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Department of Experimental and Clinical Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, University of Saarland, Homburg (Saar), Germany
(Received October 30, 2003; Accepted January 13, 2004)
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Abstract |
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 Top Abstract Materials and MethodsResults and DiscussionReferences |
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). PMMA and its N-demethylated analog para-methoxyamphetamine [IUPAC: 1-(4-methoxyphenyl)propane-2-ylazane] have been made responsible for a large number of fatalities all over the world (Felgate et al., 1998; James and Dinan, 1998; Bach et al., 1999; Kraner et al., 2001; Ling et al., 2001; Martin, 2001; Johansen et al., 2003). PMMA shares considerable pharmacological similarities with methylenedioxymethamphetamine (MDMA), but it lacks the amphetaminergic stimulant component of MDMA. It produced stimulant effects similar to N-methylbenzodioxolylbutanamine [IUPAC: [1-(1,3-benzodioxol-5-yl)butan-2-yl](methyl)azane] and dimethoxyamphetamine (1-(3,4-dimethoxyphenyl)-2-aminopropane; IUPAC: 1-(2,5-dimethoxyphenyl)propane-2-ylazane) (Glennon et al., 1988, 1997; Glennon and Higgs, 1992; Young et al., 1999; Rangisetty et al., 2001). The only data available on the effects in humans have been published by Shulgin (1991), who described its effects as somewhat different from those of MDMA. Steel et al. (1992) showed long-term (possibly neurotoxic) effects on brain serotonin neurons for PMMA, although less potent compared with neurotoxic effects of MDMA.
Previous in vivo studies in the rat have shown that PMMA is metabolized mainly by O-demethylation to para-hydroxymethamphetamine (HO-MA) (Fig. 1) (Staack et al., 2003). The aim of the current study was to identify the human hepatic cytochrome P450 (P450) enzymes involved in the O-demethylation, to determine the kinetic constants for this metabolic reaction, and to compare the metabolite formation in pooled human liver microsomes (pHLMs) and in single donor human liver microsomes with CYP2D6 poor-metabolizer genotype (PM HLMs).
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Materials and Methods |
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TopAbstractMaterials and MethodsResults and DiscussionReferences |
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Microsomal Incubations. Incubation mixtures (final volume, 50 µl) consisted of 90 mM phosphate buffer (pH 7.4), 5 mM Mg2+, 5 mM isocitrate, 1.2 mM NADP+, 0.5 U/ml isocitrate dehydrogenase, 200 U/ml superoxide dismutase, and substrate at 37°C. The substrate was added after dilution of a 250 mM methanolic stock solution in buffer. The methanol concentration did not exceed 0.4% in any of the samples. Reactions were started by addition of the ice-cold microsomes and terminated with 5 µl of 60% (w/w) HClO4. After addition of 1 µl of 0.5 mM HO-AM as internal standard (IS), the samples were centrifuged, and the supernatants were transferred to autosampler vials.
Initial Screening Studies. To investigate the involvement of particular P450 enzymes in PMMA metabolism, 100 µM PMMA and 50 pmol/ml CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, or CYP3A4 were incubated for 30 min. For incubations with CYP2A6 or CYP2C9, phosphate buffer was replaced with 45 mM or 90 mM Tris buffer, respectively.
Kinetic Studies. Duration of and protein content for all incubations were in the linear range of metabolite formation (data not shown). Kinetic constants were derived from incubations with PMMA concentrations between 2.5 and 400 µM (n = 2, each).
pHLM and cDNA-expressed P450 protein contents were 0.8 mg/ml and 20 pmol P450/ml, respectively. Incubation times were 25 and 2 min for pHLMs and cDNA-expressed P450s, respectively. Less than 20% of substrate was metabolized in all incubations. Kinetic profiles and values were first estimated by visual inspection of Eadie-Hofstee plots, since deviations from simple enzyme Michaelis-Menten kinetics could be readily seen (Clarke, 1998). Apparent Km and Vmax values were estimated by nonlinear regression according to the Michaelis-Menten equation: V = Vmax x [S]/(Km + [S]).
The kinetic data were estimated using GraphPad Prism 3.02 software (GraphPad Software Inc., San Diego, CA).
Chemical Inhibition Studies. The effect of 1 µM quinidine on HO-MA formation was assessed in incubations containing 0.8 mg of pHLM protein/ml and 40 µM PMMA. Controls contained no quinidine, but the same amount of methanol (<<0.1%) to control for any solvent effects (n = 3 each). Significance of inhibition was tested by a one-tailed unpaired t test.
Comparative Studies between pHLMs and PM HLMs. Incubations were carried out at 40 µM PMMA for 25 min using 0.8 mg of either pHLM or PM HLM protein/ml. Significance of differences in metabolite formation was tested by a one-tailed unpaired t test.
Liquid Chromatography-Mass Spectrometry (LC-MS) Conditions and Quantification of Metabolite. PMMA, HO-MA, and HO-AM were separated and quantified using an Agilent Technologies (Waldbronn, Germany) AT 1100 series atmospheric pressure chemical ionization electrospray LC-MSD, SL version, and an LC-MSD ChemStation using A.08.03 software.
