This rat study (n=180) investigated the pharmacokinetic interactions between 5-MeO-DMT (0.25mg/0.25kg) and the MAO-A inhibitor clorgyline and the MAO-A/B inhibitor pargyline, and their effects on prepulse inhibition (PPI), a phenomenon related to the ability to filter out unnecessary information. Results confirmed that the MAO inhibitors increase the accumulation of 5-MeO-DMT in the nervous system, and boost its disruptive effects on PPI by activating the 5-HT2a receptor pathway.
Introduction
Monoamine oxidase inhibitors (MAOIs) are often ingested together with tryptamine hallucinogens, but relatively little is known about the consequences of their combined use. We have shown previously that monoamine oxidase-A (MAO-A) inhibitors alter the locomotor profile of the hallucinogen 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) in rats, and enhance its interaction with 5-HT2A receptors. The goal of the present studies was to investigate the mechanism for the interaction between 5-MeO-DMT and MAOIs, and to determine whether other behavioral responses to 5-MeO-DMT are similarly affected. Hallucinogens disrupt prepulse inhibition (PPI) in rats, an effect typically mediated by 5-HT2A activation. 5-MeO-DMT also disrupts PPI but the effect is primarily attributable to 5-HT1A activation.
Methods
The present studies examined whether an MAOI can alter the respective contributions of 5-HT1A and 5-HT2A receptors to the effects of 5-MeO-DMT on PPI. A series of interaction studies using the 5-HT1A antagonist WAY-100635 and the 5-HT2A antagonist MDL 11,939 were performed to assess the respective contributions of these receptors to the behavioral effects of 5-MeO-DMT in rats pretreated with an MAOI. The effects of MAO-A inhibition on the pharmacokinetics of 5-MeO-DMT and its metabolism to bufotenine were assessed using liquid chromatography-electrospray ionization-selective reaction monitoring-tandem mass spectrometry (LC-ESI-SRM-MS/MS).
Results
5-MeO-DMT (1 mg/kg) had no effect on PPI when tested 45-min post-injection but disrupted PPI in animals pretreated with the MAO-A inhibitor clorgyline or the MAO-A/B inhibitor pargyline. The combined effect of 5-MeO-DMT and pargyline on PPI was antagonized by pretreatment with either WAY-100635 or MDL 11,939. Inhibition of MAO-A increased the level of 5-MeO-DMT in plasma and whole brain, but had no effect on the conversion of 5-MeO-DMT to bufotenine, which was found to be negligible.
Discussion
The present results confirm that 5-MeO-DMT can disrupt PPI by activating 5-HT2A, and indicate that MAOIs alter 5-MeO-DMT pharmacodynamics by increasing its accumulation in the central nervous system.
Papers cited by this study that are also in Blossom
Cakic, V., Potkonyak, J., Marshall, A. · Drug and Alcohol Dependence (2010)
Halberstadt, A. L. · Behavioural Brain Research (2014)
McKenna, D., Towers, G. H., Abbott, F. · Journal of Ethnopharmacology (1984)
Papers in Blossom that reference this study
Earlier research shows that tryptamine hallucinogens are frequently consumed together with monoamine oxidase inhibitors (MAOIs) in preparations such as ayahuasca and in recreational use, but the consequences of combined use are incompletely understood. Hallucinogens classically exert their effects via 5-HT2A receptor activation, yet indoleamines such as 5-MeO-DMT also interact with other serotonin receptor subtypes, notably 5-HT1A. Prior studies from this laboratory demonstrated that MAO-A inhibitors change the locomotor effects of 5-MeO-DMT in rats and enhance its engagement with 5-HT2A receptors; one mechanistic hypothesis is that MAO-A inhibition alters 5-MeO-DMT pharmacokinetics and/or increases its conversion to bufotenine, a potent 5-HT2A agonist. This study set out to clarify the mechanism underlying interactions between 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) and MAOIs. Specifically, Halberstadt examined whether MAOI pretreatment alters the relative contributions of 5-HT1A and 5-HT2A receptors to 5-MeO-DMT-induced disruption of prepulse inhibition (PPI) and whether MAO-A inhibition changes the pharmacokinetics of 5-MeO-DMT and its O-demethylation to bufotenine in rats. The work combined behavioural pharmacology (acoustic startle/PPI and locomotor monitoring) with quantitative LC-ESI-SRM-MS/MS measurement of drug levels in plasma and whole brain.
Adult male Sprague-Dawley rats (initial weight 250–275 g) were used. Animals were pair-housed on a reverse light–dark cycle and acclimatised for ~1 week; all procedures conformed to institutional animal care guidelines. Drugs were administered subcutaneously in a volume of 1 mL/kg. Doses reported in the paper include 5-MeO-DMT (freebase) at 0, 0.1, or 1.0 mg/kg, the non-selective MAOI pargyline at 0 or 10 mg/kg, the MAO-A inhibitor clorgyline at 0 or 0.3 mg/kg, the 5-HT1A antagonist WAY-100,635 at 1.0 mg/kg, and the 5-HT2A antagonist MDL 11,939 at 0.3 mg/kg. MDL 11,939 was prepared in acidified saline with Tween 80; other drugs were dissolved in isotonic saline. Antagonists were given 20 min prior to 5-MeO-DMT in antagonist experiments, and MAOI pretreatments were given 20 min before 5-MeO-DMT. Behavioural assessments comprised two main assays. Acoustic startle and PPI were measured in SR-LAB startle chambers using standard PULSE-ALONE (40-ms 120-dB white noise) and PREPULSE + PULSE trials with prepulses at 68, 71, and 77 dB (3, 6, and 12 dB above 65-dB background). Rats were tested 45 min after 5-MeO-DMT administration to target the time window when MAOI + 5-MeO-DMT produce delayed locomotor hyperactivity (approx. 40–70 min post-injection). For locomotor/pharmacokinetic correlation, activity was recorded in a Behavioural Pattern Monitor (BPM) and animals were removed at specified time points (10–60 min post-dose) for collection of trunk blood and whole brain for drug analysis. Experimental group sizes were provided: experiment 1 (pargyline interaction) n = 10/group (60 total), experiment 2 (clorgyline) n = 12/group (48 total), and experiment 3 (antagonist blockade) n = 10/group (60 total) with pargyline co-administration for antagonist tests. For pharmacokinetic and metabolite analysis, plasma and whole-brain homogenates were prepared and extracted using solid phase extraction. Quantification was performed by LC-ESI-SRM-MS/MS (Agilent 1100 LC coupled to Thermo TSQ Quantum Access triple quadrupole) with N-methylserotonin as internal standard. Monitored transitions were 5-MeO-DMT 219 → 174 and bufotenine 205 → 166. Calibration, recovery, accuracy, and precision metrics were reported and used to derive concentrations; Helsel's Robust Method was used to handle values below the limit of detection in some groups. Pharmacokinetic metrics included Cmax and AUC20→70 min, and statistical comparisons used ANOVA for continuous outcomes and nonparametric Tarone–Ware tests for equivalence of censored analyte levels where appropriate. Behavioural data were analysed primarily with two- or three-factor ANOVA (factors: pretreatment, treatment, and prepulse intensity as a repeated measure), with post hoc Tukey tests or Bonferroni corrections as indicated.
