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Pharmacology & Chemistry

Immunochemical monitoring of psilocybin and psilocin to identify hallucinogenic mushrooms

July 21, 2020 / Mushrooms / Psilocybin, Papers, Pharmacology & Chemistry, Publications / By

Abstract

Development of rapid and reliable immunochemical methods for monitoring psilocybin (4-phosphoryloxy-N,N-dimethyltryptamine; Pyb) and psilocin (dephosphorylated metabolite; Psi), the psychoactive compounds contained within hallucinogenic mushrooms (magic mushrooms), is desirable in order to identify these mushrooms and regulate their illicit use. Because no antibody was publicly available for this purpose, we generated two independent monoclonal antibodies (mAbs) against Pyb or Psi, and then developed enzyme-linked immunosorbent assays (ELISAs) by using them. To generate the specific antibodies, novel immunogenic conjugates were prepared by linking Pyb or Psi molecules to carrier proteins by modifying their 2-(N,N-dimethylamino)ethyl side chains. Spleen cells from mice immunized with these conjugates were fused with P3/NS1/1-Ag4-1 myeloma cells, and hybridoma clones secreting anti-Pyb and anti-Psi mAbs were established. These mAbs were characterized for their biochemical features and then applied to competitive ELISAs, which used microplates coated with Pyb or Psi linked with albumin. These ELISAs enabled the determination of Pyb or Psi with measurable ranges of ca. 0.20-20 or 0.040-2.0 μg/assay (limit of detection was 0.14 or 0.029 μg/assay), respectively. The related tryptamines were satisfactorily discriminated as exemplified by the cross-reactivity of the ELISA to determine Pyb (or Psi) with Psi (or Pyb) that were found to be 2.8 % (or <0.5 %), respectively. The Pyb and Psi contents in a dried powder of the hallucinogenic mushroom, Psilocybe cubensis, were determined to be 0.39 and 0.32 (w/w)%, respectively. The ELISAs developed using the current mAbs are promising tools for identifying illegal hallucinogenic mushrooms.

Morita, I., Oyama, H., Kiguchi, Y., Oguri, A., Fujimoto, N., Takeuchi, A., … & Kobayashi, N. (2020). Immunochemical monitoring of psilocybin and psilocin to identify hallucinogenic mushrooms. Journal of Pharmaceutical and Biomedical Analysis, 190, 113485; 10.1016/j.jpba.2020.113485

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In vitro structure-activity relationship determination of 30 psychedelic new psychoactive substances by means of β-arrestin 2 recruitment to the serotonin 2A receptor

July 5, 2020 / Papers, Pharmacology & Chemistry, Publications / By / , ,

Abstract

Serotonergic psychedelics, substances exerting their effects primarily through the serotonin 2A receptor (5-HT2AR), continue to comprise a substantial portion of reported new psychoactive substances (NPS). The exact mechanisms of action of psychedelics still remain to be elucidated further, and certain pathways remain largely unexplored on a molecular level for this group of compounds. A systematic comparison of substances belonging to different subclasses, monitoring the receptor-proximal β-arrestin 2 recruitment, is lacking. Based on a previously reported in vitro bioassay employing functional complementation of a split nanoluciferase to monitor β-arrestin 2 recruitment to the 5-HT2AR, we here report on the setup of a stable HEK 293 T cell-based bioassay. Following verification of the performance of this new stable cell system as compared to a system based on transient transfection, the stable expression system was deemed suitable for the pharmacological characterization of psychedelic NPS. Subsequently, it was applied for the in vitro assessment of the structure-activity relationship of a set of 30 substances, representing different subclasses of phenylalkylamine psychedelics, among which 12 phenethylamine derivatives (2C-X), 7 phenylisopropylamines (DOx) and 11 N-benzylderivatives (25X-NB). The resulting potency and efficacy values provide insights into the structure-activity relationship of the tested compounds, overall confirm findings observed with other reported in vitro assays, and even show a significant correlation with estimated common doses. This approach, in which a large series of psychedelic NPS belonging to different subclasses is comparatively tested, using a same assay setup, monitoring a receptor-proximal event, not only gives pharmacological insights, but may also allow prioritization of legal actions related to the most potent -and potentially dangerous- compounds.

