Effects of the Synthetic Cathinone α-Pyrrolidinobutiothiophenone (α-PBT) on Discriminative Stimulus Effects and Intracranial Self-Stimulation Thresholds in Male Rats.

Recently, the abuse of synthetic cathinones is increasing among young people. α-Pyrrolidinobutiothiophenone (α-PBT), a synthetic cathinone, is a designer drug that is freely traded online with no legal restrictions. Moreover, there is currently no scientific basis for legal regulation. Here, we examined the addictive properties of α-PBT using a drug discrimination (DD) task. We also investigated the role of α-PBT in brain stimulation reward (BSR) using an intracranial self-stimulation (ICSS) paradigm in rats. Initially, the rats were trained to discriminate between cocaine and saline. After the discrimination training criteria were met, we determined the dose-effect curves of cocaine and conducted generalization tests with α-PBT and α-pyrrolidinopentiothiophenone (α-PVT) using a cumulative dosing protocol. In a separate set of studies, we examined the dopaminergic mechanisms underlying the function of α-PBT as an interoceptive stimulus (17.8 mg/kg) by intraperitoneally injecting either the dopamine (DA) D1 antagonist SCH23390 (0.06 and 0.12 mg/kg) or the D2 antagonist eticlopride (0.05 and 0.1 mg/kg) 15 min before DD testing. Brain reward function was measured using an ICSS procedure to examine the effects of α-PBT on ICSS threshold under the frequency-rate procedure. Our results showed that α-PBT functioned as a discriminative cue similar to cocaine in rats. More importantly, SCH23390 abolished the effects of α-PBT as an interoceptive stimulus in a dose-dependent manner in rats trained to press a lever to receive cocaine. Similarly, eticlopride dose-dependently attenuated the effect of α-PBT used as a discriminative cue. Additionally, cumulative α-PBT administration dose-dependently lowered ICSS thresholds compared with those in saline-treated rats. Furthermore, α-PBT-induced potentiation of BSR was abolished by pretreatment with both SCH23390 and eticlopride. Taken together, our results suggest that α-PBT can function as a cocaine-like discriminative cue via the activation of D1 and D2 receptors. α-PBT also appears to influence BSR by reducing the brain reward threshold via changes in D1 and D2 receptors. The present study suggests that α-PBT could have addictive properties through DA D1 and D2 receptors and thus poses a threat to humans.

Identification of Gα12-vs-Gα13-coupling determinants and development of a Gα12/13-coupled designer GPCR.

G-protein-coupled receptors (GPCRs) transduce diverse signals into the cell by coupling to one or several Gα subtypes. Of the 16 Gα subtypes in human cells, Gα12 and Gα13 belong to the G12 subfamily and are reported to be functionally different. Notably, certain GPCRs display selective coupling to either Gα12 or Gα13, highlighting their significance in various cellular contexts. However, the structural basis underlying this selectivity remains unclear. Here, using a Gα12-coupled designer receptor exclusively activated by designer drugs (DREADD; G12D) as a model system, we identified residues in the α5 helix and the receptor that collaboratively determine Gα12-vs-Gα13 selectivity. Residue-swapping experiments showed that G12D distinguishes differences between Gα12 and Gα13 in the positions G.H5.09 and G.H5.23 in the α5 helix. Molecular dynamics simulations observed that I378G.H5.23 in Gα12 interacts with N1032.39, S1693.53 and Y17634.53 in G12D, while H364G.H5.09 in Gα12 interact with Q2645.71 in G12D. Screening of mutations at these positions in G12D identified G12D mutants that enhanced coupling with Gα12 and to an even greater extent with Gα13. Combined mutations, most notably the dual Y17634.53H and Q2645.71R mutant, further enhanced Gα12/13 coupling, thereby serving as a potential Gα12/13-DREADD. Such novel Gα12/13-DREADD may be useful in future efforts to develop drugs that target Gα12/13 signaling as well as to identify their therapeutic indications.

[Toxicology of synthetic cannabinoids]. / Toxicologie des cannabinoïdes de synthèse.

