The Ketamine Analogue Methoxetamine and 3- and 4-Methoxy Analogues of Phencyclidine Are High Affinity and Selective Ligands for the Glutamate NMDA Receptor

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.

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.