In vivo effects of 3,4-methylenedioxymethamphetamine (MDMA) and its deuterated form in rodents: Drug discrimination and thermoregulation.

BACKGROUND: Recent clinical studies support the use of 3,4-methylenedioxymethamphetamine (MDMA) as an adjunct treatment for posttraumatic stress disorder (PTSD). Despite these promising findings, MDMA administration in controlled settings can increase blood pressure, heart rate, and body temperature. Previous studies indicate thatO-demethylated metabolites of MDMA contribute to its adverse effects. As such, limiting the conversion of MDMA to reactive metabolites may mitigate some of its adverse effects and potentially improve its safety profile for therapeutic use. METHODS: We compared the interoceptive and hyperthermic effects of a deuterium-substituted form of MDMA (d2-MDMA) to MDMA using rodent drug discrimination and biotelemetry procedures, respectively. RESULTS: Compared to MDMA, d2-MDMA produced full substitution for a 1.5 mg/kg MDMA training stimulus with equal potency and effectiveness in the drug discrimination experiment. In addition, d2-MDMA produced increases in body temperature that were shorter-lasting and of lower magnitude compared to equivalent doses of MDMA. Last, d2-MDMA and MDMA were equally effective in reversing the hypothermic effects of the selective 5-HT2A/2C antagonist ketanserin. CONCLUSION: These findings indicate that deuterium substitution of hydrogen at the methylenedioxy ring moiety does not impact MDMA's interoceptive effects, and compared to MDMA, d2-MDMA has less potential for producing hyperthermic effects and likely has similar pharmacodynamic properties. Given that d2-MDMA produces less adverse effects than MDMA, but retains similar desirable effects that are thought to relate to the effective treatment of PTSD, additional investigations into its effects on cardiovascular functioning and pharmacokinetic properties are warranted.

Locomotor effects of 3,4-methylenedioxymethamphetamine (MDMA) and its deuterated form in mice: psychostimulant effects, stereotypy, and sensitization.

RATIONALE: There is a renewed interest in the use of 3,4-methylenedioxymethamphetamine (MDMA) for treating psychiatric conditions. Although MDMA has entered phase II clinical trials and shows promise as an adjunct treatment, there is an extensive literature detailing the potential neurotoxicity and adverse neurobehavioral effects associated with MDMA use. Previous research indicates that the adverse effects of MDMA may be due to its metabolism into reactive catechols that can enter the brain and serve directly as neurotoxicants. One approach to mitigate MDMA's potential for adverse effects is to reduce O-demethylation by deuterating the methylenedioxy ring of MDMA. There are no studies that have evaluated the effects of deuterating MDMA on behavioral outcomes. OBJECTIVES: The purpose of the present study was to assess the motor-stimulant effects of deuterated MDMA (d2-MDMA) and compare them to MDMA in male mice. METHODS: Two experiments were performed to quantify mouse locomotor activity and to vary the drug administration regimen (single bolus administration or cumulative administration). RESULTS: The results of Experiments 1 and 2 indicate that d2-MDMA is less effective at eliciting horizontal locomotion than MDMA; however, the differences between the compounds diminish as the number of cumulative administrations increase. Both d2-MDMA and MDMA can elicit sensitized responses, and these effects cross-sensitize to the prototypical drug of abuse methamphetamine. Thus, d2-MDMA functions as a locomotor stimulant similar to MDMA, but, depending on the dosing regimen, may be less susceptible to inducing sensitization to stereotyped movements. CONCLUSIONS: These findings indicate that d2-MDMA is behaviorally active and produces locomotor effects that are similar to MDMA, which warrant additional assessments of d2-MDMA's behavioral and physiological effects to determine the conditions under which this compound may serve as a relatively safer alternative to MDMA for clinical use.

The importance of small polar radiometabolites in molecular neuroimaging: A PET study with [11C]Cimbi-36 labeled in two positions.