Pottie, E., Cannaert, A., & Stove, C. P. (2020). In vitro structure–activity relationship determination of 30 psychedelic new psychoactive substances by means of β-arrestin 2 recruitment to the serotonin 2A receptor. Archives of Toxicology, 94(10), 3449-3460; 10.1007/s00204-020-02836-w
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The pharmacological interaction of compounds in ayahuasca: a systematic review

July 3, 2020 / Ayahuasca, Neuroscience, Papers, Pharmacology & Chemistry, Publications / By / , , , ,

Abstract

Ayahuasca is a South American psychoactive plant brew used as traditional medicine in spiritual and in cultural rituals. This is a review of the current understanding about the pharmacological mechanisms that may be interacting in ayahuasca. Searches were performed using PubMed, PsycINFO, and Web of Science databases and 16 papers were selected. As hypothesized, the primary narrative in existing research revolved around prevention of deamination of N,N-dimethyltryptamine (N,N-DMT, also referred to as DMT) by monoamine oxidase inhibitors (MAOIs) in ayahuasca. Two of the constituents, DMT and harmine, have been studied more than the secondary harmala alkaloids. At present, it is unclear whether the pharmacological interactions in ayahuasca act synergistically or additively to produce psychoactive drug effects. The included studies suggest that our current understanding of the preparation’s synergistic mechanisms is limited and that more complex processes may be involved; there is not yet enough data to determine any potential synergistic interaction between the known compounds in ayahuasca. Our pharmacological understanding of its compounds must be increased to avoid the potential risks of ayahuasca use.

Ruffell, S., Netzband, N., Bird, C., Young, A. H., & Juruena, M. F. (2020). The pharmacological interaction of compounds in ayahuasca: a systematic review. Brazilian Journal of Psychiatry, 42(6), 646-656.; 10.1590/1516-4446-2020-0884
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Synthesis and characterization of high-purity N,N-dimethyltryptamine hemifumarate for human clinical trials

December 6, 2021 / DMT, Papers, Pharmacology & Chemistry, Publications / By / ,

Abstract

Since 2006, there has been a resurgent interest in the pharmacology and therapeutics of psychedelic drugs. Psilocybin, the 4-phosphoryl ester of N,N-dimethyltryptamine (DMT), has been studied most often, but DMT itself is also appealing because of its brief but profound psychological effects and its presence as an endogenous substance in mammalian brain. Although there have been a few studies of ayahuasca, a DMT-containing water infusion, only one human study with pure DMT has been reported since the early 2000s. Newly planned clinical trials to assess the safety and efficacy of DMT in humans with major depressive disorders require high-purity water-soluble DMT for intravenous administration. Accordingly, we synthesized and characterized DMT hemifumarate for these upcoming studies. The synthetic approach of Speeter and Anthony was slightly modified to gain some efficiency in time. In particular, this is the first known report to use aluminum hydride, generated in situ from lithium aluminum hydride, to reduce the intermediate 2-(1H-indol-3-yl)-N,N-dimethyl-2-oxoacetamide to DMT. A quench protocol was developed to produce a good yield of exceptionally pure free base DMT upon workup, which was then converted to the hemifumarate salt. Analysis of the final product included differential scanning calorimetry, thermogravimetric analysis, gas chromatography-mass spectrometry (GC-MS), 1 H and 13 C nuclear magnetic resonance spectroscopy, high-performance liquid chromatography, residual solvent analysis by GC headspace sampling, X-ray powder diffraction analysis, and residual lithium analysis by inductively coupled plasma-mass spectrometry. The DMT hemifumarate was minimally 99.9% pure, with no significant impurities or residual solvents, thus meeting regulatory standards for administration to humans.

Cozzi, N. V., & Daley, P. F. (2020). Synthesis and characterization of high‐purity N, N‐dimethyltryptamine hemifumarate for human clinical trials. Drug Testing and Analysis, 12(10), 1483-1493.; 10.1002/dta.2889

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Stability Evaluation of DMT and Harmala Alkaloids in Ayahuasca Tea Samples

February 6, 2022 / Ayahuasca, Papers, Pharmacology & Chemistry / Leave a Comment / By / , , , , ,

