Designer benzodiazepines versus prescription benzodiazepines: can structural relation predict the next step?

Designer benzodiazepines are a part of the recently discovered abuse synthetic drugs called Novel Psychoactive Substances (NPS) which need to be controlled due to their constantly growing market. Most of them are derived from the medically approved benzodiazepines used nowadays yet, may possess stronger effects, more toxicity, and longer durations of action. Some differences have also been observed in their detection and characteristics, in addition to the variations discovered in postmortem redistribution and drug stability. All these major alterations in features can result from only minor structural modifications. For example, a classic benzodiazepine (BZD) like diazepam only lacks one fluorine atom which exists in its derivatized designer drug, diclazepam, making substantial differences in activity. For this reason, it is essential to study the designer drugs in order to identify their dangers and distinguish them thus rule out their abuse and control the spread of such drugs. This review would highlight the distinct characteristics of some of the most commonly abused designer benzodiazepine analogies in relation to their original prescription BZD compounds.

Rohypnol-induced sexual dysfunction is via suppression of hypothalamic-pituitary-testicular axis: An experimental study in rats.

Sexual activity is an essential part of reproductive functions and needed for the maintenance of fertility. Drugs, particularly substances of abuse, impair male reproductive function either by interrupting hormonal functions or through the nonhormonal pathways. This study evaluated the impact of Rohypnol use in sexual behaviour. Materials and methods: Thirty adult male Wistar rats of comparable weights (180-200 g) were randomly allocated into three groups, the control and low-dose and high-dose Rohypnol-treated groups. The control group received 0.5 ml of distilled water, while the low- and high-dose Rohypnol-treated groups received 2 mg/kg b.w and 4 mg/kg b.w of Rohypnol via oral lavage once daily for 28 days. Rohypnol significantly increased mount latency, intromission latency, ejaculation latency and post-ejaculatory interval, as well as lowered mount frequency, intromission frequency and ejaculation frequency. Rohypnol-induced sexual dysfunction was found to be associated with significant suppression of circulatory follicle-stimulating hormone, luteinising hormone, testosterone and oestrogen. The present study reveals that Rohypnol induces sexual dysfunction through suppression of hypothalamic-pituitary-testicular axis. It also implicates Rohypnol as a potential candidate for drug-induced infertility.

All solid-state miniaturized potentiometric sensors for flunitrazepam determination in beverages.

Flunitrazepam is one of the frequently used hypnotic drugs to incapacitate victims for sexual assault. Appropriate diagnostic tools should be available to victims regarding the growing concern about "date-rape drugs" and their adverse impact on society. Miniaturized screen-printed potentiometric sensors offer crucial point-of-care devices that alleviate this serious problem. In this study, all solid-state screen-printed potentiometric flunitrazepam sensors have been designed. The paper device was printed with silver and carbon ink. Formation of an aqueous layer in the interface between carbon-conducting material and ion-sensing membrane nevertheless poses low reproducibility in the solid-contact electrodes. Accordingly, poly(3,4-ethylenedioxythiophene) (PEDT) nano-dispersion was applied as a conducting hydrophobic polymer on the electrode surface to curb water accumulation. Conditioning of ion-sensing membrane in the vicinity of reference membrane has been considered carefully using special protocol. Electrochemical characteristics of the proposed PEDT-based sensor were calculated and compared favorably to PEDT-free one. The miniaturized device was successfully used for the determination of flunitrazepam in carbonated soft drinks, energy drink, and malt beverage. Statistical comparison between the proposed sensor and official method revealed no significant difference. Nevertheless, the proposed sensor provides simple and user-friendly diagnostic tool with less equipment for on-site determination of flunitrazepam.

Rapid Drop-Volume Electrochemical Detection of the "Date Rape" Drug Flunitrazepam in Spirits Using a Screen-Printed Sensor in a Dry-Reagent Format.

Flunitrazepam is an extremely potent benzodiazepine sedative which is associated with "drug-facilitated sexual assault" when administered within an alcoholic drink. This work describes a simple electrochemical method for on-site rapid detection of flunitrazepam in untreated spirits (whiskey, vodka and gin) using a single-use screen-printed sensor (featuring graphite working and auxiliary electrodes and an Ag/AgCl reference electrode) in a dry reagent format. Analysis was performed by placing a drop of sample on the sensor, which was previously coated with dry KCl, and recording selected reduction/oxidation peaks of the target compound in a cyclic voltammetry scan. The limit of quantification of flunitrazepam was at the sub-mg L-1 range. The between-sensor % relative standard deviation of the analytically useful reduction peak in a solution containing 11.4 mg L-1 flunitrazepam was 9.8% (n = 5). Quantification was performed using calibration curves constructed from pooled samples spiked with flunitrazepam with relative errors <15%. The main advantages of the methodology are that it involves no sample pretreatment (such as deoxygenation, extraction or reagent(s) addition) and requires only drop-sized volumes of the sample, thus facilitating rapid on-site screening using portable equipment.

