Comparative in vitro studies of the metabolism of six 4-substituted methamphetamines and their inhibition of cytochrome P450 2D6 by GC-MS with trifluoroacetyl derivatization

Use of new amphetamine-type stimulants (ATS) as designer drugs is a serious problem worldwide. ATS are used in tablet, capsule, and powder forms, and can be mixed with other drugs. There is little information available on how these new drugs are metabolized or their ability to inhibit the metabolism of co-administered drugs. This study aimed to investigate the metabolism of six 4-substituted analogs of methamphetamine (MA), and their potential inhibition of MA metabolism. The metabolism of MA and the 4-substituted MAs was examined in vitro using human metabolic enzymes. Metabolite analyses were performed using trifluoroacetyl derivatization and GC-MS. The experiments showed that cytochrome P450 2D6 (CYP2D6) was involved in the major metabolic pathway of MA, where it catalyzed N-demethylation of 4-fluoromethamphetamine (4-FMA), 4-chloromethamphetamine (4-CMA), 4-bromomethamphetamine (4-BMA), 4-iodomethamphetamine (4-IMA) and 4-nitromethamphetamine (4-NMA), and O-demethylation of 4-methoxymethamphetamine (4-MMA). The half maximal inhibitory concentration (IC50) values for CYP2D6 using MA as substrate were different for each of the 4-substituted MAs. The strongest inhibitors of amphetamine production from MA were, in order, 4-IMA, 4-BMA, 4-CMA, 4-MMA, 4-FMA, and 4-NMA. The same order was observed for the IC50 values for inhibition of p-hydroxymethamphetamine production from MA, except for the IC50 of 4-MMA. The IC50 values of 4-IMA were lower than the IC50 values of fluoxetine and higher than that of quinidine. The results of this study imply that the risk of illicit drug interactions fluctuates so widely that unintentional fatal drug poisonings could occur.


Introduction
Designer drugs are a serious problem worldwide, especially among young people, and in some cases they have resulted in fatal poisoning [1][2][3][4][5][6][7]. New designer drugs are created by changing the molecular structure of an existing drug. This creates a compound with similar pharmacological effects, and is performed to circumvent existing drug laws. For example, 4-methoxymethamphetamine (4-MMA) and 4-fluoromethamphetamine (4-FMA), which are 4-substituted psychoactive analogs of methamphetamine (MA), have emerged on the illicit drug market [1,2,[8][9][10][11]. Designer drugs such as illicit amphetamine-type stimulants are often co-administered with other drugs in tablet, capsule, or powder form [12][13][14][15][16][17]. The emergence of new designer drugs and co-administration of drugs have made it difficult to discriminate controlled substances in forensic samples, and have increased drug-related problems.
In general, the most common types of metabolic drugdrug interactions are inhibition and induction of drug metabolizing enzymes. These interactions can increase or decrease drug exposure when two or more drugs are co-administered, and this can enhance drug toxicity [20]. However, there is little information available about the ability of designer drugs to inhibit the metabolism of the other drugs administered. Evaluation of the potential risk of metabolic drug-drug interactions is important. In this investigation, in vitro experiments with human metabolic enzymes were used to study the major metabolites of six 4-substituted MAs and their ability to inhibit the metabolism of MA.

Synthesis of Standards
Five 4-substituted analogs of MA and five 4-substituted analogs of AP were synthesized. They were obtained by reductive amination of 4-substituted phenylacetones in methanol with sodium cyanoborohydride [21]. The synthesized compounds were ascertained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) or liquid chromatographyelectrospray ionization-time-of-flight mass spectrometry (LC-ESI-TOF-MS).
4-FMA: A mixture of 4-fluorophenylacetone (1 mmol), 40% methylamine methanol solution (1.5 mmol), acetic acid (2 mmol), and sodium cyanoborohydride (1.5 mmol) was stirred overnight at room temperature in methanol. After the reaction, methanol was evaporated in vacuo and 2 M hydrochloric acid solution was added to the residue. The aqueous solution was stirred for 0.5 h at room temperature, after which 1 M aqueous sodium hydroxide was added to make the solution alkaline. The aqueous layer was extracted with dichloromethane, and the combined organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated. Ethereal hydrochloric acid was added to the residue. The solution was allowed to stand at 5˚C until precipitation occurred. The precipitate was then filtered off, and the residue was washed with pre-chilled diethyl ether and air dried to 4-IMA: 4-iodophenylacetone was prepared by refluxing 4-iodophenylacetic acid (2 mmol) and acetic anhydride with pyridine [22]. The same procedure as that for 4-FMA hydrochloride was performed for 4-iodophenylacetone, which was used as the starting material to ob-

Microsomal Incubation and Workup for Metabolism Experiments
Incubation . The mixtures were incubated at 37˚C. Reactions were started by adding ice-cold microsomes (final concentration: 2 mg/mL for HLM, 40 pmol/mL for CYP1A2, CYP2C19, and CYP2D6, 80 pmol/mL for CYP2C9, CYP3A4, and CYP3A5) and terminated with 200 μL of methanol after 30 min of incubation. After termination, the samples were placed on wet ice and centrifuged. The supernatants were transferred to clean test tubes, and 600 mL of carbonate-bicarbonate buffer (pH 9.8) and 100 μL of 0.92 μmol/L N-butylbenzylamine methanolic (internal standard) were added. Each supernatant was extracted three times with 1 mL of 2-propanol-chloroform (1:3; v/v). The combined organic layer was transferred into a glass flask and 200 μL of 0.24 mol/L methanolic HCl was added, and the sample was evaporated to dryness under a stream of nitrogen. The residue was subjected to TFA-derivatization (Section 2.5), and then dissolved in 100 or 1000 μL of ethyl acetate. An aliquot (1 μL) of the derivatized sample was injected into the gas chromatography-mass spectrometry (GC-MS) system.