PPI and startle: When 5-MeO-DMT (1 mg/kg) was administered 45 min before testing, it did not alter PPI in vehicle-pretreated rats. However, rats pretreated with the non-selective MAOI pargyline (10 mg/kg) showed a significant reduction in PPI after 5-MeO-DMT, yielding a pretreatment × treatment interaction (F(2,54) = 17.59, p < 0.0001) and a main effect of 5-MeO-DMT (F(2,54) = 18.87, p < 0.0001). Pairwise tests indicated that 1 mg/kg 5-MeO-DMT significantly reduced PPI at all three prepulse intensities in pargyline-pretreated animals (p < 0.01). Pargyline alone reduced the startle amplitude (significant interaction between pargyline and 5-MeO-DMT on startle: F(2,54) = 5.07, p < 0.01), but subgroup analyses matched for startle magnitude demonstrated that the PPI disruption produced by pargyline + 5-MeO-DMT persisted independently of startle changes (pretreatment × treatment: F(2,36) = 10.44, p = 0.0003). A similar pattern was observed with selective MAO-A inhibition: clorgyline (0.3 mg/kg) did not alter PPI by itself, but clorgyline-pretreated rats exhibited significant PPI reduction after 1 mg/kg 5-MeO-DMT (pretreatment × treatment: F(1,44) = 24.74, p < 0.0001), with 5-MeO-DMT lowering PPI at all three prepulse intensities (p < 0.01). Startle magnitude was not significantly affected by clorgyline or 5-MeO-DMT in this experiment. Receptor antagonism: Antagonist studies tested whether 5-HT1A and 5-HT2A receptors mediated the PPI disruption produced by pargyline + 5-MeO-DMT. Pretreatment with the 5-HT1A antagonist WAY-100,635 (1 mg/kg) significantly antagonised the PPI reduction (pretreatment × treatment: F(1,36) = 73.11, p < 0.0001; post hoc p < 0.01). Similarly, the 5-HT2A antagonist MDL 11,939 (0.3 mg/kg) substantially attenuated the PPI disruption (pretreatment × treatment: F(1,36) = 23.96, p < 0.0001; post hoc p < 0.01). Startle magnitude was not meaningfully altered by MDL 11,939 and only trends were noted with WAY-100,635. Pharmacokinetics and bufotenine: Method validation indicated acceptable recovery (62.7–88.0%), reproducible calibration (mean r2 ≈ 0.99), and accuracy/precision within acceptable bounds for both analytes. After 1 mg/kg 5-MeO-DMT, plasma and brain concentrations peaked within 20 min and then declined. Clorgyline did not significantly increase plasma Cmax (vehicle 52.7 ± 11.8 ng/mL vs clorgyline 78.1 ± 9.5 ng/mL), but clorgyline significantly slowed plasma clearance, producing elevated plasma concentrations from 30–70 min and a marked increase in plasma AUC20→70 min (vehicle 12.2 ± 1.7 ng•h/mL vs clorgyline 31.4 ± 2.3 ng•h/mL; F(1,59) = 45.6, p < 0.0001). The central exposure change was striking: whole-brain Cmax increased from 30.3 ± 11.2 ng/g (vehicle) to 633.8 ± 146.5 ng/g with clorgyline (F(1,9) = 16.8, p < 0.004), and 5-MeO-DMT remained detectable for longer in brain tissue when MAO-A was inhibited. By contrast, bufotenine concentrations in plasma and brain were mostly below the limit of detection (plasma LLOQ 1–2 ng/mL; brain LLOQ 10 ng/g), and clorgyline did not produce a clear increase in bufotenine levels (plasma Cmax ~1.3 ng/mL vehicle vs 1.8 ng/mL clorgyline). Locomotor correlation: BPM monitoring confirmed the expected behavioural interaction: clorgyline pretreatment produced a significant interaction with time block on locomotor crossings (F(5,48) = 3.64, p < 0.008) and increased crossings in later time blocks compared with vehicle, consistent with delayed hyperactivity coincident with elevated central 5-MeO-DMT.
Halberstadt and colleagues interpret these results as showing that MAOI pretreatment markedly prolongs and alters the behavioural profile of 5-MeO-DMT by increasing its systemic and, especially, central accumulation. Whereas 5-MeO-DMT alone produces brief PPI and locomotor effects primarily mediated by 5-HT1A receptors, MAO-A inhibition allowed 5-MeO-DMT to disrupt PPI at 45 min post-dose and to recruit 5-HT2A receptor mechanisms; this was supported by antagonist blockade with both WAY-100,635 and MDL 11,939. Pharmacokinetic data support a mechanism of inhibited deamination: clorgyline greatly increased brain 5-MeO-DMT concentrations (whole-brain Cmax rising into the low micromolar range) and prolonged its presence in the central nervous system, while conversion to bufotenine in rats was negligible and therefore unlikely to account for the altered behavioural profile. The authors place their findings in the context of prior animal studies reporting MAOI-driven increases in blood and brain 5-MeO-DMT and note that a deuterated 5-MeO-DMT isotopologue resistant to MAO-A metabolism produces behavioural effects similar to 5-MeO-DMT combined with an MAOI, supporting a pharmacokinetic explanation. They further propose that the delayed appearance of 5-HT2A-mediated behaviours could involve downstream interactions with dopaminergic systems, since 5-HT2A activation can facilitate phasic dopamine release and produce delayed dopaminergic sensitisation; future studies are proposed to test this possibility. Regarding translational relevance, the discussion highlights toxicological concerns because MAOIs and tryptamine hallucinogens are used together in humans (for example in ayahuasca preparations). The authors point out case reports of severe toxicity and death following combined use, and suggest that MAOI-driven increases in central concentrations of 5-MeO-DMT could underlie such adverse outcomes. They also caution that while these findings for 5-MeO-DMT are likely relevant to other tryptamines that are MAO substrates (for example DMT), species differences exist: mice have shown increased O-demethylation to bufotenine in some studies, whereas this rat study found negligible bufotenine formation. The authors therefore call for further research to characterise the influence of MAOIs on DMT pharmacokinetics and on the mechanisms of action of ayahuasca. Limitations and uncertainties acknowledged in the text include species differences in metabolism (rats vs mice), the low detectability of bufotenine in rat samples which limits conclusions about its role, and the need for additional work to establish whether dopaminergic mechanisms contribute to the delayed 5-HT2A-mediated behavioural responses. The authors refrain from extending causal claims beyond the data, instead framing implications in terms of probable mechanistic pathways and public health relevance given contemporary patterns of polypharmacy with MAOIs and tryptamines.
