Introduction

Abstract Introduction Materials & Methods Results Discussion References

MeAmp (fig. 1) has recently reemerged as a significant drug abuse problem in the United States (1, 2). As approaches to treatment of addiction to this compound and its pharmacological congeners are being considered, characterization of the enzymatic nature of its metabolism becomes an important consideration. In early studies in humans, Caldwell et al. (3) showed that the urinary excretion products of this compound included the 4-hydroxy derivative (4-OH MeAmp, fig. 1) and the N-demethylation product, amphetamine (Amp, fig. 1), with 4-OH MeAmp as the dominant metabolite, representing almost 50% of all metabolites excreted (3). Metabolic oxidation of amphetamine derivatives has led to pharmacologically active species in the case of Amp (4), 4-Allyloxy MeAmp (5), and MDMA (fig. 1) (6) so the enzymatic nature of the reaction is of interest in terms of individual differences in drug effects and in potential drug interactions. As studies of the metabolism of MDMA (7), Amp (8), 4-Allyloxy MeAmp (5), and 4-Methoxy Amp (9) had shown that the oxidation of the aromatic ring and its substituents were catalyzed by CYP2D isozymes, studies investigating the role of the human isoform of this enzyme in MeAmp metabolism were initiated and the results are reported here.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Metabolic reactions of MDMA and MeAmp.

The studies used human microsomes in correlative studies with established substrates and recombinant CYP2D6, expressed in Saccharomyces cerevisae (10). The results show that 4-hydroxylation of MeAmp is indeed catalyzed by CYP2D6, the human isoform of CYP2D, and that this enzyme effects its N-demethylation as well. Kinetic studies with the expression system preparation indicated that while both reactions are catalyzed by CYP2D6, the 4-hydroxylation of MeAmp is much slower than MDMA demethylenation.

	Materials and Methods

Abstract Introduction Materials & Methods Results Discussion References

Chemicals. (+) and () MDMA hydrochloride and (+) MeAmp hydrochloride were obtained from the Research Technology Branch of the National Institute on Drug Abuse (Rockville, MD). () MeAmp hydrochloride was prepared from () ephedrine using the procedure of Kishi et al. (11). DHMA, the product of MDMA demethylenation, was prepared by modifying the procedure of Smissman and Borchardt (12). N-formyl 4-hydroxyamphetamine, prepared by procedures described earlier (5), was reduced to N-[²H₂]-Methyl-4-hydroxy-phenylisopropylamine (²H₂-4-OH MeAmp) with LiAl²H₄. [²H₃]-Amp was synthesized by the procedure of Gal (13). All other chemicals were obtained from commercial sources.

Source of CYP2D Enzymes and Preparation of Microsomes. Human livers. Samples of seven human livers were used with the approval of the Hospital Ethics Committee. Six of the livers (HL4, 5, 6, 7, 9, and 10) were from extensive CYP2D6 metabolizers and the seventh (HL3) from a poor CYP2D6 metabolizer. Clinical details of the donors are described elsewhere (14).

Yeast. The heterologous expression of human CYP2D6 in the yeast Saccharomyces cerevisiae and the preparation of microsomes have been described previously (10). The CYP2D6 wild-type cDNA employed has a valine and not methionine at position 374 (15).

Incubation Conditions. Incubation conditions for MDMA and MeAmp differed because of the instability of the MDMA metabolite, DHMA, in the presence of reactive oxygen species (6). The incubations of MDMA therefore used a reaction mixture consisting of 100 mM HEPES buffer (pH 7.4), superoxide dismutase (100 units), an NADPH generating system (0.5 mM NADP⁺, 8mM glucose-6-phosphate, 5 mM MgCl₂, and 1 unit of glucose-6-phosphate dehydrogenase), the microsome preparation (containing 33-65 pmol cytochrome P450), and substrate in a final volume of 1.0 ml (16). The reactions were initiated by the addition of the microsomal preparation and conducted at 37°C. Reactions were terminated by the addition of 0.5 ml of 7.5% (w/v) perchloric acid (HClO₄). The mixture was centrifuged and the supernatant refrigerated until HPLC analysis for DHMA.

GC/MS Analysis of Amp and 4-OH MeAmp. A 1 µL aliquot of the acetonitrile solution containing the dichloromethane extracts was injected into the GC/MS, which consisted of a Hewlett Packard 5971A system equipped with a methyl silicone (0.2 mm i.d., 0.33 micron film thickness, and 12 m length) capillary column. The gas chromatograph temperature program began at 65°C and was increased to 195°C at a rate of 25°C/min. Under these conditions, the retention time of the N,O,-bis trifluoroacetyl derivative of 4-OH MeAmp was 5.6 min and that of N-trifluoroacetylamphetamine was 4.0 min. The mass spectrometer was operated in its specific ion detection mode. Quantitation of Amp was achieved by comparing the mass spectrometer responses at M/Z = 140 and 143 (internal standard). The internal standard used in the assay was [²H₃]Amp of 99.9% isotopic purity.

