Hydrolysis of 3,4-methylenedioxymethamphetamine (MDMA) metabolite conjugates in human, squirrel monkey, and rat plasma.

Characterizing the formation of metabolites of 3,4-methylenedioxymethamphetamine (MDMA, "Ecstasy") in different species (rat, squirrel monkey, and human) may provide insight into mechanisms of MDMA neurotoxicity. Two prominent MDMA metabolites, 3,4-dihydroxymethamphetamine (HHMA) and 4-hydroxy-3-methoxymethamphetamine (HMMA), are conjugated with glucuronic or sulfuric acid, but reference standards are not available; therefore, quantification is only possible after conjugate cleavage. Different concentrations of HHMA and HMMA were obtained in human, squirrel monkey, and rat plasma specimens when acid or enzymatic cleavage was performed. Our data document that these differences are due to species-specific influences on conjugate cleavage. Acidic hydrolysis should be used for analyzing free HHMA and HMMA in human or squirrel monkey plasma, while enzymatic hydrolysis with glucuronidase or sulfatase maximizes recovery of free HHMA and HMMA in rat plasma. Optimization of cleavage conditions showed that sulfate conjugates were more readily cleaved by acid hydrolysis and glucuronides by glucuronidase.

Simultaneous liquid chromatographic-electrospray ionization mass spectrometric quantification of 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy) and its metabolites 3,4-dihydroxymethamphetamine, 4-hydroxy-3-methoxymethamphetamine and 3,4-methylenedioxyamphetamine in squirrel monkey and human plasma after acidic conjugate cleavage.

3,4-Methylenedioxymethamphetamine (MDMA, Ecstasy) is a psychoactive drug with abuse liability and neurotoxic potential. Specimen preparation of a recently presented LC-MS assay with electrospray ionization for quantifying MDMA and its main metabolites in squirrel monkey plasma was modified to include acidic hydrolysis to obtain total 3,4-dihydroxymethamphetamine and 4-hydroxy-3-methoxy-methamphetamine. Method re-validation for squirrel monkey plasma and full validation for human plasma showed selectivity for all analytes. Recoveries were > or = 71.0%. Changed specimen preparation or matrix did not affect accuracy or precision. No instability was observed after repeated freezing or in processed samples. Plasma MDMA and metabolites quantification, derived pharmacokinetic and toxicokinetic data and neurotoxicity research will benefit from this validated method.

Ambient temperature effects on 3,4-methylenedioxymethamphetamine-induced thermodysregulation and pharmacokinetics in male monkeys.

Changes in ambient temperature are known to alter both the hyperthermic and the serotonergic consequences of 3,4-methylenedioxymethamphetamine (MDMA). Metabolism of MDMA has been suggested to be a requisite for these neurotoxic effects, whereas the hyperthermic response is an important contributing variable. The aim of the present study was to investigate the interaction between ambient temperature, MDMA-induced thermodysregulation, and its metabolic disposition in monkeys. MDMA (1.5 mg/kg i.v.) was administered noncontingently at cool (18 degrees C; n = 5), room (24 degrees C; n = 7), and warm (31 degrees C; n = 7) ambient temperatures. For 240 min following MDMA administration, core temperature was recorded and blood samples were collected for analysis of MDMA and its metabolites 3,4-dihydroxymethamphetamine (HHMA), 3,4-dihydroxyamphetamine, and 3,4-methylenedioxyamphetamine (MDA). A dose of 1.5 mg/kg MDMA induced a hypothermic response at 18 degrees C, a hyperthermic response at 31 degrees C, and did not significantly change core temperature at 24 degrees C. Regardless of ambient temperature, plasma MDMA concentrations reached maximum within 5 min, and HHMA was a major metabolite. Curiously, the approximate elimination half-life (t(1/2)) of MDMA at 18 degrees C (136 min) and 31 degrees C (144 min) was increased compared with 24 degrees C (90 min) and is most likely because of volume of distribution changes induced by core temperature alterations. At 18 degrees C, there was a significantly higher MDA area under the concentration-time curve (AUC) and a trend for a lower HHMA AUC compared with 24 degrees C and 31 degrees C, suggesting that MDMA disposition was altered. Overall, induction of hypothermia in a cool environment by MDMA may alter its disposition. These results could have implications for MDMA-induced serotonergic consequences.