It is not uncommon for tryptamine hallucinogens to be administered in combination with drugs that inhibit monoamine oxidase-A (MAO A ). For example, the potent hallucinogenic beverage known as ayahuasca contains tryptamines such as N,N-dimethyltryptamine (DMT) and 5methoxy-DMT (5-MeO-DMT), as well as β-carbolines such as harmaline and harmine, which are reversible MAO A inhibitors (MAOIs). Although DMT is normally inactive orally due to first-pass metabolism, the β-carbolines contribute to the activity of ayahuasca by blocking the catabolism of DMT in the gastrointestional tract. Modern Brazilian syncretic religious groups such as União do Vegetal have adopted the use of ayahuasca as a sacrament, and this practice has now spread to Europe and North America. DMT and 5-MeO-DMT are also used recreationally in Western societies, typically by smoking, but they can also be ingested orally in combination with an MAOI. Indeed, there is evidence that tryptamine hallucinogens and MAOIs are abused together. 5-HT 2A receptor activation is responsible for mediating the characteristic subjective and behavioral effects of hallucinogens in both humans and animals. All serotonergic hallucinogens bind to 5-HT 2A receptors, but with differing selectivity. Phenylalkylamines, including mescaline and 2,5-dimethoxy-4methylamphetamine (DOM), are selective for 5-HT 2 sites, whereas indoleamines such as N,N-dimethyltryptamine (DMT), 5-MeO-DMT, psilocybin, and (+)-lysergic acid diethylamide (LSD), are non-selective for serotonin (5-HT) receptor subtypes. Previous studies in this laboratory have shown that the behavioral effects of 5-MeO-DMT are modified in animals treated with an MAOI. Rats treated with 5-MeO-DMT alone exhibit a brief reduction in locomotor activity that can be blocked by the 5-HT 1A -selective antagonist WAY-100,635, but not by a 5-HT 2A -selective antagonist. By contrast, when 5-MeO-DMT is administered to rats pretreated with a behaviorally inactive dose of an MAOI (e.g. clorgyline, pargyline, or harmaline), it produces biphasic effects, initially suppressing locomotor activity and then increasing activity at later time points. Administration of higher doses of 5-MeO-DMT alone did not reproduce this effect. The behavioral profile of 5-MeO-DMT is altered by the MAO A inhibitors harmaline and clorgyline, as well as by the MAO A/B inhibitor pargyline, whereas the selective MAO B inhibitor (-)-deprenyl is ineffective. Given those findings, it is likely that MAO A inhibition is responsible for the interaction with 5-MeO-DMT. Importantly, the late hyperactivity produced by 5-MeO-DMT in combination with an MAOI can be blocked by the selective 5-HT 2A antagonist MDL 11,939 but not by WAY-100,635). It appears MAO A inhibition markedly enhances the contribution that 5-HT 2A receptors make to the behavioral effects of 5-MeO-DMT. One possible explanation for the interaction between 5-MeO-DMT and MAOIs is that MAO A inhibition alters the pharmacokinetics of 5-MeO-DMT. The primary route of metabolism for 5-MeO-DMT is oxidative deamination by MAO A. MAOIs are known to increase blood and brain concentrations of 5-MeO-DMT. Indeed, we found that α,α,β,β-tetradeutero-5-MeO-DMT, a 5-MeO-DMT isotopologue that is resistant to metabolism by MAO A due to the kinetic isotope effect, produces a biphasic locomotor profile similar to that produced by the combination of 5-MeO-DMT and an MAOI. The finding with α,α,β,β-tetradeutero-5-MeO-DMT confirms that MAO inhibition does not directly mediate the delayed hyperactivity, which appears to be a consequence of altered 5-MeO-DMT pharmacokinetics. Nevertheless, although it is anticipated that the duration-of-action of 5-MeO-DMT would be extended by inhibiting MAO A , it is not clear why the behavioral profile of 5-MeO-DMT would be altered. It should be noted, however, that in addition to being deaminated by MAO A , 5-MeO-DMT is also O-demethylated to bufotenine (5-hydroxy-DMT) by cytochrome P450 2D6 (see Fig.). Inhibition of MAO A has been shown to enhance the conversion of 5-MeO-DMT to bufotenine in mice. Since bufotenine is a potent and highly efficacious 5-HT 2A agonist, we have theorized that that the delayed hyperactive phase induced by 5-MeO-DMT in the presence of an MAOI may occur as a consequence of the formation of bufotenine, which could potentially accumulate in the brain and induce hyperactivity by activating 5-HT 2A. The goal of the present studies was to investigate the mechanism for the interaction between 5-MeO-DMT and MAOIs, and to determine whether other behavioral responses to 5-MeO-DMT are similarly affected. Hallucinogens, including LSD and DOI, are known to inhibit prepulse inhibition (PPI) in rats by activating 5-HT 2A. 5-MeO-DMT also disrupts PPI in rats, but the effect of 5-MeO-DMT on PPI is mediated by 5-HT 1A and not by 5-HT 2A. Because MAO A inhibition can enhance the behavioral relevance of 5-MeO-DMT interactions with 5-HT 2A , the present studies assessed whether MAOI pretreatment alters the respective contributions of 5-HT 1A and 5-HT 2A receptors to the effects of 5-MeO-DMT on PPI. Additional studies were conducted to determine whether inhibition of MAO A alters the pharmacokinetics of 5-MeO-DMT and its biotransformation to bufotenine.
Male Sprague-Dawley rats (Harlan Industries, Indianapolis, IN, USA; initial weight 250-275 g) were housed in pairs in a temperature-and humidity-controlled vivarium under a 12-h reverse light-dark cycle (lights off at 0700 h). Food and water were available ad libitum (except during behavioral testing). Animals were allowed to acclimatize for approximately 1 week after arrival prior to behavioral testing and maintained in AALAC-approved facilities that meet all federal and state guidelines. Procedures were approved by the University of California San Diego (UCSD) institutional animal care and use committee. Principles of laboratory animal care were followed as well as specific laws of the United States., ammonium acetate, sodium phosphate monobasic, and sodium phosphate dibasic were purchased from Sigma-Aldrich (St. Louis, MO). Bufotenine was purchased from Cerilliant (Red Rock, TX). α-Phenyl-1-(2-phenylethyl)-4-piperidinemethanolwas purchased from Tocris Bioscience (Ellisville, MO). LC-MS grade methanol and ethyl acetate were obtained from Honeywell-Burdick & Jackson (Muskegon, MI). Blank rat plasma used to prepare the calibrators and controls were purchased from Biochemed (Winchester, VA).
