Benzodiazepine inhibits anxiogenic-like response in cocaine or ethanol withdrawn planarians.

Planarians spend less time in light versus dark environments. We hypothesized that planarians withdrawn from cocaine or ethanol would spend even less time in the light than drug-naive planarians and that a benzodiazepine would inhibit this response. Planarians pretreated in cocaine or ethanol were placed at the midline of a Petri dish containing spring water that was split evenly into dark and light compartments. Planarians withdrawn from cocaine (1, 10, 100 µmol/l) or ethanol (0.01%) spent less time in the light compartment than water controls; however, this withdrawal response to cocaine (100 µmol/l) or ethanol (0.01%) was abolished by clorazepate (0-100 µmol/l). These data suggest that planarians, similar to rodents, show benzodiazepine-sensitive, anxiogenic-like responses during cocaine or alcohol withdrawal.

Flumazenil-sensitive dose-related physical dependence in planarians produced by two benzodiazepine and one non-benzodiazepine benzodiazepine-receptor agonists.

Two benzodiazepine (midazolam and clorazepate) and one non-benzodiazepine (zolpidem) benzodiazepine-receptor agonists produced dose-related physical dependence, as evidenced by abstinence-induced decrease in planarian locomotor velocity (pLMV) when drug-exposed planarians were placed into drug-free water, but not when they were placed into drug-containing water (i.e., an abstinence-induced withdrawal, since the effect was only obtained in the removal of drug and not in the continued presence of drug). We have previously shown that the decrease in pLMV is associated with specific and transient withdrawal signs. In the present study, the selective benzodiazepine-receptor antagonist flumazenil significantly antagonized (P<0.05), by co-application, the ability of each agonist to produce the withdrawal. These results: (1) suggest that benzodiazepine-receptor agonists, for two different chemical categories, produce dose-related physical dependence manifested as abstinence-induced withdrawal in this simple and convenient model, and (2) in the absence of cloning or radioligand binding literature, suggest a possible specific interaction site (receptor?) for these compounds in planarians.

Interactions of buprenorphine and dipotassium clorazepate on anxiety and memory functions in the mouse.

Buprenorphine, a partial mu-receptor agonist widely substituted for heroin in the treatment of addiction, is often misused in combination with benzodiazepines. Improved hedonic properties may result, but only at the cost of increased buprenorphine toxicity. In order to elucidate the appeal of the benzodiazepine-buprenorphine combination, the present study looked at its neuropsycho-pharmacological effects on various emotional and cognitive parameters in the mouse. On the basis of previous dose-response studies, the regimen used was buprenorphine 0.3mg/kg, s.c. plus dipotassium clorazepate 1, 4 and 16 mg/kg, i.p. Anxiety-like behaviour was assessed using the black and white test box, and memory processes were examined via the spontaneous alternation paradigm in the Y-maze, and passive avoidance tests. Spontaneous locomotor activity was also evaluated. High doses of clorazepate impaired buprenorphine-induced hyperactivity and anxiogenic-like effects. They also increased buprenorphine-induced spontaneous alternation impairment, but did not modify its impact on long-term memory processes. These results suggest that the positive reinforcement experienced with the buprenorphine-benzodiazepine combination may be attributable, at least in part, to an increase in buprenorphine's sedative effect associated with a decrease in anxiogenicity.

Anxiolytic properties of agomelatine, an antidepressant with melatoninergic and serotonergic properties: role of 5-HT2C receptor blockade.