Validated liquid chromatographic-electrospray ionization mass spectrometric assay for simultaneous determination of 3,4-methylenedioxymethamphetamine and its metabolites 3,4-methylenedioxyamphetamine, 3,4-dihydroxymethamphetamine, and 4-hydroxy-3-methoxymethamphetamine in squirrel monkey plasma.

3,4-Methylenedioxymethamphetamine (MDMA) is a recreational drug with neurotoxic potential. Pharmacokinetic data of MDMA and its metabolites may shed light on the mechanism of MDMA neurotoxicity. An LC-MS assay with electrospray ionization (ESI) is presented for quantifying MDMA and its metabolites 3,4-methylenedioxyamphetamine (MDA), 3,4-dihydroxymethamphetamine (HHMA), and 4-hydroxy-3-methoxymethamphetamine (HMMA) in squirrel monkey plasma. The method involved enzymatic conjugate cleavage and protein precipitation. Separation was achieved within 14min. The method was validated according to international guidelines with respect to selectivity, linearity, accuracy, precision, recovery, and matrix effect. The present method should prove useful for acquiring pharmacokinetic and toxicokinetic data in squirrel monkeys.

High-performance liquid chromatography with electrochemical detection applied to the analysis of 3,4-dihydroxymethamphetamine in human plasma and urine.

Metabolic activation in the disposition of 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy") has been implicated in some of its pharmacological and toxicological effects, with the major metabolite 3,4-dihydroxymethamphetamine (HHMA) as a putative toxicant through the formation of thioether adducts. We describe the first validated method for HHMA determination based on acid hydrolysis of plasma and urine samples, further extraction by a solid-phase strong cation-exchange resin (SCX, benzenesulfonic acid), and analysis of extracts by high-performance liquid chromatography with electrochemical detection. The chromatographic separation was performed in an n-butyl-silane (C4) column and the mobile phase was a mixture of 0.1 M sodium acetate containing 0.1 M 1-octanesulphonic acid and 4 mM EDTA (pH 3.1) and acetonitrile (82:18, v/v). Compounds were monitored with an electrochemical cell (working potentials 1 and 2, +0.05 and +0.35 V, respectively, gain 60 microA). A mobile phase conditioning cell with a potential set at +0.40 V was connected between the pumping system and the injector. Calibration curves were linear within the working concentration ranges of 50-1000 microg/L for urine and plasma. Limits of detection and quantification were 10.5 and 31.8 microg/L for urine and 9.2 and 28.2 microg/L for plasma. Recoveries for HHMA and DHBA (3,4-dihydroxybenzylamine, internal standard) were close to 50% for both biological matrices. Intermediate precision and inter-day accuracy were within 3.9-6.5% and 7.4-15.3% for urine and 5.0-10.8% and 9.2-13.4% for plasma.

Determination of MDMA and its metabolites in blood and urine by gas chromatography-mass spectrometry and analysis of enantiomers by capillary electrophoresis.

A gas chromatography-mass spectrometry (GC-MS) method was used for the simultaneous quantitation of 3,4-methylenedioxymethamphetamine (MDMA) and the 3,4-methylenedioxyamphetamine (MDA), 4-hydroxy-3-methoxymethamphetamine (HMMA), and 4-hydroxy-3-methoxyamphetamine (HMA) metabolites in plasma and urine samples after the administration of 100 mg MDMA to healthy volunteers. Samples were hydrolyzed prior to a solid-phase extraction with Bond Elut Certify columns. Analytes were eluted with ethyl acetate (2% ammonium hydroxide) and analyzed as their trifluoroacyl derivatives. Linear calibration curves were obtained at plasma and urine concentration ranges of 25-400 ng/mL and 250-2000 ng/mL for MDMA and HMMA, and of 2.5-40 ng/mL and 100-1000 ng/mL for MDA and HMA. Following the same urine preparation procedure but without the derivatization step, a capillary electrophoresis (CE) method for enantiomerical resolution of compounds was developed using (2-hydroxy)propyl-beta-cyclodextrin at two different concentrations (10 and 50mM in 50mM H3PO4, pH 2.5) as chiral selector. Calibration curves for the CE method were prepared with the corresponding racemic mixture and were linear between 125 and 2000 ng/mL, 50 and 1000 ng/mL, and 125 and 1500 ng/mL for each enantiomer of MDMA, MDA, and HMMA, respectively. Stereoselective disposition of MDMA and MDA was confirmed. HMMA disposition seems to be in apparent contradiction with MDMA findings as the enantiomer ratio is close to 1 and constant over the time.