Voltammetric Electronic Tongue for the Simultaneous Determination of Three Benzodiazepines.

The presented manuscript reports the simultaneous detection of a ternary mixture of the benzodiazepines diazepam, lorazepam, and flunitrazepam using an array of voltammetric sensors and the electronic tongue principle. The electrodes used in the array were selected from a set of differently modified graphite epoxy composite electrodes; specifically, six electrodes were used incorporating metallic nanoparticles of Cu and Pt, oxide nanoparticles of CuO and WO3, plus pristine electrodes of epoxy-graphite and metallic Pt disk. Cyclic voltammetry was the technique used to obtain the voltammetric responses. Multivariate examination using Principal Component Analysis (PCA) justified the choice of sensors in order to get the proper discrimination of the benzodiazepines. Next, a quantitative model to predict the concentrations of mixtures of the three benzodiazepines was built employing the set of voltammograms, and was first processed with the Discrete Wavelet Transform, which fed an artificial neural network response model. The developed model successfully predicted the concentration of the three compounds with a normalized root mean square error (NRMSE) of 0.034 and 0.106 for the training and test subsets, respectively, and coefficient of correlation R ≥ 0.938 in the predicted vs. expected concentrations comparison graph.

Effect of flunitrazepam as an oral hypnotic on 24-hour blood pressure in healthy volunteers.

PURPOSE: The present study was carried out in order to assess the effects of chronic administration of flunitrazepam (as an oral hypnotic) on 24-h blood pressure (BP) and heart rate (HR) in healthy young adults. MATERIALS AND METHODS: Following a 2-week placebo run-in period, 28 healthy volunteers (13 males and 15 females) between 21 and 30 years were randomized to receive either flunitrazepam 1 mg or placebo (both administered once a day in the evening) for 4 weeks in two cross-over periods; each separated by a 2-week placebo period. At the end of each study period, non-invasive 24-h BP and HR ambulatory monitoring was performed. RESULTS: Flunitrazepam produced a significant decrease in nighttime systolic blood pressure (SBP) (- 6.4 mmHg) and diastolic blood pressure (DBP) (- 4.1 mmHg) (both P < 0.05 vs placebo) without affecting nocturnal HR. During the morning hours, significantly higher values of SBP (+ 7.4 mmHg, P < 0.01), DBP (+ 3.4 mmHg, P < 0.05) and HR (+ 3.9 beats/min, P < 0.05) were observed in the flunitrazepam group compared to the placebo-treated group. No significant differences were noted between the two groups during afternoon and evening hours. CONCLUSIONS: These results suggest that chronic oral administration of 1 mg flunitrazepam as a hypnotic agent causes a significant nocturnal fall in BP and a transient rebound increase of both BP and HR at awakening in the morning. Mechanisms underlying these cardiovascular effects remain unclear, although the direct vasodilatory effect, which is typical of flunitrazepam (with consequent reflex counter-regulatory responses), and the attenuation of baroreflex sensitivity are likely to play a major role.

Amnestic drugs in the odor span task: Effects of flunitrazepam, zolpidem and scopolamine.

The odor span task is an incrementing non-matching-to-sample procedure designed to provide an analysis of working memory capacity in rodents. The procedure takes place in an arena apparatus and rats are exposed to a series of odor stimuli in the form of scented lids with the selection of new stimuli reinforced. This procedure makes it possible to study drug effects as a function of the number of stimuli to remember. In the present study, the non-selective positive allosteric GABAA receptor modulator flunitrazepam impaired odor span performance at doses that did not affect a control odor discrimination. In contrast, the alpha-1 selective positive GABAA receptor modulator zolpidem and the cholinergic receptor antagonist scopolamine only impaired odor span at doses that produced more global impairment, including decreased accuracy in the control discrimination and increased response omissions in the both the odor span and control discrimination procedures. Even though the effects of flunitrazepam were selective to odor span performance, they did not depend on the number of stimuli to remember-the same degree of impairment occurred regardless of the memory load. These findings suggest that flunitrazepam interfered selectively with conditional discrimination performance rather than working memory and tentatively suggest that flunitrazepam's selective effects in the odor span task relative to the control odor discrimination are mediated by one or more non-alpha1 GABAA receptor subtypes.

