Hypnotic action of benzodiazepines: a possible mechanism

The objective of this investigation was to determine whether the effects of muscimol on benzodiazepine receptor binding relate to the hypnotic activity of nine benzodiazepines (clonazepam, triazolam, diazepam, flurazepam, nitrazepam, oxazepam, temazepam, clobazam, and chlordiazepoxide) and CL 218,872. There was no correlation between the basal receptor binding affinities of the drugs tested and their hypnotic potencies, whereas the benzodiazepine receptor agonists whose receptor bindings are strongly modulated by muscimol possess potent hypnotic activity. These results indicate that benzodiazepine receptors that couple to GABA receptors are involved in the hypnotic activity of the benzodiazepines.     

Screening method for benzodiazepines and hypnotics in hair at pg/mg level by liquid chromatography-mass spectrometry/mass spectrometry

A procedure is presented for the screening of 16 benzodiazepines and hypnotics in human hair by LC-MS/MS (alprazolam, 7-aminoclonazepam, 7-aminoflunitrazepam, bromazepam, clobazam, diazepam, lorazepam, lormetazepam, midazolam, nordiazepam, oxazepam, temazepam, tetrazepam, triazolam, zaleplon and zolpidem). The method involves decontamination of hair with methylene chloride, hair cut into small pieces, incubation of 20 mg in phosphate buffer (pH 8.4) in the presence of 1 ng diazepam-d5 used as internal standard, liquid-liquid extraction with diethyl ether/methylene chloride (10/90) and separation using liquid chromatography-tandem mass spectrometry. The limits of quantification for all benzodiazepines and hypnotics range from 0.5 to 5 pg/mg using a 20-mg hair sample. Linearity is observed from the limit of quantification of each compound to 200 pg/mg (r2 > 0.99). Coefficients of variation measured on six points and at two concentrations (10 and 50 pg/mg) range from 5 to 20% for all drugs but one. Extraction recovery, measured at the two same concentrations range from 32 to 76%. These results were found suitable to screen for 16 benzodiazepines in hair and detect them at very low concentrations, making this method suitable to monitor single dose.     

Screening for forensically relevant benzodiazepines in human hair by gas chromatography-negative ion chemical ionization-mass spectrometry

A procedure is presented for the detection in human hair of forensically relevant benzodiazepines, i.e. nordiazepam, oxazepem, bromazepam, diazepam, lorazepam, flunitrazepam, alprazolam and triazolam. The method involves decontamination of hair with methylene chloride, pulverization in a ball mill, incubation of 50 mg powdered hair in Soerensen buffer (pH 7.6) in the presence of prazepam-d₅ used as internal standard, liquid-liquid extraction with diethyl ether-chloroform (80:20, v/v) and gas chromatography-mass spectrometry using negative chemical ionization after derivatization with, N,O-bis(trimethylsilyl)trifluoroacetamide plus 1% trimethylchlorosilane. The limits of detection for all benzodiazepines ranged from 1 to 20 pg/mg using a 50-mg hair sample. Coefficients of variation and extraction recoveries, ranging from 7.4 to 25.4% and 47.6 to 90%, respectively, were found suitable for a screening procedure. One hundred and fifteen samples were submitted to this screening procedure, and specimens tested positive for nordiazepam (0.20-18.87 ng/mg, n = 42) and its major metabolite oxazepam (0.10-0.50 ng/mg, n = 14), flunitrazepam (19–148 pg/mg, n = 31), lorazepam (31–49 pg/mg, n = 4) and alprazolam (0.3-1.24 ng/mg, n = 2). Bromazepam, diazepam and triazolam were not detected.     

Simultaneous determination of twelve benzodiazepines in human serum using a new reversed-phase chromatographic column on a 2-μm porous microspherical silica gel

A high-performance liquid chromatographic method has been developed for the simultaneous analysis of twelve frequently used benzodiazepines (BZPs) (bromazepam, clonazepam, chlordiazepoxide, estazolam, etizolam, flutazoram, haloxazolam, lorazepam, nitrazepam, oxazolam, triazolam and diazepam, internal standard) by using commerically available 2 or 5 μm particle size reversed-phase columns and a microflow cell-equipped ultraviolet detector. The separation was achieved using a C₁₈ reversed-phase column (condition 1: 100×4.6 mm I.D., particle size 2 μm, TSK gel Super-ODS; condition 2: 100×4.6 mm I.D., particle size 5 μm, Hypersil ODS-C₁₈). The mobile phase was composed of methanol-5 mM NaH₂PO₄ (pH 6) (45:55, v/v), and the flow-rate was 0.65 ml/min (conditions 1 and 2). The absorbance of the eluent was monitored at 254 nm. Retention times under condition 1 were shorter than those of condition 2. When the twelve benzodiazepines were determined, sensitivity and limits of quantification were about four to ten times better under condition 1 than under condition 2. The rate of recovery and linearity in condition 1 were approximately the same as those in condition 2. These results show that a new ODS filler with a particle size of 2 μm was more sensitive, provided better separation and was more rapid than that with conventional ODS filler.     