RATIONALE: The novel antidepressant agent, agomelatine, behaves as an agonist at melatonin receptors and as an antagonist at serotonin (5-HT)(2C) receptors. OBJECTIVES: To determine whether, by virtue of its antagonist properties at 5-HT(2C) receptors, agomelatine elicits anxiolytic properties in rats. METHODS: Employing a combined neurochemical and behavioural approach, actions of agomelatine were compared to those of melatonin, the selective 5-HT(2C) receptor antagonist, SB243,213, and the benzodiazepine, clorazepate. RESULTS: In unfamiliar pairs of rats exposed to a novel environment, agomelatine enhanced the time devoted to active social interaction, an action mimicked by clorazepate and by SB243,213. In a Vogel conflict procedure, agomelatine likewise displayed dose-dependent anxiolytic activity with a maximal effect comparable to clorazepate, and SB243,213 was similarly active in this procedure. In a plus-maze procedure in which clorazepate significantly enhanced percentage entries into open arms, agomelatine revealed only modest activity and SB243,213 was inactive. Further, like SB243,213, and in contrast to clorazepate, agomelatine did not suppress ultrasonic vocalizations emitted by rats re-exposed to an environment associated with an aversive stimulus. Whereas clorazepate reduced dialysate levels of 5-HT and noradrenaline in hippocampus and frontal cortex of freely moving rats, agomelatine did not affect extracellular levels of 5-HT and elevated those of noradrenaline. SB243,213 acted similarly to agomelatine. Melatonin, which did not modify extracellular levels of 5-HT or noradrenaline, was ineffective in all models of anxiolytic activity. Furthermore, the selective melatonin antagonist, S22153, did not modify anxiolytic properties of agomelatine in either the social interaction or the Vogel Conflict tests. CONCLUSIONS: In contrast to melatonin, and reflecting blockade of 5-HT(2C) receptors, agomelatine is active in several models of anxiolytic properties in rodents. The anxiolytic profile of agomelatine differs from that of benzodiazepines from which it may also be distinguished by its contrasting influence on corticolimbic monoaminergic pathways.

Clorazepate affects cell surface regulation of delta and kappa opioid receptors, thereby altering buprenorphine-induced adaptation in the rat brain.

Concomitant abuse of buprenorphine (BPN) and benzodiazepines (BZD) may relate to a pharmacodynamic interaction between the two. The objective of the present work was to investigate the acute and chronic effects of clorazepate (CRZ) alone or in combination with BPN on selective kappa opiate tritiated ligand [3H]-U69 593 and delta opiate radioligand [3H]-deltorphine II binding in the rat brain. Bmax (maximal receptor density) and Kd (the dissociation constant) were directly determined at different brain regions of interest (ROI) selected for high densities of kappa and/or delta receptors in rats treated with BPN and/or CRZ. The agents were administered either once or for 21 consecutive days. Differences in Bmax and Kd (for both specific ligands) were related to drug treatment and receptor location. Globally, single BPN administration induced no changes in kappa or delta opiate receptor binding, whereas repeated BPN administration up-regulated kappa receptor density and decreased delta affinity. At the kappa receptor level, repeated administration of CRZ acted only on Kd, whereas the delta receptor was up-regulated. Repeated addition of CRZ to BPN had no effect on kappa receptor Bmax versus chronic controls. By significantly decreasing Bmax, CRZ nullified the effect of chronic BPN on the kappa receptor. The modifications were strongest in the nucleus accumbens, where both types of receptor occur. Treatments had region-selective effects in some brain areas, such as the amygdala, periaqueductal gray matter, hypothalamus and caudate putamen. Increased mu and delta receptor densities would be expected to provide reinforcement by enhancing reward, and impairment of kappa receptor availability would be expected to decrease aversion. The effects described are likely to influence addictive behavior among people abusing BZD and BPN.

Impaired absorption of desmethyldiazepam from clorazepate by magnesium aluminum hydroxide.

Ten healthy volunteers ingested single 15-mg doses of clorazepate dipotassium (CZP) with 60 ml of water, or with 60 ml of magnesium aluminum hydroxide (Maalox), on two occasions in a randomized, two-way crossover study. Plasma concentrations of desmethyldiazepam (DMDZ) were determined in multiple samples drawn during 48 hr after each dose. Mean kinetic variables for DMDZ in CZP-water and CZP-magnesium aluminum hydroxide treatment conditions, respectively, were: peak measured concentration, 273 and 188 ng/ml (p 0.001); time of peak concentration, 1.8 and 2.8 hr after dose (p less than 0.01); apparent absorption half-life, 14.8 and 30.7 min (p less than 0.02); area under the 48-hr plasma concentration curve, 6,028 and 5,433 ng/ml X hr (p less than 0.02). Self-rated sensations of feedling "spacey," "thinking slowed down," and of generalized sedation, were reported with both treatment conditions, but these subjective effects occurred earlier and were more profound when CZP was taken with water as opposed to magnesium aluminum hydroxide. Thus administration of single doses of CZP with usual doses of a commonly prescribed antacid reduces the rate and extent of appearance in blood of DMDZ (the compound responsible for clinical activity) and attenuates self-rated clinical effects.