Synthetic cannabinoids (CS), or synthetic endocannabinoid receptor agonists, were initially synthesized for basic research into exocannabinoid signaling pathways, as well as in clinical research for their analgesic properties. The use of CS for recreational purposes is a recent phenomenon, but one that has grown very quickly in recent years, since these molecules now represent the main category of new synthetic products (NPS). This literature review aims to bring together current data regarding the use and effects caused by CS in humans. The relationship between the structure and activity of these CSs, the pharmacology and adverse effects of these CSs and finally the different methods of analyzing CSs. A better understanding of this phenomenon is essential to raise awareness among stakeholders in the health field.

The neural basis of resting-state fMRI functional connectivity in fronto-limbic circuits revealed by chemogenetic manipulation.

Measures of fMRI resting-state functional connectivity (rs-FC) are an essential tool for basic and clinical investigations of fronto-limbic circuits. Understanding the relationship between rs-FC and the underlying patterns of neural activity in these circuits is therefore vital. Here we introduced inhibitory designer receptors exclusively activated by designer drugs (DREADDs) into the amygdala of two male macaques. We evaluated the causal effect of activating the DREADD receptors on rs-FC and neural activity within circuits connecting amygdala and frontal cortex. Activating the inhibitory DREADD increased rs-FC between amygdala and ventrolateral prefrontal cortex. Neurophysiological recordings revealed that the DREADD-induced increase in fMRI rs-FC was associated with increased local field potential coherency in the alpha band (6.5-14.5 Hz) between amygdala and ventrolateral prefrontal cortex. Thus, our multi-modal approach reveals the specific signature of neuronal activity that underlies rs-FC in fronto-limbic circuits.

Synthetic cathinones in drug-facilitated sexual assault: A case report involving the novel generation substituted cathinone N-ethylpentedrone and a review of the literature.

The use of 3,4-methylenedioxymethamphetamine (MDMA) in drug-facilitated sexual assault (DFSA) is not uncommon. Indeed, the effects associated with the use of this substance may lead to disinhibition. Several synthetic cathinones, such as mephedrone or methylone, also possess marked entactogenic properties. This manuscript aims to (i) report a DFSA case involving a novel cathinone derivative, namely N-ethyl-pentedrone (NEPD) and (ii) review previously reported DFSA cases involving synthetic cathinones. Using liquid chromatography-high-resolution mass spectrometry (LC-HRMS), NEPD was detected in both plasma and urine collected from a 36-year-old male who had been victim of DFSA. Furthermore, an exhaustive, non-period-specific English-language literature search was performed using several different electronic databases to identify DFSA cases involving synthetic cathinones. Overall, five synthetic cathinones have been associated with DFSA:methylenedioxypyrovalerone, 4-methylethcathinone, α -pyrrolidinopentiophenone, mephedrone, α -pyrrolidinohexiophenone, and methylone, which appears to be the most frequently reported. Methylone is the ß-keto analog of MDMA, with which it shares substantial pharmacological similarities. Indeed, the pharmacological effects of methylone are similar to those associated with MDMA. By contrast, little is known regarding NEPD's pharmacological effects in humans. Based on subjective reports, NEPD can produce both positive and negative effects in human. Unlike what is reported in the case of methylone or mephedrone, only a small minority of NEPD users report slightly entactogenics effects. Such properties theoretically make NEPD more suitable for use in a chemsex context than in DFSA context; even though, the boundary between these two specific forms of sexualized drug use can sometimes appear tenuous.

Designer Receptor Exclusively Activated by Designer Drug (DREADD)-Mediated Activation of the Periaqueductal Gray Restores Nociceptive Descending Inhibition After Traumatic Brain Injury in Rats.