For the in vivo experiments, drug doses are expressed as the salt form of the drug, with the exception of 5-MeO-DMT and MDL 11,939, which refer to the freebase weight. All drugs were administered subcutaneously (SC) in a volume of 1 mL/kg. 5-MeO-DMT, WAY-100,635, clorgyline, and pargyline were dissolved in isotonic saline. MDL 11,939 was dissolved in saline (pH 5.0) containing 0.75% Tween 80.
Activity was measured in the Behavioral Pattern Monitor (BPM), which assesses spatiotemporal patterns of exploratory and investigatory behavior (for details, see:. The rat BPM is a 30.5 × 61.0 × 28.0 cm black Plexiglas chamber equipped with 2.5 cm holes in the walls and floor. A 4 × 8 grid of infrared photobeams is used to detect the animal's position in an X-Y plane. Infrared photobeams in each hole are used to detect investigatory nosepokes (holepokes). Rearings are detected by touchplates on the walls. Each chamber is illuminated by a 15-W red incandescent light located above the center. The status of the photobeams and the touchplate is sampled every 55 ms, digitized, and the data stored on a PC for offline analysis.
Startle chambers (SR-LAB system, San Diego Instruments, San Diego, CA) were used to measure startle reactivity. Each startle test chamber consists of a sound-attenuated, lighted, and ventilated enclosure holding a clear nonrestrictive cylindrical Plexiglas stabilimeter, 8.2 cm in diameter. The acoustic stimuli were generated by a high-frequency loudspeaker mounted 24 cm above the Plexiglas cylinder. The peak and average amplitude of the startle response were detected by a piezoelectric accelerometer, digitized, and stored on a PC. At the onset of the startling stimulus, 100 1-ms samples were recorded, and the average amplitude was used to determine the startle response. A dynamic calibration system was used to ensure comparable stabilimeter sensitivity across test chambers, and sound levels were measured using the dB(A) scale, as described previously. Acoustic startle test sessions consisted of startle trials (PULSE-ALONE) and prepulse trials (PREPULSE + PULSE). The PULSE-ALONE trial consisted of a 40-ms 120-dB pulse of broadband white noise. PREPULSE + PULSE trials consisted of a 20-ms acoustic prepulse, an 80-ms delay, and then a 40-ms 120-dB startle pulse (100 ms onsetonset). There was an average of 15 s (range, 9-21 s) between trials. During each inter-trial interval, the movements of the rats were recorded once to measure responding when no stimulus was present (data not shown). Each startle session began with a 5-min acclimation period to a 65-dB broadband noise that was present continuously throughout the session. The startle test session included three blocks. The first and last block consisted of 5 PULSE-ALONE trials that were not used in the calculation of PPI values. The second block was designed to assess PPI; it contained 12 PULSE-ALONE trials and 30 PREPUSE + PULSE trials [10 prepulses each of 68, 71, and 77 dB (3, 6, and 12 dB above background)] presented in a pseudo-randomized order. One week after arrival, animals were tested in a brief startle/PPI session to create treatment groups matched for baseline levels of startle and PPI. 2.4. Experimental design 2.4.1. Acoustic startle Animals were placed in the startle chambers 45 min after treatment with 5-MeO-DMT. The animals were tested 45 min after drug administration because the experiments were designed to assess acoustic startle and PPI during the period of time when animals treated with pargyline and 5-MeO-DMT display locomotor hyperactivity in the BPM (i.e., 40-70 min after injection of 5-MeO-DMT; see. In experiment 1, rats (n = 10/group, 60 total) were treated with the nonselective MAO inhibitor pargyline (0 or 10 mg/kg) 20 min before administration of 5-MeO-DMT (0, 0.1, or 1.0 mg/kg). In experiment 2, rats (n = 12/group, 48 total) were treated with the MAO A inhibitor clorgyline (0 or 0.3 mg/kg) 20 min before administration of 5-MeO-DMT (0 or 1.0 mg/kg). In experiment 3, rats (n = 10/ group, 60 total) were pretreated with vehicle, the selective 5-HT 1A antagonist WAY-100,635 (1.0 mg/kg), or the selective 5-HT 2A antagonist MDL 11,939 (0.3 mg/kg), 20 min before treatment with vehicle or 1.0 mg/kg 5-MeO-DMT. For experiment 3, all the animals that were injected with 5-MeO-DMT were pretreated (20 min) with 10 mg/kg pargyline. WAY-100,635 and MDL 11,939 were tested at doses previously shown to block 5-HT 1A -and 5-HT 2A -mediated behavioral responses in Sprague-Dawley rats.
One day before the study, animals were taken to the BPM testing room, weighed, handled briefly, placed in a clear Plexiglas box (24 × 46 cm) for approximately 30 s, and then returned to their cages in the vivarium. On the testing day, rats were brought to the testing room and allowed to sit for 60 min before receiving injections. Injections were administered under red lights in the testing room. Animals were tested during the dark phase in darkness. Rats were divided into two groups (n = 30/group) and pretreated with vehicle or clorgyline (0.3 mg/kg) 20 min before administration of 1.0 mg/kg 5-MeO-DMT. The animals were placed in the BPM chambers 10 min after 5-MeO-DMT treatment, and locomotor activity was monitored for 10, 20, 30, 40, 50, or 60 min (n = 5/time point/group). The rats were immediately removed from the BPM chambers, anesthetized (isoflurane), decapitated, and trunk blood collected in cooled heparinized tubes. Plasma was isolated by centrifugation (3200×g) and stored at -40 °C. The brains were removed, flash frozen in isopentane at -80 °C, and stored at -40 °C.
The calibrators and controls were prepared in silanized, 16 × 100 mm glass culture tubes containing 1 mL blank rat plasma. Working solutions containing 0.01, 0.1, 1.0, and 10 ng/μL of 5-MeO-DMT or bufotenine were prepared in methanol for spiking the analytes. Separate sets of working solutions were used for the calibrators and controls. The calibrators were prepared at 1, 2, 5, 10, 20, 50, 100, and 200 ng/mL and were run in duplicate with one set at the beginning and one set at the end of the run; controls were prepared at 5, 15, and 160 ng/mL and run at N ≥ 3 interspersed with the other samples.