Failure of clorazepate to cause malformations or fetal wastage in the rat.

Clorazepate dipotassium (Tranxene), an anticonvulsant benzodiazepine, was tested for teratogenicity by injecting ten pregnant Long-Evans rats with 32 mg/kg of body weight intramuscularly on days 8.5, 9.5, and 10.5 of gestation. Ten control rats similarly received sterile water injections. Sixty fetuses recovered after killing five of the mothers on day 20.5 of gestation were sectioned to ascertain external and visceral malformations. Comparison with 55 control fetuses showed no statistically significant differences in external, visceral, or skeletal malformations, nor in fetal mortality, fetal and placental weight, and crown-rump length. Five clorazepate-treated rat mothers were allowed to deliver their 45 offspring for a companion study of possible behavioral effects. None of these additional 45 clorazepate-treated rats showed external malformations. Thus clorazepate caused no gross malformations in the rat under the conditions of this study.

Delayed maze-learning in rats after prenatal exposure to clorazepate.

Five pregnant Long-Evans rats were given 32 mg/kg of body weight of clorazepate dipotassium (Tranxene) intramuscularly on gestational days 8.5, 9.5, and 10.5. Five control mothers received sterile water. The control group of offspring (n = 19) and the experimental group (n = 20) were compared by means of a timetable for neurologic development and for maze learning ability. Although the experimental group was significantly slower in stomach-lifting and walking, the neurologic battery as a whole did not disclose any consistent difference. At 21 days of age, the experimental rats weighed significantly more than the control rats. On trials 10 to 14, the control group ran the maze in less than half of the time of the experimental group. The study emphasizes the need to include tests of cerebral function in addition to developmental reflexes to screen for subtle effects of teratogens, which the simpler developmental tests may miss.

Behavioural and pharmacokinetic studies in the monkey (Macaca mulatta) with diazepam, nordiazepam and related 1,4-benzodiazepines.

1. Behavioural activity (delayed differentiation and spatial delayed alternation) and pharmacokinetics of diazepam and its metabolites, N-desmethyldiazepam (nordiazepam), 3-hydroxydiazepam (temazepam) and 3-hydroxy-N-desmethyldiazepam (oxazepam), and of dipotassium clorazepate (clorazepate), were studied in the monkey (Macaca mulatta). Diazepam and its metabolites (1.8 and 3.0 mg/kg) and clorazepate (2.6 and 4.3 mg/kg) were given by intraperitoneal injection. 2. Hydroxylation of diazepam (temazepam and oxazepam) led to a loss of, or a considerable reduction in, behavioural activity, whereas activity was preserved, though modified, by demethylation (nordiazepam). It was not possible to establish change in behaviour at specific time intervals after clorazepate, but combined performance data revealed an effect. 3. The maximum mean plasma concentrations of diazepam, temazepam, oxazepam and clorazepate were observed at 0.5 h, and the maximum mean plasma concentration of nordiazepam was observed at 1 hour. Plasma concentrations of nordiazepam were the highest and decreased monoexponentially. Plasma concenqrations of the other drugs declined rapidly at first but more slowly later, and these data were analysed as biexponential models. In the analysis for metabolites, nordiazepam reached measurable levels after the injection of diazepam and clorazepate. 4. It is suggested that differences in the effects of closely related benzodiazepines may not be due solely to their plasma pharmacokinetic properties, but may arise from differences in their intrinsic activity.

Effects of clorazepate, diazepam, lorazepam, and placebo on human memory.