Microsomal Incubations for Inhibition Studies
Mixtures  TFA derivatives of the analytes were prepared by adding 100 μL of TFA and 100 μL of ethyl acetate to the sample, after which the mixture was reacted at 65˚C for 10 min. The reaction mixture was carefully evaporated and dried under a gentle nitrogen stream, and then reconstituted in 100 or 1000 μL of ethyl acetate. Subsequently, 1 μL aliquots were automatically injected into the GC-MS system within 3 h of reconstitution [23].

GC-MS
For quantification, the following target ions (m/z) were used in selected ion monitoring mode: m/z 118 for AP, m/z 154 for OHMA, and m/z 91 for the internal standard (IS) N-butylbenzylamine. Calibration curves for AP and OHMA were constructed based on the ion peak-area ratio of internal standard to AP or OHMA from 100 μL To examine the impact of the in vitro incubation matrix, inactivated incubation mixtures spiked with blank water or known concentrations (sample volume of 100 μL, final concentration of 0.01, 0.1 or 1 μmol/L in water; sample volume of 1000 μL, final concentration of 0.1, 1 or 10 μmol/L in water) of a mixture of AP and OHMA were extracted as described in Section 2.3. The obtained calibration curves were virtually the same as those for quantification. The analysis results for inactivated incubation mixtures spiked with the mixture of AP and of 2, 5, 10, 20, 50, 100, 200, 500, 1000 or 1500 μmol/L was used for the kinetic study. Kinetic data were analyzed by the Michaelis-Menten equation (

v = V max [S]/ (K m + [S])) and Hanes-Woolf equations ([S]
where v is the initial reaction velocity, V max the maximal reaction velocity, K m the Michaelis constant, and [S] is the substrate concentration. IC 50 values were calculated according to the following Equation (1): OHMA showed there was no interference from the in vitro incubation matrix (Table 1 and Figure 1).

Kinetics
The effects of the CYP2D6 protein content and incubation time were evaluated to obtain the optimum conditions for linearity of the metabolic results. The resulting conditions for the kinetics study were a 40 pmol/mL protein concentration in the microsomes and a 30 min incubation at 37˚C. Substrate MA at a final concentration a Each value was obtained from three determinations, b The peak area of the base peak ion at m/z 118 was used, c The peak area of the base peak ion at m/z 154 was used. where A and B are the higher and lower concentrations near 50% inhibition, respectively; and C and D are the percentages of control B and A, respectively.

In Vitro Metabolism of MA and 4-Substituted MAs
MA was incubated with recombinant CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP3A5 and HLM, and the main metabolites of MA, AP and OHMA, were formed (Figure 2). The production rates of the metabolites differed greatly depending on the enzyme ( Table 2).
With all of the enzymes tested, AP production from MA was greater than OHMA production. Therefore, N-demethylation of MA was superior to p-hydroxylation with these enzymes. Among the CYP isozymes tested, CYP2D6 was the dominant contributor to MA metabolism.  (Figures 3 and 4). The results suggest that when any of these six 4-substituted MAs are taken, these metabolites will be present in human body fluids and tissues obtained from the user. This study using CYP2D6 and HLM showed that 4-MMA was metabolized mainly by O-demethylation, which agrees with the results of Staack et al. [8].
The production rates of the main metabolites of MA, AP and OHMA, were measured at various MA concentrations using microsomes from cells that stably express human CYP2D6, which was the dominant contributor to the metabolism of MA. The curves for the two main metabolites were fitted to the Michaelis-Menten equation (Figure 5). The K m values for MA N-demethylation (Figure 5(a)) and p-hydroxylation (Figure 5(b)) were estimated to be 30 and 70 μmol/L, respectively. The V max values for MA N-demethylation and p-hydroxylation were 5.8 and 0.45 pmol/(min × pmol), respectively.

Comparison of Inhibitory Activity of the Six 4-Substituted MAs toward CYP2D6-Mediated Metabolism of MA
To determine the IC 50 values for CYP2D6, the percentage values of AP and OHMA production activity were plotted against the concentrations for each inhibitor (Figures 6 and 7). The percentage production activities were calculated by comparison with production activities for P and OHMA production without inhibitor. The results A      CYP2D6 by 4-MMA suggested that the production rate of OHMA from 4-MMA was much faster than the production rate of OHMA from MA. The general trend was that the strength of inhibition of CYP2D6-mediated N-demethylation and p-hydroxylation increased as the halogen group of the 4-halogenated MA became larger. This structure-activity relationship is interesting because of its relevance to the 2Cx and DOx families of drugs, which have been widely abused. These drug families include 2C-I and DOI, which are 4-halo- genated aromatic compounds like those studied here.
Comparison of the IC 50 values of the compounds in this study, suggested that the strength of drug interactions could alter greatly with a change in the substituent. There-fore, designer drugs could interact with other drugs in an unexpected manner, and carry unknown risks and unforeseen consequences.

Conclusion
This study investigated the in vitro metabolism of six 4substituted MAs and their potential inhibition of MA metabolism using human metabolic enzymes by GC-MS after TFA derivatization. The IC 50 of 4-MMA against OHMA production was the only exception to this trend. The IC 50 values were between 0.80 and >30 μmol/L for AP production and 0.84 and >30 μmol/L for OHMA production. These results show that although amphetamine-type stimulants with similar chemical structures can be metabolized by the same metabolic enzymes, they interact with co-administered compounds to varying degrees. This suggests that the risk of illicit drug interactions fluctuates so widely that drug abusers could not be aware of the potential danger of these interactions.