This survey is a compendium of genotoxicity and carcinogenicity information of benzodiazepines and benzodiazepine analogues. Data from 51 drugs were collected; 41 of them are still in the market. Of the 51 drugs, 12 (23.5%) do not have retrievable genotoxicity or carcinogenicity data. The remaining 39 (76.5%) have at least one genotoxicity or carcinogenicity test result. Of these 39, 12 (30.8%) have at least one positive finding: 9 tested positive in at least one genotoxicity assay, 8 in at least one carcinogenicity assay, and 5 gave a positive result in both at least one genotoxicity assay and at least one carcinogenicity assay. In terms of correlation between results of the various genotoxicity assays and absence of carcinogenic activity in mice and/or rats or in other species, none of the 11 non-carcinogenic drugs tested positive in one of the various genotoxicity assay systems. Concerning the predictivity of genetic toxicology findings for the result(s) of long-term carcinogenesis assays, 18 drugs had both genotoxicity and carcinogenicity data; of these 11 (61.1%) were neither genotoxic nor carcinogenic, 2 (11.1%) were carcinogenic in at least one sex of mice or rats but tested negative in genotoxicity assays, and 5 (27.8%) gave a positive response in at least one genotoxicity assay and in at least one carcinogenicity assay. Only 8 (19.5%) of the 41 marketed benzodiazepines and benzodiazepine analogues had all data required by current guidelines for testing of pharmaceuticals.

The neglected tropical disease schistosomiasis infects over 200 million people worldwide and is treated with just one broad spectrum antiparasitic drug (praziquantel). Alternative drugs are needed in the event of emerging praziquantel resistance or treatment failure. One promising lead that has shown efficacy in animal models and a human clinical trial is the benzodiazepine meclonazepam, discovered by Roche in the 1970's. Meclonazepam was not brought to market because of dose-limiting sedative side effects. However, the human target of meclonazepam that causes sedation (GABAARs) are not orthologous to the parasite targets that cause worm death. Therefore, we were interested in whether the structure of meclonazepam could be modified to produce antiparasitic benzodiazepines that do not cause host sedation. We synthesized 18 meclonazepam derivatives with modifications at different positions on the benzodiazepine ring system and tested them for in vitro antiparasitic activity. This identified five compounds that progressed to in vivo screening in a murine model, two of which cured parasite infections with comparable potency to meclonazepam. When these two compounds were administered to mice that were run on the rotarod test, both were less sedating than meclonazepam. These findings demonstrate the proof of concept that meclonazepam analogs can be designed with an improved therapeutic index, and point to the C3 position of the benzodiazepine ring system as a logical site for further structure-activity exploration to further optimize this chemical series.

Benzodiazepines are widely used in clinics and for recreational purposes, but will lead to addiction in vulnerable individuals. Addictive drugs increase the levels of dopamine and also trigger long-lasting synaptic adaptations in the mesolimbic reward system that ultimately may induce the pathological behaviour. The neural basis for the addictive nature of benzodiazepines, however, remains elusive. Here we show that benzodiazepines increase firing of dopamine neurons of the ventral tegmental area through the positive modulation of GABA(A) (gamma-aminobutyric acid type A) receptors in nearby interneurons. Such disinhibition, which relies on alpha1-containing GABA(A) receptors expressed in these cells, triggers drug-evoked synaptic plasticity in excitatory afferents onto dopamine neurons and underlies drug reinforcement. Taken together, our data provide evidence that benzodiazepines share defining pharmacological features of addictive drugs through cell-type-specific expression of alpha1-containing GABA(A) receptors in the ventral tegmental area. The data also indicate that subunit-selective benzodiazepines sparing alpha1 may be devoid of addiction liability.

Most general anaesthetics and classical benzodiazepine drugs act through positive modulation of γ-aminobutyric acid type A (GABAA) receptors to dampen neuronal activity in the brain1-5. However, direct structural information on the mechanisms of general anaesthetics at their physiological receptor sites is lacking. Here we present cryo-electron microscopy structures of GABAA receptors bound to intravenous anaesthetics, benzodiazepines and inhibitory modulators. These structures were solved in a lipidic environment and are complemented by electrophysiology and molecular dynamics simulations. Structures of GABAA receptors in complex with the anaesthetics phenobarbital, etomidate and propofol reveal both distinct and common transmembrane binding sites, which are shared in part by the benzodiazepine drug diazepam. Structures in which GABAA receptors are bound by benzodiazepine-site ligands identify an additional membrane binding site for diazepam and suggest an allosteric mechanism for anaesthetic reversal by flumazenil. This study provides a foundation for understanding how pharmacologically diverse and clinically essential drugs act through overlapping and distinct mechanisms to potentiate inhibitory signalling in the brain.

