Brain PET imaging using 11C-flumazenil and 11C-buprenorphine does not support the hypothesis of a mutual interaction between buprenorphine and benzodiazepines at the neuroreceptor level.

Among opioids, buprenorphine presents a favorable safety profile with a limited risk of respiratory depression. However, fatalities have been reported when buprenorphine is combined to a benzodiazepine. Potentiation of buprenorphine interaction with opioid receptors (ORs) with benzodiazepines, and/or vice versa, is hypothesized to explain this drug-drug interaction (DDI). The mutual DDI between buprenorphine and benzodiazepines was investigated at the neuroreceptor level in nonhuman primates (n = 4 individuals) using brain PET imaging and kinetic modelling. The binding potential (BPND) of benzodiazepine receptor (BzR) was assessed using 11C-flumazenil PET imaging before and after administration of buprenorphine (0.2 mg, i.v.). Moreover, the brain kinetics and receptor binding of buprenorphine were investigated in the same individuals using 11C-buprenorphine PET imaging before and after administration of diazepam (10 mg, i.v.). Outcome parameters were compared using a two-way ANOVA. Buprenorphine did not impact the plasma nor brain kinetics of 11C-flumazenil. 11C-flumazenil BPND was unchanged following buprenorphine exposure, in any brain region (p > 0.05). Similarly, diazepam did not impact the plasma or brain kinetics of 11C-buprenorphine. 11C-buprenorphine volume of distribution (VT) was unchanged following diazepam exposure, in any brain region (p > 0.05). To conclude, our PET imaging findings do not support a neuropharmacokinetic or neuroreceptor-related mechanism of the buprenorphine/benzodiazepine interaction.

Diazepam Binding Inhibitor Control of Eu- and Hypoglycemic Patterns of Ventromedial Hypothalamic Nucleus Glucose-Regulatory Signaling.

Pharmacological stimulation/antagonism of astrocyte glio-peptide octadecaneuropeptide signaling alters ventromedial hypothalamic nucleus (VMN) counterregulatory γ-aminobutyric acid (GABA) and nitric oxide transmission. The current research used newly developed capillary zone electrophoresis-mass spectrometry methods to investigate hypoglycemia effects on VMN octadecaneuropeptide content, along with gene knockdown tools to determine if octadecaneuropeptide signaling regulates these transmitters during eu- and/or hypoglycemia. Hypoglycemia caused dissimilar adjustments in the octadecaneuropeptide precursor, i.e., diazepam-binding-inhibitor and octadecaneuropeptide levels in dorsomedial versus ventrolateral VMN. Intra-VMN diazepam-binding-inhibitor siRNA administration decreased baseline 67 and 65 kDa glutamate decarboxylase mRNA levels in GABAergic neurons laser-microdissected from each location, but only affected hypoglycemic transcript expression in ventrolateral VMN. This knockdown therapy imposed dissimilar effects on eu- and hypoglycemic glucokinase and 5'-AMP-activated protein kinase-alpha1 (AMPKα1) and -alpha2 (AMPKα2) gene profiles in dorsomedial versus ventrolateral GABAergic neurons. Diazepam-binding-inhibitor gene silencing up-regulated baseline (dorsomedial) or hypoglycemic (ventrolateral) nitrergic neuron neuronal nitric oxide synthase mRNA profiles. Baseline nitrergic cell glucokinase mRNA was up- (ventrolateral) or down- (dorsomedial) regulated by diazepam-binding-inhibitor siRNA, but knockdown enhanced hypoglycemic profiles in both sites. Nitrergic nerve cell AMPKα1 and -α2 transcripts exhibited division-specific responses to this genetic manipulation during eu- and hypoglycemia. Results document the utility of capillary zone electrophoresis-mass spectrometric tools for quantification of ODN in small-volume brain tissue samples. Data show that hypoglycemia has dissimilar effects on ODN signaling in the two major neuroanatomical divisions of the VMN and that this glio-peptide imposes differential control of glucose-regulatory neurotransmission in the VMNdm versus VMNvl during eu- and hypoglycemia.