Prevalence and clinical correlates of flunitrazepam-related complex sleep behaviors.

AIM: Complex sleep behaviors (CSB) are often associated with the use of hypnotic drugs. This study investigated the prevalence and correlates of CSB among psychiatric patients who were given flunitrazepam. METHODS: From June 2011 to May 2012, a total of 268 psychiatric outpatients who had received flunitrazepam for at least 3 months were enrolled. Data on occurrence of CSB, demographic characteristics, flunitrazepam dosage and duration of use, psychiatric diagnoses, physical illnesses, and alcohol use were collected. Logistic regression analysis was used to examine the clinical correlates of CSB. RESULTS: Sixty-six participants (24.6%) reported experiencing CSB. Logistic regression analysis showed that a high dosage (>2 mg/day) of flunitrazepam (odds ratio [OR] = 1.941, 95% confidence interval [CI] = 1.090-3.455, P = 0.024) and alcohol use (OR = 1.948, 95%CI = 1.023-3.709, P = 0.042) were significantly associated with the occurrence of CSB. Sex, age, duration of flunitrazepam use, psychiatric diagnoses, and physical illnesses were not significantly associated with the occurrence of CSB. CONCLUSION: CSB among flunitrazepam users should be monitored routinely, especially among those receiving a high dosage who also consume alcohol.

Development of a sedation protocol using orally administered tiletamine-zolazepam-acepromazine in free-roaming dogs.

OBJECTIVE: To investigate the sedative effects in dogs of tiletamine-zolazepam-acepromazine (TZA) or ketamine-flunitrazepam (KF) administered orally and to evaluate the effectiveness of encapsulated TZA for capturing free-roaming dogs. STUDY DESIGN: Experimental study followed by a field trial. ANIMALS: Six research dogs and 27 free-roaming dogs. METHODS: In a pilot study, six research dogs were administered liquid TZA (20 mg kg-1 tiletamine-zolazepam and 2 mg kg-1 acepromazine) or liquid KF (50 mg kg-1 ketamine and 2 mg kg-1 flunitrazepam) orally: treatment 1, forcefully squirting liquid medication into the mouth; treatment 2, encapsulating liquid medication for administration in canned food; treatment 3, administering liquid medication mixed with gravy. Sedation was scored. A follow-up field trial attempted capture of 27 free-roaming dogs. RESULTS: In the pilot study, the median time (range) to lateral recumbency (% dogs) after TZA administration was: treatment 1, 47.5 (35-80) minutes (67%); treatment 2, 30 (15-65) minutes (83%); and treatment 3, 75 (45-110) minutes (100%). No dogs in KF treatment 2 or 3 achieved lateral recumbency. Based on these results, 20 free-roaming dogs were offered encapsulated TZA in canned food: TZ (20 mg kg-1) and acepromazine (2 mg kg-1). Of these, no further drugs to four dogs (one dog captured), 10 dogs were administered a second dose within 30 minutes (five dogs captured) and six dogs were administered TZ (5 mg kg-1) and xylazine (1.1-2.2 mg kg-1) intramuscularly by blow dart (six dogs captured). Seven dogs were initially offered twice the TZA dose (five dogs captured). In total, 63% free-roaming dogs were captured after administration of encapsulated TZA in canned food. CONCLUSIONS AND CLINICAL RELEVANCE: Oral administration of encapsulated TZA in canned dog food can aid in the capture of free-roaming dogs, but additional drugs may be required. The sedation onset time and medication palatability influenced the capture rate.

Activation-induced regulation of GABAA receptors: Is there a link with the molecular basis of benzodiazepine tolerance?

Benzodiazepines have been used clinically for more than 50 years to treat disorders such as insomnia, anxiety, and epilepsy, as well as to aid muscle relaxation and anesthesia. The therapeutic index for benzodiazepines if very high and the toxicity is low. However, their usefulness is limited by the development of either or both tolerance to most of their pharmacological actions and dependence. Tolerance develops at different rates depending on the pharmacological action, suggesting the existence of distinct mechanisms for each behavioral parameter. Alternatively, multiple mechanisms could coexist depending on the subtype of GABAA receptor expressed and the brain region involved. Because most of the pharmacological actions of benzodiazepines are mediated through GABAA receptor binding, adaptive alterations in the number, structure, and/or functions of these receptors may play an important role in the development of tolerance. This review is focused on the regulation of GABAA receptors induced by long-term benzodiazepine exposure and its relationship with the development of tolerance. Understanding the mechanisms behind benzodiazepine tolerance is critical for designing drugs that could maintain their efficacy during long-term treatments.