For the plasma samples, 1 mL of each sample was transferred to a silanized 16 × 100 mm glass culture tube. For brain tissue, a 10-fold diluted tissue homogenate sample was prepared for each brain. Brain tissue was weighed, transferred to a 50-mL conical polypropylene tube, shredded with a clean wood applicator stick, and nine volumes of 50 mM sodium phosphate (pH 7.0) homogenizing buffer (i.e., 13.5 mL homogenizing buffer per 1.5 g tissue) were added. Tissue was homogenized using a Vibra Cell probe homogenizer (Sonics, Newtown, CT), using 3 × 12-s pulses at 30% amplitude, and then stored frozen prior to analysis. At the time of analysis, the homogenates were thawed, and 1 mL of each sample homogenate was transferred to a new 16 × 100 mm glass culture tube. Prior to the analysis, 50 μL of 1 ng/μL NMS was added as internal standard to each sample, calibrator, and control tube. The tubes were vortexed, and 3 mL of 0.1 M sodium phosphate buffer (pH 6.0) was added to each tube. The tubes were briefly vortexed and then centrifuged (1200 ×g). The supernatants from each tube were then applied to their respective conditioned solid phase extraction column (see below).
Clean Screen ZSDAU020 solid phase extraction columns (United Chemical Technologies, Bristol, PA) were used for the extraction procedure. A separate column was utilized for each sample, standard, and control. A glass manifold device was employed for the extraction process. First, the extraction columns were conditioned by sequential addition of 3 mL of methanol, 3 mL of water, and 2 mL of 0.1 M sodium phosphate (pH 6.0), and then the supernatants from sample preparation were added. Next, the columns were sequentially washed with 3 mL of water and 3 mL of methanol. With care taken to prevent drying of the columns, the columns were eluted by the addition of 3 mL of 2% ammonium hydroxide in ethyl acetate and 4% ammonium hydroxide in methanol. The eluates from each column were collected into clean 13 × 100 mm culture tubes, and evaporated to dryness under a stream of air using a Tubovap evaporator (Caliper, Hopkinton, MA) set at 40 °C. The extracts were reconstituted with 100 μL of 10 mM ammonium acetate (pH 5.0)/methanol (80:20) and then transferred to separate 300 μL conical, polypropylene autosampler vials with snap caps.
The liquid chromatography-tandem mass spectrometry (LC-MS/ MS) system consisted of an Agilent (Santa Clara, CA) 1100 liquid chromatograph interfaced with a Thermo-Finnegan (San Jose, CA) TSQ Quantum Access triple quadrupole mass spectrometer. The system was operated by an Xcalibur® 2.0 SR2 and Tune Master version 1.4.1 operating systems (Thermo-Finnigan). A LUNA C18(2), 150 × 4.6 mm LC column (Phenomenex, Torrance, CA) at 35 °C was used. The mobile phase was 10 mM ammonium acetate (pH 5.0)/methanol (60:40) at a flow rate of 0.2 mL/min. For the MS/MS analysis, positive ion electrospray was used for ionization. The capillary temperature was 270 °C. Nitrogen was used as the sheath gas (60 units) and the aux gas (20 units). Selected reaction monitoring was used for the analysis. Ultra high purity argon was used as the collision gas for collision induced dissociation. The following transitions were monitored: 5-MeO- DMT = 219 → 174; bufotenine = 205 → 166; NMS = 191 → 160. For all compounds, the precursor ions were the protonated molecular ions. A calibration curve generated from the analysis of the calibration standards was used to extrapolate the 5-MeO-DMT and bufotenine concentrations in the analytical quality controls and the extracts prepared from of the study samples. LCquan 2.5 data analysis software (Thermo-Finnigan) was used for the quantification. 2.6. Data analysis 2.6.1. Acoustic startle The amount of PPI was calculated as a percentage score for each PREPULSE + PULSE trial type: %PPI = 100 -(((startle response for PREPULSE + PULSE trial) / (startle response for PULSE-ALONE trial)) × 100). Startle magnitude was calculated as the average response to all of the PULSE-ALONE trials. The level of spontaneous motor activity was assessed as the average response to all NOSTIM trials. PPI data were analyzed with two-or three-factor analysis of variance (ANOVA) with pretreatment and treatment as between-subjects factors and trial type (prepulse intensity) as a repeated measure. For experiments in which there was no significant interaction between drug and prepulse intensity, PPI data were collapsed across prepulse intensity and the average PPI was used as the main dependent measure. Startle magnitude and NOSTIM data were analyzed with two-factor ANOVA. Post hoc analyses were carried out using Tukey's studentized range method. Significance was demonstrated for these and other experiments by surpassing an alpha level of 0.05.
The raw data were reduced to the X and Y coordinates of the rat in the chamber. Locomotor activity was quantified by the number of crossings between any of eight equal square sectors within the BPM. Data were examined in 10-min time blocks. For each rat, only the behavioral data from the last 10-min time block (i.e., the test block immediately prior to collection of plasma and brain samples) were analyzed. Data were analyzed using two-way ANOVA with clorgyline pretreatment and time block as between-subject factors. Specific post hoc comparisons were made using Bonferroni's multiple comparisons test.
To facilitate data reporting in cases where 5-MeO-DMT or bufotenine concentrations fell below the limit of detection (bLOD), Helsel's Robust Method (implemented using the UNCENSOR program:was used to estimate group means and variance. However, simple substitution (null values) was used for groups with ≥ 4 censored values. The trapezoidal rule was used to calculate the area under the curve up to the last measured time point (AUC 20 →70 min ). The density of brain tissue in rats was assumed to be 1.04 g/mL. Statistical comparisons of raw analyte levels were made using Kaplan-Meier survival analysis; the nonparametric Tarone-Ware test (SPSS Ver. 22) was used to assess equivalence between groups. AUC and C max values were compared using one-way ANOVA.