Traumatic brain injury (TBI) patients frequently experience chronic pain that can enhance their suffering and significantly impair rehabilitative efforts. Clinical studies suggest that damage to the periaqueductal gray matter (PAG) following TBI, a principal center involved in endogenous pain control, may underlie the development of chronic pain. We hypothesized that TBI would diminish the usual pain control functions of the PAG, but that directly stimulating this center using a chemogenetic approach would restore descending pain modulation. We used a well-characterized lateral fluid percussion model (1.3 ± 0.1 atm) of TBI in male rats (n = 271) and measured hindpaw mechanical nociceptive withdrawal thresholds using von Frey filaments. To investigate the role of the PAG in pain both before and after TBI, we activated the neurons of the PAG using a Designer Receptor Exclusively Activated by Designer Drug (DREADD) viral construct. Immunohistochemical analysis of brain tissue was used to assess the location and confirm the appropriate expression of the viral constructs in the PAG. Activation of the PAG DREADD using clozapine N-oxide (CNO) caused hindpaw analgesia that could be blocked using opioid receptor antagonist, naloxone, in uninjured but not TBI rats. Due to the importance of descending serotonergic signaling in modulating nociception, we ablated spinal serotonin signaling using 5,7-DHT. This treatment strongly reduced CNO-mediated anti-nociceptive effects in TBI but not uninjured rats. To define the serotonergic receptor(s) required for the CNO-stimulated effects in TBI rats, we administered 5-HT7 (SB-269970) and 5-HT1A (WAY-100635) receptor antagonists but observed no effects. The selective 5-HT2A receptor antagonist ketanserin, however, blocked CNO's effects in the DREADD expressing TBI but not DREADD expressing sham TBI animals. Blockade of alpha-1 adrenergic receptors with prazosin also had no effect after TBI. Descending pain control originating in the PAG is mediated through opioid receptors in uninjured rats. TBI, however, fundamentally alters the descending nociceptive control circuitry such that serotonergic influences predominate, and those are mediated by the 5-HT2A receptor. These results provide further evidence that the PAG is a key target for anti-nociception after TBI.

The surge of bromazolam-related fatalities replacing other novel designer benzodiazepines-related fatalities in San Francisco.

BACKGROUND AND AIM: Bromazolam, a novel designer benzodiazepine (NBD), exhibits potent sedative, hypnotic and anxiolytic effects, raising concerns regarding its potential for misuse and fatal outcomes, particularly when combined with opioids such as fentanyl. Despite limited documented fatalities globally, its use poses a significant threat, exacerbated by under-reporting and a lack of routine testing. This study analysed NBD-related deaths in a major US city over a 4-year period. METHODS: Analysis of accidental overdose deaths involving NBDs in San Francisco, CA, USA from 2020 to 2023, was performed utilizing medico-legal death investigations including comprehensive forensic toxicology, pathology and demographic information. San Francisco conducts thorough investigations into all non-natural and sudden unexpected deaths, including routine alcohol and drug testing of decedents under its jurisdiction, including etizolam, flualprazolam, flubromazolam and bromazolam analysis. RESULTS: There was a sudden surge in bromazolam-related deaths, with 44 fatalities documented in 2023, contrasting with relatively fewer deaths related to other NBDs. Bromazolam fatalities frequently involved co-ingestion with opioids, primarily fentanyl, and stimulants such as methamphetamine and cocaine. Demographic characteristics indicated a predominance of males, with a significant proportion lacking fixed addresses. Blood concentrations of bromazolam increased during the study period, suggesting heightened availability and/or purity in the community. CONCLUSION: There was a surge in bromazolam-related deaths during 2023 in San Francisco, CA, USA, contrasting with relatively stable numbers of deaths associated with other NBDs over the preceding years. The findings underscore the urgency for enhanced death investigation, testing and reporting to facilitate targeted harm reduction strategies for individuals at risk of bromazolam-related morbidity and mortality.

Ajuste de dosis según función renal en una paciente polimedicada incluida en el servicio de reacondicionamiento de medicamentos utilizando sistemas personalizados de dosificación (SPD) / Dose adjustment according to renal function in a polymedicated patient included in the medication reconditioning service using personalized dosing systems (SPD)

Paciente de 78 años, polimedicada e incluida en el servicio de Sistema Personalizado de Dosificación de Medicamentos (SPD). Al acudir a retirar su medicación nos informa que desde hace unos meses sufre cansancio, debilidad, mareos y confusión. Se realiza una revisión de la medicación, centrada en la dosificación de los medicamentos de metabolismo o eliminación renal, en función del valor de Filtrado Glomerular estimado de la paciente (FGe). Se realiza derivación al Médico de Atención Primaria (MAP) mediante un informe, en el que se recomienda la reducción de dosis de losartán y manidipino según el valor de FGe de la paciente. El MAP redujo la dosis de los antihipertensivos. Se efectuó seguimiento del caso, que permitió observar que la paciente dejó de presentar los síntomas descritos inicialmente.(AU)

Identification of clobromazolam in Australian emergency department intoxications using data-independent high-resolution mass spectrometry and the HighResNPS.com database.