Psychotropic drug-associated electrocardiographic presentation of diffuse J-waves in hypothermia: case report and literature review.

The use of psychotropic drugs is often associated with electrocardiographic (ECG) QT-interval prolongation, but there are few reports of J-waves. This report describes the case of a schizophrenic patient under treatment with several psychotropic drugs (olanzapine, valproate, and flunitrazepam), in whom ECG J-waves diffusely appeared during a hypothermic episode. We further performed a literature review of psychotropic drug-related J-waves in hypothermia. The present case highlights the importance of recognizing psychotropic drug-related ECG J-waves on an early warning sign to ensure appropriate monitoring and/or treatment for possible life-threatening side effects of such medications.

A fatal case of poisoning with ethanol and psychotropic drugs with putrefactive changes.

We present a fatal case involving poisoning with paroxetine, flunitrazepam, and ethanol, with putrefactive changes. Quantitative toxicological analysis showed that the concentrations of paroxetine and 7-aminoflunitrazepam, a metabolite of flunitrazepam, in the femoral blood were 0.28 µg/ml and 0.17 µg/ml, respectively. We also detected an ethanol level of 2.90 mg/ml and an n-propanol level of 0.10 mg/ml. We concluded that the cause of death was due to the interaction of paroxetine, flunitrazepam, and ethanol. The effects of putrefactive changes should be considered during forensic toxicological evaluation.

Good clinical outcome after accidental intra-arterial injection of flunitrazepam tablets in 16 drug abusers with critical limb ischaemia.

OBJECTIVES: Inadvertent intra-arterial injection of flunitrazepam tablets intended for intravenous use by drug abusers has devastating effects. We report here on the clinical outcome of 16 drug abusers developing critical limb ischaemia after flunitrazepam injection. METHODS: Treatment combined immediate analgesia and anticoagulation, long-lasting local thrombolysis and vasodilatation, antibiotic prophylaxis, and physical mobilization. The immediate bolus injection of 5,000 IU heparin was followed by a continuous heparin infusion up to the target partial thromboplastin time. Under arteriographic control local intra-arterial infusion with alternating 4-h cycles of 5 mg recombinant tissue plasminogen activator followed by 5 µg prostaglandinE1 (PGE1) was performed for 24-48 hours. Subsequently, 60 µg PGE1 was applied once daily. RESULTS: Drug abusers, having been injected with 4-30 mg flunitrazepam, were treated 3-72 hours after the accident, with six of them not being treated until after 24 hours. All showed a high tissue ischaemia score. At the time of being discharged from hospital 13 patients had a normal extremity. In one patient, first receiving treatment 72 hours after injection, minor amputation of fingers was necessary. The life of the patient who injected 30 mg flunitrazepam in the leg was saved after hip disarticulation. One patient developed neurological dysfunction in the affected toes. CONCLUSIONS: Intensive treatment after inadvertent intra-arterial drug injection normalized the affected extremity in most drug abusers, even after the late onset of therapy.

Potentiation of the depressant effect of alcohol by flunitrazepam in rats: an electrocorticographic, respiratory and electrocardiographic study.

Alcohol, a widely commercialized psychotropic drug, and the benzodiazepine Flunitrazepam, an anxiolytic widely prescribed for patients with anxiety and insomnia problems, are well known drugs and both act on the central nervous system. The misuse and the association of these two drugs are public health concerns in several countries and could cause momentary, long-lasting and even lethal neurophysiological problems due to the potentiation of their adverse effects in synergy. The present study observed the result of the association of these drugs on electrophysiological responses in the brain, heart, and respiratory rate in Wistar rats. 8 experimental groups were determined: control, one alcohol group (20% at a dose of 1 ml/100 g VO), three Flunitrazepam groups (doses 0.1; 0.2 and 0.3 mg/kg) and three alcohol-Flunitrazepam groups (20% at a dose of 1 ml/100 g VO of alcohol, combined with 0.1; 0.2 and 0.3 mg/kg of Flunitrazepam, respectively). The results showed that there was a more pronounced reduction in alpha and theta wave power in the alcohol-Flunitrazepam groups, a decrease in the power of beta oscillations and greater sedation. There was a progressive decrease in respiratory rate linked to the increase of Flunitrazepam dose in the alcohol-Flunitrazepam associated administration. It was observed alteration in heart rate and Q-T interval in high doses of Flunitrazepam. Therefore, we conclude that the association alcohol-Flunitrazepam presented deepening of depressant synergistic effects according to the increase in the dose of the benzodiazepine, and this could cause alterations in low frequency brain oscillations, breathing, and hemodynamics of the patient.