Driving under the influence of alcohol, illicit drugs and certain medicines is not allowed worldwide. Roadside drug testing is considered an important tool for determining such behavior. In Spain, mandatory roadside oral fluid drug testing is carried out regularly. The aim of this study was to determine the prevalence of benzodiazepines and benzodiazepines in combination with other drugs in drivers, examine benzodiazepine concentrations in drivers, and analyze the association of these factors with age and sex.

Benzodiazepines (BZDs), the most commonly prescribed psychotropic drugs with anxiolytic action, may cause hyposalivation. It has been previously shown that BZDs can cause hypertrophy and decrease the acini cell number. In this study, we investigated the effects of BZDs and pilocarpine on rat parotid glands, specifically on acinar, ductal, and myoepithelial cells.

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.

Inappropriate utilization of prescription opioids and benzodiazepines is a public health problem. This study examined and compared user-types and trends in dispensing of these medicines, and identified associated factors related to the duration of dispensing in Australia.

The rising use of designer benzodiazepines (DBZD) is a cat-and-mouse game between organized crime and law enforcement. Non-prohibited benzodiazepines are introduced onto the global drug market and scheduled as rapidly as possible by international authorities. In response, DBZD are continuously modified to avoid legal sanctions and drug seizures and generally to increase the abuse potential of the DBZD. This results in an unpredictable fluctuation between the appearance and disappearance of DBZD in the illicit market. Thirty-one DBZD were considered for review after consulting the international early warning database, but only 3-hydroxyphenazepam, adinazolam, clonazolam, etizolam, deschloroetizolam, diclazepam, flualprazolam, flubromazepam, flubromazolam, meclonazepam, phenazepam and pyrazolam had sufficient data to contribute to this scoping review. A total of 49 reports describing 1 drug offense, 2 self-administration studies, 3 outpatient department admissions, 44 emergency department (ED) admissions, 63 driving under the influence of drugs (DUID) and 141 deaths reported between 2008 and 2021 are included in this study. Etizolam, flualprazolam flubromazolam and phenazepam were implicated in the majority of adverse-events, drug offenses and deaths. However, due to a general lack of knowledge of DBZD pharmacokinetics and toxicity, and due to a lack of validated analytical methods, total cases are much likely higher. Between 2019 and April 2020, DBZD were identified in 48% and 83% of postmortem and DUID cases reported to the UNODC, respectively, with flualprazolam, flubromazolam and etizolam as the most frequently detected substances. DBZD toxicology, public health risks and adverse events are reported.

Benzodiazepines are clinically relevant drugs that bind to GABAA neurotransmitter receptors at the α+/γ2- interfaces and thereby enhance GABA-induced chloride ion flux leading to neuronal hyperpolarization. However, the structural basis of benzodiazepine interactions with their high-affinity site at GABAA receptors is controversially debated in the literature, and in silico studies led to discrepant binding mode hypotheses. In this study, computational docking of diazepam into α+/γ2- homology models suggested that a chiral methyl group, which is known to promote preferred binding to α5-containing GABAA receptors (position 3 of the seven-membered diazepine ring), could possibly provide experimental evidence that supports or contradicts the proposed binding modes. Thus, we investigated three pairs of R and S isomers of structurally different chemotypes, namely, diazepam, imidazobenzodiazepine, and triazolam derivatives. We used radioligand displacement studies as well as two-electrode voltage clamp electrophysiology in α1β3γ2-, α2β3γ2-, α3β3γ2-, and α5β3γ2-containing GABAA receptors to determine the ligand binding and functional activity of the three chemotypes. Interestingly, both imidazobenzodiazepine isomers displayed comparable binding affinities, while for the other two chemotypes, a discrepancy in binding affinities of the different isomers was observed. Specifically, the R isomers displayed a loss of binding, whereas the S isomers remained active. These findings are in accordance with the results of our in silico studies suggesting the usage of a different binding mode of imidazobenzodiazepines compared to those of the other two tested chemotypes. Hence, we conclude that different chemically related benzodiazepine ligands interact via distinct binding modes rather than by using a common binding mode.