Renal function is a major predictor of circulating acyl-CoA-binding protein/diazepam-binding inhibitor.

Objective: Acyl-CoA-binding protein (ACBP)/diazepam-binding inhibitor has lately been described as an endocrine factor affecting food intake and lipid metabolism. ACBP is dysregulated in catabolic/malnutrition states like sepsis or systemic inflammation. However, regulation of ACBP has not been investigated in conditions with impaired kidney function, so far. Design/methods: Serum ACBP concentrations were investigated by enzyme-linked immunosorbent assay i) in a cohort of 60 individuals with kidney failure (KF) on chronic haemodialysis and compared to 60 individuals with a preserved kidney function; and ii) in a human model of acute kidney dysfunction (AKD). In addition, mACBP mRNA expression was assessed in two CKD mouse models and in two distinct groups of non-CKD mice. Further, mRNA expression of mACBP was measured in vitro in isolated, differentiated mouse adipocytes - brown and white - after exposure to the uremic agent indoxyl sulfate. Results: Median [interquartile range] serum ACBP was almost 20-fold increased in KF (514.0 [339.3] µg/l) compared to subjects without KF (26.1 [39.1] µg/l) (p<0.001). eGFR was the most important, inverse predictor of circulating ACBP in multivariate analysis (standardized ß=-0.839; p<0.001). Furthermore, AKD increased ACBP concentrations almost 3-fold (p<0.001). Increased ACBP levels were not caused by augmented mACBP mRNA expression in different tissues of CKD mice in vivo or in indoxyl sulfate-treated adipocytes in vitro. Conclusions: Circulating ACBP inversely associates with renal function, most likely through renal retention of the cytokine. Future studies need to investigate ACBP physiology in malnutrition-related disease states, such as CKD, and to adjust for markers of renal function.

Assumptions Underlying Hepatic Clearance Models: Recognizing the Influence of Saturable Protein Binding on Driving Force Concentration and Discrimination Between Models of Hepatic Clearance.

One underlying assumption of hepatic clearance models is often underappreciated. Namely, plasma protein binding is assumed to be nonsaturable within a given drug concentration range, dependent only on protein concentration and equilibrium dissociation constant. However, in vitro hepatic clearance experiments often use low albumin concentrations that may be prone to saturation effects, especially for high-clearance compounds, where the drug concentration changes rapidly. Diazepam isolated perfused rat liver literature datasets collected at varying concentrations of albumin were used to evaluate the predictive utility of four hepatic clearance models (the well-stirred, parallel tube, dispersion, and modified well-stirred model) while both ignoring and accounting for potential impact of saturable protein binding on hepatic clearance model discrimination. In agreement with previous literature findings, analyses without accounting for saturable binding showed poor clearance prediction using all four hepatic clearance models. Here we show that accounting for saturable albumin binding improves clearance predictions across the four hepatic clearance models. Additionally, the well-stirred model best reconciles the difference between the predicted and observed clearance data, suggesting that the well-stirred model is an appropriate model to describe diazepam hepatic clearance when considering appropriate binding models. SIGNIFICANCE STATEMENT: Hepatic clearance models are vital for understanding clearance. Caveats in model discrimination and plasma protein binding have sparked an ongoing scientific discussion. This study expands the understanding of the underappreciated potential for saturable plasma protein binding. Fraction unbound must correspond to relevant driving force concentration. These considerations can improve clearance predictions and address hepatic clearance model disconnects. Importantly, even though hepatic clearance models are simple approximations of complex physiological processes, they are valuable tools for clinical clearance predictions.

Tuning the neurogenesis channel.

Earlier work has implicated the neurotransmitter GABA in controlling forebrain progenitor proliferation. In this issue of Neuron, Everlien et al. (2022) demonstrate that diazepam binding inhibitor acts to keep the neurogenesis-promoting effect of GABA at bay.

Prioritization based on risk assessment to study the bioconcentration and biotransformation of pharmaceuticals in glass eels (Anguilla anguilla) from the Adour estuary (Basque Country, France).