3.1. Effect of pargyline and 5-MeO-DMT on acoustic startle and sensorimotor gating 5-MeO-DMT was previously shown to disrupt PPI when administered 10 min prior to startle testing. The current studies tested whether 5-MeO-DMT alters PPI when administered 45 min prior to testing, and examined whether the response to 5-MeO-DMT is altered in animals pretreated with 10 mg/kg pargyline. As shown in Fig., 5-MeO-DMT did not alter PPI in saline-pretreated animals but significantly reduced PPI in animals pretreated with pargyline, resulting in an interaction between pretreatment and treatment (F(2,54) = 17.59, p b 0.0001) and a main effect of 5-MeO-DMT (F(2,54) = 18.87, p b 0.0001). There was a significant main effect of prepulse intensity (F(2,108) = 162.27, p b 0.0001), and pairwise comparisons revealed that 1 mg/kg 5-MeO-DMT significantly reduced PPI at all three prepulse intensities in animals pretreated with pargyline (p b 0.01, Tukey's test). There was also significant interaction between pargyline pretreatment and prepulse intensity (F(2,108) = 5.05, p = 0.008), but pargyline did not significantly reduce PPI at any prepulse intensity compared with vehicle pretreatment. For the startle response, there was a main effect of 5-MeO-DMT (F(2,54) = 3.97, p b 0.03) and an interaction between pargyline and 5-MeO-DMT (F(2,54) = 5.07, p b 0.01). Pretreatment with pargyline significantly reduced the amplitude of the startle response (p b 0.05, Tukey's test; see Table), but the startle response was unaffected by treatment with 5-MeO-DMT. In order to confirm that the effect of pargyline and 5-MeO-DMT on PPI is independent of changes in startle magnitude, we examined the effect on PPI in subgroups of animals matched for startle level (see:. We picked 7 animals from each group that had overlapping startle responses (mean ±. Even though the magnitude of the startle response in those subgroups was unaffected by pretreatment (F(1,36) = 0.09, NS), treatment (F(2,36) = 1.21, NS), or pretreatment × treatment interactions (F(2,36) = 1.36, NS), PPI was still significantly reduced by the combination of pargyline and 5-MeO-DMT (Pretreatment × Treatment interaction: F(2,36) = 10.44, p = 0.0003). This shows that the PPI disruption produced by pargyline and 5-MeO-DMT did not occur because of changes in startle magnitude.
We also examined whether the response to 5-MeO-DMT is altered in animals pretreated with 0.3 mg/kg clorgyline. Similar to the experiment with pargyline, the animals were tested 45 min after administration of 5-MeO-DMT. 5-MeO-DMT did not alter PPI in saline-pretreated animals but significantly reduced PPI in animals pretreated with clorgyline (Pretreatment × Treatment: F(1,44) = 24.74, p b 0.0001). 5-MeO-DMT significantly reduced PPI at all three prepulse intensities in animals pretreated with clorgyline (p b 0.01, Tukey's test; Fig.). As shown in Table, the startle response was not altered by treatment with 5-MeO-DMT (Main effect: F(1,44) = 0.79, NS) or by pretreatment with clorgyline (Main effect: F(1,44) = 0.63, NS), and there was no interaction between clorgyline and 5-MeO-DMT (F(1,44) = 0.69, NS).
To determine whether the PPI reduction produced by 10 mg/kg pargyline and 1 mg/kg 5-MeO-DMT is mediated by interactions with 5-HT 1A or 5-HT 2A receptors, antagonist blockade studies were conducted with WAY-100,635 and MDL 11,939, respectively. As expected, there was a main effect of treatment (F(1,54) = 92.13, p b 0.0001), as well as an interaction between treatment and prepulse intensity (F(2,108) = 7.23, p b 0.002), and the combination of pargyline and 5-MeO-DMT significantly reduced PPI at all three prepulse intensities in vehicle-pretreated animals (p b 0.01, Tukey's test). The magnitude of the startle response was unaffected by treatment with pargyline and 5-MeO-DMT (F(1,54) = 0.26, NS; Table). The effects of pretreatment with WAY-100,635 and MDL 11,939 were assessed in a single experiment, with a shared control group, but the effect of each antagonist was analyzed independently (see below).
For PPI, there was an interaction between pretreatment and treatment (F(1,36) = 73.11, p b 0.0001). The reduction of PPI produced by 10 mg/kg pargyline and 1 mg/kg 5-MeO-DMT was significantly antagonized by pretreatment with 1 mg/kg WAY-100,635 (p b 0.01, Tukey's test; Fig.). There was also a trend toward a main effect of pretreatment (F(1,36) = 8.17, p b 0.08), but this was not confirmed by pairwise comparisons. Although there was a main effect of pretreatment on startle magnitude (F(1,36) = 5.91, p b 0.03), the reduction of startle amplitude produced by 1 mg/kg WAY-100,635 did not achieve significance (see Table).
The ability of pargyline and 5-MeO-DMT to reduce PPI was significantly attenuated by pretreatment with MDL 11,939 (Pretreatment × Treatment: F(1,36) = 23.96, p b 0.0001), and this was confirmed by post-hoc analysis (p b 0.01, Tukey's test; Fig.). Pretreatment with MDL 11,939 did not alter startle amplitude (F(1,36) = 0.25, NS).
Precision and accuracy of the method was determined from inclusion of quality control (QC) samples in 4 analytical batches that included rat plasma and brain samples. QCs prepared in rat plasma were run at n = 3 per concentration per batch. QCs were acceptable in that over 2/3 total and within concentration were within ±20% of target. a For 5-methoxy-DMT, 3 low and 1 medium QCs were treated as outliers and not used in calculation of precision and accuracy. b For bufotenine, 1 medium QC was treated as an outlier and not used in calculation of precision and accuracy.
3.4. Effect of clorgyline pretreatment on 5-MeO-DMT and bufotenine levels in plasma and brain
Several different extraction procedures were investigated, including liquid-liquid extraction and precipitation with methanol, but were associated with poor recovery and sensitivity (data not shown). Previous studies measuring 5-MeO-DMT and bufotenine levels in biological fluids used solid phase extraction to prepare the samples. We used a different solid phase extraction method for the simultaneous detection of 5-MeO-DMT and bufotenine in plasma and brain, using liquid chromatographyelectrospray ionization-selective reaction monitoring-tandem mass spectrometry (LC-ESI-SRM-MS/MS), with N-methylserotonin (NMS) as the internal standard. Performance characteristics were collected over the course of three pre-study and four study sample analytical runs. The method was selective as determined from examination of analytical results for 7 blank plasma samples fortified with internal standard; the mean detectable signal (peak area ratio) at the retention time of analytes did not exceed 0.25% and 14.7% of mean LLOQ peak area heights for 5-MeO-DMT and bufotenine, respectively. Chromatography of 5-MeO-DMT, bufotenine, and NMS was subject to ion suppression. The suppression, however, was uniform across the chromatographic effusion window of the analytes (3.5-4.5 min). Retention times were not affected by ion suppression, with little variation within or between runs (mean 5-; the coefficient of variation (CV) for retention times relative to the NMS were 3.51% and 0.99% for 5-MeO-DMT and bufotenine, respectively. There was relatively high extraction recovery (62.7-88.0%) for all analytes. Calibration was reproducible with the use of eight calibration points in duplicate; back-calculated concentrations were within 5% of target with %CVs within 12.9% for both analytes; mean r 2 values were 0.990 for 5-MeO-DMT and 0.994 for bufotenine. With the accuracy (93.3-104.7% for 5-MeO-DMT and 96.3-107.9% for bufotenine) and precision (%CV not exceeding 17.8 for 5-MeO-DMT and 19.4 for bufotenine) achieved (see Table; data specific for study sample runs), the conditions were adequate for reliable analysis.
