Cytochrome P450 enzymes contribute to the metabolism of LSD to nor-LSD and 2-oxo-3-hydroxy-LSD: Implications for clinical LSD use

This cell-based (in vitro) study investigated how the cytochrome P450 (CYPs) enzymes contribute to the metabolism of LSD to nor-LSD and 2-oxo-3-hydroxy-LSD and its potential for clinical LSD use. The study found that the human liver converted only small quantities of LSD to nor-LSD and O-H-LSD, however, several CYPs substantially contributed to the process. The review concluded that there is a link between genetic polymorphisms and drug interactions and it could therefore affect the pharmacodynamics and pharmacokinetics of LSD. Also, it was found that nor-LSD potentially may have hallucinogenic activity similar to LSD, while O-H-LSD is inactive.

Abstract

“In recent years, experimental research on lysergic acid diethylamide (LSD) in humans has gained new momentum. In humans, LSD is metabolized rapidly into several metabolites but knowledge of the involved metabolizing enzymes is limited. The aim of the current study was to identify the cytochrome P450 (CYP) isoforms involved in the metabolism of LSD to 6-norlysergic acid diethylamide (nor-LSD) and 2-oxo-3-hydroxy-LSD (O-H-LSD) in vitro, in order to evaluate potential effects of enzyme polymorphisms or prescription drugs on LSD pharmacokinetics. Additionally, interactions of LSD and both metabolites with 5-hydroxytryptamine (5-HT) receptors were assessed. LSD was incubated with human liver microsomes over 4 h and the production of nor-LSD and O-H-LSD was quantified by liquid chromatography tandem mass spectrometry. Metabolism was inhibited by the addition of specific CYP inhibitors. Additionally, recombinant CYPs were used to verify the inhibition results obtained with microsomes and induction of metabolism was investigated in human hepatocyte-derived cells. Radioligand binding and calcium mobilization assays were used to determine 5-HT receptor affinities and activities, respectively. Human liver microsomes displayed minor metabolite formation (<1% metabolized) over 4 h. CYP2D6, 2E1, and 3A4 significantly contributed to the formation of nor-LSD, and CYP1A2, 2C9, 2E1, and 3A4 were significantly involved in the formation of O-H-LSD. These findings could be verified using recombinant CYPs. Enzyme induction with rifampicin distinctly increased the formation of both metabolites, whereas treatment with omeprazole only slightly increased formation of nor-LSD. LSD and nor-LSD were pharmacologically active at the 5-HT1A, 5-HT2A, 5-HT2B, and 5-HT2C receptors. Nor-LSD mainly differed from the parent compound by having a lower affinity to the 5-HT2C receptor. O-H-LSD displayed substantially weaker affinity and activity at serotonergic receptors in comparison to LSD. To conclude, human liver microsomes converted only small amounts of LSD to nor-LSD and O-H-LSD but several CYPs significantly contributed. Genetic polymorphisms and drug interactions could therefore influence pharmacokinetics and pharmacodynamics of LSD. Nor-LSD likely has hallucinogenic activity similar to LSD, whereas O-H-LSD is inactive. Drug-drug interaction studies in humans are required to further assess the clinical relevance of these findings.“

Authors: Dino Luethi, Marius C. Hoener, Stephan Krähenbühl, Matthias E. Liechti & Urs Duthaler

Summary

In recent years, research on lysergic acid diethylamide (LSD) in humans has gained new momentum. The current study aimed to identify the CYP isoforms involved in the metabolism of LSD to 6-nor-LSD and 2-oxo-3-hydroxy-LSD in vitro.

1. Introduction

Lysergic acid diethylamide (LSD) was first synthesized in 1938 and was used in psychiatric research during the 1950s and 1960s. It gained popularity among recreational users and was largely suppressed in the 1970s, but has slowly found its way back into academic research and psychotherapy.

The two main LSD metabolites nor-LSD and O-H-LSD are metabolized by human liver microsomes and primary human hepatocytes, and several CYPs are involved in this metabolism. Further study of CYPs metabolizing LSD is necessary to determine potential influences of CYP inhibition and induction. There is currently not absolute clarity about the psychoactive activity of nor-LSD and O-H-LSD in humans. However, experiments in rats suggest a clearly decreased activity for nor-LSD compared to LSD.

(+)-N-3-benzylnirvanol, ketoconazole, 4-methylpyrazole hydrochloride, quinidine sulphate salt, sulfaphenazole, and ticlopidine hydrochloride were obtained from Sigma-Aldrich, and furafylline was purchased from Toronto Research Chemicals.

2.5. Drug-metabolizing enzyme systems

Drug-metabolizing enzyme systems were obtained from Corning Life Sciences B.V. (Amsterdam, The Netherlands) and stored at 80 °C.

2.7. Chemicals and reagents

Gradient grade acetonitrile, HPLC grade methanol, HPLC grade water, formic acid, bovine serum albumin, and dimethyl sulfoxide were purchased from Merck, Sigma-Aldrich, and Corning Life Sciences, respectively.

2.8. CYP inhibition studies

Calibration standards were prepared by enriching 0.1 M potassium phosphate buffer (pH 7.4) containing 1.5% BSA. The reaction mixture was then incubated at 37 °C and 600 rounds per min for 0.5, 1, 2, 3, and 4 h, and the samples were stored at 80 °C until analysis.