The presence of contaminants of emerging concern in the aquatic environment directly impacts water-living organisms and can alter their living functions. These compounds are often metabolized and excreted, but they can also be accumulated and spread through the food chain. The metabolized contaminants can also lead to the formation of new compounds with unknown toxicity and bioaccumulation potential. In this work, we have studied the occurrence, bioconcentration, and biotransformation of CECs in glass eels (Anguilla anguilla) using UHPLC-HRMS. To select the target CECs, we first carried out an environmental risk assessment of the WWTP effluent that releases directly into the Adour estuary (Bayonne, Basque Country, France). The risk quotients of every detected contaminant were calculated and three ecotoxicologically relevant contaminants were chosen to perform the exposure experiment: propranolol, diazepam, and irbesartan. An experiment of 14 days consisting of 7 days of exposure and 7 days of depuration was carried out to measure the bioconcentration of the chosen compounds. The quantitative results of the concentrations in glass eel showed that diazepam and irbesartan reached BCF ≈10 on day 7, but both compounds were eliminated after 7 days of depuration. On the other hand, propranolol's concentration remains constant all along with the experiment, and its presence can be detected even in the non-exposed control group, which might suggest environmental contamination. Two additional suspect screening strategies were used to identify metabolization products of the target compounds and other xenobiotics already present in wild glass eels. Only one metabolite was identified, nordiazepam, a well-known diazepam metabolite, probably due to the low metabolic rate of glass eels at this stage. The xenobiotic screening confirmed the presence of more xenobiotics in wild glass eels, prominent among them, the pharmaceuticals exemestane, primidone, iloprost, and norethandrolone.

Yokukansan suppresses neuroinflammation in the hippocampus of mice and decreases the duration of lipopolysaccharide- and diazepam-mediated loss of righting reflex induced by pentobarbital.

Neuroinflammation is associated with the development of hypoactive delirium, which results in poor clinical outcomes. Drugs effective against hypoactive sur have not yet been established. Yokukansan has an anti-neuroinflammatory effect, making it potentially effective against hypoactive delirium. This study aimed to examine the effect of Yokukansan on the pentobarbital-induced loss of righting reflex duration extended with lipopolysaccharide (LPS)-induced neuroinflammation and diazepam-induced gamma-aminobutyric acid receptor stimulation in a mouse model. The active ingredients in Yokukansan and its anti-neuroinflammatory effect on the hippocampus were also investigated. Furthermore, we examined the in vitro anti-inflammatory effects of Yokukansan on LPS-stimulated BV2 cells, a murine microglial cell line. Findings revealed that treatment with Yokukansan significantly decreased the duration of pentobarbital-induced loss of righting reflex by attenuating the LPS-induced increase in interleukin-6 and tumor necrosis factor-alpha levels in the hippocampus. Moreover, treatment with Yokukansan significantly decreased the number of ionized calcium-binding adapter molecule-1-positive cells in the hippocampal dentate gyrus after 24 h of LPS administration. In addition, glycyrrhizic acid, an active ingredient in Yokukansan, partially decreased the duration of pentobarbital-induced loss of righting reflex. Treatment with Yokukansan also suppressed the expression of inducible nitric oxide, interleukin-6, and tumor necrosis factor mRNA in LPS-stimulated BV2 cells. Thus, these findings suggest that Yokukansan and glycyrrhizic acid may be effective therapeutic agents for treating neuroinflammation-induced hypoactive delirium.

Inhibiting with-no-lysine kinases enhances K+/Cl- cotransporter 2 activity and limits status epilepticus.

