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Markus R Meyer

34 papers in the library · 768 citations · publishing 2009-2026

Papers

Metabolism of designer drugs of abuse: an updated review.

Current Drug Metabolism June 1, 2010 Markus R Meyer, Hans H Maurer 112 citations

This review updates a 2005 paper on how the body metabolizes new designer drugs that have appeared on the black market. It covers 2C compounds (phenethylamines like 2C-B, 2C-I, 2C-D, 2C-E, 2C-T-2, and 2C-T-7), beta-keto drugs (butylone, ethylone, methylone, mephedrone), pyrrolidinophenones (MPBP and PVP), phencyclidine-derived drugs (PCPr, PCEEA, PCMPA, PCMEA), tryptamines (5-MeO-DIPT), and fentanyl analogs (alpha-MF and 3-MF). The review focuses on studies that identified human or animal metabolites formed in vivo or in vitro and the roles of cytochrome P450 and monoamine oxidase enzymes in their metabolism.

Absorption, Distribution, Metabolism and Excretion Pharmacogenomics of Drugs of Abuse

Pharmacogenomics February 1, 2011 Markus R Meyer, Hans H Maurer 107 citations

The effects of drugs of abuse and other foreign substances depend on an individual's genetic makeup and the specific enzymes that break down those substances. This article summarizes current knowledge about the enzymes—such as cytochrome P450, glucuronyltransferases, esterases, and reductases—involved in metabolizing frequently abused opioids (oxycodone, hydrocodone, methadone, fentanyl, buprenorphine, tramadol, heroin, morphine, codeine), anesthetics (GHB, propofol, ketamine, phencyclidine), cognitive enhancers (methylphenidate, modafinil), plant-derived hallucinogens (LSD, salvinorin A, psilocybin, psilocin), and nicotine. Understanding these metabolic pathways helps predict drug interactions, explain individual differences in drug response, assess toxicity, and improve drug testing interpretation.

Studies on the metabolism and toxicological detection of the new psychoactive designer drug 2-(4-iodo-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine (25I-NBOMe) in human and rat urine using GC-MS, LC-MSn, and LC-HR-MS/MS

Analytical and Bioanalytical Chemistry June 24, 2015 Achim T. Caspar, Andreas G. Helfer, Julian A Michely et al. 69 citations

25I-NBOMe, a potent hallucinogenic drug that activates 5-HT2A receptors, is extensively metabolized in rats. After administration to male Wistar rats, 68 metabolites were identified in urine using liquid chromatography-high-resolution tandem mass spectrometry. The main metabolic pathways include O-demethylation, O,O-bis-demethylation, hydroxylation, and combinations, followed by glucuronidation and sulfation. Detection of intake is possible through metabolites using LC-MS methods but not by GC-MS standard urine screening. Initial tests indicate that CYP1A2 and CYP3A4 are involved in hydroxylation, while CYP2C9 and CYP2C19 mediate O-demethylation, suggesting potential drug-drug interactions.

Metabolism of the new psychoactive substances N,N-diallyltryptamine (DALT) and 5-methoxy-DALT and their detectability in urine by GC-MS, LC-MSn, and LC-HR-MS-MS.

Analytical and Bioanalytical Chemistry October 1, 2015 Julian A Michely, Andreas G. Helfer, Simon D. Brandt et al. 49 citations

N,N-Diallyltryptamine (DALT) and 5-methoxy-DALT (5-MeO-DALT) are synthetic tryptamines with psychoactive effects. In rats, after high-dose administration, their metabolism involves aromatic and aliphatic hydroxylations, N-dealkylation, N-oxidation, and combinations; 5-MeO-DALT also undergoes O-demethylation, followed by extensive glucuronidation or sulfation. The main cytochrome P450 enzymes for DALT are CYP2C19, CYP2D6, and CYP3A4; for 5-MeO-DALT, CYP1A2, CYP2C19, CYP2D6, and CYP3A4. For detecting low-dose consumption in rat urine, LC-MS(n) and LC-HR-MS-MS are suitable; the most abundant markers are a ring hydroxy metabolite of DALT, the N,O-bis-dealkyl metabolite of 5-MeO-DALT, and their glucuronides. GC-MS can screen DALT only via its main metabolites.