Healthy adults (N = 10) were given oral doses of lorazepam (1 and 2 mg), diazepam (5 and 10 mg), clorazepate (7.5 and 15 mg), or placebo and tested 30, 60, 90, and 120 minutes later on a word-recall memory task. All subjects received each drug dose once and placebo twice in randomized order at weekly intervals. Testing was double-blind. Lorazepam was found to have a significantly greater effect on memory than placebo. Diazepam and clorazepate did not differ significantly from placebo in their effect on word recall. High doses of lorazepam produced more pronounced memory effects than did low doses; neither diazepam nor clorazepate was found to exert a dose-related effect on memory.

Lack of amnestic effects of clorazepate on geriatric recall.

Significant amnestic effects in young adults have been found with the short-acting sedative-hypnotic triazolam and the intermediate-acting lorazepam, but not with the longer-acting clorazepate. The effects of placebo and clorazepate 3.75 and 7.5 mg were compared in 43 nonanxious geriatric subjects. Results were consistent with earlier studies, and no significant impairment of immediate or delayed recall was found.

Intravenous buspirone self-administration in rhesus monkeys.

Rhesus monkeys trained to press levers for intravenous cocaine injections were tested with saline and various doses of buspirone, chlordiazepoxide, and clorazepate. Buspirone was not self-administered at rates significantly above saline control levels. Chlordiazepoxide and clorazepate also failed to serve as reinforcers under these conditions. The lack of reinforcing properties, taken together with the results of other preclinical behavioral studies, suggests a low potential for recreational use of buspirone in man.

Differential amnestic properties of short- and long-acting benzodiazepines.

It has been demonstrated previously that orally administered lorazepam can cause anterograde amnesia in young adults. In this study, the effects of 7.5 and 15 mg clorazepate and 1 and 2 mg lorazepam on recall were compared in 74 healthy adults. Word list presentation tests were administered to subjects at selected intervals to measure immediate and delayed recall. Statistically significant memory impairment was found with 2 mg lorazepam during both immediate and delayed recall testing. Clorazepate produced no statistically significant amnestic effects. The data suggest that benzodiazepines differ in their potential for causing memory impairment.

Plasma concentrations and clinical effects after single oral doses of prazepam, clorazepate, and diazepam.

In a double-blind parallel-group pharmacokinetic and pharmacodynamic study, 31 healthy volunteers received single oral doses of prazepam (10 mg), clorazepate (7.5 mg), or diazepam (5 mg). Appearance in plasma of diazepam and of desmethyldiazepam was rapid after administration of diazepam and clorazepate, respectively, with peak plasma concentrations reached within an average of 1 hour. After oral prazepam, however, desmethyldiazepam appeared in blood slowly, with the highest mean concentration at 6 hours postdosage. Clinical self-ratings of fatigue and of "feeling spacey" were significantly different among groups, with changes over baseline being more marked with clorazepate and diazepam than with prazepam. Thus, differences in absorption rate of orally administered benzodiazepines can lead to differences in the intensity of single-dose effects, despite administration of doses that are equivalent in terms of long-term anxiolytic efficacy.

Self-rated sedation and plasma concentrations of desmethyldiazepam following single doses of clorazepate.

Plasma concentrations of desmethyldiazepam (DMDZ) and intensity of self-rated sedation (SRS) were measured at multiple points in time during 6 h after a single 15 mg oral dose of clorazepate dipotassium. Mean plasma DMDZ levels and mean SRS scores both became maximal at 1.0--2.5 h after drug dosage. By 6 h, however, mean SRS had returned to the predrug baseline score while mean DMDZ concentration fell only slighty from the maximum value. Disappearance of SRS despite persistence of high DMDZ levels might be due to adaptation or tolerance. If this is the case, subjective effects of benzodiazepines may depend upon duration of drug exposure as well as dose and concentration.

Pharmacokinetics of N-desmethyldiazepam in healthy volunteers after single daily doses of dipotassium chlorazepate.