First-in-line benzodiazepine treatment fails to terminate seizures in about 30% of epilepsy patients, highlighting a need for novel anti-seizure strategies. It is emerging that impaired K+/Cl- cotransporter 2 (KCC2) activity leads to deficits in GABAergic inhibition and increased seizure vulnerability in patients. In neurons, the with-no-lysine (WNK) kinase-STE20/SPS1-related proline/alanine-rich (SPAK) kinase signalling pathway inhibits KCC2 activity via T1007 phosphorylation. Here, we exploit the selective WNK kinase inhibitor WNK463 to test the effects of pharmacological WNK inhibition on KCC2 function, GABAergic inhibition, and epileptiform activity. Immunoprecipitation and western blotting analysis revealed that WNK463 reduces KCC2-T1007 phosphorylation in vitro and in vivo. Using patch-clamp recordings in primary rat neurons, we further observed that WNK463 hyperpolarized the Cl- reversal potential, and enhanced KCC2-mediated Cl- extrusion. In the 4-aminopyridine slice model of acute seizures, WNK463 administration reduced the frequency and number of seizure-like events. In vivo, C57BL/6 mice that received intrahippocampal WNK463 experienced delayed onset of kainic acid-induced status epilepticus, less epileptiform EEG activity, and did not develop pharmaco-resistance to diazepam. Our findings demonstrate that acute WNK463 treatment potentiates KCC2 activity in neurons and limits seizure burden in two well-established models of seizures and epilepsy. In summary, our work suggests that agents which act to increase KCC2 activity may be useful adjunct therapeutics to alleviate diazepam-resistant status epilepticus.

Overcoming the challenges of developing an intranasal diazepam rescue therapy for the treatment of seizure clusters.

Seizure clusters must be treated quickly and effectively to prevent progression to prolonged seizures and status epilepticus. Rescue therapy for seizure clusters has focused on the use of benzodiazepines. Although intravenous benzodiazepine administration is the primary route in hospitals and emergency departments, seizure clusters typically occur in out-of-hospital settings, where a more portable product that can be easily administered by nonmedical caregivers is needed. Thus, other methods of administration have been examined, including rectal, intranasal, intramuscular, and buccal routes. Following US Food and Drug Administration (FDA) approval in 1997, rectal diazepam became the mainstay of out-of-hospital treatment for seizure clusters in the United States. However, social acceptability and consistent bioavailability present limitations. Intranasal formulations have potential advantages for rescue therapies, including ease of administration and faster onset of action. A midazolam nasal spray was approved by the FDA in 2019 for patients aged 12 years or older. In early 2020, the FDA approved a diazepam nasal spray for patients aged 6 years or older, which has a different formulation than the midazolam nasal product and enhances aspects of bioavailability. Benzodiazepines, including diazepam, present significant challenges in developing a suitable intranasal formulation. Diazepam nasal spray contains dodecyl maltoside (DDM) as an absorption enhancer and vitamin E to increase solubility in an easy-to-use portable device. In a Phase 1 study, absolute bioavailability of the diazepam nasal spray was 97% compared with intravenous diazepam. Subsequently, the nasal spray demonstrated less variability in bioavailability than rectal gel (percentage of geometric coefficient of variation of area under the curve = 42%-66% for diazepam nasal spray compared with 87%-172% for rectal gel). The diazepam nasal spray safety profile is consistent with that expected for rectal diazepam, with low rates of nasal discomfort (≤6%). To further improve the efficacy of rescue therapy, investigation of novel intranasal benzodiazepine formulations is underway.

How does α1Histidine102 affect the binding of modulators to α1ß2γ2 GABAA receptors? molecular insights from in silico experiments.

The activation of GABAA receptors by the neurotransmitter gamma-aminobutyric acid mediates the rapid inhibition response in the central nervous system of mammals. Many neurological and mental health disorders arise from alterations in the structure or function of these pentameric ion channels. GABAA receptors are targets for numerous drugs, including benzodiazepines, which bind to α1ß2γ2 GABAA receptors with high affinity to a site in the extracellular domain, between subunits α1 and γ2. It has been established experimentally that the binding of these drugs depends on the presence of one particular amino acid in the α1 subunit: histidine 102. However, the specific role it plays in the intermolecular interaction has not been elucidated. In this work, we applied in silico methods to understand whether certain protonation and rotamer states of α1His102 facilitate the binding of modulators. We analysed diazepam binding, a benzodiazepine, and the antagonist flumazenil to the GABAA receptor using molecular dynamics simulations and adaptive biasing force simulations. The binding free energy follows changes in the protonation state for both ligands, and rotameric states of α1His102 were specific for the different compounds, suggesting distinct preferences for positive allosteric modulators and antagonists. Moreover, in the presence of diazepam and favoured by a neutral tautomer, we identified a water molecule that links loops A, B, and C and may be relevant to the modulation mechanism.

