EPHENIDINE: A NEW PSYCHOACTIVE AGENT WITH KETAMINE-LIKE NMDA RECEPTOR ANTAGONIST PROPERTIES

: To avoid legislation based on chemical structure, research chemicals, frequently used for recreational purposes, are continually being synthesized. N-Ethyl-1,2-diphenylethanamine (ephenidine) is a diarylethylamine that has recently become popular with recreational users searching for dissociative hallucinogenic effects. In the present study, the pharmacological basis of its neural actions has been investigated, initially by assessing its profile in central nervous system receptor binding assays and subsequently in targeted electrophysiological studies. Ephenidine was a potent inhibitor of 3H-MK-801 binding (Ki: 66 nM), implying that it acts at the PCP site of the N-methyl-D-aspartate (NMDA) receptor. It also showed modest activity at dopamine (379 nM) and noradrenaline (841 nM) transporters and at sigma 1 (629 nM) and sigma 2 (722 nM) binding sites. In experiments of extracellular recording of field excitatory postsynaptic potentials (fEPSPs) from area CA1 of rat hippocampal slices, ephenidine, 1 and 10 µM, respectively, produced a 25% and a near maximal inhibition of the NMDA receptor mediated fEPSP after 4 h superfusion. By contrast, ephenidine (50 µM) did not affect the AMPA receptor mediated fEPSPs. ABSTRACT: To avoid legislation based on chemical structure, research chemicals, frequently used for recreational purposes, are continually being synthesized. N -Ethyl-1,2-diphenylethanamine (ephenidine) is a diarylethylamine that has recently become popular with recreational users searching for dissociative hallucinogenic effects. In the present study, the pharmacological basis of its neural actions has been investigated, initially by assessing its profile in central nervous system receptor binding assays and subsequently in targeted electrophysiological studies. Ephenidine was a potent inhibitor of 3 H-MK-801 binding (Ki: 66 nM), implying that it acts at the PCP site of the N-methyl-D-aspartate (NMDA) receptor. It also showed modest activity at dopamine (379 nM) and noradrenaline (841 nM) transporters and at sigma 1 (629 nM) and sigma 2 (722 nM) binding sites. In experiments of extracellular recording of field excitatory postsynaptic potentials (fEPSPs) from area CA1 of rat hippocampal slices, ephenidine, 1 and 10 µ M, respectively, produced a 25% and a near maximal inhibition of the NMDA receptor mediated fEPSP after 4 h superfusion. By contrast, ephenidine (50 µ M) did not affect the AMPA receptor mediated fEPSPs. In whole cell patch clamp recordings, from hippocampal pyramidal cells, ephenidine (10 µ M) blocked NMDA receptor-mediated EPSCs in a highly voltage-dependent manner. Additionally, ephenidine, 10 µ M, blocked the induction of long term potentiation (LTP) in CA1 induced by theta burst stimulation. The present data show that the new psychoactive substance, ephenidine, is a selective NMDA receptor antagonist with a voltage-dependent profile similar to ketamine. Such properties help explain the dissociative, cognitive and hallucinogenic effects in man. the effects of ephenidine with those of ketamine on synaptic transmission in hippocampal brain slices both extracellular and whole-cell the selectivity of ephenidine comparing its potency at displacing MK-801 binding with its actions on a wide range of CNS receptors The data show that ephenidine is a relatively selective, voltage-dependent NMDA antagonist that potently blocks LTP. These observations can explain the psychotomimetic effects of ephenidine and predict a range of side-effects including memory impairments.


