Pere N. Roset

Hydroxytyrosol Disposition in Humans

BACKGROUND Animal and in vitro studies suggest that phenolic compounds in virgin olive oil are effective antioxidants. In animal and in vitro studies, hydroxytyrosol and its metabolites have been shown to be strong antioxidants. One of the prerequisites to assess their in vivo physiologic significance is to determine their presence in human plasma. METHODS We developed an analytical method for both hydroxytyrosol and 3-O-methyl-hydroxytyrosol in plasma. The administered dose of phenolic compounds was estimated from methanolic extracts of virgin olive oil after subjecting them to different hydrolytic treatments. Plasma and urine samples were collected from 0 to 12 h before and after 25 mL of virgin olive oil intake, a dose close to that used as daily intake in Mediterranean countries. Samples were analyzed by capillary gas chromatography-mass spectrometry before and after being subjected to acidic and enzymatic hydrolytic treatments. RESULTS Calibration curves were linear (r >0.99). Analytical recoveries were 42-60%. Limits of quantification were <1.5 mg/L. Plasma hydroxytyrosol and 3-O-methyl-hydroxytyrosol increased as a response to virgin olive oil administration, reaching maximum concentrations at 32 and 53 min, respectively (P <0.001 for quadratic trend). The estimated hydroxytyrosol elimination half-life was 2.43 h. Free forms of these phenolic compounds were not detected in plasma samples. CONCLUSIONS The proposed analytical method permits quantification of hydroxytyrosol and 3-O-methyl-hydroxytyrosol in plasma after real-life doses of virgin olive oil. From our results, approximately 98% of hydroxytyrosol appears to be present in plasma and urine in conjugated forms, mainly glucuronoconjugates, suggesting extensive first-pass intestinal/hepatic metabolism of the ingested hydroxytyrosol.

Pharmacology of MDMA in Humans

MDMA given at recreational doses (range tested 50 to 150 mg) to healthy volunteers, produced mydriasis and marked increases in systolic and diastolic blood pressure, heart rate, and pupillary diameter. MDMA induced changes on oral temperature. The time course of this observation was biphasic, as a slight decrease at 1 h and a slight increase at 2 and 4 h were observed. MDMA induced a slight dose‐dependent impairment on psychomotor performance. MDMA produced a marked rise in plasma cortisol and prolactin concentrations. The elimination half‐life of MDMA was about 8‐9 h. Drug concentrations increased, and a parallel increase in physiologic and hormonal measures was observed. Both peak concentrations and peak effects were obtained between 1 and 2 h and decreased to baseline values 4‐6 h after drug administration.

Pharmacological Interaction between 3,4-Methylenedioxymethamphetamine (Ecstasy) and Paroxetine: Pharmacological Effects and Pharmacokinetics

3,4-Methylenedioxymethamphetamine (MDMA, “ecstasy”) is increasingly used by young people for its euphoric and empathic effects. MDMA can be used in combination with other drugs such as selective serotonin reuptake inhibitors. A clinical trial was designed where subjects pretreated with paroxetine, one of the most potent inhibitors of both 5-hydroxytryptamine reuptake and CYP2D6 activity, were challenged with a single dose of MDMA. The aim of the study was to evaluate the pharmacodynamic and pharmacokinetic interaction between paroxetine and MDMA in humans. A randomized, double-blind, crossover, placebo-controlled trial was conducted in 12 healthy male subjects. Variables included physiological parameters, psychomotor performance, subjective effects, and pharmacokinetics. Subjects received 20 mg/day paroxetine (or placebo) orally for the 3 days before MDMA challenge (100 mg oral). MDMA alone produced the prototypical effects of the drug. Pretreatment with paroxetine was associated with marked decreases of both physiological and subjective effects of MDMA, despite a 30% increase in MDMA plasma concentrations. The decreases of 3-methoxy-4-hydroxymethamphetamine plasma concentrations suggest a metabolic interaction of paroxetine and MDMA. These data show that pretreatment with paroxetine significantly attenuates MDMA-related physiological and psychological effects. It seems that paroxetine could interact with MDMA at pharmacodynamic (serotonin transporter) and pharmacokinetic (CYP2D6 metabolism) levels. Marked decrease in the effects of MDMA could lead users to take higher doses of MDMA and to produce potential life-threatening toxic effects.