Driving Impairment Cases Involving Etizolam and Flubromazolam.

This study describes 12 cases of drivers stopped for impaired driving, where a designer benzodiazepine was detected, specifically etizolam or flubromazolam. Etizolam was detected in three cases, with blood concentrations ranging from 40 to 330 ng/mL. Two of these cases had low concentrations of methamphetamine and/or amphetamine, and in the third case tetrahydrocannabinol (THC) was detected. Flubromazolam was detected in nine cases; in all cases, at least one other drug was detected, with THC being the most prevalent. The mean blood concentration of flubromazolam was 16.3 ng/mL and had a median concentration of 17.0 ng/mL, ranging from 7.0 to 31 ng/mL. The low concentrations of designer benzodiazepines that produce pharmacological effects may allow many of these drugs to go undetected using routine testing in laboratories; therefore, it is necessary to include these novel compounds within validated analytical methods to reduce the chance of reporting false negative results. The prevalence in which laboratories are detecting the presence of novel benzodiazepines in impaired drivers illustrates the increased threat to public safety. These case studies demonstrate the importance of investigating agencies and forensic laboratories to be vigilant in monitoring the emerging novel psychoactive substances in their region.

Safety and tolerability associated with chronic intermittent use of diazepam buccal film in adult, adolescent, and pediatric patients with epilepsy.

OBJECTIVE: Diazepam buccal film (DBF) is in development for treatment of patients experiencing bouts of increased seizure activity. We assessed safety, tolerability, and usability of self- or caregiver-administered DBF in the outpatient setting. METHODS: Patients aged 2-65 years needing treatment with a rescue benzodiazepine at least once monthly were eligible for the study. DBF (5-17.5 mg) was dispensed based on age and body weight. Patients/caregivers administered DBF for up to five seizure episodes per month. Adverse events (AEs) and usability assessments were recorded after the first dose, then every 3 months. RESULTS: Onehundred eighteen patients who used ≥1 DBF dose (adults, n = 82; adolescents, n = 19; children, n = 17) were enrolled. Eleven treatment-related AEs (10 being mild or moderate in severity) occurred in nine (7.6%) patients over a mean of 243 days of follow-up. No patient discontinued participation because of AEs. Mild local buccal discomfort, buccal swelling, and cheek skin sensitivity were reported by one patient each. Twenty-two serious AEs were reported; one was treatment-related. The three deaths reported, all unrelated to DBF, resulted from seizures or seizure with brain malignancy. Self-administration by adults was attempted on 23.6% (188/795) of use occasions. Administration of DBF occurred under ictal or peri-ictal conditions on 49.5% (538/1087) of use occasions, and DBF was successfully administered on a first or second attempt on 96.6% (1050/1087) of use occasions. Overall, patients received their dose of DBF on 99.2% (1078/1087) of use occasions. A second DBF dose was required within 24 hours after the first dose on 8.5% (92/1087) of use occasions. SIGNIFICANCE: In this observational study of chronic intermittent use, DBF was easy to administer, safe, and well tolerated in adult, adolescent, and pediatric patients with epilepsy experiencing seizure emergencies. DBF can be readily self-administered by adults with epilepsy, as well as successfully administered by a caregiver in seizure emergencies.

Shared structural mechanisms of general anaesthetics and benzodiazepines.

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.

Anti-Epileptic Effects of FABP3 Ligand MF1 through the Benzodiazepine Recognition Site of the GABAA Receptor.

