toxicity are discussed below. Neurotoxicity will be discussed in two sections; the first concerning serotonergic axon damage and the second concerning neuronal cell death. Finally, the issue of reproductive and developmental toxicity will be briefly mentioned.
Hyperthermia
As discussed above, MDMA administered in a controlled setting produces only a slight increase in body temperature. However, hyperthermia is one of the most commonly reported serious adverse events in ecstasy users. Peripheral vasoconstriction (Pedersen and Blessing 2002), non-shivering heat production and possible effects on heat- production related uncoupling proteins (Mills et al. 2003; Sprague et al. 2003), and activity at serotonin or norepinephrine receptors (Fantegrossi et al. 2003; Fantegrossi et al. 2004) all may play a role in generating hyperthermia. Hyperthermia may be dose dependent, as suggested by case series of people who took ecstasy in the same London area nightclub on the same evening (Greene et al. 2003). Studies in rats and mice suggest that crowded housing (Fantegrossi et al. 2003) and high ambient temperature (see for example Brown and Kiyatkin 2004; Green et al. 2004; Malberg et al. 1998) promotes a hyperthermic reaction to MDMA. It is expected that hyperthermia will be very unlikely in the proposed study setting, since the participant will be in a room maintained at a comfortable temperature and he or she will not experience crowding. The investigators will periodically measure body temperature with an automatic temperature sensor and telemetry device worn on the skin, and the investigators will also measure ambient temperature during the course of the study.
Hepatotoxicity
Because hepatotoxicity has been noted in ecstasy users, five in vitro and two in vivo studies have examined the hepatotoxicity of MDMA. These studies show that MDMA can impair liver cell viability, but that this is very unlikely to occur in the proposed clinical study. The peak liver exposure to MDMA in the proposed clinical study should be approximately one-eleventh the concentration shown to impair cell viability in these in vitro studies.
In one study, MDMA caused increases in ALT, AST, and LDH activities in rat hepatocytes (Beitia et al. 2000). These increases were statistically significant with high concentrations of MDMA (1 mM for six hours) or lower concentrations for prolonged exposures (0.1 mM for 24 hours). Further evidence of MDMA-induced toxicity to hepatocytes came from moderate decreases in ATP (after three, but not one, hour incubation with 0.1 mM MDMA). A second in vitro study examined the possible pro- fibrogenic effects of MDMA on the liver by measuring expression of procollagen mRNA in a cell line of hepatic stellate cells (Varela-Rey et al. 1999). These cells produce the collagen characteristics of a fibrotic liver. Expression of !1(I) procollagen mRNA was significantly increased by 0.5, but not 0.1, mM MDMA for 24 hr. This effect required sustained exposures, as 1 mM MDMA for 8 h did not increase mRNA expression. A third in vitro study using mouse hepatocytes showed that MDMA increases lipid peroxidation and loss of cell viability produced by hyperthermic conditions (Carvalho et al. 2001). 1.6 mM MDMA slightly but significantly decreased cell viability, yet it did not affect lipid peroxidation over 60 to 180 min under normothermic (37 C) conditions. When temperature was raised to 41 C, the hepatotoxicity of MDMA was dramatically increased. At this temperature, 1.6 mM MDMA approximately doubled lipid peroxidation after 180 min and decreased cell viability after as little as 60 minutes. A lower concentration, 0.8 mM MDMA, also decreased cell viability after 180 min at 41 C but not at 37 C. The fourth study incubated rat liver cells with 0.5 to 5 mM MDMA for 8 or 24 hours and assayed for apoptosis with cell staining, Western blot for apoptosis- related factors, and bax assays (Montiel-Duarte et al. 2002). Higher concentrations increased signs of apoptosis, but at doses that are about eleven times those usually seen in humans. The fifth study examined the effects of 3 hours of incubation with 0.8 to 1.6 mM MDMA metabolites MDA and alpha-MeDA on liver cells (Carvalho et al. 2003) and found that alpha-MeDA but not MDA increased signs of liver peroxidation at the higher concentrations.
