This study (2013) determined the pharmacological profiles of novel ketamine and phencyclidine analogues and compared them to the parent substances.
In this paper we determined the pharmacological profiles of novel ketamine and phencyclidine analogues currently used as ‘designer drugs’ and compared them to the parent substances via the resources of the National Institute of Mental Health Psychoactive Drug Screening Program. The ketamine analogues methoxetamine ((RS)-2-(ethylamino)-2-(3-methoxyphenyl)cyclohexanone) and 3-MeO-PCE (N-ethyl-1-(3-methoxyphenyl)cyclohexanamine) and the 3- and 4-methoxy analogues of phencyclidine, (1-[1-(3-methoxyphenyl)cyclohexyl]piperidine and 1-[1-(4-methoxyphenyl)cyclohexyl]piperidine), were all high affinity ligands for the PCP-site on the glutamate NMDA receptor. In addition methoxetamine and PCP and its analogues displayed appreciable affinities for the serotonin transporter, whilst the PCP analogues exhibited high affinities for sigma receptors. Antagonism of the NMDA receptor is thought to be the key pharmacological feature underlying the actions of dissociative anaesthetics. The novel ketamine and PCP analogues had significant affinities for the NMDA receptor in radioligand binding assays, which may explain their psychotomimetic effects in human users. Additional actions on other targets could be important for delineating side-effects.
Novel synthetic psychoactive drugs sold via the internet have raised concerns because many lack formal toxicology profiles. Roth and colleagues describe three classes of emerging ‘‘designer’’ dissociative drugs: methoxetamine (an analogue of ketamine), 3-methoxy-eticyclidine (3-MeO-PCE, a deoxy-analogue of methoxetamine), and two methoxy-substituted phencyclidine analogues (3-MeO-PCP and 4-MeO-PCP). These compounds have been used as legal alternatives to controlled dissociatives and reportedly produce effects in humans similar to ketamine and PCP, including euphoria, dissociation, hallucinations and, in some cases, sympathomimetic toxicity and reversible cerebellar injury. Ketamine and PCP exert major actions as non-competitive antagonists at the NMDA (N-methyl-D-aspartate) subtype of glutamate receptors, and the behavioural similarity of the novel analogues has led to the hypothesis that they act at the same target(s). The introduction also notes established harms of chronic ketamine use (for example ulcerative cystitis) and emphasises the current lack of knowledge about long-term effects of the new analogues. This study set out to characterise the neurochemical profiles of methoxetamine and several methoxy-substituted PCP analogues and to compare them with ketamine, PCP and other reference compounds. Using the resources of the National Institute of Mental Health Psychoactive Drug Screening Program (NIMH-PDSP), the investigators obtained receptor binding and functional data across a broad panel of central nervous system targets. Chemical identity and purity of the tested samples were confirmed prior to pharmacological screening, allowing comparison of affinity profiles and identification of additional off-target interactions that might underlie adverse effects or distinguish these analogues from their parent drugs.
Papers in Blossom that reference this study
du Jardin, K. G., Müller, H. K., Elfving, B. et al. · Progress in Neuro-Psychopharmacology and Biological Psychiatry (2016)
Chemical characterisation and samples: The study tested six compounds: ketamine, PCP, methoxetamine, 3-MeO-PCP, 4-MeO-PCP and 3-MeO-PCE. Roth and colleagues report that 4-MeO-PCP was purchased from an online supplier and its identity was confirmed by one- and two-dimensional nuclear magnetic resonance (NMR), high-resolution electrospray ionisation mass spectrometry (HRESIMS), infrared spectroscopy and elemental analysis. Methoxetamine, 3-methoxy-PCP and 3-methoxy-PCE were analysed by proton NMR, mass spectrometry and infrared spectroscopy; purities were determined by high-performance liquid chromatography with diode-array detection and corrected for residual water (Karl Fischer) and residual solvent (proton NMR). Certified purities reported in the extracted text were 98.3% (methoxetamine), 99.1% (3-methoxy-PCP) and 99.0% (3-methoxy-PCE). Pharmacological profiling: Receptor binding and functional assays were performed through the NIMH-PDSP using their standard procedures (the paper refers to on-line protocol details). Compounds were initially screened in quadruplicate at a fixed concentration reported in the extraction as 10 mM; targets producing greater than 50% inhibition in the primary screen were advanced to Ki determinations. Ki values were obtained by 12-point concentration–response studies in triplicate. The extracted text states that a total of 57 molecular targets relevant to CNS drug action were included in the radioligand binding panel. Where the prose did not specify further procedural details the authors referred readers to PDSP protocols and to tables/figures for full Ki values; the extracted text does not include the full assay protocols or the complete numerical dataset.