Analysis of DHMA. The concentration of DHMA, the demethylenation product of MDMA, was determined in a 20 µL aliquot of the quenched incubation mixture by HPLC analysis using a Biophase ODS column, 4.6 × 250 mm (Bioanalytical Systems Inc., West Lafayette, IN) and an electrochemical detector (LC-4; Bioanalytical Systems Inc.). The mobile phase was 0.1 M citrate buffer (pH 3.5) containing 1 mM octyl sodium sulfate, acetonitrile and methanol in a volume ratio of 8:1:1 at a flow rate of 0.7 ml/min. The working electrode (LC-4) was set at 0.7 V relative to a Ag/AgCl reference electrode. The retention time for the catechol was 11.3 min. Concentrations were determined by comparison with a standard curve generated from authentic compound carried through the analysis (16).

Analysis of -Hydroxymetoprolol (-HM). The preparation of samples and the chromatography were carried out using modifications of previously described methods (17). After centrifugation of the precipitated microsomal protein, 0.45 ml of the supernatant was transferred to glass tubes containing 0.5 ml of 4 M NaOH and 5 ml of methyl-t-butyl ether. The tubes were vortex mixed for 2 min and then centrifuged at 1,500 rpm for 10 min. The organic layer was transferred to a glass test tube and evaporated to dryness under a nitrogen stream at room temperature. The residue was dissolved in 200 µL of mobile phase which had been adjusted to pH 3, of which 100 µL were injected onto the HPLC column.

Kinetics. Estimates of the enzymatic parameters for MeAmp and MDMA metabolism were obtained by nonlinear regression procedures using the AR program, a regression program, of the BMDP Statistical Software System (18).

	Results

Abstract Introduction Materials & Methods Results Discussion References

Six preparations of microsomes from human livers were compared for their ability to catalyze MeAmp metabolism (4-hydroxylation and N-demethylation) at a substrate concentration of 100 µM (fig. 2). The activities of MDMA demethylenation, at a substrate concentration of 10 µM (7), and metroprolol -hydroxylation (19), at a substrate concentration of 40 µM, of these preparations were determined also. The correlations between the amphetamine reactions and metroprolol -hydroxylation are shown in fig. 3, and values of the correlation coefficients for all of the reactions are shown in table 1. Both MDMA demethylenation and MeAmp 4-hydroxylation (the (+) isomer more than the ()) correlated with metoprolol -hydroxylation activity, and MDMA demethylenation and MeAmp hydroxylation correlated highly with each other. However, the N-demethylation of (+) MeAmp exhibited a lower but significant correlation with MDMA demethylenation and with MeAmp 4-hydroxylation.

View larger version (38K): [in this window] [in a new window]   Fig. 2.   Metabolism of MDMA and MeAmp by human liver microsomes. Reaction conditions are described in Methods. The substrate concentrations were 10 µM for MDMA demethylenation and 100 µM for MeAmp N-demethylation and 4-hydroxylation. The values represent the means ± SD of three determinations.

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Correlation between metoprolol -hydroxylation and MDMA, MeAmp oxidation by human livers. The rates shown are expressed as pico mols/min·(mg protein)1 with substrate concentrations of 10 µM (MDMA), 100 µM (MeAmp), and 40 µM (metoprolol).

The demethylenation of MDMA by human EM liver microsome preparations was also inhibited by quinidine (1µM), a selective and potent inhibitor of CYP2D6, to a level similar to that observed in the PM (HL3) preparation (fig. 4), consistent with previous work (7) and the metoprolol data that indicated participation by this enzyme in the reaction. These observations, indicated that CYP2D6 in human liver catalyzes MDMA demethylenation and, based on the correlative studies, the 4-hydroxylation of MeAmp. The correlation suggested also that CYP2D6 may also catalyze the N-demethylation of the (+) enantiomer of this substrate.

View larger version (50K): [in this window] [in a new window]   Fig. 4.   Effect of quinidine on MDMA demethylenation. MDMA (10 µM) was incubated with liver microsomes from four human subjects in the presence or absence of 1 µM quinidine according to the procedures described in Methods. The values are the means ± SD of three determinations.