Metabolic fate and detectability of the new psychoactive substances 2-(4-bromo-2,5-dimethoxyphenyl)- N- [(2-methoxyphenyl)methyl]ethanamine (25B-NBOMe) and 2-(4-chloro-2,5-dimethoxyphenyl)- N- [(2-methoxyphenyl)methyl]ethanamine (25C-NBOMe) in human and rat urine by GC–MS, LC–MS n , and LC–HR–MS/MS approaches

Journal of Pharmaceutical and Biomedical Analysis November 27, 2016 Achim T. Caspar, Simon D. Brandt, Andreas E. Stoever et al. 43 citations

25B-NBOMe and 25C-NBOMe, potent 5-HT2A receptor agonists linked to hallucinogenic effects and severe intoxications, are extensively metabolized in rats and humans. Using LC-HR-MS/MS, 66 metabolites were identified for 25B-NBOMe and 69 for 25C-NBOMe, primarily through O-demethylation, O,O-bis-demethylation, hydroxylation, and subsequent glucuronidation and sulfation. After low-dose administration to rats, both substances were detectable mainly via their metabolites using LC-based screening approaches. In an authentic human urine sample from an acute intoxication, 25B-NBOMe and its metabolites were detected by GC-MS as well. Initial screening showed CYP1A2 and CYP3A4 involvement in hydroxylation, and CYP2C9 and CYP2C19 in O-demethylation.

A qualitative/quantitative approach for the detection of 37 tryptamine-derived designer drugs, 5 β-carbolines, ibogaine, and yohimbine in human urine and plasma using standard urine screening and multi-analyte approaches.

Analytical and Bioanalytical Chemistry January 1, 2014 Markus R Meyer, Achim T. Caspar, Simon D. Brandt et al. 39 citations

A new laboratory method using liquid chromatography and linear ion trap mass spectrometry can detect 37 synthetic tryptamines plus five β-carbolines, ibogaine, and yohimbine in human urine and plasma. The method is selective for all tested substances, with detection limits in urine between 10 and 100 ng/mL and in plasma between 1 and 100 ng/mL. Validated quantification in plasma was achieved for 33 of the 44 analytes. This addresses the previous scarcity of analytical data on detecting these emerging designer drugs in human biosamples.

Enantioselectivity in the Methylation of the Catecholic Phase I Metabolites of Methylenedioxy Designer Drugs and Their Capability To Inhibit Catechol-O-methyltransferase-Catalyzed Dopamine 3-Methylation

Chemical Research in Toxicology May 22, 2009 Markus R Meyer, Hans H Maurer 39 citations

The designer drugs MDMA, MDEA, and MBDB are chiral compounds whose metabolism is enantioselective, favoring the S-enantiomer. This study investigated whether the elimination of their catecholamine metabolites via O-methylation by catechol-O-methyltransferase (COMT) is also enantioselective. Using human liver cytosol and microsomes, the S-enantiomers of all three catecholamines were preferentially O-methylated by both soluble and membrane-bound COMT. The membrane-bound COMT had 10-fold higher affinity for substrates, while the soluble form had 10-fold higher turnover rate. All tested catechols uncompetitively inhibited dopamine methylation. Enantioselective elimination may contribute to different pharmacokinetic properties of the enantiomers.

Pharmacological and biotransformation studies of 1-acyl-substituted derivatives of -lysergic acid diethylamide (LSD)

Neuropharmacology November 19, 2019 Adam L. Halberstadt, Muhammad Chatha, Adam K. Klein et al. 37 citations

The ergoline d-lysergic acid diethylamide (LSD) is one of the most potent psychedelic drugs. 1-Acetyl-LSD (ALD-52) and other 1-acyl-substituted LSD derivatives, including 1-propanoyl-LSD (1P-LSD) and 1-butanoyl-LSD (1B-LSD), have appeared as designer drugs assumed to act as prodrugs for LSD. Competitive binding studies and calcium mobilization assays showed 1-acyl-substitution reduced affinity for most monoamine receptors, including 5-HT2A sites, by one to two orders of magnitude, and these derivatives had weak efficacy or acted as antagonists in Ca2+-mobilization assays. Despite this, they induced head twitches in mice with relatively high potency. High levels of LSD were detected in rat plasma after subcutaneous administration of ALD-52 and 1P-LSD, demonstrating rapid and efficient deacylation in vivo, consistent with the prediction that these compounds serve as prodrugs for LSD.