The proliferation of novel psychoactive substances (NPSs) continues to challenge toxicology laboratories. In particular, the United Nations Office on Drugs and Crime considers designer benzodiazepines to be a current primary threat among all NPSs. Herein, we report detection of a new emerging designer benzodiazepine, clobromazolam, using high-resolution mass spectrometry and untargeted data acquisition in combination with a "suspect screening" method built from the crowd-sourced HighResNPS.com database. Our laboratory first detected clobromazolam in emergency department presenting intoxications included within the Emerging Drugs Network of Australia-Victoria project in the state of Victoria, Australia, from April 2022 to March 2023. Clobromazolam was the most frequent designer benzodiazepine detected in this cohort (100/993 cases, 10%). No patients reported intentional administration of clobromazolam, although over half reported exposure to alprazolam, which was detected in only 7% of cases. Polydrug use was prevalent (98%), with phenazepam (45%), methylamphetamine (71%) and other benzodiazepines (60%) most frequently co-detected. This is the first case series published in the literature concerning clobromazolam in clinical patients. The identification of clobromazolam in patients presenting to emergency departments in Victoria demonstrates how high-resolution mass spectrometry coupled with the HighResNPS.com database can be a valuable tool to assist toxicology laboratories in keeping abreast of emerging psychoactive drug use.

[Detoxification after use of new psychoactive substances purchased online]. / Avrusning etter bruk av nye rusmidler fra internett.

Background: The knowledge base on new psychoactive substances (NPS) is generally limited. This introduces new challenges and increased unpredictability in substance abuse treatment. Case presentation: A man in his thirties was submitted to detoxification after reportedly using flubromazolam, a high potency designer benzodiazepine, which he had purchased on the dark web. Extensive drug testing of serum, urine and hair, and the remains in a dropper bottle delivered by the patient, did not reveal flubromazolam or possible metabolites, but did reveal several common drugs of abuse, and 8-aminoclonazolam, a metabolite of clonazolam, another designer benzodiazepine sold on the dark web. The detoxification was uncomplicated. An excessive treatment protocol based on the patient's information, involving high preparedness and increased resources, both clinically and analytically, turned out to be unnecessary. Interpretation: The drug use and clinical course in this case proved to be more common than the unit prepared for. The case history illustrates both the challenges with users of NPS and the general unpredictability in substance abuse treatment.

Effects of the designer drug 4-methylamphetamine on core temperature and serotonin levels in the striatum and hippocampus of rats.

New psychoactive substances (NPS) are typically synthesized in clandestine laboratories in an attempt to chemically modify already federally regulated drugs in an effort to circumvent the law. Drugs derived from a phenethylamine pharmacophore, such as 4-chloroamphetamine and 3,4-methylenedioxymethamphetamine (MDMA), reliably induce thermogenesis and serotonergic deficits in the striatum and hippocampus of rodents. 4-methylamphetamine (4-MA), a relative newcomer to the NPS scene, was originally investigated in the mid-1900 s as a potential anorexigenic agent. With its phenethylamine pharmacophore, 4-MA was hypothesized to produce similar toxicological alterations as its chemical analogs. In the present study, three doses (1.0, 2.5, and 5.0 mg/kg, ip.) of 4-MA were administered to rats twice daily for two days. Core temperature data were calculated and analyzed as temperature area under the curve (TAUC). On the second day of dosing, a hypothermic response to 4-MA (2.5 and 5.0 mg/kg) was noted between 0.5 and 2.0 h post-treatment. Only the highest dose of 4-MA decreased body weight on the second day of treatment and maintained this reduction in weight for seven days after treatment ceased. None of the doses of 4-MA evaluated significantly altered serotonin levels in the hippocampus or striatum seven days after final treatment. The present findings demonstrate that the 4-methyl substitution to amphetamine generates a pharmacological and toxicological profile that differs from other similar phenethylamine analogs.

Metabolic characterization of 25X-NBOH and 25X-NBOMe phenethylamines based on UHPLC-Q-Exactive Orbitrap MS in human liver microsomes.