Roth and colleagues screened six compounds across a panel of 57 CNS-relevant molecular targets using radioligand binding assays. The primary screen was used to identify targets showing greater than 50% inhibition at the tested concentration, and those targets were followed up with full concentration–response Ki determinations. All novel analogues showed significant affinity for the PCP binding site on the NMDA receptor. Methoxetamine was reported to have an affinity for the NMDA receptor comparable to or higher than ketamine. The 3-methoxy phencyclidine analogue, 3-MeO-PCP, was particularly potent at the NMDA site, with a reported Ki of 20 nM; the authors place this value among the most potent known NMDA antagonists and compare it to the MK-801 benchmark (reported Ki = 4.8 nM in the extraction). Several compounds displayed additional noteworthy interactions: PCP, methoxetamine and the PCP analogues showed appreciable affinity for sigma receptors, whereas ketamine showed no significant effect at sigma-1 or sigma-2 receptors when tested at the reported screening concentration (10 mM). Methoxetamine, 4-MeO-PCP and 3-MeO-PCE exhibited submicromolar affinities for the serotonin transporter (SERT). By contrast, the extracted text reports no appreciable affinity for the human dopamine transporter (hDAT) in binding assays at the tested concentration, although the exact numerical value for the tested concentration is unclear in the extraction. The paper notes that full Ki values and a three-dimensional summary mesh plot are provided in tables and figures referenced in the text; those detailed numerical tables are not present in the extracted material provided here.
The investigators interpret their screening results as indicating that the novel methoxy-substituted ketamine and PCP analogues share the principal pharmacological profile of ketamine and PCP as ligands at the NMDA receptor. In particular, methoxetamine demonstrated NMDA affinity comparable to or exceeding that of ketamine, and 3-MeO-PCP displayed especially high potency (Ki = 20 nM), placing it among the more potent NMDA antagonists identified to date. Structure–activity relationships are discussed: the addition of a 3-methoxyl group on the phenyl ring appears to modulate target binding, enhancing affinity at SERT in some compounds and correlating with strong NMDA affinity in the 3-methoxy PCP analogue. The authors contrast their findings with some prior reports that implicated the dopamine transporter and sigma receptors in the pharmacology of dissociative anaesthetics. In this screening, no appreciable affinity for hDAT was observed in binding assays at the tested concentration (the extraction does not clearly state the numerical test concentration), and ketamine did not bind sigma receptors under the same conditions, although other analogues did. The authors suggest assay differences (for example substrates or uptake assays versus radioligand displacement assays) may account for discrepancies between studies. Potential clinical implications are raised cautiously: because NMDA antagonism is thought to underlie the psychotomimetic and dissociative effects of ketamine and PCP, the strong NMDA affinity of these analogues may explain similar effects reported in human users and suggests a risk of psychiatric sequelae with abuse. The paper also notes case reports of acute methoxetamine toxicity, including a ‘‘dissociative catatonic’’ presentation with sympathomimetic signs and reversible cerebellar toxicity, and reiterates that chronic harms observed with ketamine (for example ulcerative cystitis and dependence) have not yet been established for methoxetamine. Finally, the authors observe that while these substances pose risks, chemical analogues of ketamine might also have pharmaceutical interest given ketamine’s rapid antidepressant effects; they acknowledge limitations of their screening approach, including an inability to distinguish NMDA receptor subtypes and the constraints inherent in radioligand binding panels, and they anticipate that further analogues will continue to emerge and require characterisation.