Benzodiazepine-Mediated Structural Changes in the Multidrug Transporter P-Glycoprotein: An Intrinsic Fluorescence Quenching Analysis

P-glycoprotein expressed in Pichia pastoris was used to study the drug binding sites of different benzodiazepines. The effect of bromazepam, chlordiazepoxide, diazepam and flurazepam on P-glycoprotein structure was investigated by measuring the intrinsic fluorescence of the transporter tryptophan residues. Purified mouse mdr1a transporter in mixed micelles of 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid and 1,2-dimiristoyl-sn-glycerol-3-phosphocholine emitted fluorescence at 340 nm indicative of the fluorophores in a relatively apolar environment. Acrylamide and iodide ion were used as collisional quenchers toward distinct regions of the transporter, the protein and the interface protein-surface, respectively. Binding of ATP induced conformational changes at the protein surface level in accordance with the location of the nucleotide binding sites. Bromazepam interaction with the transporter was located at the protein-surface interface, diazepam at the membrane region and chlordiazepoxide at the protein surface. Only the flurazepam interaction site was not detected by the quenchers used. All benzodiazepines were able to elicit reorientation of the protein fluorophores on the P-glycoprotein—ATP complex.     

Direct injection micellar liquid chromatographic determination of benzodiazepines in serum

A simple micellar liquid chromatographic (MLC) procedure is reported for the determination of several benzodiazepines in serum: bromazepam, diazepam, flunitrazepam, halazepam, medazepam, nitrazepam, oxazepam and tetrazepam. The optimization studies have been made in C(18) and C(8) columns, using solutions containing sodium dodecyl sulphate (SDS) modified with butanol or pentanol as mobile phases. The method proposed for the determination of the benzodiazepines uses a hybrid micellar mobile phase of 0.06 M SDS-5% butanol-0.01 M phosphate buffer (pH 7) at 25 degrees C, and UV detection (230 nm) in a C(18) column. The serum samples were injected directly, without any pretreatment, and eluted in less than 22 min, in accordance with their relative polarities, as indicated by their octanol-water partition coefficients. The limits of detection (ng ml(-1)) were within the ranges of 2-6 and 4-18 for aqueous and serum samples, respectively. Repeatability and intermediate precision were tested for three different concentrations of the drugs, and RSD (%) was below 10 for most of the assays. The MLC results were compared with those obtained from a conventional HPLC method using methanol-water 5:5 (v/v) which requires a previous extraction procedure.     

The decomposition of benzodiazepines during analysis by capillary gas chromatography/mass spectrometry

A capillary gas chromatography column directly interfaced to a mass spectrometer was used for the analysis of sixteen benzodiazepines. The thermal stability of the drugs was found to be related to their chemical structure. Nine of the benzodiazepines were thermally unstable indicating that care should be taken in the interpretation of gas chromatographic data from this class of drugs. The unstable benzodiazepines were: ketazolam which decomposes to diazepam; N-4 oxides (chlordiazepoxide and demoxepam) which lose an oxygen radical; aromatic 7-nitro compounds (nitrazepam and clonazepam) which are partially reduced to the corresponding amine; alpha-hydroxy ketones (lorazepam and oxazepam) which decompose with the loss of water and N-methyl-alpha-hydroxy ketones (lormetazepam and temazepam) which partially decompose with the loss of a hydrogen molecule to produce the corresponding alpha, beta-diketones. Few problems were encountered in distinguishing the drugs by their mass spectra, the exceptions being ketazolam which decomposes to diazepam and demoxepam which decomposes to desmethyldiazepam. In general, good spectra were obtained from 20-50 ng of drug injected. However, for those compounds where the decompositions were not quantitative (nitrazepam, clonazepam, lormetazepam, temazepam) detection limits were poor.     

