Monoamine transporter and receptor interaction profiles of novel psychoactive substances: Para-halogenated amphetamines and pyrovalerone cathinones

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Abstract

The pharmacology of novel psychoactive substances is mostly unknown. We evaluated the transporter and receptor interaction profiles of a series of para-(4)-substituted amphetamines and pyrovalerone cathinones. We tested the potency of these compounds to inhibit the norepinephrine (NE), dopamine (DA), and serotonin (5-HT) transporters (NET, DAT, and SERT, respectively) using human embryonic kidney 293 cells that express the respective human transporters. We also tested the substance-induced efflux of NE, DA, and 5-HT from monoamine-loaded cells, binding affinities to monoamine receptors, and 5-HT2B receptor activation. Para-(4)-substituted amphetamines, including 4-methylmethcathinone (mephedrone), 4-ethylmethcathinone, 4-fluoroamphetamine, 4-fluoromethamphetamine, 4-fluoromethcatinone (flephedrone), and 4-bromomethcathinone, were relatively more serotonergic (lower DAT:SERT ratio) compared with their analogs amphetamine, methamphetamine, and methcathinone. The 4-methyl, 4-ethyl, and 4-bromo groups resulted in enhanced serotonergic properties compared with the 4-fluoro group. The para-substituted amphetamines released NE and DA. 4-Fluoramphetamine, 4-flouromethamphetamine, 4-methylmethcathinone, and 4-ethylmethcathinone also released 5-HT similarly to 3,4-methylenedioxymethamphetamine. The pyrovalerone cathinones 3,4-methylenedioxypyrovalerone, pyrovalerone, α-pyrrolidinovalerophenone, 3,4-methylenedioxy-α-pyrrolidinopropiophenone, and 3,4-methylenedioxy-α-pyrrolidinobutiophenone potently inhibited the NET and DAT but not the SERT. Naphyrone was the only pyrovalerone that also inhibited the SERT. The pyrovalerone cathinones did not release monoamines. Most of the para-substituted amphetamines exhibited affinity for the 5-HT2A receptor but no relevant activation of the 5-HT2B receptor. All the cathinones exhibited reduced trace amine-associated receptor 1 binding compared with the non-β-keto-amphetamines. In conclusion, para-substituted amphetamines exhibited enhanced direct and indirect serotonergic agonist properties and are likely associated with more MDMA-like effects. The pharmacological profile of the pyrovalerone cathinones predicts pronounced stimulant effects and high abuse liability.

Introduction

Novel psychoactive substances (“designer drugs”) are newly misused psychotropic drugs that may pose a threat to public health that is comparable to previously listed drugs of abuse. Novel psychoactive substances are typically sold through the Internet (i.e., “Internet drugs”) and misbranded as “research chemicals,” “bath salts,”, and “plant food” and labeled “not for human consumption.” The substances are typically chemically slightly different from already scheduled drugs to circumvent regulations and are therefore also termed “legal highs”. Over the last few years, we have seen an unprecedented growth in the number of new psychoactive substances on the illicit drug market. More than 300 novel substances have been detected since 2005 (European Monitoring Center for Drugs and Drug Addiction, 2014a). Currently, more than one new substance is identified in one of the EU countries every week (European Monitoring Center for Drugs and Drug Addiction, 2014a). In most cases, pharmacological data are not available for the newly misused substances. Many novel psychoactive substances are amphetamine derivatives that can be expected to interact with the norepinephrine (NE), dopamine (DA), and serotonin (5-hydroxytryptamine [5-HT]) transporters (NET, DAT, and SERT, respectively) to inhibit monoamine transport or induce transporter-mediated monoamine release. However, chemical substitutions at the amphetamine core structure may significantly alter the absolute or relative potency of these newly designed substances at the NET and DAT relative to the SERT (Baumann et al., 2012, Blough et al., 2014, Cozzi et al., 2013, Eshleman et al., 2013, Iversen et al., 2013, Simmler et al., 2013, Simmler et al., 2014a, Simmler et al., 2014b). Consequently, more noradrenergic and dopaminergic substances may have greater sympathomimetic and reinforcing properties (Simmler et al., 2013). Conversely, more serotonergic substances are likely associated with more MDMA-like properties, including empathogenic effects, serotonin syndrome, and hyperpyrexia (Simmler et al., 2013, Simmler et al., 2014a). In addition, novel amphetamines may directly activate monoamine receptors. Characterizing the primary pharmacodynamic properties of novel designer amphetamines in vitro provides a basis for further preclinical studies and the evaluation of potential clinical effects, abuse potential, and acute toxicity of these novel substances. Such data are useful for clinical toxicologists and regulatory agencies for scheduling purposes. Therefore, the aim of the present study was to determine the effects of a series of para-(4)-substituted amphetamines and of a series of pyrovalerone cathinones on monoamine uptake and release and interactions with various monoamine receptors.