2.9. CYP1A2 and 3A4 induction studies

Primary human hepatocytes derived HepatoCells were seeded in a collagen I 24-well clear flat bottom tissue culture-treated polystyrene plate and incubated with omeprazole, rifampicin, or vehicle control for 3 days. The medium was replaced every 24 h with medium containing tizanidine, midazolam, or LSD for 72 h.

2.10. LC-MS/MS instrumentation and settings

A negative and a positive mode method was developed, which used water and acetonitrile as mobile phases. The analytes were separated using a stepwise gradient program, with the flow rate of pump C being decreased to 0 mL/min within the first 0.5 min of each run.

The mass transitions, retention times, and specific potentials of the analytes were determined by scheduled multiple reaction monitoring (MRM) with a detection window of 20 s.

LSD, O-H-LSD, and nor-LSD were analyzed on an analytical column using 2% mobile B (methanol plus 0.1% formic acid) and 98% mobile A (10 mM ammonium bicarbonate). The method was terminated by flushing the column for 1 min with 95% mobile B and reconditioning it for another 0.5 min with 2% B.

Analyte stock solutions of 10 mM were prepared in DMSO or acetonitrile. Calibration lines were prepared in PBS (pH 7.4) and used to estimate the analyte concentration in unknown samples by linear regression.

2.11. Binding affinities at serotonergic 5-HT receptors

LSD, O-H-LSD, and nor-LSD were tested at human serotonergic 5-HT1A, 5-HT2A, and 5-HT2C receptors. The specific binding of the drugs to the target receptors was determined by measuring the displacement of the ligand by the drugs.

2.12. Agonist activity at the 5-HT2A receptor

Human 5-HT2A receptor activity was assessed in receptor-transfected NIH-3 T3 cells by adding dye, incubating for 1 h, and measuring the increase in fluorescence for 51 s. The EC50 values were derived from the concentration-response curves using nonlinear regression.

2.13. Agonist activity at the 5-HT2B receptor

Human 5-HT2B receptor activity was determined in receptor-transfected HEK 293 cells. The cells were incubated at 37 °C with LSD or its metabolites for 2 h, and the increase in fluorescence was measured.

3.1. Microsomal stability

LSD concentrations decreased significantly after 4 h incubation with HLM, and 11.3 nM nor-LSD and 2.1 nM O-H-LSD were formed. No metabolite formation was observed after incubation in the absence of microsomes.

3.2. CYPs enzymes contributing to LSD metabolism in human liver microsomes

After microsomal incubations of 1000 nM LSD, the formation rates of nor-LSD and O-H-LSD were measured. CYP inhibitors Quinidine, 4-methylpyrazole, ketoconazole, and sulfaphenazole decreased the formation rates of nor-LSD and O-H-LSD, respectively.

3.3. Recombinant human CYPs contributing to LSD metabolism

Inhibition of rhCYP2D6, 2E1, and 3A4 decreased the formation of nor-LSD and O-H-LSD, respectively. Thus, incubations in recombinant human cytochromes could confirm the observed metabolism in human liver microsomes.

3.4. Induction of CYP1A2 and 3A4 in primary hepatocytes-derived cells

Over the course of 4 h, the amount of LSD was decreased by 26% in non-induced HepatoCells, by 27% in CYP1A2 induced cells, and by 33% in CYP3A4 induced cells.

3.5. Affinity and activity at serotonergic 5-HT receptors

LSD displayed potent affinity at the serotonin 5-HT1A, 5-HT2A, and 5-HT2C receptors, and partially activated the 5-HT2A and 5-HT2B receptors. Nor-LSD displayed higher activation potency and efficacy than LSD at both receptors, and O-H-LSD did not activate either receptor in the investigated concentration range.

4. Discussion

While partial agonism at 5-HT2A and 5-HT2B receptors was observed at high concentrations of O-H-LSD, the activity and efficacy of nor-LSD at both receptors was higher compared with LSD. This suggests a significant role of the 5-HT2C receptor in the psychedelic effects of LSD.

LSD is a highly potent psychedelic and common doses in the range of 100 – 200 g result in peak plasma concentrations in the low nanomolar range. The metabolism of LSD involves microsomal enzymes, but only 1% and 0.2% of the parent compound were detected as nor-LSD and O-H-LSD, respectively.

Experiments using recombinant CYPs confirmed the results of the human liver microsomes assays, and CYP induction with rifampicin in HepatoCells increased the formation of nor-LSD and O-H-LSD. However, CYP induction with omeprazole resulted in a slight increase in nor-LSD but not O-H-LSD.

The contribution of the CYPs identified in the present study could have clinical consequences. For instance, CYP3A4 is the most important CYP contributing to the metabolism of LSD, but several food and herbal products can inhibit or induce CYP3A4, and genetic polymorphisms can affect CYP function.

In this study several CYPs involved in LSD metabolism were identified, but the reason for the discrepancy between LSD decrease and metabolite formation remains unclear. Additional metabolites and investigation of non-microsomal metabolic pathways are needed in order to draw firmer conclusions.