Cell‐Mediated Immune Response in MDMA Users After Repeated Dose Administration

Abstract: Acute administration of 3,4‐methylenedioxymethamphetamine (MDMA, “ecstasy”) produces time‐dependent immune dysfunction in humans. Recreational use of MDMA generally includes repeated drug consumption, often in association with other drugs, such as alcohol and cannabis. In the laboratory setting, repeated MDMA administration to healthy MDMA consumers produced a time‐dependent immune dysfunction similar to that observed with the ingestion of a single dose, and the first of the two administrations paralleled the time‐course of MDMA‐induced cortisol stimulation kinetics and MDMA plasma concentrations. A significant decrease in CD4 T‐helper cells with simultaneous increase in natural killer (NK) cell and a decrease in functional responsiveness of lymphocytes to mitogenic stimulation was observed. Response to the second dose was either long‐lasting compared with the first dose or disproportionate and did not show any parallelism with cortisol and MDMA plasma concentrations. This circumstance extended the critical period during which immunocompetence is highly impaired as a result of MDMA use. Accumulation of MDMA in the body of a poor metabolizer induced higher immunomodulatory effects with statistically significant differences in NK cell function compared with extensive metabolizers. When basal values of lymphocyte subsets were examined in a population of recreational MDMA users participating in different clinical trials, alterations in several immunological parameters were observed. The absolute number of lymphocytes, in particular T lymphocytes and CD4 T‐helper cell subsets, showed a trend toward reduced values, although cell counts were within normal limits. By contrast, NK cells in MDMA consumers were reduced to one‐third of those from healthy persons. A statistically significant decrease in affected immune parameters was recorded during a 2‐year observation period in a subgroup of recreational MDMA users. These permanent alterations in immunologic homeostasis may result in impairment of general health and subsequent increased susceptibility to infection and immune‐related disorders.

Effects of repeated doses of MDMA ("ecstasy") on cell-mediated immune response in humans.

Cell-mediated immune response after the administration of two repeated doses of 100 mg 3,4-methylenedioxymethamphetamine (MDMA) at 4-hour and 24-hour intervals was evaluated in two randomised, double-blind and cross-over clinical trials conducted in healthy male MDMA consumers. MDMA produced a time-dependent decrease in the CD4/CD8 T-cell ratio due to a decrease in the number of CD4 T-helper cells, a decrease in the functional responsiveness of lymphocytes to mitogenic stimulation, and a simultaneous increase in natural killer cells. In case of two 100 mg MDMA doses given 4 hour apart, immune alterations produced by the first dose were strengthened by the second one. At 24 hours after treatment, statistically significant residual effects were observed for all the altered immune parameters after the administration of two MDMA doses if compared to single dose and placebo. In the second clinical trial, the second 100 mg MDMA dose given 24 hours after the first dose produced immunological changes significantly greater than those induced by the initial drug administration and which seemed to show a delayed onset. Significant residual effects were observed for all the immune parameters as late as 48 hours after the second dose. These results show that repeated administration of MDMA with both a short and a long time interval between doses extends the critical period following MDMA administration, already observed after a single dose, in which immunocompetence is severely compromised.