Recently, we developed the fatty acid-binding protein 3 (FABP3) ligand MF1 (4-(2-(1-(2-chlorophenyl)-5-phenyl-1H-pyrazol-3-yl)phenoxy) butanoic acid) as a therapeutic candidate for α-synucleinopathies. MF1 shows affinity towards γ-aminobutyric acid type-A (GABAA) receptor, but its effect on the receptor remains unclear. Here, we investigate the pharmacological properties of MF1 on the GABAA receptor overexpressed in Neuro2A cells. While MF1 (1-100 µm) alone failed to evoke GABA currents, MF1 (1 µm) promoted GABA currents during GABA exposure (1 and 10 µm). MF1-promoted GABA currents were blocked by flumazenil (10 µm) treatment, suggesting that MF1 enhances receptor function via the benzodiazepine recognition site. Acute and chronic administration of MF1 (0.1, 0.3 and 1.0 mg/kg, p.o.) significantly attenuated status epilepticus (SE) and the mortality rate in pilocarpine (PILO: 300 mg/kg, i.p.)-treated mice, similar to diazepam (DZP: 5.0 mg/kg, i.p.). The anti-epileptic effects of DZP (5.0 mg/kg, i.p.) and MF1 (0.3 mg/kg, p.o.) were completely abolished by flumazenil (25 mg/kg, i.p.) treatment. Pentylenetetrazol (PTZ: 90 mg/kg, i.p.)-induced seizures in mice were suppressed by DZP (5.0 mg/kg, i.p.), but not MF1. Collectively, this suggests that MF1 is a mild enhancer of the GABAA receptor and exercises anti-epileptic effects through the receptor's benzodiazepine recognition site in PILO-induced SE models.

Anxiolytic Activity and Brain Modulation Pattern of the α-Casozepine-Derived Pentapeptide YLGYL in Mice.

α-Casozepine (α-CZP) is an anxiolytic-like bioactive decapeptide derived from bovine αs1­casein. The N-terminal peptide YLGYL was previously identified after proteolysis of the original peptide in an in vitro digestion model. Its putative anxiolytic-like properties were evaluated in a Swiss mice model using a light/dark box (LDB) after an intraperitoneal injection (0.5 mg/kg). The effect of YLGYL on c-Fos expression in brain regions linked to anxiety regulation was afterwards evaluated via immunofluorescence and compared to those of α-CZP and diazepam, a reference anxiolytic benzodiazepine. YLGYL elicited some anxiolytic-like properties in the LDB, similar to α­CZP and diazepam. The two peptides displayed some strong differences compared with diazepam in terms of c-Fos expression modulation in the prefontal cortex, the amygdala, the nucleus of the tractus solitarius, the periaqueductal grey, and the raphe magnus nucleus, implying a potentially different mode of action. Additionally, YLGYL modulated c-Fos expression in the amygdala and in one of the raphe nuclei, displaying a somewhat similar pattern of activation as α-­CZP. Nevertheless, some differences were also spotted between the two peptides, making it possible to formulate the hypothesis that these peptides could act differently on anxiety regulation. Taken together, these results showed that YLGYL could contribute to the in vivo overall action of α­CZP.

High Doses of Drugs Extensively Metabolized by CYP3A4 Were Needed to Reach Therapeutic Concentrations in Two Patients Taking Inducers.

INTRODUCTION: In the last 20 years of clinical practice, the senior author has identified these 2 rare cases in which the patients needed extremely high doses of drugs metabolized by CYP3A4 to reach and maintain serum therapeutic concentrations. METHODS: The high metabolic ability of these 2 patients was demonstrated by the low concentration-to-dose ratios (C/D ratios) of several drugs metabolized by CYP3A4. RESULTS: Case 1 was characterized by a history of high carbamazepine doses (up to 2,000mg/day) and needed 170 mg/day of diazepam in 2 days to cooperate with dental cleaning. The high activity of the CYP3A4 isoenzyme was manifested by fast metabolism for quetiapine and diazepam, which took more than 1 year to normalize after the inducer, phenytoin, was stopped. Case 2 was also very sensitive to CYP3A4 inducers as indicated by very low C/D ratios for carbamazepine, risperidone and paliperidone. The carbamazepine (2,800 mg/day) and risperidone (20 mg/day) dosages for this second patient are the highest doses ever seen for these drugs by the senior author. Risperidone induction appeared to last for many months and metabolism was definitively normal 3 years after stopping carbamazepine. On the other hand, olanzapine C/D ratios were normal for induction. CONCLUSIONS: The literature has never described similar cases of very high doses of drugs metabolized by CYP3A4. We speculate that these 2 patients may have unusual genetic profiles at the nuclear receptor levels; these receptors regulate induction of drugs.