Separation of 1,4-benzodiazepines by micellar elektrokinetic capillary chromatography

In this work the applicability of micellar elektrokinetic capillary chromatography (MECC) for the determination of benzodiazepines (BZD) has been studied. The applied method was used for the simultaneous separation of 8 BZDs (alprazolam, bromazepam, chlordiazepoxide, diazepam, flunitrazepam, medazepam, oxazepam, nitrazepam), and also for the study of stability in acidic medium. A fast and reliable method has been developed; using a separation buffer composed of sodium tetraborate 25 mM (pH 9.5), SDS (50 mM) and methanol (at least 12%) as an organic modifier.     

In vitro and in vivo inhibition by zopiclone of benzodiazepine binding to rodent brain receptors.

Up to now the only drugs known to be able to inhibit the binding of benzodiazepines to rodent brain receptors are members of this chemical family.Zopiclone (RP 27 267), a new drug with a pharmacological profile similar to that of chlordiazepoxide and nitrazepam but entirely different chemically from benzodiazepines, has been tested for its ability to inhibit benzodiazepine binding. $\text{In vitro}$ and $\text{in vivo}$ studies have shown that zopiclone is able to inhibit the binding of [³H] diazepam and [³H] flunitrazepam to brain receptors. The potency of zopiclone is quite comparable to that of diazepam and nitrazepam $\text{in vitro}$ and to that of chlordiazepoxide $\text{in vivo}$.These results confirm the pharmacological similarities existing between zopiclone and the benzodiazepines.     

Benzodiazepines and Tricyclic Antidepressant Plasma Levels

Twelve patients acted successfully as subjects to study what effect if any the benzodiazepines nitrazepam, diazepam, oxazepam, and chlordiazepoxide might have on steady-state plasma levels of nortriptyline and amitriptyline. No significant detectable effect was discovered. In view of the known interaction effects of other alternative tranquillizing drugs and hypnotics it seems reasonable to choose benzodiazepines wherever possible when anxiolytics or hypnotics need to be added during treatment of depression with tricyclic antidepressants.     

Rapid and sensitive detection of benzodiazepines and zopiclone in serum using high-performance thin-layer chromatography

We developed a rapid and sensitive method of identifying benzodiazepines and zopiclone in human serum using high-performance thin-layer chromatography (HPTLC). These drugs were developed and separated on plates within 8–11 min and detected by means of UV radiation and colour. Each drug was accurately identified by means of the values of R_F × 100 and the spot colour in three systems. The detection limit of the benzodiazepines in serum was 0.1-0.4 μg/ml, except for cloxazolam and haloxazolam. The sensitivity was increased about ten-fold over the conventional method. These results suggested that the HPTLC system is useful for the initial detection and identification of these drugs in emergencies.     

The temporal dimensions of anticonvulsant action of some newer benzodiazepines against metrazol induced seizures in mice and rats

In a time-distribution study, the anticonvulsant effects of four benzodiazepine compounds were compared with those of three standard antiepileptics against metrazol-induced seizures in mice and rats. Ethosuximide and trimethadione had the shortest duration of action in mice, but protected the rats up to 6 hr. Phenobarbitone, diazepam, flurazepam and nitrazepam protected the mice up to 12 hr, but the rats were effectively protected only up to 3-4 hr. Clonazepam, the most potent and effective agent, protected the mice from clonic-tonic seizures up to 18-20 hr and the rats up to 6-7 hr. Comparison of the PD50 from clonic seizure at the peak-effect hours revealed that the benzodiazepines were 16 to 96 times more potent than phenobarbitone on a molar basis, while phenobarbitone itself was 12 to 26 times more potent than ethosuximide and trimethadione. Tonic seizures and mortality were largely suppressed by all drugs until 18-20 hr in mice and 6-7 hr in rats. Seizure latency and mortality patterns varied from drug to drug but not in a dose-dependent manner.     

Effect of tranquilizers of the 1,4-benzodiazepine series on the xanthine oxidase activity of the rat brain

Experiments in vivo and in vitro on 90 rats were made to study the influence of 1,4-benzodiazepine tranquilizers (phenazepam, nitrazepam and diazepam) on cerebral xanthine oxidase activity. Phenazepam, nitrazepam and diazepam in the dose of 5 mg per 200 g bw were shown to reduce xanthine oxidase activity by 80.4%, 64.3% and 55.8%, respectively 2 h after intraperitoneal injection. 6 h after the injection of benzodiazepines the enzyme activity grows, but control values are achieved only after nitrazepam injection. In vitro experiments revealed direct influence of the tranquilizers on xanthine oxidase. Phenazepam inhibits xanthine oxidase activity in concentration as long as 10(-10) M (to 36.6%), and practically completely in 10(-6) M concentration. Nitrazepam and diazepam inhibit xanthine oxidase activity within concentration range between 10(-8) M (to 51.5% and 33.2%, respectively), and 10(-4) M (practically completely). The inhibition of xanthine oxidase activity is shown to be caused by the competition between hypoxanthine, the reaction substrate, and tranquilizer, to bind with the active site of the enzyme.     