Para-(4)-phenyl-substituted amphetamines, which have emerged in recent years, include 4-methylmethcathinone (mephedrone) and 4-ethylmethcathinone and particularly several para-halogenated compounds, including 4-fluoroamphetamine, 4-fluoromethamphetamine, 4-fluoromethcatinone (flephedrone), and 4-bromomethcathinone. 4-Methylmethcathinone has been the most popular and still is a very commonly misused cathinone in the EU (Elliott and Evans, 2014, Helander et al., 2014, Rust et al., 2012, Winstock et al., 2011). 4-Ethylmethcatinone was detected in 2011 in the EU (European Monitoring Center for Drugs and Drug Addiction, 2011), and its use is discussed in Internet user forums. Similarly, the use of 4-bromomethcathinone is also discussed in user forums, but no scientific data are available. 4-Fluoroamphetamine appeared in 2007 in the EU, followed later by 4-fluoromethamphetamine and 4-fluoroephedrine. 4-Fluoroephedrine may serve as a precursor for the synthesis of 4-fluoromethamphetamine. 4-Fluoroamphetamine and 4-fluoromethamphetamine have also been detected in patients with acute toxicity associated with novel psychoactive substances and forensic cases (Helander et al., 2014, Johansen and Hansen, 2012, Rohrich et al., 2012, Rust et al., 2012). Users report that the subjective effects of 4-methlylmethcathinone (Carhart-Harris et al., 2011) and 4-fluoroamphetamine (Erowid, 2014) are comparable to those of MDMA. Pharmacological information is available only for some of these novel substances, including 4-methylmethcatinone (Baumann et al., 2012, Eshleman et al., 2013, Simmler et al., 2013), 4-fluoroamphetamine (Marona-Lewicka et al., 1995), and 4-fluoromethcathinone (Eshleman et al., 2013, Simmler et al., 2013). Because 4-fluoroamphetamine and MDMA are relatively more serotonergic than amphetamine and methamphetamine (Marona-Lewicka et al., 1995, Simmler et al., 2013), we hypothesized that a substitution at the 4-position as a characteristic of these novel para-substituted substances would also result in a shift toward more serotonergic than dopaminergic pharmacology. Thus, such para-substituted substances may also be designed to mimic the effects of MDMA.

Pyrovalerone cathinones include 3,4,-methylenedioxypyrovalerone (MDPV), pyrovalerone, α-pyrrolidinovalerophenone (α-PVP), naphyrone, 3,4-methylenedioxy-α-pyrrolidinopropiophenone (MDPPP), 3,4-methylenedioxy-α-pyrrolidinobutiophenone (MDPBP), α-pyrrolidinopropiophenone (α-PPP), and α-pyrrolidinobutiophenone (α-PBP). All these cathinones are characterized by a pyrrolidine ring structure, making them different structurally and possibly also pharmacologically from other synthetic cathinones (Marusich et al., 2014, Simmler et al., 2013). Among the pyrovalerone cathinones, MDPV is currently the most widely detected and used, both in the EU (European Monitoring Center for Drugs and Drug Addiction, 2014b, Helander et al., 2014, Zuba and Byrska, 2013) and US (Leffler et al., 2014, Marinetti and Antonides, 2013, Spiller et al., 2011). In fact, MDPV has become the most frequently detected and used of all cathinones (“bath salts”) in some EU countries (Helander et al., 2014, Zuba and Byrska, 2013) and the US (Leffler et al., 2014). More recently, a second generation of MDPV-like cathinones, including α-PVP, MDPPP, and MDPBP, has been detected and/or used in several EU countries (Eiden et al., 2013, Helander et al., 2014, Westphal et al., 2011, Zuba and Byrska, 2013) and the US (Elliott and Evans, 2014, Smollin et al., 2011, Thornton et al., 2012). MDPV has been associated with severe clinical toxicity (Spiller et al., 2011) and a high potential for addiction (Aarde et al., 2013). Similarly, α-PVP has recently been associated with cases of severe acute psychosis and cardiac arrest (Eiden et al., 2013). Pharmacologically, both MDPV and α-PVP are very potent inhibitors of the NET and DAT but not SERT (Baumann et al., 2013, Marusich et al., 2014, Meltzer et al., 2006, Simmler et al., 2013). Of the second generation MDPV analogs, α-PPP and α-PBP also inhibit the NET and DAT similarly to MDPV (Marusich et al., 2014), but no data are available on MDPBP and MDPPP. We hypothesized that these and other cathinones with a pyrovalerone structure would inhibit the NET and DAT but not SERT, similar to MDPV (Marusich et al., 2014, Meltzer et al., 2006, Simmler et al., 2013). Naphyrone also potently inhibits the SERT, unlike other pyrovalerone cathinones, and this exemplifies the necessity to pharmacologically assess each substance individually to avoid drawing false conclusions from structural relationships with previously assessed analogs. We predicted that these pyrovalerone cathinones are distinct from other cathinones, in which they are pure uptake inhibitors and do not act as substrate releasers as previously shown for MDPV (Baumann et al., 2013, Simmler et al., 2013).