Suicide attempt during ramelteon addition to flunitrazepam: A case report.

Background: Benzodiazepines, such as flunitrazepam, may be at risk of disinhibition, leading to aggressiveness, impulsivity, self-harm, and possibly suicide attempts, whereas ramelteon may be far from disinhibition. Case Presentation: In a 43-year-old female with primary insomnia, flunitrazepam alone did not induce any type of disinhibition, but the addition of ramelteon to flunitrazepam brought about disinhibition, leading to aggressiveness and finally to her suicide attempt. Her disinhibition rapidly subsided as soon as ramelteon alone was discontinued. Conclusion: These findings suggest that a suicide attempt may occur during ramelteon and flunitrazepam combination in a susceptible patient.

Flunitrazepam and its metabolites compromise zebrafish nervous system functionality: An integrated microbiome, metabolome, and genomic analysis.

The psychotropic drug flunitrazepam (FLZ) is frequently detected in aquatic environments, yet its neurotoxicity to aquatic organisms has not received sufficient attention. In this study, microbiome, metabolome, and genome analyses were conducted to study the effects of FLZ and its metabolite 7-aminoflunitrazepam (7-FLZ) on the zebrafish nervous system and understand their toxic mechanisms. The results demonstrated that drug exposure induced gut dysbiosis, decreased short-chain fatty acids and promoted the production of lipopolysaccharides (LPS). LPS entered the brain and interacted with Toll-like receptors to cause neuroinflammation by upregulating the expression of proinflammatory cytokines TNFα and NF-κB. The increased ratio of S-adenosylmethionine to S-adenosylhomocysteine in brain tissues indicated abnormal expression of Dnmt1 gene. Whole-genome bisulfite sequencing displayed an increase in differentially methylated regions (DMRs) associated-genes and pertinent biological pathways encompassed the MAPK signaling pathway, calcium signaling pathway, and Wnt signaling pathway. Correlation analysis confirmed connections between gut microbiota, their metabolites, inflammatory factors, and DNA methylation-related markers in brain tissue. These findings indicate that while the toxicity is somewhat reduced in metabolized products, both FLZ and 7-FLZ can induce DNA methylation in brain tissue and ultimately affect the biological function of the nervous system by disrupting gut microbiota and their metabolites.

Neurotoxic effects induced by flunitrazepam and its metabolites in zebrafish: Oxidative stress, apoptosis, and histone hypoacetylation.

Benzodiazepines (BZDs) have been widely detected in aquatic environments, but their neurotoxic effects and potential mechanisms are still unclear. This study focuses on flunitrazepam (FLZ) and its metabolite, 7-aminoflunitrazepam (7-FLZ), as representative psychotropic BZD. We investigated their neurotoxic effects on adult zebrafish following a 30-day exposure to environmentally relevant concentrations. The findings reveal that exposure to these drugs induces anxiety-like and aggressive behaviors in zebrafish. Additionally, notable morphological damage to brain tissue and mitochondrial structures was observed. Through TUNEL staining, an increase in apoptotic cells was detected in the brain tissue of the exposed group, accompanied by marked elevations in ROS and caspase-3/9 levels. The upregulation of apoptosis-related genes Bax, p53, and Bcl-2 confirmed the occurrence of apoptosis. Furthermore, exposure to the drugs resulted in decreased acetylation levels of brain histones H3 and H4. The upregulation of histone deacetylation enzyme genes (HDAC1, HDAC3, HDAC4, and HDAC6) supported this result. Molecular docking results suggest that compared to 7-FLZ, FLZ has a higher binding affinity with HDAC3 and HDAC4, explaining why it causes lower histone acetylation levels. This study in zebrafish elucidates the neurotoxicity and molecular mechanisms induced by FLZ and 7-FLZ, which is significant for further understanding the impact of BZDs on human health and assessing their ecological risks.

Coloration Phenomenon of Flunitrazepam on the Tongue of an Elderly Patient: A Case Report.