Contribution of cytochrome P450 2D6 to 3,4- methylenedioxymethamphetamine disposition in humans : Use of paroxetine as a metabolic inhibitor probe

AbstractBackground: 3,4-Methylenedioxymethamphetamine (MDMA) is a synthetic amphetamine derivative typically used for recreational purposes. The participation of cytochrome P450 (CYP) 2D6 in the oxidative metabolism of MDMA may suggest an increased risk of acute toxicity in CYP2D6 poor metabolisers. This study was aimed at assessing the contribution of CYP2D6 to MDMA disposition in vivo using paroxetine as a metabolic probe inhibitor. Paroxetine, a CYP2D6 inhibitor, was repeatedly administered before MDMA administration. Study design: This was a randomised, double-blind, crossover, placebo-controlled trial conducted in seven healthy male volunteers who were CYP2D6 extensive metabolisers. Treatment conditions (paroxetine/MDMA and placebo/ MDMA) were randomly assigned. Each volunteer participated in two 3-day sessions. On days 1, 2 and 3 subjects received a single oral dose of paroxetine or placebo 20mg. On the third day, a single oral dose of MDMA 100mg was administered in both paroxetine and placebo conditions. Methods: Plasma concentration-time profiles and urinary recoveries of MDMA and its metabolites were measured, as well as plasma concentrations of paroxetine, (3S,4R)-4-(4-fluorophenyl)-3-(3,4-methylenedioxyphenoxymethyl)-piperidine, and (3S,4R)-4-(4-fluorophenyl)-3-(3-methoxy-4-hydroxyphenoxymethyl)-piperidine (HM-paroxetine). Results: Paroxetine given before MDMA resulted in significant increases of MDMA area under the plasma concentration-time curve from 0 to 27 hours (AUC27) [23%], AUC from zero to infinity (AUC∞) [27%] and maximum plasma concentration (Cmax) [17%], without significant differences in MDMA time to reach Cmax (tmax). MDMA elimination-related pharmacokinetic parameters showed a significant reduction of MDMA elimination rate constant (Ke) [−14%] and plasmatic clearance (CLP) [−29%]. In the case of 3,4-dihydroxymethamphetamine (HHMA), a 21% decrease in Cmax with no significant differences in AUC27, AUC∞, Ke and elimination half-life) were found. 4-Hydroxy-3-methoxymethamphetamine (HMMA) showed a decrease in plasma concentrations with a reduction in AUC27 (−28%), AUC∞ (−20%) and Cmax (−46%). In the case of 3,4-methylenedioxyamphetamine (MDA) an increase in Cmax (17%) and AUC27 (16%) was found. Following paroxetine pretreatment, the urinary recovery (0–45 hours) of MDMA increased by 11%; HHMA and HMMA urinary recoveries were 27% and 16% lower, respectively compared with placebo. The ratio of Cmax values of paroxetine and its metabolite on days 1 and 3 showed a 3-fold reduction, with no differences in tmax. Discussion and conclusion: The contribution of CYP2D6 to MDMA metabolism in humans is not >30%, therefore other CYP isoenzymes may contribute to O-demethylenation of MDMA. Accordingly, the relevance of genetic polymorphism in CYP2D6 activity on MDMA effects and MDMA-induced acute toxicity should be examined as well as the interactions of other CYP2D6 substrates with MDMA, once the enzyme is inhibited. The pharmacokinetics of HM-paroxetine in humans after the administration of repeated doses is reported for the first time in this study.

MDMA (ecstasy) pharmacokinetics in a CYP2D6 poor metaboliser and in nine CYP2D6 extensive metabolisers.

(CYP)isoform 2D6 [2]. HHMA is further metabolised to 4-hydroxy, 3-methoxymethamphetamine (HMMA) bycatechol- O-methyltransferase (COMT). HMMA in-duces vasopressin secretion to a higher extent thanMDMA and has been postulated to contribute to hyp-onatraemia observed in some MDMA acute intoxica-tions [4]. The CYP2D6 gene is highly polymorphic andmany variations affect the expression or activity of theenzyme. Approximately 7–10% of European Caucasianspresent a metabolic deficiency and are termed poormetabolisers (PM) [12]. Evidence that individuals pos-sessingacompromiseinCYP2D6activitywouldbemoresusceptible to acute toxic effects of MDMA has beenlacking. A previous metabolic bioactivation of MDMA,regulated partially by this enzyme, is needed to elicitneurotoxic effects in the central nervous system. It ispostulated that the chemical species involved inserotonergic neurotoxicity are catechol thiol conjugatemetabolites of the drug (HHMA) [8]. The functionalpolymorphisms in CYP2D6 could influence the devel-opment of MDMA neurotoxicity. During a trial de-signed to study the pharmacology of two consecutivedoses of MDMA [3], one subject was found to possessthe CYP2D6*4/*4 genotype and was classified as a PM.The objective of this paper was to compare the results ofthis subject with those of nine others previously pub-lished. Furthermore, the paper includes pharmacokineticdata on MDMA metabolites not previously published.