We tested whether the substances inhibit the human NET, DAT, and SERT. We also determined the transporter-mediated release of NE, DA, and 5-HT and characterized the binding affinities of the compounds for monoamine transporters, α1- and α2-adrenergic receptors, dopamine D1–D3 receptors, serotonin 5-HT1A, 5-HT2A, and 5-HT2C receptors, the histamine H1 receptor, and trace amine-associated receptor 1 (TAAR1). For example, 5-HT2A receptors mediate the effects of hallucinogens (Nichols, 2004) and TAAR1 play a role in the addictive properties of psychoactive substances (Pei et al., 2014). Furthermore, some novel psychoactive substances have been reported to bind to the serotonin 5-HT2B receptor (Iversen et al., 2013), which has been implicated in endocardial fibrosis induced by serotonergic substances. Therefore, we also tested functional activity at the 5-HT2B receptor.

Some of the substances, including MDMA, amphetamine, methamphetamine, methcathinone, mephedrone, flephedrone, MDPV, naphyrone, and pyrovalerone, have previously been characterized using the same assays as those used in the present study (Simmler et al., 2013), but we retested them herein because of their structural similarity to the other substances that were evaluated, to our knowledge, for the first time.

Section snippets

Drugs

MDMA, d-amphetamine, d-methamphetamine, methcathinone, 4-methylmethcathinone, 4-fluoromethcathione, 4-fluoroamphetamine, 4-fluoroephedrine, ephedrine, MDPBP, MDPPP, MDPV, pyrovalerone, and α-PVP were purchased from Lipomed (Arlesheim, Switzerland). 4-Fluoromethamphetamine, 4-etylmethcathinone, and 4-bromomethcathinone were purchased from Cayman Chemicals (Ann Arbor, MI, USA). Naphyrone was synthesized as previously described (Simmler et al., 2013). All the drugs were obtained as racemic

Monoamine uptake transporter inhibition

The monoamine transporter inhibition profiles are shown in Figure 2, Figure 3, and the corresponding IC50 values and DAT:SERT inhibition ratios are listed in Table 1. In all cases, the para-(4) substitution (Figure 1A) reduced the potency of the amphetamines to inhibit both NET and DAT compared with the non-para-(4)-substituted amphetamines (Table 1). In contrast, the potency to inhibit the SERT increased for all of the substituted amphetamines, with the exception of 4-fluoromethcathinone

Discussion

The goal of the present study was to describe the mechanism of action of two series of novel psychoactive substances: para-(4)-substituted (mostly halogenated) amphetamines and pyrovalerone cathinones. All the para-(4)-substituted amphetamines evaluated in this study exhibited more serotonergic properties than their non-substituted amphetamine analogs. In particular, 4-bromomethcatinone, 4-ethylmethcathinone, and 4-methylmethcathinone were more potent SERT inhibitors than DAT inhibitors,

Conclusion

Para-(4)-substituted amphetamines are more serotonergic than their non-substituted analogs, likely resulting in more MDMA-like serotonergic subjective and acute toxic effects. Pyrovalerone cathinones are potent NET and DAT inhibitors that are likely associated with significant stimulant-type effects and toxicity and a high risk of addiction.

Conflict of interest

The authors do not have any conflicts of interest to declare for this work.

Contributors

AR, MCH and MEL designed the study. MEL and MCH obtained funding. AR, MCH and MEL conducted experiments, analyzed data and wrote the manuscript. All the authors reviewed and approved the manuscript.

Role of funding source

This work was supported by the Federal Office of Public Health (No. 13.006497) and the joint Translational Medicine Hub Innovation Fund of F. Hoffmann-La Roche and the University of Basel.

Acknowledgments

The authors thank Daniele Buchy for performing the experiments, Linda Simmler for critical comments on the manuscript, and Michael Arends for text editing.

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