The recent emergence of novel synthetic psychoactive drugs and their sale through internet sites has raised concerns about the potential harms associated with compounds which lack any formal toxicology profiles. Among the novel psychoactive substances that have emerged in recent years are methoxetamine ((RS)-2-(ethylamino)-2-(3-methoxyphenyl)cyclohexanone), which is an analogue of ketamine ((RS)-2-(2-chlorophenyl)-2-(methylamino)cyclohexanone), methoxetamine's close deoxy-analogue 3-methoxyeticyclidine ('3-MeO-PCE', N-ethyl-1-(3-methoxyphenyl)cyclohexanamine), and both the 3-and 4-methoxy analogues of phencyclidine, namely 1-[1-(3-methoxyphenyl)cyclohexyl]piperidine and 1-[1-(4-methoxyphenyl)cyclohexyl]piperidine (Figure). Methoxetamine, also known as 'MXE', 'MXE-powder', 'METH-O', and 'MEXXY' has gained some prominence in the United Kingdom as a legal alternative to ketamine. Phencyclidine (PCP) and the related compounds eticyclidine (PCE), rolicyclidine and tenocyclidine are controlled substances, but recently 3methoxy-PCP, 4-methoxy-PCP, and 3-methoxy-PCE have emerged as legally available alternatives to PCP. Pharmacologically, ketamine's main action is on glutamatergic transmission, the major excitatory neurotransmitter system in the brain. It is a non-competitive antagonist at one of the three glutamate receptor subtypes, the N-methyl-D-aspartate (NMDA) receptor. The NMDA receptor is also considered to be a key pharmacological target for phencyclidine. Although there is little information available on the novel ketamine and PCP analogues, their behavioural effects in human subjects resemble those induced by ketamine and PCP, characteristic of dissociative anaesthetics. The wanted effects include euphoria, empathy, dissociation from the physical body, hallucinations, but these may be accompanied by adverse side effects, dizziness, confusion, psychomotor agitation, and cognitive impairment. The clinically reported symptoms of acute toxicology of methoxetamine include a 'dissociative catatonic' state similar to that seen with ketamine, accompanied by sympathomimetic toxicity, with significant tachycardia and hypertension. Reversible cerebellar toxicity has also been reported in three cases of methoxetamine overdose. A major physical harm associated with chronic ketamine use is ulcerative cystitis, leading to significant damage to bladder function, and evidence of dependence, but it is not yet known whether methoxetamine will also prove to be associated with these adverse side effects. In the present study the resources of the National Institute of Mental Health Psychoactive Drug Screening Program (NIMH-PDSP) were used to obtain neurochemical profiles of methoxetamine and the novel PCP analogues and to compare these with those of ketamine and PCP and other reference compounds. The results confirmed that all of the novel analogues had significant affinity for NMDA receptors, and revealed other effects possibly mediated by monoamine transporter targets and sigma receptors. Chemical identity of these materials was established using proton Nuclear Magnetic Resonance, Mass Spectrometry and Infrared Spectroscopy. Purities were established using High Performance Liquid Chromatography with Diode Array Detection, and corrected for any residual water (by Karl Fischer) and residual solvent (by proton NMR). Certified purities were 98.3% (methoxetamine), 99.1% (3-methoxy-PCP) and 99.0% (3-methoxy-PCE) 1 .
4-Methoxyphencyclidine (4-MeO-PCP; 1-[1-(4-methoxyphenyl)cyclohexyl]piperidine) was purchased from a UK-based website (Mandala supplies). The chemical identity was confirmed by 1-and 2-dimensional Nuclear Magnetic Resonance (NMR) spectroscopy, High-Resolution Electrospray Ionization Mass Spectrometry (HRESIMS), Infrared Spectroscopy (IR) and elemental analysis which confirmed the presence of the free base form. The 1 H-NMR and IR spectral were data were identical to those of synthetic 4-MeO-PCP from the literature. Ketamine and phencyclidine were from the NIMH-PDSP. Chemical structures are shown in Figure.