Metabolism of the tryptamine-derived new psychoactive substances 5-MeO-2-Me-DALT, 5-MeO-2-Me-ALCHT, and 5-MeO-2-Me-DIPT and their detectability in urine studied by GC-MS, LC-MSn , and LC-HR-MS/MS.

Drug Testing and Analysis January 1, 2018 Achim T. Caspar, Jonas B Gaab, Julian A Michely et al. 33 citations

Three new psychoactive tryptamines—5-MeO-2-Me-DALT, 5-MeO-2-Me-ALCHT, and 5-MeO-2-Me-DIPT—are mainly broken down in the body through O-demethylation, hydroxylation, and N-dealkylation, followed by glucuronidation or sulfation. In rats given 20 mg/kg doses, 5-MeO-2-Me-DALT produced 24 phase I and 12 phase II metabolites, 5-MeO-2-Me-ALCHT produced 24 phase I and 14 phase II metabolites, and 5-MeO-2-Me-DIPT produced 20 phase I and 11 phase II metabolites. Human liver enzyme incubations suggest the same major metabolic pathways occur in humans. CYP1A2, CYP2C19, CYP2D6, and CYP3A4 catalyze hydroxylation; CYP2C19 and CYP2D6 catalyze O-demethylation; and CYP2C19, CYP2D6, and CYP3A4 catalyze N-dealkylation. Liquid chromatography-based urine screening detected intake of all three compounds after low doses (0.1–1 mg/kg), whereas gas chromatography-based screening did not.

Urinary Excretion Kinetics of 3,4-Methylenedioxymethamphetamine (MDMA, Ecstasy) and Its Phase I and Phase II Metabolites in Humans following Controlled MDMA Administration

Clinical Chemistry October 7, 2011 Andrea E. Schwaninger, Markus R Meyer, Allan J. Barnes et al. 33 citations

After oral MDMA (ecstasy) intake, human urine contains mostly sulfate and glucuronide conjugates of MDMA metabolites, with sulfates present at higher concentrations than glucuronides. More than 90% of the metabolites DHMA and HMMA were excreted as conjugates. HMMA sulfate had the longest detection window in urine. The ratio of HMMA sulfate to glucuronide was 2.0, and the ratio of DHMA 3-sulfate to 4-sulfate was 5.3 during the first 24 hours, matching predictions from earlier lab experiments. These findings can improve direct urine analysis for MDMA and its metabolites in clinical and forensic toxicology.

Screening for illicit drugs in pooled human urine and urinated soil samples and studies on the stability of urinary excretion products of cocaine, MDMA, and MDEA in wastewater by hyphenated mass spectrometry techniques

Drug Testing and Analysis February 17, 2016 Marie Mardal, Juliet Kinyua, Pedram Ramin et al. 31 citations

Wastewater-based epidemiology can track community drug use, but biomarkers are often diluted. Pooled urine and urinated soil from festivals were screened for illicit drug excretion products. Cocaine and ecstasy-like compounds were most frequent. A method was developed to quantify their excretion products. Hydroxymethoxymethamphetamine (HMMA), MDMA, MDA, HMMA sulfate, benzoylecgonine, and cocaethylene retained 85–102% of initial concentration after 8 hours, while cocaine and ecgonine methyl ester dropped to 74% and 67%, respectively. HMMA increased over 24 hours, likely from conjugate cleavage and MDMA biotransformation. HMMA is suggested as a stable analytical target for MDMA consumption in wastewater.

Interactions of phenethylamine-derived psychoactive substances of the 2C-series with human monoamine oxidases.

Drug Testing and Analysis February 1, 2019 Lea Wagmann, Simon D. Brandt, Alexander Stratford et al. 26 citations

Thirteen of 17 phenethylamine-derived designer drugs (12 from the 2C-series and five FLY analogs) inhibited monoamine oxidase A (MAO-A), and 11 inhibited monoamine oxidase B (MAO-B) in an in vitro assay using heterologously expressed enzymes and hydrophilic interaction liquid chromatography-high resolution tandem mass spectrometry. For the seven drugs where MAO-A IC50 values were determined, values ranged from 10 to 125 μM; for the nine drugs with MAO-B IC50 values, the range was 1.7 to 180 μM. Because clinical information on most test drugs is lacking, a pharmacological contribution of MAO inhibition cannot be excluded, and further studies are warranted.