To characterize the effect of MAO A inhibition on the pharmacokinetics and metabolism of 5-MeO-DMT, rats were administered 1 mg/kg 5-MeO-DMT after pretreatment with vehicle or clorgyline (0.3 mg/kg), and 5-MeO-DMT and bufotenine levels in plasma and whole brain were quantified. The concentration of 5-MeO-DMT in plasma is shown in Table; the concentration in brain is shown in Table. The level of 5-MeO-DMT in plasma and brain peaked within the first 20 min after SC administration and then rapidly declined. Clorgyline did not significantly increase the plasma C max of 5-MeO-DMT (vehicle = 52.7 ± 11.8 ng/mL; clorgyline = 78.1 ± 9.5 ng/mL), but the clearance of 5-MeO-DMT from plasma was slowed by clorgyline, as evidenced by the fact that the concentration of 5-MeO-DMT in plasma was significantly elevated 30-70 min after administration in animals pretreated with clorgyline (see Table). Furthermore, clorgyline significantly increased the plasma AUC 20→70 min for 5-MeO-DMT (vehicle = 12.2 ± 1.7 ng•h/ mL, clorgyline = 31.4 ± 2.3 ng•h/mL; F(1,59) = 45.6, p b 0.0001). In contrast to the negligible effect of clorgyline on the plasma C max , clorgyline significantly increased the C max of 5-MeO-DMT in whole brain (vehicle: 30.3 ± 11.2 ng/g; clorgyline: 633.8 ± 146.5 ng/g; F(1,9) = 16.8, p b 0.004). As shown in Table, the concentration of 5-MeO-DMT in whole brain was markedly elevated in animals pretreated with clorgyline, and 5-MeO-DMT was also detectable for a longer period of time in the presence of an MAO inhibitor. It does not appear that clorgyline pretreatment significantly altered the concentration of bufotenine in plasma or brain because most of the samples were bLOD (plasma = 1-2 ng/mL; brain = 10 ng/g). The plasma C max for bufotenine was 1.3 ± 0.1 ng/mL in rats pretreated with vehicle, and 1.8 ± 0.3 ng/mL in rats pretreated with clorgyline (data not shown). To confirm that the expected behavioral interaction occurred between 0.3 mg/kg clorgyline and 1 mg/kg 5-MeO-DMT, locomotor activity was assessed in the BPM prior to collection of analytical samples (see Fig.). There was a significant main effect of clorgyline (F(1,48) = 7.25, p b 0.01) and a significant interaction between clorgyline and time block (F(5,48) = 3.64, p b 0.008). Importantly, compared with vehicle pretreatment, rats pretreated with clorgyline made significantly more crossings (a measure of locomotor activity) during the fifth 10-min
These studies demonstrate that pretreatment with an MAOI markedly prolongs the behavioral response to 5-MeO-DMT. Although 5-MeO-DMT has previously been shown to disrupt PPI by activating 5-HT 1A receptors, the effect on PPI has a relatively short duration, and the present results confirm that 5-MeO-DMT does not alter PPI 45 min post-injection. By contrast, 5-MeO-DMT disrupted PPI at that time point in rats pretreated with an MAOI. The effect of combined treatment with 5-MeO-DMT and the MAOI pargyline on PPI was antagonized by pretreatment with the 5-HT 1A antagonist WAY-100,635 and the 5-HT 2A antagonist MDL 11,939. Based on the findings with WAY-100,635 and MDL 11,939, it appears that the PPI disruption produced by 5-MeO-DMT in animals pretreated and an MAOI is dependent on activation of 5-HT 1A and 5-HT 2A receptors. These findings confirm that 5-MeO-DMT, like other hallucinogens, can alter PPI in rats by activating 5-HT 2A receptors. Analysis of 5-MeO-DMT pharmacokinetics revealed that systemic and central exposure to the drug is increased and prolonged after inhibition of MAO A , and showed that there is minimal conversion of 5-MeO-DMT to bufotenine in rats. Pretreatment with the MAO A inhibitor clorgyline dramatically altered the pharmacokinetics of 5-MeO-DMT. These effects probably occurred because clorgyline blocked the metabolism of 5-MeO-DMT. 5-MeO-DMT is metabolized by deamination and clorgyline was administered at a dose that is known to inhibit MAO A . Using 5-HT deamination as a measure of MAO A activity ex vivo,determined that clorgyline inhibits MAO A in liver and brain by approximately 60% and 90%, respectively, when administered to rats at 0.3 mg/kg SC. Compared to the present results, previous studies in rats and mice have yielded similar findings regarding the effects of MAOIs on 5-MeO-DMT pharmacokinetics. Sitaram and coworkersexamined the effect of pretreatment with the non-selective MAOI iproniazid phosphate on tissue levels of 5-MeO-DMT in Sprague-Dawley rats. After treatment with 5-MeO-DMT (10 mg/kg IP), blood and brain levels averaged 0.03 nmol/g (6.9 ng/mL) and 0.08 nmol/g (17.5 ng/g), respectively, when samples were collected 45 min post-injection. By contrast, in rats pretreated with iproniazid, the concentration of 5-MeO-DMT in blood was 1.2 nmol/g (275 ng/mL) and the concentration in brain was 7.8 nmol/ g (1703 ng/g). In summary, iproniazid increased 5-MeO-DMT blood levels 40-fold and brain levels almost 100-fold. One of the more interesting findings with 5-MeO-DMT is that its pharmacodynamics can be altered by inhibition of MAO. Normally, 5-MeO-DMT produces brief alterations of locomotor activity and PPI that are mediated by 5-HT 1A and not by 5-HT 2A receptors. Conversely, in rats pretreated with an MAO A inhibitor, the action of 5-MeO-DMT is prolonged and involves 5-HT 2A activation. Yu and colleagues have shownthat there is a marked increase in the Odemethylation of 5-MeO-DMT in mice pretreated with an MAOI, resulting in elevated and prolonged systemic exposure to bufotenine. Based on their findings, we hypothesized that if MAOIs produce similar effects on the metabolism of 5-MeO-DMT in rats, the behavioral interaction between MAOIs and 5-MeO-DMT could be a consequence of increased formation of bufotenine, a potent and highly efficacious 5-HT 2A agonist. The present results, however, reveal that there is negligible conversion of 5-MeO-DMT to bufotenine in rats, even in the presence of an MAOI. Similarly, a previous study failed to detect any bufotenine in the brains of rats treated with 5-MeO-DMT and iproniazid. Importantly, we found that inhibition of MAO A did produce a significant increase in the central accumulation of 5-MeO-DMT, and micromolar concentrations were present in the brain for at least 60 min post-injection. Therefore, it appears that MAOIs alter the behavioral response to 5-MeO-DMT by blocking its deamination, which allows 5-MeO-DMT to