The Designer Methcathinone Analogs, Mephedrone and Methylone, Are Substrates for Monoamine Transporters in Brain Tissue
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Neuropsychopharmacology (2012) 37, 1192–1203 & 2012 American College of Neuropsychopharmacology. All rights reserved 0893-133X/12 www.neuropsychopharmacology.org The Designer Methcathinone Analogs, Mephedrone and Methylone, are Substrates for Monoamine Transporters in Brain Tissue ,1 1 1 1 2 Michael H Baumann* , Mario A Ayestas Jr , John S Partilla , Jacqueline R Sink , Alexander T Shulgin , 2 3 1 4 5 Paul F Daley , Simon D Brandt , Richard B Rothman , Arnold E Ruoho and Nicholas V Cozzi 1 Translational Pharmacology Section, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, 2 3 MD, USA; Alexander Shulgin Research Institute, Lafayette, CA, USA; School of Pharmacy and Biomolecular Sciences, Liverpool John Moores 4 University, Liverpool, UK; Department of Neuroscience and the UW Eye Research Institute, University of Wisconsin School of Medicine and 5 Public Health, Madison, WI, USA; Neuropharmacology Laboratory, Department of Cell and Regenerative Biology, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA The nonmedical use of ‘designer’ cathinone analogs, such as 4-methylmethcathinone (mephedrone) and 3,4-methylenedioxymethcathi- none (methylone), is increasing worldwide, yet little information is available regarding the mechanism of action for these drugs. Here, we employed in vitro and in vivo methods to compare neurobiological effects of mephedrone and methylone with those produced by the structurally related compounds, 3,4-methylenedioxymethamphetamine (MDMA) and methamphetamine. In vitro release assays using rat brain synaptosomes revealed that mephedrone and methylone are nonselective substrates for plasma membrane monoamine transporters, similar to MDMA in potency and selectivity. In vivo microdialysis in rat nucleus accumbens showed that i.v. administration of 0.3 and 1.0 mg/kg of mephedrone or methylone produces dose-related increases in extracellular dopamine and serotonin (5-HT), with the magnitude of effect on 5-HT being greater. Both methcathinone analogs were weak motor stimulants when compared with methamphetamine. Repeated administrations of mephedrone or methylone (3.0 and 10.0 mg/kg, s.c., 3 doses) caused hyperthermia but no long-term change in cortical or striatal amines, whereas similar treatment with MDMA (2.5 and 7.5 mg/kg, s.c., 3 doses) evoked robust hyperthermia and persistent depletion of cortical and striatal 5-HT. Our data demonstrate that designer methcathinone analogs are substrates for monoamine transporters, with a profile of transmitter-releasing activity comparable to MDMA. Dopaminergic effects of mephedrone and methylone may contribute to their addictive potential, but this hypothesis awaits confirmation. Given the widespread use of mephedrone and methylone, determining the consequences of repeated drug exposure warrants further study. Neuropsychopharmacology (2012) 37, 1192–1203; doi:10.1038/npp.2011.304; published online 14 December 2011 Keywords: dopamine; MDMA; mesolimbic; microdialysis; serotonin (5-HT); transporters INTRODUCTION analogs of N-methylcathinone (ie, methcathinone) have become commercially available for purchase over the Cathinone (2-amino-1-phenylpropan-1-one) is a naturally Internet and in retail shops (Bossong et al, 2005; Brandt occurring b-keto analog of amphetamine that has served as et al, 2010; Karila and Reynaud, 2010; Vardakou et al, 2011; a structural template for the discovery of compounds with a Winstock et al, 2011). These so-called ‘designer’ methcathi- range of pharmacological activities (Carroll et al, 2010; nones are used outside of medical settings for personal Damaj et al, 2004; Meltzer et al, 2006). For example, experimentation, mood elevation, and as ‘legal highs’. The cathinone derivatives that display therapeutic properties European Monitoring Center for Drugs and Drug Addiction include the antidepressant bupropion and the anorectic reported that in 2010 alone, 15 out of 40 newly encountered agent diethylpropion. Recently, a number of synthetic psychoactive substances were found to be cathinone derivatives or isomers (EMCDDA, 2010). Two of the most *Correspondence: Dr MH Baumann, Translational Pharmacology popular methcathinone derivatives are 4-methylmethcathi- Section, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, 333 Cassell Drive, Suite 4500, none (mephedrone) and 3,4-methylenedioxymethcathinone Baltimore, MD 21224, USA, Tel: + 1 443 740 2660, Fax: + 1 443 740 (methylone) (Figure 1). Reports describing serious cardio- 2241, E-mail: [email protected] vascular and neurological side-effects of mephedrone have Received 30 August 2011; revised 28 October 2011; accepted 29 increased