3,4-Dihydroxymethamphetamine (HHMA). A major in vivo 3,4-methylenedioxymethamphetamine (MDMA) metabolite in humans.

There is evidence that some heavy users of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) show signs of neurotoxicity (a cognitive dysfunction, a larger incidence of psychopathology). It has been postulated that the catechol intermediates of methylenedioxyamphetamines such as 3,4-dihydroxymethamphetamine (HHMA), a metabolite of MDMA, may play a role in their neurotoxicity by formation of thioether adducts. This study describes the first validated method for HHMA determination in plasma and urine by strong cation-exchange solid-phase extraction high-performance liquid chromatography/electrochemical detection (HPLC/ED) analysis. The method has been applied for the determination of HHMA in plasma and urine samples from a clinical study in healthy volunteers of MDMA and provides preliminary kinetic data on this metabolite. HHMA appeared to be a major MDMA metabolite with plasma concentrations as high as the parent compound. Thus, HHMA C(max) (154.5 microg/L) and AUC(0-24h)(1990.9 microg/L h) were similar to those obtained in previously published reports for MDMA (181.6 microg/L and 1465.9 microg/L h, respectively). The 24-h urinary recovery of HHMA accounted for 17.7% of the MDMA dose administered and increases the total 24 h recovery of MDMA and metabolites to 58% of the 100 mg dose administered. The determination of HHMA in plasma and urine samples is of interest in order to establish its relevance in MDMA metabolism and its possible contribution to MDMA neurotoxicity in humans. Its validation showed appropriate accuracy and precision for its use in pharmacokinetic studies.

Determination of catecholamines in sheep plasma by high-performance liquid chromatography with electrochemical detection: comparison of deoxyepinephrine and 3,4-dihydroxybenzylamine as internal standard.

3,4-Dihydroxybenzylamine (DHBA) is commonly used as the internal standard in HPLC catecholamine assays. Sheep are frequently used in studies of cardiovascular physiology and in such studies measurement of catecholamines is important. The recovery of DHBA from sheep plasma is, however variable and poor. Therefore, we have developed a reliable and sensitive HPLC-ED method with alumina extraction for measurement of catecholamines in sheep plasma using deoxyepinephrine (epinine) as the internal standard. Separation was performed on a muBondapak C18 column (300x3.9 mm, 10 microm) with a mobile phase containing 2% acetonitrile and 98% buffer (0.05% sodium acetate-0.02% EDTA-0.013% sodium heptanesulfonate), pH 3.25. The extraction of epinine from water, human plasma, dog plasma and sheep plasma did not differ (p>0.05), but extraction of DHBA from sheep plasma was significantly impaired (p<0.0001). The R2 of regression curves (n=5) of norepinephrine (NE) (25.02 pg/ml-1.00 ng/ml) and epinephrine (E) (25.82 pg/ml-1.03 ng/ml), using epinine as internal standard were greater than 0.99. The intra- and inter-day coefficients of variation were 2.11-11.15 and 0.88-12.60% for NE and 1.12-10.91 and 2.88-12.60% for E, respectively. The detection limits for NE and E are 12 pg/ml. The technique described has the advantage that it allows the simultaneous determination of both endogenous and [3H]norepinephrine in sheep plasma using a sensitive and reproducible HPLC technique.

Analysis of 3,4-methylenedioxymethamphetamine (MDMA) and its metabolites in plasma and urine by HPLC-DAD and GC-MS.