accumulate in the brain at concentrations sufficient to activate 5-HT 2A receptors for an extended period of time. The fact that α,α,β,β-tetradeutero-5-MeO-DMT, a 5-MeO-DMT isotopologue that is resistant to metabolism by MAO A , produces a behavioral profile identical to that of 5-MeO-DMT in combination with an MAO A inhibitorsupports the conclusion that MAOIs alter the response to 5-MeO-DMT by blocking its deamination. Interactions between serotonergic and dopaminergic signaling may contribute to the delayed 5-HT 2A -mediated behavioral response induced by 5-MeO-DMT and an MAOI. 5-HT 2A receptors are expressed as heteroreceptors on dopaminergic terminals; evidence indicates that 5-HT 2A can facilitate phasic dopamine release. Activation of 5-HT 2A has been observed to produce a delayed sensitization of the dopaminergic system (Marona-Lewicka and. It is possible that the high level of 5-HT 2A activation produced by 5-MeO-DMT in the presence of an MAOI could induce a delayed sensitization of dopamine release, resulting in hyperactivity and disruption of PPI. The ability of 5-HT 2A agonists to produce delayed effects on dopaminergic transmission would explain why 5-HT 2A antagonists block the behavioral response to 5-MeO-DMT at ~45 min post-treatment, but not at earlier time-points, despite the fact that 5-MeO-DMT would occupy 5-HT 2A receptors in the central nervous system immediately after administration. Future studies will examine whether the dopaminergic system contributes to the behavioral interactions between 5-MeO-DMT and MAOI. The present findings confirm that 5-MeO-DMT can activate 5-HT 2A receptors in vivo. We found the combined effect of 5-MeO-DMT and pargyline on PPI was partially dependent on 5-HT 2A receptor activation. Along the same lines, we had previously shown that in the presence of clorgyline, 5-MeO-DMT induces delayed hyperactivity via 5-HT 2A receptor activation. It has also been reported that 5-MeO-DMT can elicit the head twitch response (HTR), a 5-HT 2Amediated behavior, in rats at doses ranging from 0.2-1 mg/kg. Furthermore, although the discriminative stimulus effects of 5-MeO-DMT are primarily mediated by 5-HT 1A receptors, 5-MeO-DMT can produce full substitution in rats trained to discriminate the 5-HT 2 agonist DOM. Given that 5-MeO-DMT activates 5-HT 2A receptors in rats in vivo, it is surprising that the receptor normally does not contribute to the effects of 5-MeO-DMT on PPI and locomotor activity. One possible explanation is that activation of the 5-HT 1A receptor by 5-MeO-DMT may attenuate its ability to induce behavioral responses via 5-HT 2A. It is well known that the 5-HT 1A receptor inhibits 5-HT 2A -mediated behavioral responses. Reports indicate that the concentration of 5-MeO-DMT required to activate 5-HT 2A is on the order of several hundred nanomolaror in the low micromolar range. Therefore, the peak level of 5-MeO-DMT in the brain (144.3 nM) may not produce sufficient 5-HT 2A receptor activation to overcome the relatively potent countervailing effects mediated by 5-HT 1A . It would appear, however, that in the presence of an MAO inhibitor 5-MeO-DMT accumulates in the brain at levels that are adequate to increase locomotor activity and disrupt PPI via 5-HT 2A . 5-MeO-DMT can induce lateral head-weaving, tremor, and other symptoms of serotonin syndrome in rats (Grahame. It has been reported that MAOIs can markedly intensify and prolong the serotonin syndrome induced by 5-MeO-DMT (Grahame-. It is unlikely, however, that 5-MeO-DMT-induced serotonin syndrome interfered with the assessment of the startle response in the present studies. First, the duration of the serotonin syndrome after combined administration of 5-MeO-DMT (1 mg/kg IP) and an MAOI is only 25-30 min, which is shorter than the interval between drug treatment and startle testing in the present studies. Second, we have confirmed that the magnitude of the startle response is not significantly altered by treatment with pargyline and 5-MeO-DMT, demonstrating that the serotonin syndrome did not interfere with expression of the startle response. The interaction between MAOIs and tryptamine hallucinogens is important from a toxicological perspective because these substances are used concurrently by humans. Use of ayahuasca, a hallucinogenic beverage containing DMT and MAOIs such harmaline and harmine, is currently increasing worldwide. Furthermore, according to published accounts, 5-MeO-DMT can produce intense hallucinogenic effects when combined with harmaline and other MAOIs. The knowledge that MAOIs can alter the psychoactive effects of 5-MeO-DMT and other tryptamines has inspired recreational drug users to experiment with polypharmacy, sometimes with tragic consequences. In one case, a man was found dead after ingesting 5-MeO-DMT, DMT, and β-carbolines in an ayahuasca-like preparation. In another instance, a 17-year old college student became extremely agitated and exhibited signs of hyperthermia, tachycardia, and rhabdomyolysis after consuming 5-MeO-DMT and an extract containing harmaline. The toxicity occurring in those cases may have been a consequence of MAOI effects on blood and brain levels of 5-MeO-DMT. 5-MeO-DMT inhibits 5-HT uptake at low micromolar concentrations, an effect that could contribute to interactions with MAO A inhibitors and other 5-HT agonists. The present results indicate that inhibition of MAO would likely enhance the hallucinogenic effects of 5-MeO-DMT in humans by increasing 5-HT 2A activation, but the high concentrations present in the brain would also interact with a variety of other targets, potentially producing adverse effects and toxicity. It is not clear to what extent the present findings with 5-MeO-DMT are relevant to DMT, but it is important to note that the latter drug is also deaminated by MAO. Although it is generally assumed that MAOIs contribute to the action of ayahuasca by allowing DMT to take effect orally, it is possible that MAO inhibition can also increase the accumulation of DMT in the central nervous system, similar to our findings with 5-MeO-DMT. Further studies are required to fully characterize the influence of MAOIs on the pharmacokinetics of DMT and the mechanism of action of ayahuasca.
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Lima da Cruz, R. V., Moulin, T. C., Petiz, L. L. et al. · Frontiers in Molecular Neuroscience (2018)