[11C]Cimbi-36, a 5-HT2A receptor agonist PET radioligand, contains three methoxy groups amenable to [11C]-labeling. In pigs, [11C]Cimbi-36 yields a polar (M1) and a less polar (M2) radiometabolite fraction, while changing the labeling to [11C]Cimbi-36_5 yields only the M1 fraction. We investigate whether changing the labeling position of [11C]Cimbi-36 eliminates M2 in humans, and if this changes the signal-to-background ratio. Six healthy volunteers each underwent two dynamic PET scans; after injection of [11C]Cimbi-36, both the M1 and M2 fraction appeared in plasma, whereas only the M1 appeared after [11C]Cimbi-36_5 injection. [11C]Cimbi-36_5 generated higher uptake than [11C]Cimbi-36 in both neocortex and cerebellum. With the simplified reference tissue model mean neocortical non-displaceable binding potential for [11C]Cimbi-36 was 1.38 ± 0.07, whereas for [11C]Cimbi-36_5, it was 1.18 ± 0.14. This significant difference can be explained by higher non-displaceable binding caused by demethylation products in the M1 fraction such as [11C]formaldehyde and/or [11C]carbon dioxide/bicarbonate. Although often considered without any impact on binding measures, we show that small polar radiometabolites can substantially decrease the signal-to-background ratio of PET radioligands for neuroimaging. Further, we find that [11C]Cimbi-36 has a better signal-to-background ratio than [11C]Cimbi-36_5, and thus will be more sensitive to changes in 5-HT2A receptor levels in the brain.

Synthesis, radiofluorination, and preliminary evaluation of the potential 5-HT2A receptor agonists [18 F]Cimbi-92 and [18 F]Cimbi-150.

An agonist PET tracer is of key interest for the imaging of the 5-HT2A receptor, as exemplified by the previously reported success of [11 C]Cimbi-36. Fluorine-18 holds several advantages over carbon-11, making it the radionuclide of choice for clinical purposes. In this respect, an 18 F-labelled agonist 5-HT2A receptor (5-HT2A R) tracer is highly sought after. Herein, we report a 2-step, 1-pot labelling methodology of 2 tracer candidates. Both ligands display high in vitro affinities for the 5-HT2A R. The compounds were synthesised from easily accessible labelling precursors, and radiolabelled in acceptable radiochemical yields, sufficient for in vivo studies in domestic pigs. PET images partially conformed to the expected brain distribution of the 5-HT2A R; a notable exception however being significant uptake in the striatum and thalamus. Additionally, a within-scan displacement challenge with a 5-HT2A R antagonist was unsuccessful, indicating that the tracers cannot be considered optimal for neuroimaging of the 5-HT2A R.

Detailed Characterization of the In Vitro Pharmacological and Pharmacokinetic Properties of N-(2-Hydroxybenzyl)-2,5-Dimethoxy-4-Cyanophenylethylamine (25CN-NBOH), a Highly Selective and Brain-Penetrant 5-HT2A Receptor Agonist.

Therapeutic interest in augmentation of 5-hydroxytryptamine2A (5-HT2A) receptor signaling has been renewed by the effectiveness of psychedelic drugs in the treatment of various psychiatric conditions. In this study, we have further characterized the pharmacological properties of the recently developed 5-HT2 receptor agonist N-2-hydroxybenzyl)-2,5-dimethoxy-4-cyanophenylethylamine (25CN-NBOH) and three structural analogs at recombinant 5-HT2A, 5-HT2B, and 5-HT2C receptors and investigated the pharmacokinetic properties of the compound. 25CN-NBOH displayed robust 5-HT2A selectivity in [3H]ketanserin/[3H]mesulergine, [3H]lysergic acid diethylamide and [3H]Cimbi-36 binding assays (Ki2C/Ki2A ratio range of 52-81; Ki2B/Ki2A ratio of 37). Moreover, in inositol phosphate and intracellular Ca2+ mobilization assays 25CN-NBOH exhibited 30- to 180-fold 5-HT2A/5-HT2C selectivities and 54-fold 5-HT2A/5-HT2B selectivity as measured by Δlog(Rmax/EC50) values. In an off-target screening 25CN-NBOH (10 µM) displayed either substantially weaker activity or inactivity at a plethora of other receptors, transporters, and kinases. In a toxicological screening, 25CN-NBOH (100 µM) displayed a benign acute cellular toxicological profile. 25CN-NBOH displayed high in vitro permeability (Papp = 29 × 10-6 cm/s) and low P-glycoprotein-mediated efflux in a conventional model of cellular transport barriers. In vivo, administration of 25CN-NBOH (3 mg/kg, s.c.) in C57BL/6 mice mice produced plasma and brain concentrations of the free (unbound) compound of ∼200 nM within 15 minutes, further supporting that 25CN-NBOH rapidly penetrates the blood-brain barrier and is not subjected to significant efflux. In conclusion, 25CN-NBOH appears to be a superior selective and brain-penetrant 5-HT2A receptor agonist compared with (±)-2,5-dimethoxy-4-iodoamphetamine (DOI), and thus we propose that the compound could be a valuable tool for future investigations of physiologic functions mediated by this receptor.