In Europe, the compound 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy, Adam), in addition to cannabis, is the most abused illicit drug at all-night "techno" parties. Methods for the determination of MDMA and its metabolites, 4-hydroxy-3-methoxymethamphetamine (HMMA), 3,4-dihydroxy-methamphetamine (HHMA), 3,4-methylenedioxyamphetamine (MDA), 4-hydroxy-3-methoxyamphetamine (HMA), and 3,4-dihydroxyamphetamine (HHA), in biological fluids were established. Plasma and urine samples were collected from two patients in a controlled clinical study over periods of 9 and 22 h, respectively. MDMA and MDA were determined in plasma and urine by reversed-phase high-performance liquid chromatography with diode array detection (HPLC-DAD) after solid-phase extraction on cation-exchange columns. Acidic or enzymatic hydrolysis was necessary to detect HMMA, HMA, HHMA, and HHA, which are mainly excreted as glucuronides. Gas chromatography-mass spectrometry (GC-MS) was used for confirmation. Sample extraction and on-disc derivatization with heptafluorobutyric anhydride (HFBA) were performed on Toxi-Lab SPEC solid-phase extraction concentrators. After administration of a single oral dose of 1.5 mg/kg body weight MDMA, peak plasma levels of 331 ng/ml MDMA and 15 ng/mL MDA were measured after 2 h and 6.3 h, respectively. Peak concentrations of 28.1 micrograms/mL MDMA in urine appeared after 21.5 h. Up to 2.3 micrograms/mL MDA, 35.1 micrograms/mL HMMA, and 2.1 micrograms/mL HMA were measured within 16-21.5 h. Conjugated HMMA and HHMA are the main urinary metabolites of MDMA.

High activity of semicarbazide-sensitive amine oxidase (SSAO): an important source of errors in the determination of the concentration of dopamine in pig plasma.

We noted rapid breakdown at 4 degrees and 20 degrees C of dopamine (DA) (but not of (nor)epinephrine and epinine) in pig plasma, but not in human plasma. The enzyme responsible appears to be a semicarbazide-sensitive amine oxidase (SSAO) because the breakdown can be inhibited by semicarbazide, but not by pargyline, clorgyline, EDTA, or (extra) glutathione. Among catecholamines tested, only DA and 3,4-dihydroxybenzylamine (DHBA, the internal standard of most catecholamine assays using high-performance liquid chromatography (HPLC) with electrochemical detection) were good substrates for the pig plasma SSAO. At 37 degrees C, especially after prolonged storage, all catecholamines break down. This breakdown results from autoxidation since it can be prevented by addition of extra glutathione (but not by semicarbazide) for all catecholamines except DA and DHBA. Breakdown at 37 degrees C of these two compounds cannot be prevented by addition of extra glutathione or semicarbazide, but only by addition of both. For reliable measurements of DA concentrations in pig plasma, blood should be collected in tubes containing not only glutathione, but also semicarbazide. The possibility of similarly high plasma SSAO activity in other species should be investigated further.

Effects of ibopamine on postural hypotension in pure autonomic failure.

We wished to determine whether ibopamine, a dopaminergic prodrug with weak agonist activity on alpha- and beta-adrenoceptors, improves orthostatic tolerance in autonomic insufficiency. Three subjects with severe orthostatic hypotension resulting from pure autonomic failure (PAF) were studied. Direct arterial blood pressure (ABP) and heart rate (HR) were recorded continuously. Orthostatic tolerance was evaluated by 60 degrees passive head-up tilting. Tilting was performed before and after a single oral 100-mg dose of ibopamine. Blood samples for measurement of plasma catecholamines, free epinine (the active metabolite of ibopamine), and conjugated epinine were taken at regular intervals. In all 3 subjects, orthostatic tolerance was greatly improved by ibopamine. This improvement occurred as soon as 10-30 min after administration of ibopamine and lasted 20-50 min. alpha-Adrenoceptor blockade with phentolamine abolished the effect of ibopamine. The interindividual pharmacokinetics of ibopamine varied considerably: Peak plasma concentrations of ibopamine in the three subjects were 2.8, 4.5, and 35.4 ng/ml, respectively. The high level of epinine in one patient was associated with severe hypertension and tachycardia. Ibopamine may be a valuable new pharmacologic treatment for orthostatic hypotension in PAF, but in light of the highly variable interindividual pharmacokinetics further studies must be performed before use of the compound can be advocated in this disorder.

