Elsevier

Toxicology Letters

Volumes 112–113, 15 March 2000, Pages 133-142
Toxicology Letters

Toxicokinetics and analytical toxicology of amphetamine-derived designer drugs (‘Ecstasy’)

https://doi.org/10.1016/S0378-4274(99)00207-6Get rights and content

Abstract

The phase I and II metabolites of the designer drugs methylenedioxyamphetamine (MDA), R,S-methylenedioxymethamphetamine (MDMA), R,S-methylenedioxyethylamphetamine (MDE), R,S-benzodioxazolylbutanamine (BDB) and R,S-N-methyl-benzodioxazolylbutanamine (MBDB) were identified by gas chromatography-mass spectrometry (GC-MS) or liquid chromotography-mass spectrometry (LC-MS) in urine and liver microsomes of humans and rats. Two overlapping pathways could be postulated: (1) demethylenation followed by catechol-O-methyl-transferase (COMT) catalyzed methylation and/or glucuronidation/sulfatation; (2) N-dealkylation, deamination and only for MDA, MDMA, MDE oxidation to the corresponding benzoic acid derivatives conjugated with glycine. Demethylenation was mainly catalyzed by CYP2D1/6 or CYP3A2/4, but also by CYP independent mechanisms. In humans, MDMA and MBDB could also be demethylenated by CYP1A2. N-demethylation was mainly catalyzed by CYP1A2, N-deethylation by CYP3A2/4. Based on these studies, GC-MS procedures were developed for the toxicological analysis in urine and plasma. Finally, toxicokinetic parameters are reviewed.

Introduction

The designer drugs methylenedioxyamphetamine (MDA), R,S-1-(3′,4′-methylenedioxyphenyl)-2-propanamine, ‘Love Pills’, R,S-methylenedioxymethamphetamine, ‘Adam’, ‘Ecstasy’ (MDMA) and R,S-methylenedioxyethylamphetamine; ‘Eve’ (MDE) as well as benzodioxazolylbutanamine (BDB); R,S-1-(1′,3′-benzodioxazol-5′-yl)-2-butanamine or R,S-1-(3′,4′-methylenedioxyphenyl)-2-butanamine and R,S-N-methyl-benzodioxazolylbutanamine (MBDB) are so-called entactogens (Nichols, 1986), producing feelings of euphoria and energy and a desire to socialize. This may explain their current popularity as ‘rave drugs’ (Hegadoren et al., 1999). Although these drugs have the reputation of being safe, several experimental studies in rats and humans and epidemiological studies indicated risks to humans. Recent reviews summarize the current knowledge on hepatotoxicity (Jones and Simpson, 1999) and neurotoxicity, psychopathology and abuse potential of such designer drugs (Hegadoren et al., 1999). Since metabolites are claimed to be responsible for the hyperthermic, the neuro- and/or hepatotoxic effects (Hiramatsu et al., 1990, Carvalho et al., 1996), detailed knowledge on the metabolism is necessary.

In vivo studies in humans showed two main metabolic pathways: demethylenation and N-dealkylation (Ensslin et al., 1996a, Maurer, 1996). In vitro studies in rats and humans were described indicating that the demethylenation of MDMA to the toxic catechols and/or their oxidation products was catalyzed by polymorphic CYP2D1/6 (Tucker et al., 1994, Lin et al., 1997, Wu et al., 1997). However, since CYP2D1/6 deficient rats could also form such catechols (Colado et al., 1995), other CYP isoenzymes and/or other enzymatic and/or nonenzymatic mechanisms should be responsible for the demethylenation in vivo (Kumagai et al., 1991). Therefore, it was studied which metabolic pathway is catalyzed in humans or rats by the main CYP isoenzymes CYP1A2, CYP2C11/9, CYP2D1/6, CYP3A2/4 and/or by cytosolic enzymes. A comparison of human and rat metabolism was necessary to prove the applicability of toxicological data from rats (Chu et al., 1996) to risk assessment in humans.

Designer drugs may lead to more or less severe intoxications (Walubo and Seger, 1999) and impairment to drive a car (Moeller and Hartung, 1997). Therefore, in clinical and forensic toxicology, these illicit drugs are often to be analyzed. Commercial amphetamine immunoassays (e.g. radioimmunoassay, Cody, 1990), enzyme immunoassays (Kunsman et al., 1990) or fluorescence polarization immunoassays (FPIA) (Kunsman et al., 1990, Ensslin et al., 1996b) were successfully used for screening of methylenedioxyamphetamine derivatives. For example, intake of 140 mg of MDE led to FPIA positive results up to 60 h after ingestion (Ensslin et al., 1996a). However, more specific procedures are necessary for confirmation and for differentiation, since non-scheduled therapeutics may also lead to positive results (Kraemer and Maurer, 1998). Confirmation and quantification methods using gas chromatography-mass spectrometry (GC-MS), HPLC, CE or TLC were recently reviewed (Kraemer and Maurer, 1998).

In the following, in vivo and in vitro studies on the metabolism of designer drugs necessary for the toxicological risk assessment will be presented as well as GC-MS screening, identification and quantification procedures necessary for clinical and forensic toxicology. For toxicological interpretation of the analytical results, toxicokinetic parameters were selected from the literature.

Section snippets

Chemicals and reagents

All chemicals were obtained from E. Merck, Darmstadt, Germany, Sigma–Aldrich, Deisenhofen, Germany, or Biomol, Hamburg, Germany, and deuterated standards from Promochem, Wesel, Germany.

GC-MS and liquid chromatography-mass spectrometry (LC-MS) apparatus

Hewlett Packard (HP, Waldbronn, Germany) 5890 Series II GC combined with an HP 5989B MS Engine (for details see Ref. Kraemer et al., 1997). MS conditions: full scan mode or single ion monitoring (SIM) mode; electron-impact (EI) mode or positive chemical ionization (PCI) mode using methane. Quantification in

Metabolism of designer drugs studied in human and rat urine

The metabolites detected in urine were identified by interpretation of the mass spectra and, if available, by comparison with synthesized reference substances (Ensslin et al., 1996b). PCI was used for confirmation of the molecular mass. As shown in Fig. 1, the methylenedioxyphenylalkylamine designer drugs undergo two overlapping metabolic pathways: O-demethylenation of the methylenedioxy group to dihydroxy derivatives followed by methylation of one of the hydroxy groups and successive

Conclusions

Comparison of rat and human metabolism (in vitro and in vivo) of designer drugs resulted in only minor differences concerning N-dealkylation and demethylenation. The toxicologically relevant demethylenation is catalyzed not only by the polymorphic CYP2D1/6, but also by the non-polymorphic main isoenzyme CYP3A as well as by CYP independent mechanisms. Therefore, possible hepatotoxic and neurotoxic effects due to demethylenation seem to be independent from genetic polymorphism.

The GC-MS

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