In vivo studies in mice indicate that ambient temperature and oxidative stress may be involved in liver toxicity. Carvalho and colleagues assayed liver sections from mice given a single i.p. injection of 5, 10 or 20 mg/kg MDMA in either a 20 C or a 30 C environment for antioxidant enzyme levels, signs of lipid peroxidation, and cell histology (Carvalho et al. 2002). The researchers found that increases in MDMA dose and ambient temperature during MDMA administration affected degree of apparent oxidative stress and detectable liver abnormalities. In the second study, repeated injections of 10 mg/kg, but not 5 mg/kg, of the MDMA enantiomer S-(+)-MDMA (4 s.c. every 2 h), produced some hepatic necrosis (Johnson et al. 2002), with more pronounced effects in mice fed a vitamin E deficient diet than in mice receiving sufficient amounts of vitamin E. Hepatotoxicity has not yet been reported to occur in any of the clinical studies where MDMA was administered to research subjects, and the drug exposures that can damage liver cells would not occur in the currently proposed clinical study. The lowest concentration that impaired cell functioning in these studies (0.1mM or ~19.3 mg/l MDMA) affected indices of cell viability after 24, but not 6, hours in the study by Beitia et al (2000). This same concentration had no significant pro-fibrogenic effect after 24 hr in the study by Varela-Rey et al (1999). This lowest toxic concentration is approximately 82 times higher than the expected peak MDMA plasma level (236.4 57.97 "g/l MDMA) after 125 mg, the proposed dose in this study. Liver exposure to drugs is often higher than plasma levels. In an autopsy of a deceased ecstasy user, liver MDMA concentration was 7.2 times higher than femoral blood MDMA concentration (Rohrig and Prouty 1992). Thus, the peak liver exposure to MDMA in a clinical setting should be approximately one-eleventh the concentration shown to impair cell viability in these studies. This peak concentration would only be briefly sustained. Therefore it is unlikely that MDMA exposures in clinical studies will approach those demonstrated in these studies to impair rat liver cell viability or induce procollagen mRNA. Higher ambient temperatures appear to amplify the degree and likelihood of hepatoxicity, and since study participants will receive MDMA in a comfortable room and the investigators will monitor ambient temperature during the course of the study, it seems especially unlikely that MDMA will induce hepatoxicity. Nonetheless, people with significant liver disease will be excluded from the study, and participants will be monitored for hepatotoxicity with liver panels performed before MDMA administration and at the time of medical examination follow-up ("Day 36" - see Table 1 above).
Neurotoxicity
Extensive studies in animals indicate that high or repeated dose MDMA exposure can oxidatively damage serotonergic axons originating in the dorsal raphe nucleus of the brainstem. This is associated with decreases in serotonin, serotonin metabolites, and serotonin transporter. Although some regrowth occurs, seemingly permanent redistribution of axons was noted in a study with squirrel monkeys (Hatzidimitriou et al. 1999). These serotonergic changes have not been associated with lasting behavioral impairment in the vast majority of animal studies, despite dramatic serotonin depletions. The great volume of research addressing MDMA neurotoxicity is discussed in more detail in the Investigator's Brochure and subsequent updates of the Investigator's Brochure (Baggott et al. 2001; Baggott and Jerome 2003; Jerome 2004).
A study published in 2004 comparing MDMA administration (3 7.5 mg/kg doses given i.p.) with the serotonin neurotoxin 5,7-DHT in rats found that DHT, but not MDMA, reduced serotonin transporter and brain serotonin while increasing levels of glial fibrillary acidic protein (GFAP), a marker of neuronal injury (Wang et al. 2004). MDMA lowered brain serotonin without altering levels of serotonin transporter or GFAP, suggesting a dissociation between brain serotonin levels and other presumed markers of neurotoxicity, and an investigation of neurons from the substantia nigra of mice given four 5 mg/kg doses every 2 hours found signs of oxidative stress, such as increased signs of DNA fragmentation and ubiquitin-positive whorls, but no signs of cell death (Fornai et al. 2003). However, in contrast, raphe neurons taken three weeks after rats received twice-daily s.c. doses of MDMA on four consecutive days were much less able to transport radioactively labeled praline, used as a measure of axonal neurotoxicity (Callahan et al. 2001). Examining and considering these and other research findings continues to demonstrate the contentious nature of findings relating to MDMA neurotoxicity.
We have carefully considered the risks of such neurotoxicity and conclude that they are minimal in the proposed study. This conclusion is supported by empirical and toxicokinetic evidence and is consistent with the lack of toxicity in previous clinical MDMA studies. A series of letters in the journal Neuropsychopharmacology discussed the risks of neurotoxicity in MDMA studies (Gijsman et al. 1999; Lieberman and Aghajanian 1999; McCann and Ricaurte 2001; Vollenweider et al. 1999a; Vollenweider et al. 2001), leading two of the journal editors to conclude that there is no evidence that the MDMA exposures in the studies of Vollenweider and colleagues (similar to those currently proposed) were neurotoxic (Aghajanian and Lieberman 2001). Finally, studies in rhesus monkeys suggest that use of interspecies scaling to arrive at dosing in previous studies produced inappropriately high doses of MDMA.