Protein Binding and Hepatic Clearance: Re-Examining the Discrimination between Models of Hepatic Clearance with Diazepam in the Isolated Perfused Rat Liver Preparation.

This study re-examined the hepatic extraction for diazepam, the only drug for which isolated perfused rat liver (IPRL) studies have been reported not to be consistent with the well stirred model of organ elimination when only entering and exiting liver concentration measurements are available. First, the time dependency of diazepam equilibrium fraction unbound measurements from 4 to 24 hours was tested, reporting the continuing increases with time. The results showed that the time dependency of equilibrium protein-binding measurements for very highly bound drugs may be an issue that is not readily overcome. When examining C out/C in (F obs) measurements for diazepam when no protein is added to the incubation media, IPRL outcomes were consistent with previous reports showing marked underpredictability of in vivo clearance from in vitro measures of elimination in the absence of protein for very highly bound drugs, which is markedly diminished in the presence of albumin. F obs for diazepam at additional low concentrations of protein that would allow discrimination of the models of hepatic elimination produced results that were not consistent with the dispersion and parallel-tube models. Therefore, although the outcomes of this study were similar to those reported by Rowland and co-workers, when no protein is added to the perfusion media, these IPRL results for diazepam cannot be reasonably interpreted as proving that hepatic organ elimination is model-independent or as supporting the dispersion and parallel-tube models of organ elimination. SIGNIFICANCE STATEMENT: The only drug experiments for which isolated perfusion rat liver studies do not support hepatic clearance being best described by the well stirred model have been carried out with diazepam at zero protein concentration. This study repeated those studies, confirming the previous results at zero protein concentration, but the addition of low protein-binding conditions capable of differentiating the various models of hepatic elimination are more consistent with the well stirred model of hepatic elimination. These experimental studies do not support the preference for alternate models of hepatic elimination or the proposal that hepatic organ clearance is model-independent.

Formation of biologically active benzodiazepine metabolites in Arabidopsis thaliana cell cultures and vegetable plants under hydroponic conditions.

The use of recycled water for agricultural irrigation comes with the concern of exposure to crops by contaminants of emerging concerns (CECs). The concentration of CECs in plant tissues will depend on uptake, translocation and metabolism in plants. However, relatively little is known about plant metabolism of CECs, particularly under chronic exposure conditions. In this study, metabolism of the pharmaceutical diazepam was investigated in Arabidopsis thaliana cells and cucumber (Cucumis sativus) and radish (Raphanus sativus) seedlings grown in hydroponic solution following acute (7 d)/high concentration (1 mg L-1), and chronic (28 d)/low concentration (1 µg L-1) exposures. Liquid chromatography paired with mass spectrometry, 14C tracing, and enzyme extractions, were used to characterize the metabolic phases. The three major metabolites of diazepam - nordiazepam, temazepam and oxazepam - were detected as Phase I metabolites, with the longevity corresponding to that of human metabolism. Nordiazepam was the most prevalent metabolite at the end of the 5 d incubation in A. thaliana cells and 7 d, 28 d seedling cultivations. At the end of 7 d cultivation, non-extractable residues (Phase III) in radish and cucumber seedlings accounted for 14% and 33% of the added 14C-diazepam, respectively. By the end of 28 d incubation, the non-extractable radioactivity fraction further increased to 47% and 61%, indicating Phase III metabolism as an important destination for diazepam. Significant changes to glycosyltransferase activity were detected in both cucumber and radish seedlings exposed to diazepam. Findings of this study highlight the need to consider the formation of bioactive transformation intermediates and different phases of metabolism to achieve a comprehensive understanding of risks of CECs in agroecosystems.