Biotransformation and detectability of the new psychoactive substances N,N-diallyltryptamine (DALT) derivatives 5-fluoro-DALT, 7-methyl-DALT, and 5,6-methylenedioxy-DALT in urine using GC-MS, LC-MSn, and LC-HR-MS/MS.

Analytical and Bioanalytical Chemistry February 1, 2017 Julian A Michely, Simon D. Brandt, Markus R Meyer et al. 23 citations

Derivatives of N,N-diallyltryptamine (DALT) are new psychoactive substances. Their metabolism and detectability were studied in rat urine and human liver microsomes using liquid chromatography-high resolution-tandem mass spectrometry. Main metabolic pathways include aromatic and aliphatic hydroxylations, N-dealkylation, N-oxidation, and combinations; carboxylation was detected for 7-Me-DALT and O-demethylenation for 5,6-MD-DALT. Phase I metabolites were extensively glucuronidated or sulfated, catalyzed by several CYP isoenzymes. GC-MS could not reliably monitor consumption, but LC-MSn and LC-HR-MS/MS approaches were suitable, especially for detecting 5-F-DALT and 7-Me-DALT at low doses. The most abundant targets for each compound are specified.

Stereoselective urinary MDMA (ecstasy) and metabolites excretion kinetics following controlled MDMA administration to humans

Biochemical Pharmacology September 29, 2011 Andrea E. Schwaninger, Markus R Meyer, Allan J. Barnes et al. 23 citations

The R- and S-enantiomers of MDMA are eliminated differently in human urine. After controlled oral doses of 1.0 and 1.6 mg/kg, urine from ten participants was analyzed. Over five days, a median of 21% of the measured compounds were excreted as R-stereoisomers and 17% as S-stereoisomers. Significantly more R-enantiomers of MDMA, DHMA, and HMMA sulfate were excreted, while more S-stereoisomers of HMMA and HMMA glucuronide were excreted. No significant differences appeared for MDA and DHMA sulfate. The ratio of R- to S-stereoisomers changed steadily over the first 48 hours, suggesting it could help estimate time of MDMA ingestion in clinical and forensic toxicology.

Phenethylamine-derived new psychoactive substances 2C-E-FLY, 2C-EF-FLY, and 2C-T-7-FLY: Investigations on their metabolic fate including isoenzyme activities and their toxicological detectability in urine screenings.

Drug Testing and Analysis October 1, 2019 Lea Wagmann, Nora Hempel, Lilian H. J. Richter et al. 20 citations

Three new psychoactive substances of the 2C-FLY series—2C-E-FLY, 2C-EF-FLY, and 2C-T-7-FLY—were studied to determine how the body metabolizes them and how they can be detected in toxicological tests. Using rat urine and human liver S9 fractions analyzed by LC-HRMS/MS, 32 metabolites were identified. Main metabolic steps were hydroxylation and N-acetylation, catalyzed by CYP2D6, CYP3A4, FMO3, NAT1, and NAT2. Deamination by MAO-A and B was also observed. Polymorphisms or drug interactions may cause interindividual differences. Standard urine screening approaches using GC-MS, LC-MSn, and LC-HRMS/MS were suitable for detecting intake, but common metabolites of 2C-E-FLY and 2C-EF-FLY must be considered when interpreting results.

Nano liquid chromatography-high-resolution mass spectrometry for the identification of metabolites of the two new psychoactive substances N-(ortho-methoxybenzyl)-3,4-dimethoxyamphetamine and N-(ortho-methoxybenzyl)-4-methylmethamphetamine.

Talanta October 1, 2018 Achim T. Caspar, Markus R Meyer, Folker Westphal et al. 17 citations

Two new hallucinogens, 3,4-DMA-NBOMe and 4-MMA-NBOMe, are extensively metabolized in rats and human liver preparations. Using nano liquid chromatography with high-resolution mass spectrometry, 38 metabolites of 3,4-DMA-NBOMe and 33 metabolites of 4-MMA-NBOMe were identified. The main metabolic pathways are O-demethylation and glucuronic acid conjugation for 3,4-DMA-NBOMe, and oxidation of the tolyl group to carboxylic acid for 4-MMA-NBOMe. The nanoLC approach performed comparably to conventional UHPLC. Standard urine screening methods could detect an estimated low user dose only through metabolites. Suggested screening targets include O-demethyl- and O,O-bis-demethyl-3,4-DMA-NBOMe and their glucuronides, and carboxy-4-MMA-NBOMe and its glucuronide and N-demethyl-carboxy-4-MMA-NBOMe.