at an alarming rate (CDC, 2011; James et al, 2010; October 2011 Wood et al, 2010, 2011), and deaths attributable to overdose Methcathinone analogs and monoamine transporters MH Baumann et al 1193 long-term consequences of repeated high-dose drug admin- istration (Baumann et al, 2008). Our findings indicate that mephedrone and methylone are potent substrates for monoamine transporters in rat brain, with a profile of dopamine- and 5-HT-releasing activity, which more closely resembles MDMA than methamphetamine. However, im- portant differences were noted among the effects of mephedrone, methylone, and MDMA after high-dose administration. Further studies are warranted to elucidate the mechanistic distinctions among these various drugs. Figure 1 Chemical structures of designer methcathinone analogs and related compounds. MATERIALS AND METHODS Drugs and Reagents from mephedrone alone or in combination with other drugs have been documented (Dickson et al, 2010; Lusthof et al, Mephedrone and methylone were synthesized in racemic 2011; Maskell et al, 2011). form as HCl salts in our laboratories. Chemical and Although it might be inferred from structural similarities structural analysis included proton nuclear magnetic with cathinone that designer analogs of methcathinone resonance, gas chromatography/mass spectrometry, thin target monoamine transporters (Cozzi and Foley, 2003; layer chromatography, and melting point determination. All Rothman et al, 2003) or nicotinic acetylcholine receptors data confirmed the expected structures. ( + )-3,4-Methyle- (Carroll et al, 2010; Damaj et al, 2004), only limited nedioxymethamphetamine HCl (MDMA) and ( + )-metham- information is available regarding the pharmacodynamic phetamine HCl (methamphetamine) were obtained from the mechanisms of these drugs. Cozzi et al (1999) demonstrated Pharmacy at the National Institute on Drug Abuse (NIDA), that methylone blocks the reuptake of norepinephrine, Intramural Research Program (IRP) in Baltimore, MD. dopamine and serotonin (5-HT) in vitro, whereas (Nagai [3H]Serotonin ([3H]5-HT, specific activity ¼ 20 Ci/mmol) et al, 2007) found that methylone evokes the release of was purchased from Perkin Elmer (Shelton, CT) whereas radiolabeled monoamines from rat brain synaptosomes. [3H]1-methyl-4-phenylpyridinium ([3H]MPP + , specific The in vitro results indicate that methylone interacts with activity ¼ 85 Ci/mmol) was purchased from American Radi- plasma membrane transporters, but the precise nature of olabeled Chemicals (St Louis, MO). All other chemicals and this interaction requires clarification. Dal Cason et al (1997) reagents used for the in vitro assays, microdialysis methods, used a drug discrimination paradigm to demonstrate that and high-pressure liquid chromatography with electroche- methylone substitutes for 3,4-methylenedioxymethamphet- mical detection (HPLC-ECD) were acquired from Sigma- amine (MDMA) in rats trained to distinguish MDMA Aldrich (St Louis, MO). from saline, but fails to substitute for the psychedelic drug 2-amino-1-(2,5-dimethoxy-4-methylphenyl)propane (DOM) Animals and Surgery in rats trained to discriminate DOM from saline. A recent study by Kehr et al (2011) showed that mephedrone Male Sprague–Dawley rats weighing 300–350 g were housed increases extracellular dopamine and 5-HT in rat nucleus under conditions of controlled temperature (22±2 1C) and accumbens, and these effects could be related to inhibition humidity (45±5%) with food and water freely available. of monoamine uptake (Hadlock et al, 2011). Importantly, Rats were maintained in facilities accredited by the pharmacokinetic studies in rats and humans have shown Association for the Assessment and Accreditation of that mephedrone and methylone are metabolized in a Laboratory Animal Care, and procedures were carried out manner similar to ring-substituted amphetamines (Kamata in accordance with the Animal Care and Use Committee of et al, 2006; Meyer et al, 2010), suggesting the possibility that the NIDA IRP. Lights were on from 0700 to 1900 hours and bioactive metabolites might be formed in vivo. experiments were carried out between 0900 and 1400 hours. Given the lack of empirical data and the complex Rats were double-housed on receipt and allowed at least structure–activity relationships of cathinone analogs (Car- 2 weeks to acclimate to the vivarium conditions before roll et al, 2010; Dal Cason et al, 1997; Damaj et al, 2004; being used in experiments. For in vitro assays, rats were Nagai et al, 2007), we sought to examine the mechanism(s) euthanized with CO2 and decapitated. Brains were rapidly of action for mephedrone and methylone. To this end, we removed and tissue was dissected on ice. For in vivo used in vitro and in vivo methods to compare the microdialysis experiments, rats received