Copper-psychoactive drug complexes: a voltammetric approach to complexation by 1,4-benzodiazepines

Copper complexation by the 1,4-benzodiazepines medazepam, diazepam, flurazepam, nitrazepam, and clonazepam was investigated using differential pulse polarography and cyclic voltammetry at a mercury electrode in 0.10 M KNO3 and pH 7.0 +/- 0.1. Because the 1,4-benzodiazepines are easily reduced at a mercury electrode through the two-electron reduction of the 4,5-azomethine functional group, copper reduction, as well as that of the ligands, was analyzed under varying experimental conditions. In most situations adsorption phenomena occurred and their influence on voltammetric signals had to be carefully analyzed. The voltammetric behavior was then interpreted in terms of complex formation. The results showed that all benzodiazepines can act as ligands toward copper(II) ions, forming 1:1 and 1:2 complexes with similar stabilities. The stoichiometric acidity constants of the benzodiazepines under study were also determined by potentiometric titration in water-ethanol medium and 0.10 M KNO3 and then extrapolated to 0% concentration of ethanol.     

Validation of a simultaneous analytical method for the detection of 27 benzodiazepines and metabolites and zolpidem in hair using LC-MS/MS and its application to human and rat hair

Benzodiazepines and zolpidem are controlled in many countries due to their inherent adverse effects of a high degree of tolerance and dependence. Recently, as some of these drugs have become distributed illegally and available through media such as the Internet, their abuse is becoming a serious social problem. Hair is a useful specimen to prove chronic drug use. In the present study, a simultaneous analytical method for the detection of 27 benzodiazepines and metabolites and zolpidem in hair was established and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The drugs and their metabolites in hair were extracted using methanol, filtered and injected on the LC-MS/MS. The following validation parameters of the method were satisfactory: selectivity, linearity, matrix effect, recovery, process efficiency, intra- and inter-assay precision and accuracy and processed sample stability. The limit of detection (LOD) and the limit of quantification (LOQ) were the total drug detected from the sample. The LODs ranged from 0.005 ng (zolpidem) to 0.5 ng (bromazepam and chlordiazepoxide) and the LOQs were 0.25 ng in every analyte except for bromazepam and chlordiazepoxide, for which they were 0.5 ng. The developed method was successfully applied to five legal cases involving use of benzodiazepines and zolpidem and to an animal study on drug incorporation into hair. Diazepam and its three metabolites, as well as lorazepam, were detected in hair from both the multiple- and single-dose administration groups of lean Zucker rats. The concentration of diazepam was higher than those of its metabolites in both dark grey and white hair from the multiple-dose administration groups, with the mean concentration ranges from 0.16 to 0.51 ng/mg and from 0.10 to 0.24 ng/mg, respectively. The mean concentration ranges of lorazepam were from 0.05 to 0.37 ng/mg in dark grey hair and from 0.11 to 0.45 ng/mg in white hair from the multiple-dose administration groups. Hair pigmentation did not have any significant effect on the degree of the deposition of drugs and their metabolites in hair.     

Simultaneous determination of nitrazepam and its metabolites in urine by micellar electrokinetic capillary chromatography

We applied micellar electrokinetic capillary chromatography to simultaneous separation and determination of nitrazepam and its major metabolites, 7-aminonitrazepam and 7-acetamidonitrazepam, in spiked urine. Prior to electrophoresis, the three compounds were successfully extracted from the spiked urine with commercial disposable solid-phase cartridges. The optimum running buffer for the separation was prepared by combining 85 parts of 60 mM sodium dodecyl sulphate—6 mM phosphate—borate, adjusted to pH 8.5, with 15 parts of methanol. The separation order, completed within 25 min, was 7-aminonitrazepam > 7-acetamidonitrazepam > nitrazepam, at an applied potential of 20 kV. We obtained reproducible electropherograms in successive repetitions, and few other peaks or interferences appeared in the electropherogram. The detection limits of the three compounds were 50–100 pg (0.1–0.2 μg/ml of analyte in spiked urine), and the recoveries were 78.9–100.8% for 1 μg/ml and 84.1–100.3% for 5 μg/ml. The application of this method to forensic or clinical samples is demonstrated.     