Analytical characterization of N,N-diallyltryptamine (DALT) and 16 ring-substituted derivatives.

Drug Testing and Analysis January 1, 2017 Simon D. Brandt, Pierce V. Kavanagh, Geraldine Dowling et al. 16 citations

Many N,N-dialkylated tryptamines have psychoactive properties in humans, and the number of derivatives has grown across research areas. Some are used in medicinal products, others as recreational drugs, and sometimes these uses overlap. 5-Methoxy-N,N-diallyltryptamine (5-MeO-DALT) recently emerged as a new psychoactive substance, while 4-acetoxy-DALT and unsubstituted DALT have been detected only recently. This report describes the analytical characterization of 17 N,N-diallyltryptamines (DALTs), including 15 prepared via microwave-accelerated synthesis. The compounds were characterized using NMR, GC-MS, mass spectrometry, photodiode array detection, and GC solid-state infrared analysis. The resulting spectral data are provided to help researchers identify newly emerging substances and explore clinical and non-clinical uses.

Biotransformation and detectability of the designer drug 2,5-dimethoxy-4-propylphenethylamine (2C-P) studied in urine by GC-MS, LC-MS(n), and LC-high-resolution-MS(n).

Analytical and Bioanalytical Chemistry January 1, 2015 Carina S. D. Wink, Markus R Meyer, Tina Braun et al. 13 citations

2C-P is a hallucinogenic designer drug from the phenethylamine class. This work identified its phase I and II metabolites and tested detectability in urine. Proposed metabolic pathways include N-acetylation, deamination with reduction to alcohol or oxidation to carbonic acid, mono- and bis-hydroxylation, mono- and bis-O-demethylation followed by glucuronidation or sulfation, and combinations. A common user's dose of 2C-P was reliably detectable in urine using standard GC-MS and LC-MS(n) screening methods, supporting its identification in clinical and forensic cases.

Metabolism and cytotoxicity studies of the two hallucinogens 1cP-LSD and 4-AcO-DET in human liver and zebrafish larvae models using LC-HRMS/MS and a high-content screening assay.

Journal of Pharmaceutical and Biomedical Analysis August 1, 2024 Tanja M Gampfer, Victoria Schütz, Philip Schippers et al. 11 citations

Two new hallucinogenic drugs, 1cP-LSD and 4-AcO-DET, were metabolized in human liver S9 fraction and in zebrafish larvae, with several phase I and phase II metabolites identified. Some metabolites were unique to zebrafish larvae. Neither compound showed toxic effects on human liver cells, though 4-AcO-DET combined with a CYP inhibitor altered two cellular parameters at concentrations far above expected in vivo levels. The authors suggest further testing with other liver cell lines that express more CYP enzymes.

Can the Intake of a Synthetic Tryptamine be Detected Only by Blood Plasma Analysis? A Clinical Toxicology Case Involving 4-HO-MET

Journal of Analytical Toxicology June 3, 2021 Lea Wagmann, Sascha K. Manier, Markus R Meyer 11 citations

A non-fatal clinical case involving the synthetic tryptamine 4-HO-MET (metocin or methylcybin) was analyzed using blood plasma. Liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-HRMS-MS) detected the parent compound and four metabolites—N-demethyl-, oxo-, hydroxy-4-HO-MET, and the N-oxide—while gas chromatography-mass spectrometry did not detect it. The plasma concentration of 4-HO-MET was 193 ng/mL. These findings provide data for clinical and forensic toxicologists interpreting future cases involving synthetic tryptamines, particularly when only blood samples are available.

Bioisosteric analogs of MDMA: Improving the pharmacological profile?

