In vitro monoamine oxidase inhibition potential of alpha-methyltryptamine analog new psychoactive substances for assessing possible toxic risks
Introduction
New psychoactive substances (NPS) are emerging drugs that are mainly consumed as legal and easy available substitutes for traditional and controlled drugs of abuse (Brandt et al., 2014, Meyer, 2016). The detection of synthetic tryptamines including alpha-methyltryptamine (AMT) has been frequently reported to the EU Early Warning System coordinated by the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) and continuously monitored as part of the EMCDDA’s toxicovigilance system (EMCDDA, 2015). AMT (α-MT, 3-IT, IT-290) has been investigated in the 1960s as a potential antidepressant and its popularity as a drug of abuse came to light in the 1990s due to its hallucinogenic and stimulant properties (Araujo et al., 2015). In late 2011, an isomer of AMT, 5-(2-aminopropyl)indole (5-IT, 5-API), appeared on the European drug market (EMCDDA, 2014). After intake of AMT or 5-IT, symptoms related to monoaminergic toxicity were described, such as restlessness, agitation, disorientation, shivering, sweating, mydriasis, vomiting, tachycardia, or hyperthermia (EMCDDA, 2015, Shulgin and Shulgin, 1997). Both were also involved in several fatalities (Boland et al., 2005, Elliott and Evans, 2014). A key factor involved in the occurrence of clinical features included the inhibition of monoamine oxidase (MAO) enzymes, followed by increased monoamine levels inducing a serotonin syndrome (Boyer and Shannon, 2005, EMCDDA, 2014). In 1968, AMT, 5-IT, and four positional isomers were identified as inhibitors of guinea pig MAO (Cerletti et al., 1968). More recently, 5-IT was confirmed as highly selective and potent inhibitor of recombinant human MAO-A (Herraiz and Brandt, 2014).
Furthermore, tryptamines were identified in NPS products combining different NPS groups (UNODC, 2016). Thus, the risk of encountering monoaminergic (side) effects and drug–drug interactions is very likely although the extent to which this might occur is difficult to predict, given that systematic data are not available. In contrast to authorized medicines, NPS are marketed and consumed without preclinical or clinical studies. Procedures employed for various substrates, mostly therapeutic drugs, have been described for in vitro monitoring of MAO activity (Tipton et al., 2006). Examples of drugs of abuse tested for human MAO inhibition activity include MDMA and its metabolites, and 5-IT (Herraiz and Brandt, 2014, Steuer et al., 2016).
Therefore, the aim of the present study was to develop a MAO inhibition assay based on hydrophilic interaction liquid chromatography–high resolution-tandem mass spectrometry (HILIC–HR-MS/MS), to investigate the in vitro inhibition potential of AMT and 13 ring-substituted analogs (Fig. 1) on recombinant human MAO-A or B, and to determine their IC50 values.
Section snippets
Chemicals and enzymes
Harmine was obtained from THC Pharm (Frankfurt, Germany), amphetamine-d5 and AMT succinate from LGC Standards (Wesel, Germany), harmaline, yohimbine, selegiline, kynuramine (KYN), 4-hydroxyquinoline (4-OHC), ammonium acetate, potassium dihydrogenphosphate, and dipotassium hydrogenphosphate from Sigma-Aldrich (Taufkirchen, Germany), formic acid (MS grade) from Fluka (Neu-Ulm, Germany), acetonitrile, methanol (both LC–MS grade), and all other chemicals from VWR (Darmstadt, Germany). 5-IT was
Method validation
The analytical procedure was based on HILIC–HR-MS/MS in t-SIM mode with a subsequent dd-MS2 mode. The method was successfully validated in accordance to the criteria of the (EMA, 2011). The method was selective at LLOQ levels, with a response of interfering components less than 20% or 5% compared to the response of the analyte at the LLOQ and the response of the IS, respectively. No carry-over problems could be observed and the LLOQ for 4-OHC was defined as 50 nM. The mean for within-run and
Discussion
MAO activity was assessed using KYN, a non-selective substrate for MAO-A and B, which was transformed to the corresponding aldehyde, followed by non-enzymatic condensation to 4-OHC (Fig. 2) (Weissbach et al., 1960). 4-OHC formation was analyzed using HILIC–HR-MS/MS. Although literature described many MAO activity assays (Tipton et al., 2006), Herraiz and Chaparro found that the needed selectivity for analyzing 4-OHC could only be achieved by chromatographic separation before detection (Herraiz
Conclusion
The presented study was the first to describe the MAO inhibition for a broad range of NPS of the alpha-methylated tryptamine type. The workup and analysis based on HILIC–HR-MS/MS were validated according to international guidelines. Due to its high sensitivity, only minimal amounts of recombinant MAO enzymes were needed, thus, reducing the risk of non-specific protein binding, as well as material costs. All tested AMT analogs inhibited MAO-A activity, whereas four compounds inhibited MAO-B as
Conflict of interest
The authors declare that they have no conflict of interest.
Acknowledgements
The authors like to thank Achim T. Caspar, Julia Dinger, Andreas G. Helfer, Sascha K. Manier, Julian A. Michely, Lilian H. J. Richter, and Armin A. Weber for their support and fruitful discussion.
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