It is unclear whether benzodiazepines increase the risk of suicide. The aim of this study was to test the hypothesis that benzodiazepines are associated with an increased risk of suicide, by comparing psychopharmacological interventions between psychiatric patients who committed suicide and a group of matched controls.

Benzodiazepines are frequently prescribed in patients with Alzheimer's disease. Unfortunately, studies evaluating their benefits and risks in these patients are limited.

Benzodiazepines are important therapeutic drugs, but they are often abused and co-abused with opioids. Clinical evidence suggests that benzodiazepines can inhibit respiration, and when combined with the respiratory-depressive effects of opioids, may increase likelihood of death. In this study we used oxygen sensors coupled with high-speed amperometry and multi-site thermorecording to examine how intravenous (iv) midazolam, a potent benzodiazepine, modulates the brain hypoxic and temperature effects of iv heroin in freely-moving rats. Oxygen levels and brain temperature were assessed with high temporal resolution in the nucleus accumbens (NAc), an important structure in the motivational-reinforcement circuit. When administered alone, midazolam (2 mg/kg) modestly decreased NAc temperature but had no evident effects on oxygen levels in this structure. In contrast, heroin (0.4 mg/kg) induced a strong decrease in NAc oxygen that was followed by a weaker, rebound-like oxygen increase. Midazolam pretreatment did not affect heroin-induced brain hypoxia but potentiated the initial hypothermia induced by heroin. However, co-administration of these drugs potentiated the heroin-induced oxygen decrease and enhanced heroin-induced brain hypothermia. Co-administration of heroin and midazolam also resulted in enhanced locomotor inhibition and loss of motor control. This effect caused some rats to collapse, resulting in nose and mouth occlusion, which caused a secondary hypoxic phase. These results could have important implications for human drug users, as the combined use of benzodiazepines with potent opioids not only results in sustained brain hypoxia but creates conditions of loss of motor control which could result in asphyxia and death. This article is part of the Special Issue entitled 'New Vistas in Opioid Pharmacology'.

In previous work our group described the synthesis and the activity on rat cerebellum granule cell GABAA receptors of new 1,5-benzodiazepine compounds. Here we are describing the synthesis of new triazolobenzodiazepines (mainly 1,5-benzodiazepine derivatives) and the evaluation of their biological activity in terms of effects on those GABAA receptors. Their effects were compared to those of 1,4-benzodiazepine agonists and some known 1,5-benzodiazepines. The activities were evaluated for the two GABAA receptor populations present in cerebellar granule cells, one mediating phasic inhibition and the other one mediating tonic inhibition. Some of the compounds displayed a profile of agonist at the component mediating phasic inhibition. This agonistic activity was prevented by the benzodiazepine site antagonist flumazenil. Interestingly, the active compounds displayed an agonistic activity at these receptors significantly greater than that of "classical" 1,4-benzodiazepine agonists, such as diazepam, flunitrazepam and alprazolam.

Social behavior represents an integral part of behavioral repertoire of rats particularly sensitive to pharmacological and environmental influences. The aim of the present study was to investigate whether early postnatal clonazepam (CZP) exposure can induce age-dependent changes related to expression of social behavior. The drug was administered from postnatal day (P) 7 until P11 at daily doses of 0.1, 0.5 and 1.0 mg/kg i.p. We designed three experiments to assess whether exposure to CZP affects social behavior in respect to the age of rats and the test circumstances, specifically their familiarity with test conditions during adolescence (P32), social behavior in juveniles and adolescents (P18-P42) and social behavior in a resident-intruder paradigm. The frequency and duration of a various patterns of social behavior related to play and social investigation not related to play were evaluated. The results showed that CZP postnatal exposure decreased social play behavior regardless of age and familiarity or unfamiliarity of experimental environment but did not affect the social investigation per se. When rats were confronted with an intruder in their home cages intense wrestling and inhibition of genital investigation were found. In conclusion, these findings show that short-term CZP postnatal exposure inhibits social play behavior and alters specific patterns of social behavior in an age and environment related manner.