Abstract

Ayahuasca tea is a hallucinogenic beverage used for religious purposes in Brazil and many other countries that has therapeutic potential in the treatment of some mental health disorders. In the context of psychedelic research, quantification of the tea’s main alkaloids prior to its administration in animal or human studies is essential. For this reason, this study aims to provide information regarding the stability of the main ayahuasca alkaloids (dimethyltryptamine, DMT; harmine, HRM; tetrahydroharmine, THH; harmaline, HRL) in three different conditions: (1) A year stored in a refrigerator either in plastic or glass containers, (2) seven days at 37 °C to reproduce usual mail transportation, and (3) after three freeze-thaw cycles. Samples were quantified after a dilute-and-shoot procedure using liquid chromatography tandem mass spectrometry (LC-ESI-MS/MS). There was no significant degradation of DMT concentration over time in all tested conditions. Harmala alkaloids (THH, HRL, and HRM) showed important variations after long-term and high-temperature storages. Although DMT has proven to be stable in all studied conditions, the harmala alkaloids revealed intense degradation and even concentration increment. This may be caused by degradation, alkaloid inter-conversion, and leaching from tea precipitate material. Therefore, ayahuasca quantification before administration in controlled sets is mandatory.

de Oliveira Silveira, G., Guimarães Dos Santos, R., Rebello Lourenço, F., Novak Rossi, G., Hallak, J., & Yonamine, M. (2020). Stability Evaluation of DMT and Harmala Alkaloids in Ayahuasca Tea Samples. Molecules (Basel, Switzerland), 25(9), 2072. https://doi.org/10.3390/molecules25092072

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

January 13, 2020 / Anxiety Disorders / PTSD, MDMA, Papers, Pharmacology & Chemistry, Psychiatry & Medicine, Publications / By / , , ,

Abstract

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.

Berquist, M. D., Leth-Petersen, S., Kristensen, J. L., & Fantegrossi, W. E. (2020). In vivo effects of 3, 4-methylenedioxymethamphetamine (MDMA) and its deuterated form in rodents: drug discrimination and thermoregulation. Drug and Alcohol Dependence, 107850., 10.1016/j.drugalcdep.2020.107850
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4-MeO-PCP and 3-MeO-PCMo, new dissociative drugs, produce rewarding and reinforcing effects through activation of mesolimbic dopamine pathway and alteration of accumbal CREB, deltaFosB, and BDNF levels

December 11, 2019 / Neuroscience, Papers, Pharmacology & Chemistry, Psychiatry & Medicine, Publications, Substance Use Disorder / By / , , , , , ,

Abstract

Rationale: A high number of synthetic dissociative drugs continue to be available through online stores, leading to their misuse. Recent inclusions in this category are 4-MeO-PCP and 3-MeO-PCMo, analogs of phencyclidine. Although the dissociative effects of these drugs and their recreational use have been reported, no studies have investigated their abuse potential.

Objectives: To examine their rewarding and reinforcing effects and explore the mechanistic correlations.

Methods: We used conditioned place preference (CPP), self-administration, and locomotor sensitization tests to assess the rewarding and reinforcing effects of the drugs. We explored their mechanism of action by pretreating dopamine receptor (DR) D1 antagonist SCH23390 and DRD2 antagonist haloperidol during CPP test and investigated the effects of 4-MeO-PCP and 3-MeO-PCMo on dopamine-related proteins in the ventral tegmental area and nucleus accumbens. We also measured the levels of dopamine, phosphorylated cyclic-AMP response element-binding (p-CREB) protein, deltaFosB, and brain-derived neurotrophic factor (BDNF) in the nucleus accumbens. Additionally, we examined the effects of both drugs on brain wave activity using electroencephalography.

Results: While both 4-MeO-PCP and 3-MeO-PCMo induced CPP and self-administration, only 4-MeO-PCP elicited locomotor sensitization. SCH23390 and haloperidol inhibited the acquisition of drug CPP. 4-MeO-PCP and 3-MeO-PCMo altered the levels of tyrosine hydroxylase, DRD1, DRD2, and dopamine, as well as that of p-CREB, deltaFosB, and BDNF. All drugs increased the delta and gamma wave activity, whereas pretreatment with SCH23390 and haloperidol inhibited it.

Conclusion: Our results indicate that 4-MeO-PCP and 3-MeO-PCMo induce rewarding and reinforcing effects that are probably mediated by the mesolimbic dopamine system, suggesting an abuse liability in humans.