Effects of benzodiazepines on single unit activity in the substantia nigra pars reticulata

Intravenous administration of two benzodiazepines, flurazepam and diazepam, had an inhibitory effect on the firing rates of neurons of the substantia nigra pars reticulata, a brain region with an identified GABAergic innervation. Diazepam was more potent than flurazepam. Bicuculline and picrotoxin, two drugs which block GABAergic transmission, and caffeine and theophylline, two methylxanthines which inhibit benzodiazepine binding, all reversed the inhibition produced by diazepam. The action of theophylline was less consistent than that of caffeine. Similarly, Ro 15–1788, an imidazodiazepine which putatively functions as a specific benzodiazepine antagonist, reversed the diazepam-induced inhibition. These findings are consistent with previous reports which suggest that the benzodiazepines may act through a GABAergic mechanism. In a separate group of experiments, caffeine or Ro 15–1788 was administered alone. While caffeine excited all reticulata cells tested. Ro 15–1788, the more specific benzodiazepine antagonist, generally had little excitatory effect. These results suggest: 1) that cells of the substantia nigra pars reticulata may not receive a substantial, tonic inhibition mediated by an endogenous benzodiazepine-like substance; and 2) that the methylxanthines may increase reticulata cell firing, at least in part, through mechanisms unrelated to the blockade of benzodiazepine receptors.     

Benzodiazepine - specific and nonspecific tolerance following chronic flurazepam treatment

Rats were given a flurazepam solution as their only water source for 4 weeks. The drug concentration was adjusted so the rats would consume 100–150 mg/kg daily. This treatment is known to cause a reduction in the number of specific benzodiazepine binding sites (receptor down-regulation) and tolerance to the locomotor impairment caused by the injection of a large test dose of flurazepam. Both the tolerance and the receptor down-regulation disappear within 24 hours after the end of chronic treatment. After 4 weeks of flurazepam treatment, rats were tested for locomotor impairment and loss of the righting response caused by pentobarbital, ethanol or diazepam. There was a small tolerance to pentobarbital. This lasted at least 4 days, but had disappeared by 7 days. Rats also had a small tolerance to ethanol, which disappeared between 24 and 48 hours after the end of chronic flurazepam treatment. In contrast, there was a large tolerance to diazepam, but this was gone by 24 hours after the end of chronic treatment. It appears that two types of tolerance develop during benzodiazepine treatment: (1) tolerance specific for benzodiazepines possibly mediated by receptor down-regulation, and (2) nonspecific tolerance, possibly analogous to that which develops during chronic barbiturate treatment.     

Genetic toxicology of four commonly used benzodiazepines: A review

Benzodiazepines are a group of drugs which have been extensively used for their activities as an anti-anxiety, sedative, muscle relaxant and anti-convulsant. Benzodiazepines at present are the most commonly prescribed drugs. Some of these drugs are teratogenic and also carcinogenic in experimental animals. The wide human exposure to this group of drugs throughout the world is of great concern for human health. In the present review, we have attempted to evaluate and update the mutagenic and genotoxic effects of four of the most commonly used benzodiazepines, i.e., chlordiazepoxide (CDZ), diazepam (DZ), nitrazepam (NZ) and oxazepam (OZ) based on available literature.     

Effect of GABA agonists on the neurotoxicity and anticonvulsant activity of benzodiazepines

Progabide (50 mg/kg, i.p.), a GABA receptor agonist, significantly decreases the median minimal neurotoxic dose (TD50) of clobazam, chlordiazepoxide, and diazepam; the receptor binding of these substances is highly enhanced by muscimol. Progabide has no significant effect on the TD50 of clonazepam and triazolam; the receptor bindings of these substances is either only slightly enhanced or not altered by muscimol. Progabide also significantly decreases the median antimaximal electroshock dose (MES ED50) of all the benzodiazepines tested. However, progabide has no effect on the median antipentylenetetrazol dose (PTZ ED50) of the benzodiazepines. Likewise, THIP (2.5 mg/kg, i.p.) significantly decreases the TD50 of chlordiazepoxide but not that of triazolam. THIP significantly decreases the MES ED50 of chlordiazepoxide and triazolam but has no effect on the PTZ ED50 of these two substances. The above data suggest that benzodiazepine receptors linked to GABA receptors contribute to the minimal neurotoxicity and anti-MES activity but not to the anti-PTZ activity of benzodiazepines.