ABSTRACT:
To avoid legislation based on chemical structure, research chemicals, frequently used for recreational purposes, are continually being synthesized. N-Ethyl-1,2-diphenylethanamine (ephenidine) is a diarylethylamine that has recently become popular with recreational users searching for dissociative hallucinogenic effects.
In the present study, the pharmacological basis of its neural actions has been investigated, initially by assessing its profile in central nervous system receptor binding assays and subsequently in targeted electrophysiological studies. Ephenidine was a potent inhibitor of 3 H-MK-801 binding (Ki: 66 nM), implying that it acts at the PCP site of the N-methyl-D-aspartate (NMDA) receptor. It also showed modest activity at dopamine (379 nM) and noradrenaline (841 nM) transporters and at sigma 1 (629 nM) and sigma 2 (722 nM) binding sites. In experiments of extracellular recording of field excitatory postsynaptic potentials (fEPSPs) from area CA1 of rat hippocampal slices, ephenidine, 1 and 10 µM, respectively, produced a 25% and a near maximal inhibition of the NMDA receptor mediated fEPSP after 4 h superfusion. By contrast, ephenidine (50 µM) did not affect the AMPA receptor mediated fEPSPs.
In whole cell patch clamp recordings, from hippocampal pyramidal cells, ephenidine (10 µM) blocked NMDA receptor-mediated EPSCs in a highly voltagedependent manner. Additionally, ephenidine, 10 µM, blocked the induction of long term potentiation (LTP) in CA1 induced by theta burst stimulation.
The present data show that the new psychoactive substance, ephenidine, is a selective NMDA receptor antagonist with a voltage-dependent profile similar to ketamine. Such properties help explain the dissociative, cognitive and hallucinogenic effects in man.
Ephenidine, like ketamine, blocks the NMDA receptor in a highly voltagedependent manner.
NMDA receptor antagonism likely underlies the psychoactive effects of ephenidine.

INTRODUCTION
Shortly after their development as potential general anesthetics for veterinary and human use (Greifenstein et al., 1958;McCarthy et al., 1965;Domino et al., 1965), both phencyclidine (PCP) and ketamine were widely abused throughout the world for their dissociative effects (Petersen and Stillman, 1978;Jansen, 2000). Although PCP is still abused as a 'street drug' in the USA, its misuse has been reduced particularly in Europe because of severe and long lasting psychotomimetic effects, including lethality (Moeller et al., 2008) whereas the shorter-acting ketamine has remained a popular recreational drug (Freese et al., 2002;Nutt et al., 2007;Morris and Wallach, 2014), although not without dangers (Morgan and Curran, 2012). However, legislation has been enacted in many countries in an attempt to prevent their use and sale, which in turn has resulted in a burgeoning of new chemicals with dissociative properties (Roth et al., 2013;Morris and Wallach, 2014). Interestingly, the most common structures, like phencyclidine, are tricyclic compounds and include various 1,2diarylethylamines e.g. diphenidine and 2-methoxydiphenidine (Morris and Wallach, 2014 (Anis et al., 1983) and other dissociative hallucinogens (Lodge and Mercier, 2015), these tricyclic 1,2-diarylethylamines have proved to be potent and selective NMDA antagonists (Wallach et al., 2016).
Recently, ephenidine, a two ringed N-ethyl-1,2-diphenylethylamine, has become available and anecdotally appears popular with users of dissociative research chemicals e.g. 'finally a worthy alternative to ketamine …',  , 1989). However, no suggestion of the relationship to NMDA receptor antagonism was made nor were its selectivity, its mode of action and its potential to affect synaptic function and plasticity explored. We have therefore addressed these and further compared the effects of ephenidine with those of ketamine on synaptic transmission in hippocampal brain slices using both extracellular and whole-cell recording techniques. We have also examined the selectivity of ephenidine by comparing its potency at displacing MK-801 binding with its actions on a wide range of CNS receptors The data show that ephenidine is a relatively selective, voltage-dependent NMDA antagonist that potently blocks LTP. These observations can explain the psychotomimetic effects of ephenidine and predict a range of side-effects including memory impairments.

Preparation of ephenidine.
Full details of the synthesis and analytical characterization of ephenidine (N-ethyl-1,2-diphenylethylamine) are given in Supplement 1.

Receptor binding experiments.
The binding affinity (Ki) of ephenidine to the MK-801 binding site of the NMDA receptor was determined as described by Reynolds and Sharma (1999).