The types and quantities of new psychoactive substances synthesized based on structural modifications have increased rapidly in recent years and pose a great challenge to clinical and forensic laboratories. N-benzyl derivatives of phenethylamines, 25B-NBOH, 25E-NBOH, 25H-NBOH, and 25iP-NBOMe have begun to flow into the black market and have caused several poisoning cases and even fatal cases. The aim of this study was to avoid false negative results by detecting the parent drug and its metabolites to extend the detection window in biological matrices and provide basic data for the simultaneous determination of illegal drugs and metabolites in forensic and emergency cases. To facilitate the comparison of metabolic characteristics, we divided the four compounds into two groups of types, 25X-NBOH and 25X-NBOMe. The in vitro phase I and phase II metabolism of these four compounds was investigated by incubating 10 mg mL-1 pooled human liver microsomes with co-substrates for 180 min at 37 â„ƒ, and then analyzing the reaction mixture using ultrahigh-performance liquid chromatography-quadrupole/electrostatic field orbitrap mass spectrometry. In total, 70 metabolites were obtained for the four compounds. The major biotransformations were O-demethylation, hydroxylation, dehydrogenation, N-dehydroxybenzyl, N-demethoxybenzyl, oxidate transformation to ketone and carboxylate, glucuronidation, and their combination reactions. We recommended the major metabolites with high peak area ratio as biomarkers, B2-1 (56.61%), B2-2 (17.43%) and B6 (17.78%) for 25B-NBOH, E2-1 (42.81%), E2-2 (34.90%) and E8-2 (10.18%) for 25E-NBOH, H5 (49.28%), H2-1 (21.54%), and H1 (18.37%) for 25H-NBOH, P3-1 (10.94%), P3-2 (33.18%), P3-3 (14.85%) and P12-2 (23.00%) for 25iP-NBOMe. This is a study to evaluate their metabolic characteristics in detail. Comparative analysis of the N-benzyl derivatives of phenethylamines provided basic data for elucidating their pharmacology and toxicity. Timely analysis of the metabolic profiles of compounds with abuse potential will facilitate the early development of regulatory measures.

Post mortem blood bromazolam concentrations and co-findings in 96 coronial cases within England and Wales.

Bromazolam is a newly emerging benzodiazepine drug which is not licensed for medicinal use. It may be sourced as a New Psychoactive Substance (NPS) for its desired effects or be consumed unknowingly via counterfeit Xanax® or Valium® preparations. As part of our Coronial workload, we observed an increase in the detection of bromazolam from September 2021 to November 2022. We report a series of 96 cases in which bromazolam was quantitated by high resolution accurate mass - mass spectrometry (HRAM - MS) in post-mortem blood. The mean (SD) post-mortem blood bromazolam concentration from our case series was 64.6 ( ± 79.4) µg/L (range <1-425 µg/L). Routine toxicological screening results have also been reported; the most commonly encountered drugs taken in combination with bromazolam were cocaine, gabapentinoids and diazepam. In 48% of cases at least one further designer benzodiazepine drug was also present (etizolam, flualprazolam, flubromazolam, flubromazepam). It is essential that laboratories providing toxicological investigations are aware of the limitations of their assays; and inclusion of bromazolam within targeted screening panels using LC-MS/MS is encouraged. Bromazolam has not been associated with death in isolation from resulting toxic concentrations; however, it is likely to enhance adverse clinical effects when taken in combination with stimulant and/or centrally-acting depressant drugs (poly-drug deaths). Bromazolam, similar to other benzodiazepines, may also impair cognition and decision making skills.

Clozapine N-oxide, compound 21, and JHU37160 do not influence effortful reward-seeking behavior in mice.

RATIONALE: Clozapine N-oxide (CNO) has been developed as a ligand to selectively activate designer receptors exclusively activated by designer drugs (DREADDs). However, previous studies have revealed that peripherally injected CNO is reverse-metabolized into clozapine, which, in addition to activating DREADDs, acts as an antagonist at various neurotransmitter receptors, suggesting potential off-target effects of CNO on animal physiology and behaviors. Recently, second-generation DREADD agonists compound 21 (C21) and JHU37160 (J60) have been developed, but their off-target effects are not fully understood. OBJECTIVES: The present studies assessed the effect of novel DREADD ligands on reward-seeking behavior. METHODS: We first tested the possible effect of acute i.p. injection of low-to-moderate (0.1, 0.3, 1, 3 mg/kg) of CNO, C21, and J60 on motivated reward-seeking behavior in wild-type mice. We then examined whether a high dose (10 mg/kg) of these drugs might be able to alter responding. RESULTS: Low-to-moderate doses of all drugs and a high dose of CNO or C21 did not alter operant lick responding for a reward under a progressive ratio schedule of reinforcement, in which the number of operant lick responses to obtain a reward increases after each reward collection. However, high-dose J60 resulted in a total lack of responding that was later observed in an open field arena to be due to a sedative effect. CONCLUSIONS: This study provides definitive evidence that commonly used doses of CNO, C21, and J60 have negligible off-target effects on motivated reward-seeking but urges caution when using high doses of J60 due to sedative effects.