Diazepam-induced loss of inhibitory synapses mediated by PLCδ/ Ca2+/calcineurin signalling downstream of GABAA receptors.

Benzodiazepines facilitate the inhibitory actions of GABA by binding to γ-aminobutyric acid type A receptors (GABAARs), GABA-gated chloride/bicarbonate channels, which are the key mediators of transmission at inhibitory synapses in the brain. This activity underpins potent anxiolytic, anticonvulsant and hypnotic effects of benzodiazepines in patients. However, extended benzodiazepine treatments lead to development of tolerance, a process which, despite its important therapeutic implications, remains poorly characterised. Here we report that prolonged exposure to diazepam, the most widely used benzodiazepine in clinic, leads to a gradual disruption of neuronal inhibitory GABAergic synapses. The loss of synapses and the preceding, time- and dose-dependent decrease in surface levels of GABAARs, mediated by dynamin-dependent internalisation, were blocked by Ro 15-1788, a competitive benzodiazepine antagonist, and bicuculline, a competitive GABA antagonist, indicating that prolonged enhancement of GABAAR activity by diazepam is integral to the underlying molecular mechanism. Characterisation of this mechanism has revealed a metabotropic-type signalling downstream of GABAARs, involving mobilisation of Ca2+ from the intracellular stores and activation of the Ca2+/calmodulin-dependent phosphatase calcineurin, which, in turn, dephosphorylates GABAARs and promotes their endocytosis, leading to disassembly of inhibitory synapses. Furthermore, functional coupling between GABAARs and Ca2+ stores was sensitive to phospholipase C (PLC) inhibition by U73122, and regulated by PLCδ, a PLC isoform found in direct association with GABAARs. Thus, a PLCδ/Ca2+/calcineurin signalling cascade converts the initial enhancement of GABAARs by benzodiazepines to a long-term downregulation of GABAergic synapses, this potentially underpinning the development of pharmacological and behavioural tolerance to these widely prescribed drugs.

Flavones-bound in benzodiazepine site on GABAA receptor: Concomitant anxiolytic-like and cognitive-enhancing effects produced by Isovitexin and 6-C-glycoside-Diosmetin.

Increasing evidence suggests that flavones can modulate memory and anxiety-like behaviour. However, these therapeutic effects are inconsistent and induce of adverse effects, which have been associated with interactions at the Benzodiazepine (BZ)-binding site. To improve our understanding of flavone effects on memory and anxiety, we employed a plus-maze discriminative avoidance task. Furthermore, we evaluated the potential of the compounds in modulating GABAA receptors via BZ-binding site using molecular modelling studies. Adult male Wistar rats were treated 30 min before training session with Vicenin-2 (0.1 and 0.25 mg/kg), Vitexin (0.1 and 0.25 mg/kg), Isovitexin (0.1 and 0.25 mg/kg) and 0.1 mg/kg 6-C-glycoside-Diosmetin, vehicle and a GABAA receptor agonist. The analysis of the time spent in the non-aversive vs aversive enclosed arms during the test session and percentage of time in the open arms within the training session revealed that treatment with Isovitexin and 6-C-glycoside-Diosmetin had memory-enhancing and anxiolytic-like effects (P < 0.001). In contrast, treatment with a higher dose of Diazepam impaired short-and long-term memory when it alleviated anxiety level. Docking studies revealed that flavones docked in a very similar way to that observed to the Diazepam, except by a lack of interaction in residue α1His101 in the BZ-binding site on GABAA receptors, which may be related to memory-enhancing effect. The occurrence of the α1His101 interaction could justify the memory-impairing observed following Diazepam treatment. These findings provide the first evidence that Isovitexin and 6-C-glycoside-Diosmetin could exert their memory-enhancing and anxiolytic-like effects via GABAA receptor modulation, which likely occurs via their benzodiazepine-binding site.