Abiero, A., Botanas, C. J., Custodio, R. J., Sayson, L. V., Kim, M., Lee, H. J., … & Cheong, J. H. (2020). 4-MeO-PCP and 3-MeO-PCMo, new dissociative drugs, produce rewarding and reinforcing effects through activation of mesolimbic dopamine pathway and alteration of accumbal CREB, deltaFosB, and BDNF levels. Psychopharmacology, 237(3), 757-772; 10.1007/s00213-019-05412-y

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Prospective examination of synthetic 5-methoxy-N,N-dimethyltryptamine inhalation: effects on salivary IL-6, cortisol levels, affect, and non-judgment

December 10, 2019 / DMT, Neuroscience, Papers, Pharmacology & Chemistry, Psychiatry & Medicine, Publications / By / , , , , ,

Abstract

Rationale

5-methoxy-N,N-dimethyltryptamine is a psychotropic substance found in various plant and animal species and is synthetically produced. 5-methoxy-N,N-dimethyltryptamine is used in naturalistic settings for spiritual exploration, recreation, or to address negative affect and mood problems. However, scientific knowledge on the effects of 5-methoxy-N,N-dimethyltryptamine in humans is scarce.

Objectives

The first objective was to assess the effects of inhalation of vaporized synthetic 5-methoxy-N,N-dimethyltryptamine on neuroendocrine markers. The second objective was to assess effects of the substance on affect and mindfulness. In addition, we assessed whether ratings of subjective measures were associated with changes in stress biomarkers (i.e., cortisol) and immune response (i.e., IL-6, CRP, IL-1β), as well as the acute psychedelic experience.

Methods

Assessments (baseline, immediately post-session, and 7-day follow-up) were made in 11 participants. Salivary samples were collected at baseline and post-session and analyzed by high-sensitivity enzyme-linked immunosorbent assay (ELISA).

Results

5-methoxy-N,N-dimethyltryptamine significantly increased cortisol levels and decreased IL-6 concentrations in saliva immediately post-session. These changes were not correlated to ratings of mental health or the psychedelic experience. Relative to baseline, ratings of non-judgment significantly increased, and ratings of depression decreased immediately post-session and at follow-up. Ratings of anxiety and stress decreased from baseline to 7-day follow-up. Participant ratings of the psychedelic experience correlated negatively with ratings of affect and positively with ratings of non-judgment.

Conclusion

Inhalation of vaporized synthetic 5-methoxy-N,N-dimethyltryptamine produced significant changes in inflammatory markers, improved affect, and non-judgment in volunteers. Future research should examine the effect of 5-methoxy-N,N-dimethyltryptamineamine with healthy volunteers in a controlled laboratory setting.

Uthaug, M. V., Lancelotta, R., Szabo, A., Davis, A. K., Riba, J., & Ramaekers, J. G. (2019). Prospective examination of synthetic 5-methoxy-N, N-dimethyltryptamine inhalation: effects on salivary IL-6, cortisol levels, affect, and non-judgment. Psychopharmacology, 1-13., 10.1007/s00213-019-05414-w
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Injury-Triggered Blueing Reactions of Psilocybe “Magic” Mushrooms

February 6, 2022 / Mushrooms / Psilocybin, Papers, Pharmacology & Chemistry / Leave a Comment / By / , , , , , , ,

Abstract

Upon injury, psychotropic psilocybin-producing mushrooms instantly develop an intense blue color, the chemical basis and mode of formation of which has remained elusive. We report two enzymes from Psilocybe cubensis that carry out a two-step cascade to prepare psilocybin for oxidative oligomerization that leads to blue products. The phosphatase PsiP removes the 4-O-phosphate group to yield psilocin, while PsiL oxidizes its 4-hydroxy group. The PsiL reaction was monitored by in situ 13 C NMR spectroscopy, which indicated that oxidative coupling of psilocyl residues occurs primarily via C-5. MS and IR spectroscopy indicated the formation of a heterogeneous mixture of preferentially psilocyl 3- to 13-mers and suggest multiple oligomerization routes, depending on oxidative power and substrate concentration. The results also imply that phosphate ester of psilocybin serves a reversible protective function.

Lenz, C., Wick, J., Braga, D., García-Altares, M., Lackner, G., Hertweck, C., Gressler, M., & Hoffmeister, D. (2020). Injury-Triggered Blueing Reactions of Psilocybe “Magic” Mushrooms. Angewandte Chemie (International ed. in English), 59(4), 1450–1454. https://doi.org/10.1002/anie.201910175

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

November 15, 2019 / MDMA, Neuroscience, Papers, Pharmacology & Chemistry, Publications / By / , , ,

Abstract

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.
Berquist, M. D., Leth-Petersen, S., Kristensen, J. L., & Fantegrossi, W. E. (2020). Locomotor effects of 3, 4-methylenedioxymethamphetamine (MDMA) and its deuterated form in mice: psychostimulant effects, stereotypy, and sensitization. Psychopharmacology, 237(2), 431-442; https://doi.org/10.1007/s00213-019-05380-3

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