Vollenweider and colleagues recently measured serotonin transporter density using positron emission tomography (PET) with [11C]McN5652 before and after a single clinical MDMA exposure (Vollenweider et al. 2000, data presented at the 2000 conference of the German Society for Psychiatry, Psychotherapy and Neuromedicine). Vollenweider and colleagues were unable to detect any lasting effect of 1.5 or 1.7 mg/kg MDMA in a pilot study with six MDMA-naive healthy volunteers and in a second study with additional volunteers (n = 8). This ligand and measurement technique had been previously reported by another group to be sensitive to apparent serotonin transporter changes in illicit ecstasy users with at least 70 drug exposures (McCann et al. 1998). This measurement technique was validated in a study using a baboon exposed to a neurotoxic MDMA regimen (Scheffel et al. 1998), and this validation study found that PET tended to overestimate serotonin transporter changes in most cases. A more recent study in humans that employed this ligand also found reduced levels of serotonin transporter in current ecstasy users, but did not report as large a reduction in serotonin transporter as did the original study (Buchert et al. 2003; Buchert et al. 2004). Given the small sample size in the study by Vollenweider et al., it is possible that a modest change in SERT density could have gone undetected. However, very little variance in ligand binding was found in baseline measures of ligand binding.
Imaging studies in repeated ecstasy users have consistently found lower serotonin transporter levels, but these findings are also qualified by degree of exposure and period of abstinence. One study using the same ligand used by McCann and colleagues (Buchert et al. 2003; Buchert et al. 2004) and one study using a different ligand (Reneman et al. 2001) both found that serotonin transporter levels returned to normal in people who abstained from ecstasy for a year to a year and a half. Both studies also found greater reductions in serotonin transporter, greater numbers of affected areas, in women. Reneman and colleagues (2001) also compared people reporting at least 50 exposures with people who reported fewer than 50 exposures, and they found that moderate ecstasy users (those reporting use on fewer than 50 occasions) did not have significant reductions in serotonin transporter sites. These findings suggest that effects on serotonin transporter may be at least partly dependent on degree of use and time since last exposure. Because of findings in humans and non-human animals, the possibility of neurotoxicity will be discussed with all volunteers, even though strong evidence from studies in humans and non-human animals suggests that the risk of neurotoxicity posed by participating in this study is low.
Interspecies pharmacokinetic comparisons support the safety of 125 mg MDMA in humans. Vollenweider et al. (2001) compare published pharmacokinetic data for humans and rats and conclude that human exposure to MDMA after 125 mg is significantly less than the lowest known consistently neurotoxic MDMA dose in Sprague-Dawley rats, 20 mg/kg, sc, (Battaglia et al. 1988; Commins et al. 1987). At these doses, human MDMA plasma AUC are approximately 30% of the rat AUC. Similarly, human Cmax are approximately 10% of rat Cmax.
We note that this comparison is limited by several considerations. First, it is not known whether rats and humans have different vulnerability to the same MDMA exposure. Second, it is not known whether metabolites of MDMA contribute to neurotoxicity. If they do, then the margin of safety for 125 mg MDMA should be even wider because formation of metabolites is more extensive in rodents than in humans. Third, rats and humans may differ in the brain concentration of drug produced by a given blood concentration. In rats, MDMA concentrations in the brain are 7 to 10 times higher than in plasma (Chu et al. 1996). In a human fatality, postmortem MDMA concentrations were about 6 times higher in the brain than in the plasma (Rohrig and Prouty 1992), although postmortem drug redistribution may have occurred. If these data are reliable, rats may have similar peak brain levels to humans when plasma levels are the same. Fourth, neurotoxicity in rodents appears to be increased by hyperthermia in many studies.
Finally, the threshold for neurotoxicity is not well established in rats. The threshold for neurotoxicity in Sprague-Dawley rats appears to be above 10 mg/kg (Battaglia et al. 1988) and below 20 mg/kg (Commins et al. 1987). Therefore, a conservative comparison indicates that human MDMA exposure (measured as plasma AUC) after 125 mg is likely between 30% and 60% of the exposure required for neurotoxicity in rats. Because of non- linear pharmacokinetics and possible differences in rat versus human MDMA disposition, at least one researcher has concluded that using interspecies scaling is not recommended for calculating equivalent doses in neurotoxicity studies (De la Torre and Farre 2004). We think that the margin of safety is probably wider due to the presence of hyperthermia and increased formation of toxic metabolites in animal studies but not in clinical MDMA trials.
