Study of the in vitro and in vivo metabolism of 4-HO-MET

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Tryptamines can occur naturally in plants, mushrooms, microbes, and amphibians. Synthetic tryptamines are sold as new psychoactive substances (NPS) because of their hallucinogenic effects. When it comes to NPS, metabolism studies are of crucial importance, due to the lack of pharmacological and toxicological data. Different approaches can be taken to study in vitro and in vivo metabolism of xenobiotica. The zygomycete fungus Cunninghamella elegans (C. elegans) can be used as a microbial model for the study of drug metabolism. The current study investigated the biotransformation of four naturally occurring and synthetic tryptamines [N,N-Dimethyltryptamine (DMT), 4-hydroxy-Nmethyl-N-ethyltryptamine (4-HO-MET), N,N-di allyl-5-methoxy tryptamine (5-MeO-DALT) and 5-methoxy-N-methyl-N-isoporpoyltryptamine (5-MeO-MiPT)] in C. elegans after incubation for 72 hours. Metabolites were identified using liquid chromatography-high resolution-tandem mass spectrometry (LC-HR-MS/MS) with a quadrupole time-of-flight (QqTOF) instrument. Results were compared to already published data on these substances. C. elegans was capable of producing all major biotransformation steps: hydroxylation, N-oxide formation, carboxylation, deamination, and demethylation. On average 63% of phase I metabolites found in the literature could also be detected in C. elegans. Additionally, metabolites specific for C. elegans were identified. Therefore, C. elegans is a suitable complementary model to other in vitro or in vivo methods to study the metabolism of naturally occurring or synthetic tryptamines. KEYWORDS fungi Cunninghamella elegans, metabolism, NPS, tryptamines 1 | INTRODUCTIONMetabolism studies are of essential importance in clinical and forensic toxicology, especially when designer drugs or new psychoactive substances (NPS) are the target substrate as very little is known about their pharmacology and toxicology. When a drug of abuse is consumed, the compound is metabolized. General unknown screening analysis should be able to detect metabolites besides parent drugs, the latter which might not even be detectable a few hours after drug consumption. Hence, when developing methods for the detection of a substance of abuse, the main metabolites are used as analytical targets. 1 Therefore, the pharmacological processes and biotransformation of a drug need to be studied. Furthermore, the information about biotransformation of a substance can be used to evaluate its safety and efficacy, because some metabolites have non-negligible psychoactive properties themselves. [2][3][4] Metabolism studies can involve different in vivo and in vitro methods. In vivo methods on humans are difficult to apply because human samples from forensic or clinical cases are rare and human studies raise major ethical concerns (especially in the absence of preclinical safety data). 5 Alternative in vivo methods are animal studies but these are costly and time consuming and require ethical approval.Furthermore, one cannot neglect interspecies differences. In vitro

Section: Dmtcontrasting

confidence: 51%

“…28 For the metabolite M6, two isomers were found eluting at 1.92 and 4.74 minutes. 28 For the metabolite M6, two isomers were found eluting at 1.92 and 4.74 minutes.…”

Section: -Ho-metmentioning

confidence: 95%

Section: Dmtmentioning

confidence: 95%

Section: Dmtmentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Investigating the ability of the microbial model Cunninghamella elegans for the metabolism of synthetic tryptamines

Grafinger

Wilke

König

et al. 2018

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Tentative identification of in vitro metabolites of O‐acetylpsilocin (psilacetin, 4‐AcO‐DMT) by UHPLC‐Q‐Orbitrap MS

Zhai

Li²,

Zhao³

et al. 2022

4-Acetoxy-N,N-dimethyltryptamine (4-AcO-DMT, psilacetin, O-acetylpsilocin) is a synthetic tryptamine with psychedelic properties. Psilacetin may also act as precursor drug of psilocin, similar to psilocybin, but little is known about its metabolism. In this study, the phase I and phase II in vitro metabolism of 4-AcO-DMT was investigated with pooled human liver microsomes, and the reaction mixture was analyzed using liquid chromatography-quadrupole/electrostatic field orbitrap mass spectrometry.Fifteen metabolites were formed after incubation of pooled human liver microsomes with 4-AcO-DMT (12 phase I metabolites and 3 phase II metabolites). The proposed metabolite structures were based on accurate mass analysis and MS/MS fragmentation patterns. The biotransformations included hydrolysis, hydroxylation, N-demethylation, oxidation, and conjugation with glucuronic acid. The hydrolysis metabolite was the most abundant compound. For the development of new methods for the identification of 4-AcO-DMT consumption, the beta-hydroxylation metabolite of 4-AcO-DMT (M2-1) is recommended as a biomarker. The data reported in this work might be applicable to metabolic transformation of 4-AcO-DMT in vivo and also forensically helpful.

Metabolite markers for three synthetic tryptamines N‐ethyl‐N‐propyltryptamine, 4‐hydroxy‐N‐ethyl‐N‐propyltryptamine, and 5‐methoxy‐N‐ethyl‐N‐propyltryptamine

Bergh,

Bogen,

Grafinger

et al. 2024

N‐Ethyl‐N‐propyltryptamine (EPT), 4‐hydroxy‐N‐ethyl‐N‐propyltryptamine (4‐OH‐EPT), and 5‐methoxy‐N‐ethyl‐N‐propyltryptamine (5‐MeO‐EPT) are new psychoactive substances classified as tryptamines, sold online. Many tryptamines metabolize rapidly, and identifying the appropriate metabolites to reveal intake is essential. While the metabolism of 4‐OH‐EPT and 5‐MeO‐EPT are not previously described, EPT is known to form metabolites by indole ring hydroxylation among others. Based on general knowledge of metabolic patterns, 5‐MeO‐EPT is also expected to form ring hydroxylated EPT (5‐OH‐EPT). In the present study, the aim was to characterize the major metabolites of EPT, 4‐OH‐EPT, and 5‐MeO‐EPT, to provide markers for substance identification in forensic casework. The tryptamines were incubated with pooled human liver microsomes at 37°C for up to 4 h. The generated metabolites were separated and detected by ultra‐high performance liquid chromatography–quadrupole time‐of‐flight mass spectrometry analysis. The major in vitro EPT metabolites were formed by hydroxylation, N‐dealkylation, and carbonylation. In comparison, 4‐OH‐EPT metabolism was dominated by double bond formation, N‐dealkylation, hydroxylation, and carbonylation in vitro and hydroxylation or carbonylation combined with double bond loss, carbonylation, N‐dealkylation, and hydroxylation in vivo. 5‐MeO‐EPT was metabolized by O‐demethylation, hydroxylation, and N‐dealkylation in vitro. The usefulness of the characterized metabolites in forensic casework was demonstrated by identification of unique metabolites for 4‐OH‐EPT in a human postmortem blood sample with suspected EPT or 4‐OH‐EPT intoxication.