Patients with dementia may forget to take their oral medications or may accidentally take too much. Furthermore, there are cases where people lick the medicine without recognizing it as a medicine or accidentally ingest it. An 88-year-old woman with a history of insomnia presented to the hospital, complaining of her weakness and mild loss of consciousness. Although her blood tests, imaging studies, and neurological findings were unremarkable, we noticed that her tongue was blue and determined that she had mistakenly taken flunitrazepam. This accidental ingestion was diagnosed as the cause of the symptoms. Patients with dementia report that they may take medicine by licking it, and some oral medicines have a coloring effect.

Flunitrazepam and its metabolites induced brain toxicity: Insights from molecular dynamics simulation and transcriptomic analysis.

Psychoactive drugs frequently contaminate aquatic environments after human consumption, raising concerns about their residues and ecological harm. This study investigates the effects of flunitrazepam (FLZ) and its metabolite 7-aminoflunitrazepam (7-FLZ), benzodiazepine-class psychoactive drugs, on brain accumulation, blood-brain barrier (BBB), and neuroinflammation of the model organism zebrafish. Molecular dynamics simulation and transcriptome sequencing were used to uncover their toxic mechanisms. Results demonstrate that both FLZ and 7-FLZ can accumulate in the brain, increasing Evans blue levels by 3.4 and 0.8 times, respectively. This increase results from abnormal expression of tight junction proteins, particularly ZO-1 and Occludin, leading to elevated BBB permeability. Furthermore, FLZ and 7-FLZ can also induce neuroinflammation, upregulating TNFα by 91% and 39%, respectively, leading to pathological changes and disrupted intracellular ion balance. Molecular dynamics simulation reveals conformational changes in ZO-1 and Occludin proteins, with FLZ exhibiting stronger binding forces and greater toxicity. Weighted gene co-expression network analysis identifies four modules correlated with BBB permeability and neuroinflammation. KEGG enrichment analysis of genes within these modules reveals pathways like protein processing in the endoplasmic reticulum, NOD-like receptor signaling pathway, and arginine and proline metabolism. This study enhances understanding of FLZ and 7-FLZ neurotoxicity and assesses environmental risks of psychoactive substances. ENVIRONMENTAL IMPLICATION: With the increasing prevalence of mental disorders and the discharge of psychoactive drugs into water, even low drug concentrations (ng/L-µg/L) can pose neurological risks. This study, utilizing molecular dynamic (MD) simulations and transcriptome sequencing, investigate the neurotoxicity and mechanisms of flunitrazepam and 7-aminoflunitrazepam. It reveals that they disrupt the blood-brain barrier in zebrafish and induce neuroinflammation primarily by inducing conformational changes in tight junction proteins. MD simulations are valuable for understanding pollutant-protein interactions. This research offers invaluable insights for the environmental risk assessment of psychoactive drugs and informs the development of strategies aimed at prevention and mitigation.

Flunitrazepam induces neurotoxicity in zebrafish through microbiota-gut-brain axis.

The abuse of psychoactive substances has led to their frequent detection in the environment, with unknown effects on the nervous system. In this study, zebrafish were exposed to benzodiazepine drug flunitrazepam (FLZ, 0.2 and 5 µg/L) for 30 days to assess its neurotoxicity. Results revealed that FLZ disrupted the balance of gut microbiota and caused an increase in pathogenic bacteria, such as Paracoccus and Aeromonas, leading to pathological damage to the intestine. The upregulation of intestinal pro-inflammatory factors, IL-1ß and TNF-α, by 2.4 and 6.3 times, respectively, along with the downregulation of tight junction proteins, Occludin and zonula occludens 1 (ZO-1), by 80 % and 50 %, increased in intestinal permeability. Moreover, untargeted metabolomics demonstrated that FLZ interfered with intestinal nucleotide metabolism and amino acid biosynthesis. FLZ could also increase the levels of lipopolysaccharide (LPS) and malondialdehyde (MDA) in the brain by 0.9 and 3.4 times, respectively, leading to pathological changes in brain tissue. Furthermore, FLZ significantly disturbed nucleotide metabolism and amino acid biosynthesis and metabolism pathways in the brain. Correlation analysis between gut microbiota and neurochemicals confirmed that FLZ can induce neurotoxicity through the microbiota-gut-brain axis. These findings elucidate the molecular mechanisms of psychoactive drugs on microbiota-gut-brain axis and provide a theoretical basis for the ecological environmental risk assessment of various psychoactive substances.