Flualprazolam and flubromazolam: Blood concentrations and prevalence of two novel psychoactive substances in forensic case work in Ontario, Canada.

Flualprazolam and flubromazolam are synthetic benzodiazepines that have not been approved for use in humans. They are categorized as novel psychoactive substances (NPS), and have been increasingly encountered in forensic case work. This report examines information from cases analyzed for flualprazolam and flubromazolam between July 1 and December 31, 2021 to identify the prevalence, trends and demographic data associated with these novel drugs in Ontario, Canada. Flualprazolam was identified in blood, serum or liver in 395 death investigations, 108 impaired driving and five sexual assault cases. Among all case types, blood concentrations were determined in 123 individuals aged 19-66 years. In impaired driving and sexual assault cases, flualprazolam blood concentrations ranged from <1.3 to 227 ng/mL (median 11.0 ng/mL), whereas a range of 3-59 ng/mL (median 6.8 ng/mL) was reported in death investigations. Flubromazolam was identified in blood, serum or liver in 137 death investigations, 55 impaired driving and one sexual assault case. Blood concentrations ranged from <1.3 to 323 ng/mL in 65 individuals, aged 14-61 years. In impaired driving and sexual assault cases, flubromazolam blood concentrations ranged from <1.3 to 323 ng/mL (median 7.7 ng/mL), which overlapped with the range of 2-220 ng/mL (median 8.0 ng/mL) reported in death investigations. Other drugs were frequently detected with flualprazolam and flubromazolam with opioids identified in more than 89% of positive flualprazolam and flubromazolam cases. These results demonstrated the prevalence of flualprazolam and flubromazolam in Ontario, Canada. Trends showed that over the 6-month period, as the number of flubromazolam cases decreased, the incidences of flualprazolam increased. An overlap in concentrations of these drugs was observed in both death investigations and cases involving living individuals. These data provide valuable information for the scientific community regarding the use of these drugs in antemortem and postmortem casework.

Hallucinating hallucinogens.

Fighting the designer drug epidemic with generative AI.

A head-to-head comparison of two DREADD agonists for suppressing operant behavior in rats via VTA dopamine neuron inhibition.

RATIONALE: Designer receptors exclusively activated by designer drugs (DREADDs) are a tool for "remote control" of defined neuronal populations during behavior. These receptors are inert unless bound by an experimenter-administered designer drug, commonly clozapine-n-oxide (CNO). However, questions have emerged about the suitability of CNO as a systemically administered DREADD agonist. OBJECTIVES: Second-generation agonists such as JHU37160 (J60) have been developed, which may have more favorable properties than CNO. Here we sought to directly compare effects of CNO (0, 1, 5, & 10 mg/kg, i.p.) and J60 (0, 0.03, 0.3, & 3 mg/kg, i.p.) on operant food pursuit. METHODS: Male and female TH:Cre + rats and their wildtype (WT) littermates received cre-dependent hM4Di-mCherry vector injections into ventral tegmental area (VTA), causing inhibitory DREADD expression in VTA dopamine neurons of TH:Cre + rats. All rats were trained to stably lever press for palatable food on a fixed ratio 10 schedule, and doses of both agonists were tested on separate days in counterbalanced order. RESULTS: All three CNO doses reduced operant rewards earned in rats with DREADDs, and no CNO dose had behavioral effects in WT controls. The highest J60 dose tested significantly reduced responding in DREADD rats, but this dose also increased responding in WTs, indicating non-specific effects. The magnitude of CNO and J60 effects in TH:Cre + rats were correlated and were present in both sexes. CONCLUSIONS: Findings demonstrate the usefulness of directly comparing DREADD agonists when optimizing behavioral chemogenetics, and highlight the importance of proper controls, regardless of the DREADD agonist employed.