LC conditions. Gradient elution was achieved on a Supelco Discovery HS F5 column (15 cm x 2.1 mm, 3 µm; Supelco, Bellefonte, PA). The mobile phase consisted of ammonium formate (5 mM, adjusted to pH 3 with formic acid) (eluent A) and acetonitrile (eluent B). The gradient and the flow rate were as follows: 0 to 3.5 min 20% B (flow, 0.4 ml/min), 3.5 to 7.5 min 90% B (flow, 0.6 ml/min), 7.5 to 9 min 20% B (flow, 0.9 ml/min), and 9 to 10 min 20% B (flow, 0.4 ml/min). The injection volume was 7 µl.
Electrospray conditions. The following atmospheric pressure chemical ionization inlet conditions were applied: drying gas (7000 ml/min, 300°C) and nebulizer pressure (25 psi) (both nitrogen); capillary voltage, 4000 V; drying gas temperature set at 300°C, vaporizer temperature set at 400°C; corona current was 5.0 µA; positive selected-ion monitoring mode; and fragmentor voltage, 100 V.
MS conditions. For quantification, the following target ions (m/z) were used in the selected-ion monitoring mode: 152 for HO-AM (IS), 166 for HO-MA, and 180 for PMMA.
Metabolite quantification. Calibration curves were constructed plotting peak area ratios (HO-MA versus IS) of spiked calibrators versus their concentrations (0.5, 1, 2.5, 5, 7.5, 10, 15, 20 µM). Quantification was carried out using a simple linear regression model.
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Results and Discussion |
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TopAbstractMaterials and MethodsResults and DiscussionReferences |
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Initial Screening Studies. Among the nine P450 enzymes tested for possible HO-MA formation from PMMA, CYP2D6 was the only P450 capable of catalyzing this reaction.
Kinetic Studies. All incubations were carried out at initial rate conditions, a prerequisite for Michaelis-Menten kinetics. CYP2D6 showed a typical hyperbolic metabolite formation profile (Fig. 2A). The apparent Km value for CYP2D6 was determined to be 4.6 ± 1.0 µM, and Vmax was calculated to be 92.0 ± 3.7 pmol/min/pmol P450. The kinetic parameter for incubations using pHLMs was determined to be: apparent Km, 42.0 ± 4.0 µM and Vmax, 412.5 ± 10.8 pmol/min/mg protein. The Michaelis-Menten and Eadie-Hofstee plots (Fig. 2, B and C) for the incubations with pHLMs did not indicate biphasic kinetics, which is consistent with the finding of the initial screening studies that only one P450 was involved in the O-demethylation of PMMA. Apparent Km values for CYP2D6 and for pHLM differed somewhat from one another. This may be due to the fact that nonspecific protein binding was greater in pHLMs (0.8 mg/ml) compared with CYP2D6 microsomes (0.3 mg/ml). Such nonspecific binding to phospholipid membranes has been described for lipophilic and cationic compounds at pH 7.4, which should be the case for PMMA (Margolis and Obach, 2003).
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Chemical Inhibition Studies. To underline the importance of CYP2D6 in PMMA metabolism, the CYP2D6 specific inhibitor quinidine (1 µM) was added to incubation mixtures, and the rate of metabolite formation was compared with incubations without the inhibitor. The concentration of the inhibitor was based on average literature data (Clarke 1998). The inhibition experiments were performed with 40 µM PMMA, a substrate concentration that corresponds to the calculated Km value in pHLM. HO-MA formation was significantly inhibited by 67.2 ± 0.6% (p < 0.0001), which was further evidence of the involvement of CYP2D6 in the monitored reaction.
Comparative Studies between pHLMs and PM HLMs. To point out the importance of CYP2D6 in PMMA O-demethylation and to demonstrate differences in CYP2D6 poor metabolizer (PM) and CYP2D6 extensive metabolizer (EM), the metabolite formation rate of pHLM was compared with that of PM HLM. The metabolite formation rate in PM HLMs was significantly lower (87.3 ± 1.1%; p < 0.0001) than that of pHLMs. This remarkable difference in metabolite formation between PM and EM is further proof of the involvement of CYP2D6 in the major metabolic step of PMMA. The study design used has already successfully been applied for investigations on the metabolism of other designer drugs (Springer et al., 2003a,b).
The results of the current study are in accordance with the results of studies on the involvement of CYP2D6 in the O-demethylation of other methoxy-substituted amphetamine derivatives (Coutts et al., 1994; Wu et al., 1997; Bach et al., 1999). Our studies showed that CYP2D6 is the major P450 involved in O-demethylation of PMMA, and significant differences between PMs and EMs could be shown in vitro with PMs exhibiting lower metabolic clearances. PMMA might also be susceptible to drug-drug interactions with potent CYP2D6 inhibitory drugs leading to a decreased clearance of PMMA and elevated plasma concentrations. Of course, it cannot be concluded that these genetic polymorphisms and drug interactions are actually of clinical relevance. This would require in vivo studies in humans. Furthermore, it would be of particular interest to study whether a correlation between the reported fatalities and their CYP2D6 genotype exists.
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Acknowledgments |
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Footnotes |
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Address correspondence to: Prof. Dr. Hans H. Maurer, Department of Experimental and Clinical Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, University of Saarland, Building 46, D-66421 Homburg (Saar), Germany. E-mail: hans.maurer{at}uniklinik-saarland.de
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References |
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TopAbstractMaterials and MethodsResults and DiscussionReferences |
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)-pyrrolidinopropiophenone, a novel scheduled designer drug, in human liver microsomes. Drug Metab Dispos 31: 979-982.
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