In conclusion, the lack of apparent toxicity in previous clinical MDMA studies, evidence of unaltered serotonin transporter density after similar doses, and toxicokinetic comparisons suggest that the doses of MDMA used in this study are unlikely to produce measurable neurotoxicity or significant adverse functional consequences. MDMA-Induced Neuronal Apoptosis (Programmed Cell Death)
Two in vitro studies have suggested that MDMA may trigger programmed neuronal cell death (apoptosis) under certain conditions. This phenomenon has not been verified in vivo. No cell death occurs in regions containing the cell bodies of serotonergic neurons after MDMA exposure (Fischer et al. 1995; Hatzidimitriou et al. 1999; O'Hearn et al. 1988). However, one study detected evidence of non-serotonergic cell body damage in the rat somatosensory cortex after 80 mg/kg MDMA (Commins et al. 1987). It is theoretically possible that this damage was due to apoptosis. MDMA-induced apoptosis appears to require high concentrations and exposure times. It is unlikely that 125 mg MDMA in the currently proposed clinical study will trigger programmed cell death in neurons. In the currently proposed study, the peak brain concentration of MDMA is estimated to be approximately 6% of a concentration that produced no toxicity after 96 hr of exposure in vitro.
Forty-eight hours of incubation with MDMA dose-dependently decreased survival of cultured human placental serotonergic cells (Simantov and Tauber 1997). This decreased cell viability was accompanied by DNA fragmentation and cell cycle arrest (in the G2M phase). Forty-eight hour exposure to 0.4 mM MDMA decreased cell survival by 1.4 4%, while 1.2 mM MDMA decreased cell survival by 61 9%. In another study, the effects of MDMA on cultured rat neocortical neurons were studied at concentrations of 125 to 1000 M MDMA and exposure times of 1, 24, and 96 hours (Stumm et al. 1999). Cell survival was decreased by 34.2 11.4% at 96 hours after an average exposure of 500 M MDMA, but not after 125 M MDMA. Stumm et al. also noted DNA fragmentation and altered expression of the bcl-xLS gene, which supports the interpretation that programmed cell death had occurred. The degree of cytotoxicity noted for MDMA in this study was comparable to the toxicity produced by other structurally related amphetamines.
A study that used fluoro-jade staining to examine brain sections from rats killed 3 days after receiving 10, 20 or 40 mg/kg MDMA found increased staining in most brain areas in rats given 40 mg/kg MDMA, and in some brain areas in some rats given 20 mg/kg MDMA (Schmued et al. 2003). Increased signs of neuronal degeneration were strongly associated with hyperthermia, suggesting a role of dose and body temperature in producing these effects. However, as discussed earlier, another study examining substantia nigra in mice given a total dose of 20 mg/kg (four doses of 5 mg/kg) found signs of oxidative stress, but failed to find signs of frank cell death (Fornai et al. 2003). It is unlikely that MDMA exposures in the currently proposed clinical study will approach those demonstrated to trigger programmed cell death in neurons. If MDMA levels in the brain are about 6 times higher than in plasma (Rohrig and Prouty 1992), then 125 mg MDMA should produce peak plasma levels of 236.4 57.97 "g/l MDMA (de la Torre et al. 2000b) and peak brain levels of 1.4 0.3 mg/L. This estimated peak level is significantly less than the lowest drug concentration used in either apoptosis study. While 0.4 mM MDMA or 77.3 mg/L had modest effects in the first study, 125 M or 24.2 mg/L had no significant effect in the second study. Peak plasma levels after a supplemental dose of 62.5 mg follows 125 mg are liable to be somewhat higher, but they are not likely to approach levels in brain that produced cell death. Given these concentration differences and the long exposure times used in these studies, it does not seem likely that human oral doses of MDMA would be sufficient to induce programmed cell death in neurons. Additionally, body temperature is only slightly elevated in humans given MDMA in clinical settings, further reducing any possible effects due to hyperthermia.
Reproductive and Developmental Toxicity
As discussed in the Investigator's Brochure, one of two studies of polydrug-using ecstasy users found a possibly increased incidence of developmental abnormalities when pregnant women used illicit drugs including ecstasy (McElhatton et al. 1999). There is some contention as to whether the developmental abnormalities reported in the study conducted by McEllhatton and colleagues are, in fact, the result of "ecstasy" consumption. Neonatal rats given repeated doses of MDMA show signs of lower brain serotonin and showed impairments in learning and memory, with the neonatal period in rats considered equivalent to the third trimester of pregnancy in humans. In one study, rats given the very high, repeated dose regimen of 20 mg/kg MDMA twice daily from Day 11 to Day 20 performed less well on a task assessing spatial learning and memory (Williams et al. 2003), and had lower brain serotonin and greater increases in the dopamine metabolite homovanillic acid (HVA) in frontal cortex, hippocampus and striatum (Koprich et al. 2003A). Maternal administration has produced contradictory results. Rats born to dams given twice-daily injections of 15 mg/kg for 7 consecutive days were less active in a novel environment (Koprich et al. 2000B), yet lower brain serotonin was not detected in rats born to dams given twice-daily injections of 20 mg/kg MDMA for four days (Kelly et al. 2002). Pregnant women will be excluded from participation in the proposed study and urine pregnancy tests will be performed before each drug administration.