Lack of desensitization of alpha- and beta-adrenoceptor function during chronic treatment of healthy volunteers with ibopamine, an orally active dopamine receptor agonist.

In 18 healthy volunteers, in a double-blind placebo-controlled study, we investigated of whether 14 days treatment with a therapeutic dose of ibopamine (3 x 100 mg/day p.o.), respectively its active metabolite epinine, would desensitize lymphocyte beta 2- or platelet alpha 2-adrenoceptors, or alpha 1- and beta-adrenoceptor mediated (phenylephrine- and isoprenaline infusions, respectively), changes in systolic and diastolic blood pressure and heart rate. Ibopamine-treatment, which resulted in peak plasma epinine concentrations of 4-5 nmol.l-1, neither affected resting heart rate or blood pressure, nor any of the alpha- or beta-adrenoceptor parameters measured. Since in man in general long-term administration of alpha- and beta-adrenoceptor agonists desensitizes alpha- and beta-adrenoceptors, the lack of any alpha- and beta-adrenoceptor desensitizing effect of ibopamine suggests that, in the dose employed (3 x 100 mg per day), ibopamine does not exert alpha- or beta-adrenoceptor agonistic effect in humans.

Effects of ibopamine on exercise-induced increase in norepinephrine in normal men.

The effects of 100 mg ibopamine, an orally active aselective dopamine (DA) agonist, on plasma catecholamines was evaluated in 8 healthy men during sympathetic stimulation by graded exercise in a single-blind, placebo-controlled cross-over study. The exercise consisted of progressive cycling activity less than or equal to 90% of the previously determined VO2max. Graded exercise resulted in an increase in systolic and mean blood pressure (SBP, MBP), heart rate, norepinephrine (NE) and epinephrine level, with a decrease in diastolic BP (DBP). The increase in NE was significantly blunted by ibopamine as compared with placebo. No differences for BP, heart rate (HR), or epinephrine between placebo- and ibopamine study day were noted. In previous studies, ibopamine decreased resting plasma NE in patients with congestive heart failure (CHF), whereas plasma NE was not altered by ibopamine in healthy volunteers. This different outcome in both categories might therefore be explained by the absence of substantial sympathetic stimulation in normal humans at rest. Because it is reasonable to assume that the effect of ibopamine on systemic and local hemodynamics is negligible as compared with the effect of exercise in the healthy volunteers, the plasma decrease caused by ibopamine is probably related to stimulation of DA2-receptors. In conclusion, ibopamine blunts the increase of plasma NE during graded exercise in healthy men.

Simultaneous determination of free catecholamines and epinine and estimation of total epinine and dopamine in plasma and urine by high-performance liquid chromatography with fluorimetric detection.

Epinine (N-methyldopamine) is the pharmacologically active hydrolysis product of the prodrug ibopamine, which is currently being widely studied for the treatment of congestive heart failure. This paper reports a sensitive and reliable method for the simultaneous determination of free catecholamines and epinine in plasma and urine. The compounds are isolated from plasma or urine by a specific liquid-liquid extraction, derivatized with the selective fluorogenic agent 1,2-diphenylethylenediamine, and quantitated by high-performance liquid chromatography with gradient elution and fluorimetric detection. The limits of detection for the derivatized catecholamines and epinine are 0.3-0.6 pg of injected compound. Intra- and inter-assay coefficients of variation of all four compounds are good (1-8%), as are the accuracy and linearity. A method is also reported for the determination of total dopamine and epinine in plasma and urine based on the same principle. This method, in which deconjugation is accomplished by acid hydrolysis at 95 degrees C, also shows good sensitivity and reproducibility.

Efficacy of ibopamine treatment in patients with advanced heart failure: purpose of a new therapeutic scheme with multiple daily administrations.