Journal of Neurochemistry September 1, 2024 Ana Sofia Alberto-Silva, Selina Hemmer, Hailey A. Bock et al. 10 citations

Three new chemical variants of MDMA—ODMA, TDMA, and SeDMA—show similar activity at serotonin, dopamine, and norepinephrine transporters but reduced activity at 5-HT2A/2B/2C receptors compared to MDMA. They also differ in liver metabolism, with N-demethylation as the only shared route and no phase II metabolites formed. TDMA showed faster clearance. The analogs interacted more weakly with organic cation transporters and plasma membrane monoamine transporter. These bioisosteres may offer therapeutic alternatives to MDMA with a reduced off-target profile, but further studies are needed to determine if they pose lower risks.

In Vivo and In Vitro Metabolic Fate and Urinary Detectability of Five Deschloroketamine Derivatives Studied by Means of Hyphenated Mass Spectrometry.

Metabolites May 8, 2024 Fabian Frankenfeld, Lea Wagmann, Anush Abelian et al. 3 citations

Five deschloroketamine derivatives—deschloro-N-cyclopropyl-ketamine, deschloro-N-ethyl-ketamine, deschloro-N-isopropyl-ketamine, deschloro-N-propyl-ketamine, and deschloroketamine—are primarily metabolized through N-dealkylation, hydroxylation, multiple oxidations, and combinations, plus glucuronidation and N-acetylation. In total, 29 phase I and 10 phase II metabolites were detected in rat urine after a 2 mg/kg body weight dose, using liquid chromatography high-resolution tandem mass spectrometry and gas chromatography-mass spectrometry. For the LC-HRMS/MS standard urine screening approach, compound-specific metabolites were identified and confirmed in pooled human liver microsomes for all derivatives except deschloro-N-cyclopropyl-ketamine. The GC-MS approach detected only non-specific acetylated N-dealkylation metabolites.

Studies on the Stability and Microbial Biotransformation of Five Deschloroketamine Derivatives as Prerequisite for Wastewater-Based Epidemiology Screening.

Drug Testing and Analysis August 1, 2025 Fabian Frankenfeld, Lea Wagmann, Markus R Meyer 2 citations

Five deschloroketamine derivatives and most of their metabolites remain sufficiently stable in raw wastewater to serve as analytical targets for wastewater-based epidemiology. After incubating the parent compounds, rat urine, or rat feces in untreated influent wastewater for 24 hours, all parent compounds, seven Phase I metabolites, and one Phase II metabolite were detected in rat feces. Peak areas of Phase II N- and O-glucuronides decreased markedly, consistent with earlier findings that Phase II conjugates are unstable in wastewater and unsuitable as biomarkers. The results indicate that the parent drugs and their Phase I metabolites can be used for wastewater surveillance of these new psychoactive substances.

Metabolic fate of drugs of abuse and new psychoactive substances: A pilot study on a novel workflow using a zebrafish embryo model combined with human microdosing.

British Journal of Clinical Pharmacology June 16, 2025 Wellenberg K Simon, Tanja M Gampfer, Wagmann Lea et al. 1 citation

A workflow using zebrafish embryos (ZEs) followed by human microdosing (HMD) can identify human urine biomarkers for drugs of abuse and new psychoactive substances. Metabolites of amphetamine, cocaine, LSD, MDMA, methamphetamine, THC, MDMB-CHMICA, and MDPPP were first identified in ZEs exposed via immersion or injection, then compared with known human metabolites and confirmed by HMD. Both methods identified main human urine metabolites, except for LSD (due to low dose) and cannabinoids (due to low oral bioavailability). ZEs produced more metabolites, including conjugates, than HMD. The approach provides quick, reliable data for urinary drug screening, though challenges remain with HMD, including different administration routes and low-dose detectability.

Correction to "Next-Generation MDMA Analogue SDMA: Pharmacological and Metabolic Insights".

ACS Chem Neurosci March 4, 2026 Nina Kastner, Núria Nadal‐gratacós, Selina Hemmer et al. correction

A correction notice clarifies that two errors in the original paper do not affect the accuracy of the results, interpretations, or conclusions. The first correction addresses a presentation issue in Table 1, confirming the data are correct. The second correction clarifies that thigmotaxis, a measure of anxiety-like behavior, was evaluated in rats given SDA or SDMA compounds. Compared to saline, only the 10 mg/kg dose of SDA significantly increased thigmotaxis, meaning the rats spent less time in the center of the arena.