Ki determinations, receptor binding profiles and functional assays were provided by the National Institute of Mental Health's Psychoactive Drug Screening Program essentially as previously described; full methodological details are found on-line at:In brief, compounds were initially screened in quadruplicate at a fixed concentration of 10 mM. Compounds which yielded inhibition of binding .50% were subjected to Ki determinations via 12-point concentration-response studies in triplicate as described[17] andCH%20Protocol%20Book.pdf. All compounds were screened against the targets listed in Table.
A total of 6 compounds (ketamine, PCP, methoxetamine, 3-MeO-PCP, 4-MeO-PCP and, 3-MeO-PCE; chemical structures in Figure, were screened at 57 molecular targets relevant to CNS drug action (Table) in quadruplicate at 10 mM via radioligand binding assays. Where initial screening results disclosed significant inhibitory activity (.50% inhibitory activity), Ki determinations were performed as previously detailed. Representative Ki value determinations are summarized in Table. Figureshows a summary of the final pKi data in a three dimensional mesh plot format (see Tablefor Ki values) while Figure
The results obtained in receptor screening reveal that the novel analogues share the profile of ketamine and PCP as ligands for the glutamate NMDA receptor. Although one previous study reported that a number of ketamine and PCP analogues, including 4-MeO-PCP, were active as NMDA receptor antagonists, using both GluN2A and GluN2B receptor subtypes, this study did not include methoxetamine or the 3-MeO-PCP and 3-MeO-PCE analogues. The present screening approach cannot distinguish between NMDA receptor subtypes, but did reveal methoxetamine to have an affinity for the NMDA receptor comparable to or higher than the parent compound ketamine. The methoxy analogues of PCP also had appreciable affinities for the NMDA receptor, and 3-MeO-PCP in particular proved particularly active, with a Ki of 20 nM placing it among the most potent known NMDA antagonists (cf MK-801 Ki = 4.8 nM). Some indications of the relationship between chemical structure and function can be discerned. Thus, methoxetamine is ketamine without the 2-chloro but with a '3-methoxyl' group on the phenyl ring and with an N-ethyl rather than N-methyl substituent, whilst 3-MeO-phencyclidine is phencyclidine with a 3-methoxyl substituent on the phenyl ring. The addition of the 3-methoxyl moiety to the phenyl ring thus appears to enhance the affinity for the serotonin transporter. A potential role for glutamatergic mechanisms in schizophrenia was first proposed based on the observation that psychotomimetic drugs such as PCP and ketamine induce psychotic symptoms and neurocognitive disturbances similar to those of schizophrenia by blocking glutamate actions at NMDA receptors. While previous reports have implicated the dopamine transporter (DAT) and sigma receptors in the behavioural pharmacology of ketamine and PCP analogues, the present findings do not support these suggestions. Nishimura et alfound only weak effects of ketamine isomers on rat brain DAT (Ki = 50-390 mM) while Chaudieu et alreported submicromolar potency for PCP and some related analogues. However, in the present study no appreciable affinity was observed for any compound at a concentration of ,10 mM for hDAT in binding assays. The poor correlation with the results of Chaudieu et allikely reflects the fact that the substrate can bind to different sites on the transporter than the radioligands used for displacement assays. Although PCP, methoxetamine and the PCP analogues had appreciable affinity for the sigma receptors (Table), (Table), ketamine had no significant effect on either sigma -1 or sigma 2 receptors when tested at 10 mM, suggesting that while interactions with these receptors might contribute to the profile of some dissociative anaesthetics, this is not a common property which all share. Similarly, while methoxetamine, 4-MeO-PCP and 3-MeO-PCE displayed submicromolar affinities for the serotonin transporter (SERT), this is not a universal property of these drugs. Other publications have described a variety of other synthetic analogues of ketamine and PCP, so it is likely that many other chemical analogues of this family of drugs will be found to possess the characteristic dissociative anaesthetic properties of ketamine and PCP. These results imply that abuse of these ketamine and PCP analogues could be associated with significant psychiatric sequelae. On the other hand, analogues of ketamine are also of pharmaceutical interest, following the discovery of the rapid antidepressant properties of ketamine.
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