Electrophysiology in hippocampal slices.
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RESULTS.
Receptor binding data. The data from the NIMH PDSP assay ( showed less than 50% displacement with 10 µM ephenidine.

Electrophysiology in hippocampal slices.
In initial experiments, superfusion of hippocampal slices with NBQX, picrotoxin and CGP55845 containing aCSF to reduce AMPA, GABA-A and GABA-B receptor-mediated events resulted in fEPSPs, which were slowly reduced by 30 µM ephenidine (Fig 2A). The rapid reduction of such fEPSPs in the presence of 1 and 10 µM D-AP5 ( Fig 3A) and slower reduction in presence of 1 and 10 µM ketamine ( Fig 3B) confirms their dependence on NMDA receptors. To investigate further the sensitivity of the fEPSP to ephenidine, in the presence of the same cocktail of GABA and AMPA receptor antagonists, it was superfused at 10 µM, which slowly reduced the fEPSP reaching a near maximal effect after 4 h. By contrast, the effect of 1 µM ephenidine on this fEPSP was limited to approximate 25% inhibition after 4 h perfusion (Fig 3C). This slow antagonism by ephenidine is typical of other use-dependent uncompetitive NMDA receptor antagonists, e.g. ketamine (Fig 3B) and suggests a similar mode of action (see Wallach et al., 2016).
To study the selectivity of ephenidine against the AMPA subtype of glutamate receptor, the effect of ephenidine was studied on the early component of the fEPSP in the absence of NBQX (Fig 2B). One hour of superfusion with 50 µM ephenidine produced no significant reduction of the peak amplitude of the fEPSP, although it can be seen from the individual recordings that the late NMDA receptor-mediated component is considerably reduced (Fig 2B). Switching the superfusion to one containing 3 µM NBQX resulted in a rapid block of the remaining fEPSP (Fig 2B), demonstrating its dependence on AMPA receptors.
To compare the effect of ephenidine on current-voltage relationship of NMDA receptor mediated synaptic currents, patch clamp recordings were made in the presence of the above cocktail of drugs and in the absence of added Mg 2+ ions before adding 2mM Mg 2+ (Fig 4A) or 30 µM ephenidine (Fig 4B) to the aCSF for 30 min. Ephenidine showed a profile typical of a channel blocker, i.e. reduction of inward current at negative holding potentials. The rectification index (RI), expressed as the ratio of currents at holding potentials of +40 mV to -40 mV, was also used to quantify this profile. In separate studies where slices were bathed in drug-containing aCSF before patch clamping, RIs were calculated from similar current-voltage measurements. For example, at -40 mV, the mean current in 30 µM ephenidine was -48 ± 8 pA and at +40 mV was 133 ± 12 pA. The mean of the individual RI values was 3.6 ± 0.3, which compares with an RI of 0.6 ± 0.01 at the same holding potentials in control aCSF with no added Mg 2+ ions ( Fig 4C). Thus, it appears from the RI values ( Fig 4C) that 30 µM ephenidine falls between 2mM Mg 2+ (RI = 4.2 ± 0.3) and 10 µM ketamine (RI = 2.0 ± 0.2). This is in contrast to the competitive antagonist, D-AP5, which, as expected, reduced both inward and outward current, so that its RI (0.8 ± 0.01) remains similar to the Mg 2+ -free control value (Fig 4C).
Other NMDA antagonists block the form of synaptic plasticity known as long-term potentiation (LTP), although interestingly this ability varies according to the nature of different channel blockers (Frankiewicz et al., 1996). We therefore tested ephenidine in a standard LTP protocol. Theta burst stimulation (TBS) applied to the Schaffer collateral pathway provided a robust potentiation of the fEPSP slope (Fig 5) in control slices interleaved between slices incubated with 30 (n =3; data not shown) or 10 µM (n =5; Fig 5) ephenidine. At both concentrations, ephenidine completely blocked the induction of LTP.