Eighteen patients with severe unstable heart failure who presented with hemodynamic deterioration after withdrawal of i.v. dopamine were treated with high doses of ibopamine (100 mg eight times a day, orally) in association with digitalis, diuretic, and vasodilator therapy. Ibopamine, used at these dosages, could effectively substitute for the i.v. infusion of dopamine in 15 of the 18 studied patients. Each patient was studied by right heart Swan-Ganz catheterization and two-dimensional echocardiography before and during ibopamine infusion, after dopamine withdrawal, and after acute ibopamine administration; measurements were then made after 3 months of chronic ibopamine treatment (100 mg eight times a day). Blood samples for plasma dopamine and epinine concentrations were obtained at the time of the hemodynamic measurements. Both drugs did not induce any significant change in arterial pressure whereas heart rate was slightly reduced after dopamine and chronic ibopamine. Cardiac index and stroke volume index were significantly increased by dopamine (from 1.81 to 2.24 L/min/m2 and from 20.3 +/- 6.5 to 27.7 +/- 8.1 ml/beat/m2) and acute and chronic ibopamine (from 1.80 to 2.15 and 2.23 L/min/m2 and from 22.4 +/- 6.8 to 28.2 +/- 7.5 and 30.3 +/- 7.0 ml/beat/m2, respectively); these changes were attended by a decrease in systemic vascular resistance; pulmonary pressures were not significantly modified. No significant change of the echocardiographic parameters was noted. During dopamine infusion, mean dopamine plasma concentrations were 211.8 +/- 32.4 pmol/ml; after acute and repeated administration of ibopamine, free epinine plasma concentrations averaged 45.1 +/- 14.5 and 62.2 +/- 12.7 pmol/ml, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Ibopamine kinetics after single and multiple dosing in patients with congestive heart failure.

The pharmacokinetics of ibopamine after single and multiple dosing was studied in 20 patients with congestive heart failure (CHF) of NYHA functional class II. Ibopamine 100 mg was given 3 times a day for 7 days in 6 patients and for 30 days in the other 14 patients. Plasma pharmacokinetics of total (mainly conjugated) and free epinine was studied after the first dose and on the 3rd, 7th and 30th days of treatment. The urinary recoveries of total epinine, HVA and DOPAC were measured in 5 patients for 24 h after ibopamine ingestion on the 1st and 30th days. Plasma pharmacokinetics of ibopamine did not vary during the repeated administration of the drug. In the course of the treatment, total epinine elimination t1/2 showed no significant variations. The build-up of Cmax, Cmin and AUC of total epinine observed after multiple dosing was as expected on the basis of the interval adopted between the doses of ibopamine and of the elimination t1/2 of total epinine. Pharmacokinetic parameters of free epinine did not show significant variations during the course of the treatment. The amounts of HVA and DOPAC recovered in urine on the 1st and 30th days of treatment were similar while the amount of total epinine was greater on the 30th day, the increment mainly reflecting a partial carry over of the less rapidly excreted conjugated epinine from the last previous doses. The results obtained for free epinine plasma levels and for the urinary recoveries of ibopamine metabolites thus indicated that no saturation of the enzymes involved in ibopamine metabolism occurred.

Ibopamine (SK&F 100168) pharmacokinetics in relation to the timing of meals.

The effect of the timing of a standard meal relative to a single oral dose of 200 mg ibopamine, on the appearance of its pharmacologically active metabolite, epinine, in plasma was investigated in a randomised crossover study in 12 healthy volunteers. After a 12 h fast, ibopamine was administered either in the fasting state (no meal), or 1 h before, 0.5 h before, immediately after, 2 h after or 3 h after a standardised meal. Blood samples taken immediately before and at intervals for 3 h after dosing were analysed for free epinine. Maximum concentration (Cmax), time to Cmax(tmax), and area under the concentration-time curve (AUC) for free epinine in plasma were calculated. When compared with the fasting state, Cmax and AUC0-3h were significantly reduced when ibopamine was given immediately after or 2 h after a meal. AUC was also reduced for ibopamine given 0.5 h before a meal. tmax was significantly delayed when ibopamine was given immediately after, or 2 or 3 h after a meal. Thus, administration of ibopamine with or shortly after a meal reduced the rate and extent of appearance of free epinine in plasma. The clinical significance of reduced epinine levels on acute dosing in the presence of food is unknown.