Immunomodulating properties of MDMA alone and in combination with alcohol: a pilot study.

Cell-mediated immune response after the administration of MDMA alone and in combination with alcohol was evaluated in a randomized, double-blind, double-dummy, cross-over pilot clinical trial conducted in four healthy MDMA consumers who received single oral doses of 75 mg MDMA (n = 2) or 100 mg MDMA (n = 2), alcohol (0.8 mg/kg), MDMA and alcohol, or placebo. Acute MDMA treatment produced a time-dependent immune dysfunction associated with MDMA plasma concentrations. Although total leukocyte count remained unchanged, there was a decrease in the CD4 T/CD8 T-cell ratio as well as in the percentage of mature T lymphocytes, probably because of a decrease in both the percentage and absolute number of T helper cells. The decrease in CD4 T-cell counts and in the functional responsiveness of lymphocytes to mitogenic stimulation was dose-dependent. The correlation between MDMA pharmacokinetics and the profile of MDMA-induced immune dysfunction suggests that alteration of the immune system may be mediated by the central nervous system. Alcohol consumption produced a decrease in T helper cells, B lymphocytes, and PHA-induced lymphocyte proliferation. Combined MDMA and alcohol produced the greatest suppressive effect on CD4 T-cell count and PHA-stimulated lymphoproliferation. Immune function was partially restored at 24 hours. These results provide the first evidence that recreational use of MDMA alone or in combination with alcohol alters the immunological status.

Immunomodulating Activity of MDMA

MDMA (3,4‐methylenedioxymethamphetamine) use can cause neurochemical, behavioral and endocrine alterations, similar to those produced by exposure to acute stress, suggesting its potential as a “chemical stressor.” It is known that stressful stimuli can produce a depression of immune function and an alteration in immune cells distribution. In vitro exposure to MDMA resulted in a modulation of several immune functional parameters such as T‐cell regulatory function, cytotoxic T‐lymphocyte activity, natural killer cell activity and macrophage function.

Abuse liability of flunitrazepam among methadone-maintained patients

Abstract Abuse liability and acute subjective and psychomotor effects of flunitrazepam were assessed in ten methadone-maintained males with history of benzodiazepine and alcohol use, who voluntarily participated in a double-blind, controlled, cross-over, randomized clinical trial. There were six experimental sessions in which a single oral dose of flunitrazepam 1, 2, and 4 mg; triazolam 0.5 and 0.75 mg; and placebo was given. Evaluations included physiological measures; psychomotor performance tasks (simple reaction time, Digit Symbol Substitution Test, balance task, Maddox-wing device); and self-administered subjective effects questionnaires [Addiction Research Center Inventory (ARCI), Profile of Mood States (POMS), a series of visual analog scales (VAS)]. All drugs but flunitrazepam 1 mg caused an impairment of psychomotor tasks. Effects were more evident with the highest doses of both drugs. Only flunitrazepam 4 mg produced a significant decrease in balance time. Triazolam 0.75 mg induced increases in sedation measured by ARCI-PCAG, depression in POMS, and VAS-drowsiness scores. Flunitrazepam 4 mg caused euphoria-related effects as measured by increases in ARCI-MBG and “high” scores in the VAS. Our findings of flunitrazepam-induced euphoria in methadone-maintained subjects together with epidemiological evidence of flunitrazepam abuse by opioid dependents, suggest that it may be included in the group of benzodiazepines with a relatively high abuse potential.