DISCUSSION.
We have shown that ephenidine, an abused psychoactive substance (Brandt et al., 2014) with dissociative effects in man, is a relatively selective, potent and voltage-dependent NMDA receptor antagonist and hence can be classified as an uncompetitive channel blocking compound.
Although ephenidine's use was detected in Germany in 2008 (Westphal et al., 2010), its availability from internet retailers has only been a more recent phenomenon. Ephenidine appears in older chemical (Goodson et al., 1946) and pharmacological (Tainter et al., 1943) literature. In a brief medicinal chemistry report, relating it to MK-801 as an anticonvulsant, its affinity for the PCP binding site was given as 257 nM (Thurkauf et al., 1989) and information regarding its rapid metabolism has also recently appeared (Wink et al., 2014;. However, its ability to affect NMDA receptor function had not been explored. The voltage-dependent effects, we report here for ephenidine (Fig 4), are similar to earlier reports on ketamine (MacDonald et al., 1987;, memantine and MK-801 (Wong et al., 1986;Chen et al., 1992;Frankiewicz et al., 1996) and Mg 2+ ions (Nowak et al., 1984;Mayer et al., 1984), in which outward currents were the less affected. Interestingly the nature of the voltagedependency block of NMDA receptors has been related to therapeutic potential (Frankiewicz et al., 1996). In the present experiments, due to slow wash-in times of ephenidine, it was not possible to achieve a full equilibrium block. However, ephenidine is clearly a highly voltage-dependent blocker of synaptic NMDA receptors and has a potency slightly greater than ketamine at resting membrane potentials. When compared with other uncompetitive NMDA antagonists in the same protocol for studying NMDA-fEPSPs (Wallach et al., 2016), the potency and pharmacodynamic properties of ephenidine are closer to those of ketamine than of phencyclidine.
Perhaps because of these pharmacodynamic properties or for some pharmacokinetic reason, ephenidine appears anecdotally to be popular amongst those interested in the dissociative state (see Introduction). The subjective effects reported appear dose-dependent and include dissociative-like effects including mood and thought alteration and complex visual hallucinations at doses between 100-500 mg (see Introduction).
Many voltage-dependent uncompetitive NMDA antagonists, such as ketamine, PCP and dextromethorphan, also show schizophrenia-like effects in man (Lodge and Mercier, 2015). Although ephenidine is not described as a psychotomimetic agent in the scientific literature, anecdotal descriptions of the dissociative effects of ephenidine (see above) suggest that, in high enough doses, it mimics some of the symptoms of schizophrenia. Casual users of ephenidine may put themselves in danger particularly as a result of the dissociative and analgesic effects of the NMDA receptor antagonism, as has been similarly described for ketamine abuse (Morgan and Curran, 2012). The block of the induction of LTP with 10 µM ephenidine predicts cognitive disruption and amnesia (Bliss and Collingridge, 1993). Our data also suggests that at even higher doses, interaction with monoamine transporters and sigma receptors may contribute to the behavioural effects of ephenidine.
Beside the analgesic and neuroprotective properties of ketamine (see Lodge and Mercier, 2015), an exciting novel therapeutic target for ketamine has emerged. Thus, ketamine and some other NMDA receptor antagonists have recently been proposed as rapidly acting anti-depressants (Berman et al., 2000;Zarate et al., 2006;Price et al., 2009). During depressed mood and during stress, memories of unpleasant events may be established through long-term potentiation (LTP) and long-term depression (LTD). Treatment with NMDA receptor antagonists, inhibitors of LTP and LTD, may block this cycle of pathological plasticity and change mood (Collingridge et al., 2010). Not all NMDA receptor antagonists have this property. Thus MK-801, another channel blocker with slower pharmacodynamic and pharmacokinetic properties than ketamine (see Wallach et al., 2016), does not show sustained anti-depressant-like effects (Maeng et al., 2008). It remains to be seen whether ephenidine, with pharmacodynamic and LTP blocking properties similar to ketamine, will have the appropriate profile for such a therapeutic indication.   Fig 1 with legend (0803).docx     Fig 3 with legend (0803).docx