Characterization of the hepatic cytochrome P450 enzymes involved in the metabolism of 25I-NBOMe and 25I-NBOH.

The dimethoxyphenyl-N-((2-methoxyphenyl)methyl)ethanamine (NBOMe) compounds are potent serotonin 5-HT2A receptor agonists and have recently been subject to recreational use due to their hallucinogenic effects. Use of NBOMe compounds has been known since 2011, and several non-fatal and fatal intoxication cases have been reported in the scientific literature. The aim of this study was to determine the importance of the different cytochrome P450 enzymes (CYP) involved in the metabolism of 2-(4-iodo-2,5-dimethoxyphenyl)-N-(2methoxybenzyl)ethanamine (25I-NBOMe) and 2-[[2-(4-iodo-2,5dimethoxyphenyl)ethylamino]methyl]phenol (25I-NBOH) and to characterize the metabolites. The following approaches were used to identify the main enzymes involved in primary metabolism: incubation with a panel of CYP and monoamine oxidase (MAO) enzymes and incubation in pooled human liver microsomes (HLM) with and without specific CYP chemical inhibitors. The study was further substantiated by an evaluation of 25I-NBOMe and 25I-NBOH metabolism in single donor HLM. The metabolism pathways of 25I-NBOMe and 25I-NBOH were NADPHdependent with intrinsic clearance values of (CLint) of 70.1 and 118.7 mL/min/kg, respectively. The biotransformations included hydroxylation, O-demethylation, N-dealkylation, dehydrogenation, and combinations thereof. The most abundant metabolites were all identified by retention time and spectrum matching with synthesized reference standards. The major CYP enzymes involved in the metabolism of 25I-NBOMe and 25INBOH were identified as CYP3A4 and CYP2D6, respectively. The compound 25I-NBOH was also liable to direct glucuronidation, which may diminish the impact of CYP2D6 genetic polymorphism. Users of 25I-NBOMe may be subject to drug-drug interactions (DDI) if 25I-NBOMe is taken with a strong CYP3A4 inhibitor. Copyright © 2016 John Wiley & Sons, Ltd.

5-HT2A/5-HT2C Receptor Pharmacology and Intrinsic Clearance of N-Benzylphenethylamines Modified at the Primary Site of Metabolism.

The toxic hallucinogen 25B-NBOMe is very rapidly degraded by human liver microsomes and has low oral bioavailability. Herein we report on the synthesis, microsomal stability, and 5-HT2A/5-HT2C receptor profile of novel analogues of 25B-NBOMe modified at the primary site of metabolism. Although microsomal stability could be increased while maintaining potent 5-HT2 receptor agonist properties, all analogues had an intrinsic clearance above 1.3 L/kg/h predictive of high first-pass metabolism.

Metabolic Fate of Hallucinogenic NBOMes.

2,5-Dimethoxy-N-benzylphenethylamines (NBOMes) are very potent 5-HT2AR agonists. Illicit use of these psychedelic compounds has emerged in recent years, and several fatalities have been linked to their recreational use. In its [(11)C]-labeled form, one NBOMe (25B-NBOMe) was recently developed as a PET-ligand for clinical investigations of 5HT2AR ([(11)C]Cimbi-36). Herein, we have identified the phase I and phase II metabolites of 25B-NBOMe in pigs as well as in humans. We find that the primary route of metabolism is 5'-demethylation, followed by conjugation to glucuronic acid. Carbon-11 labeling of 25B-NBOMe in three different positions followed by in vivo evaluation in pigs and humans corroborated these findings.

Correlating the metabolic stability of psychedelic 5-HT2A agonists with anecdotal reports of human oral bioavailability.