Long-term sedative use is prevalent and associated with significant morbidity, including adverse events such as falls, cognitive impairment, and sedation. The development of dependence can pose significant challenges when discontinuation is attempted as withdrawal symptoms often develop. We conducted a scoping review to map and characterize the literature and determine opportunities for future research regarding deprescribing strategies for long-term benzodiazepine and Z-drug (zopiclone, zolpidem, and zaleplon) use in community-dwelling adults.

Cancer remains the second major cause of death in the world. Thus, there is a pressing need to identify potential synthetic route for the development of novel anticancer agents which will serve as lead compounds to effectively combat this life-threatening epidemic. Pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) have sparked a great interest as lead compounds because of their cancerostatic and anti-infective properties. The twisted molecular structure of PBD analogs provides both helical and chiral elements. In an effort to expand novel PBDs that interact with the key exocyclic amino group of the DNA-guanine base, we hypothesized that construction of a fused cyclic active system, would likely serve as an electrophilic site when compared to traditional electrophilic C11-N10 imine group. To examine our theory, we report herein the synthesis and cell viability/cytotoxicity of a series of PBD analogs using NCI-60 cell lines screening. Thus, compounds 1-13 were synthesized and fully characterized. The selected PBDs were found to have marginal inhibition of growth, up to 30%, for certain cell lines.

Inhibitory A type γ-aminobutyric acid receptors (GABAARs) play a pivotal role in orchestrating various brain functions and represent an important molecular target in neurological and psychiatric diseases, necessitating the need for the discovery and development of novel modulators. Here, we show that a natural compound curcumol, acts as an allosteric enhancer of GABAARs in a manner distinct from benzodiazepines. Curcumol markedly facilitated GABA-activated currents and shifted the GABA concentration-response curve to the left in cultured hippocampal neurons. When co-applied with the classical benzodiazepine diazepam, curcumol further potentiated GABA-induced currents. In contrast, in the presence of a saturating concentration of menthol, a positive modulator for GABAAR, curcumol failed to further enhance GABA-induced currents, suggesting shared mechanisms underlying these two agents on GABAARs. Moreover, the benzodiazepine antagonist flumazenil did not alter the enhancement of GABA response by curcumol and menthol, but abolished that by DZP. Finally, mutations at the β2 or γ2 subunit predominantly eliminated modulation of recombinant GABAARs by curcumol and menthol, or diazepam, respectively. Curcumol may therefore exert its actions on GABAARs at sites distinct from benzodiazepine sites. These findings shed light on the future development of new therapeutics drugs targeting GABAARs.

Benzodiazepine (BZD) poisoning is a common medical condition often accompanied by respiratory arrest, aspiration pneumonitis, and may rarely result in death. Although it is not studied well, hypoglycemia is a real and life-threatening condition that may occur in BZD poisoning. Thus, the present study was shown to determine the prevalence of hypoglycemia among patients with BZD poisoning compared to toxicity with other drugs.

Antiretroviral therapy (ART) lowers human immunodeficiency virus type 1 (HIV-1) viral load to undetectable levels, but does not eliminate the latent reservoir. One of the factors controlling the latent reservoir is transcriptional silencing of the integrated HIV-1 long terminal repeat (LTR). The molecular mechanisms that control HIV-1 transcription are not completely understood. We have previously shown that RUNX1, a host transcription factor, may play a role in the establishment and maintenance of HIV-1 latency. Prior work has demonstrated that inhibition of RUNX1 by the benzodiazepine (BDZ) Ro5-3335 synergizes with suberanilohydroxamic acid (SAHA) to activate HIV-1 transcription. In this current work, we examine the effect of RUNX1 inhibition on the chromatin state of the integrated HIV-1 LTR. Using chromatin immunoprecipitation (ChIP), we found that Ro5-3335 significantly increased the occupancy of STAT5 at the HIV-1 LTR. We also screened other BDZs for their ability to regulate HIV-1 transcription and demonstrate their ability to increase transcription and alter chromatin at the LTR without negatively affecting Tat activity. These findings shed further light on the mechanism by which RUNX proteins control HIV-1 transcription and suggest that BDZ compounds might be useful in activating HIV-1 transcription through STAT5 recruitment to the HIV-1 LTR.