Subsequent incubation of MeAmp with microsomes from yeast expressing CYP2D6 revealed formation of both 4-OH MeAmp and Amp. When the kinetic behavior of (+) and () MeAmp was examined over a substrate range of 0.005 to 0.5 mM, kinetic patterns consistent with a single enzyme were observed (fig. 5) with the parameters shown in table 2. The K_m values for the two reactions were in the micromolar range and the V_max values showed a different stereoselectivity, i.e. the value for N-demethylation was higher for the (+) enantiomer, whereas that for 4-hydroxylation was higher for the () enantiomer. Kinetic parameters for MDMA demethylenation, obtained in the same preparation, are also shown. N-demethylation of this substrate was not detected under these conditions.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Eadie-Hofstee plots of the 4-hydroxylation and N-demethylation of MeAmp enantiomers by microsomal preparations of yeast expressing CYP2D6. The reactions were carried out as described in Methods with substrate concentration varying from 5-500 µM. The lines are generated from parameter estimates obtained by nonlinear regression analysis of the kinetic data.

	Discussion

Abstract Introduction Materials & Methods Results Discussion References

The studies correlating MeAmp metabolism with MDMA demethylenation and metoprolol -hydroxylation, together with direct experiments with microsomes from yeast preparations expressing CYP2D6, show that MeAmp is a substrate that undergoes 4-hydroxylation and N-demethylation by this enzyme.

The hydroxylation of the aromatic ring of Amp is pharmacologically important, as it leads to 4-hydroxynorephedrine, a compound implicated in the tolerance to the peripheral actions of Amp (20, 21). The role of 4-hydroxylation in the central actions of Amp and MeAmp is not well established, but 4-OH Amp (22) and 4-OH MeAmp (23) accumulate in the striatum after administration of the parent drugs. The genetic polymorphism associated with CYP2D6 (17) and its role in the metabolism of other drugs such as the tricyclic antidepressants (24), serotonin uptake blockers (25), and beta blockers (17, 26) has resulted in significant individual differences in responses to some of these compounds. In addition, however, therapy with two or more CYP2D6 substrates can lead to significant bioavailability differences of each. As tricyclic antidepressants (27) and serotonin uptake blockers have been suggested as potential therapeutic approaches to MeAmp addiction, evaluation of the effectiveness of the treatment will require that pharmacokinetic interactions be considered.

Catalysis of N-dealkylation reactions by CYP2D6 has been reported for imipramine (28) -blockers (26, 29) and the MeAmp analog, deprenyl (30), but was not a significant reaction with the ring substituted analog, MDMA. The lower correlation of CYP2D6 activity with MeAmp N-demethylation compared with MeAmp 4-hydroxylation and MDMA demethylenation (table 1) is consistent with the participation of additional cytochromes P450 in MeAmp N-demethylation. The variable nature of CYP2D6 contribution to propranolol N-dealkylation has been reported earlier. The contribution of CYP2D6 to the catalysis of MeAmp N-demethylation in the liver preparations examined appears to be stereoselective, as the metabolism of the (+)-isomer correlates much more strongly with CYP2D6 activity than does the ()-isomer. This difference is not reflected in the kinetics for N-demethylation by recombinant CYP2D6, although the reaction does favor the (+)-isomer.

N-demethylation of MeAmp and deprenyl, which have single basic nitrogen atoms, appears to be a reaction inconsistent with the observation that most reactions catalyzed by CYP2D6 occur on carbon atoms at sites 5-7Å from the basic nitrogen atom (31). To account for this inconsistency, Grace et al. (30) suggest the possibility of alternative positioning in the active site, together with significant concentrations of the free base which undergoes electron transfer (30). Since deprenyl, which is N-propargyl MeAmp, is a weaker base than MeAmp, its free base would be present at higher concentrations than that of MeAmp. Consistent with this notion, the k_cat reported by them is about 10 times that found here for MeAmp. However, the stereoselectivity observed for deprenyl N-demethylation is opposite that found for MeAmp, so the positioning of the two substrates may be different. Alternatively, Guengerich (32) has suggested an N-dealkylation mechanism involving single electron transfer from the nitrogen atom, which may be operative over the range of about 5Å. A related question requiring further study is why MeAmp but not MDMA (7) undergoes significant N-demethylation by CYP2D6. One possibility may relate to the relative rates of oxidation. The high turnover of MDMA demethylenation relative to 4-hydroxylation of MeAmp may preclude alternate orientation within the active site. Although MeAmp and MDMA are both substrates for the same cytochrome P450 enzyme, the rates by which they are oxidized differ markedly, with MDMA oxidation taking place at almost 100 times the rate of MeAmp oxidation (see V_max/K_m, table 2). The basis for the difference is not clear, but oxidation of alkyl groups of ethers on the aromatic ring by CYP2D seems to be faster; we have observed that 4-alloxy MeAmp, also a substrate for CYP2D enzymes, is cleaved rapidly to 4-OH MeAmp in rat liver microsomes. The slow oxidation of the aromatic ring of MeAmp may contribute to the N-demethylation of this substrate by allowing its reorientation in the catalytic site, i.e. a form of "metabolic switching."