2,5-Dimethoxyphenethylamines and their N-benzylated derivatives are potent 5-HT2A agonists with psychedelic effects in humans. The N-benzylated derivatives are among the most selective 5-HT2A agonists currently available and their usage as biochemical and brain imaging tools is increasing, yet very little is known about the relationships between the structure of the ligands and their pharmacokinetic profile. In order to evaluate the potential of these compounds for in vivo applications we have determined the microsomal stability of 11 phenethylamines and 27 N-benzylated derivatives thereof using human liver microsomes. We found that the N-benzylated phenethylamines have much higher intrinsic clearance than the parent phenethylamines. We hypothesize that their low hepatic stability renders them orally inactive due to first pass metabolism, which is supported by anecdotal data from recreational use of these compounds.

Synthesis and structure-activity relationships of N-benzyl phenethylamines as 5-HT2A/2C agonists.

N-Benzyl substitution of 5-HT2A receptor agonists of the phenethylamine structural class of psychedelics (such as 4-bromo-2,5-dimethoxyphenethylamine, often referred to as 2C-B) confer a significant increase in binding affinity as well as functional activity of the receptor. We have prepared a series of 48 compounds with structural variations in both the phenethylamine and N-benzyl part of the molecule to determine the effects on receptor binding affinity and functional activity at 5-HT2A and 5-HT2C receptors. The compounds generally had high affinity for the 5-HT2A receptor with 8b having the highest affinity at 0.29 nM but with several other compounds also exhibiting subnanomolar binding affinities. The functional activity of the compounds was distributed over a wider range with 1b being the most potent at 0.074 nM. Most of the compounds exhibited low to moderate selectivity (1- to 40-fold) for the 5-HT2A receptor in the binding assays, although one compound 6b showed an impressive 100-fold selectivity for the 5-HT2A receptor. In the functional assay, selectivity was generally higher with 1b being more than 400-fold selective for the 5-HT2A receptor.

Structure-activity relationships of constrained phenylethylamine ligands for the serotonin 5-HT2 receptors.

Serotonergic ligands have proven effective drugs in the treatment of migraine, pain, obesity, and a wide range of psychiatric and neurological disorders. There is a clinical need for more highly 5-HT2 receptor subtype-selective ligands and the most attention has been given to the phenethylamine class. Conformationally constrained phenethylamine analogs have demonstrated that for optimal activity the free lone pair electrons of the 2-oxygen must be oriented syn and the 5-oxygen lone pairs anti relative to the ethylamine moiety. Also the ethyl linker has been constrained providing information about the bioactive conformation of the amine functionality. However, combined 1,2-constriction by cyclization has only been tested with one compound. Here, we present three new 1,2-cyclized phenylethylamines, 9-11, and describe their synthetic routes. Ligand docking in the 5-HT2B crystal structure showed that the 1,2-heterocyclized compounds can be accommodated in the binding site. Conformational analysis showed that 11 can only bind in a higher-energy conformation, which would explain its absent or low affinity. The amine and 2-oxygen interactions with D3.32 and S3.36, respectively, can form but shift the placement of the core scaffold. The constraints in 9-11 resulted in docking poses with the 4-bromine in closer vicinity to 5.46, which is polar only in the human 5-HT2A subtype, for which 9-11 have the lowest affinity. The new ligands, conformational analysis and docking expand the structure-activity relationships of constrained phenethylamines and contributes towards the development of 5-HT2 receptor subtype-selective ligands.

N-Methylanilide and N-methylbenzamide derivatives as phosphodiesterase 10A (PDE10A) inhibitors.

PDE10A is a recently identified phosphodiesterase with a quite remarkable localization since the protein is abundant only in brain tissue. Based on this unique localization, research has focused extensively on using PDE10A modulators as a novel therapeutic approach for dysfunction in the basal ganglia circuit including Parkinson's disease, Huntington's disease, schizophrenia, addiction and obsessive compulsive disorder. Medicinal chemistry efforts identified the N-methyl-N-[4-(quinolin-2-ylmethoxy)-phenyl]-isonicotinamide (8) as a nanomolar PDE10A inhibitor. A subsequent Lead-optimization program identified analogous N-methylanilides and their corresponding N-methylbenzamides (29) as potent PDE10A inhibitors, concurrently some interesting and unexpected binding modes were identified.