Dipotassium chlorazepate was administered to 12 healthy volunteers (8 males and 4 females), aged 22-38 years, as a single daily dose of 20 mg for 14 days. Plasma concentrations of N-desmethyldiazepam were monitored with a gas-chromatographic method during the medication period and for 5 days after withdrawal of the drug. The plasma half-life (t1/2), the elimination coefficient (Kbeta), the concentration (Css), and the apparent volume of distribution (Vbeta) were calculated at steady state, and the mean values +/- SEM were 53 +/- 6 h, 0.0147 +/- 0.0013 h-1, 884 +/- 73 ng/ml, and 1.13 +/- 0.08 1/kg, respectively. A moderate interindividual variability was observed regarding these parameters. There was no tendency toward a biexponential elimination. A significant difference in the apparent volume of distribution was found when males and females were compared.

Individual variations in the effects of flurazepam, clorazepate, L-dopa and thyrotropin-releasing hormone on REM sleep in man.

The comporative effects of flurazepam, clorazepate, L-dopa, and thyrotropin-releasing hormone (TRH) on REM sleep were investigated in normal, healthy adults. A single dose of each drug was given orally to the subjects 30 min before bedtime. A dose of 30 mg flurazepam significantly decreased REM sleep-time when compared to the mean baseline record. No change was noted in REM sleep-time on the clorazepate (15 mg) night, L-dopa (1000 mg) night, or TRH (2 mg) night, when compared to the mean baseline record. Because large individual variations were found in REM sleep time on each drug night, and in percentage increase in REM sleep following partial differential REM deprivation (PDRD), correlation was investigated between them. The percentage decrease in REM sleep during flurazepam was found to have a significant negative correlation with the percentage increase in REM sleep after PDRD in individual subjects. Although there was no significant change in REM sleep on TRH night when compared to the mean baseline record, a similar significant negative correlation was noted. On the L-dopa night, there was a tendency toward a negative correlation between them. No significant correlation was noted on the clorazepate night.

Comparison studies of chlorazepate administered as a divided daily dose and as a single dose at night.

The residual effects of dipotassium chlorazepate administered as either a single daily dose of 20 mg at bedtime or a divided daily dose (5 + 5 + 10 mg) were studied in a placebo-controlled, double-blind trial comprising 12 out-patients. The following tests were used to determine changes in perceptual wakefulness, performance ability, fine motor skills, and coordination: critical flicker fusion test, car driving in a simulator, and the "bead and needle tests." In addition, the patients underwent a clinical assessment and also filled out a self-rating scale for judging factors related to the tests. No significant differences were found between the dosage schedules or between the active medication and the placebo. The clinical results were not dependent on the dosage schedule.

Clorazepate synchronizes cultured rat C6 glioma in the early G1 phase of the cell cycle.

A water soluble benzodiazepine, clorazepate, has been used to establish the point of benzodiazepine proliferative arrest in the rat C6 glioma. Clorazepate inhibited C6 proliferation in a dose-dependent manner with an IC50 value of 280 microM, as judged by a nuclei counting procedure. Release of cells from a 48 h exposure to 350 microM clorazepate, at which over 70% of the cells were arrested, resulted in a synchronous entry into S phase 8-9 h later, as evidenced by a sharp increase in the incorporation of [3H]thymidine. This restriction point was demonstrated to be 2-3 h into the G1 phase by measuring the length of G1 in synchronized populations of C6 cells obtained by selection of mitotic figures from an asynchronous culture. Synchronous arrest of C6 by clorazepate required an exposure period of 24-36 h, approximately twice the doubling time of the cell line. A morphological study confirmed an early G1 point of proliferative arrest. Clorazepate synchronized cells exhibited a uniform morphology with the majority of cells assuming a configuration representative of anchorage-dependent cells in an early phase of attachment. The majority of cells were somewhat rounded and attached to the substratum by cytoplasmic 'skirts' with punctate structures which may represent focal adhesion points.

Behavioural evidence for partial agonist properties of RO 15-1788, a benzodiazepine receptor antagonist.

Rats trained to discriminate pentylenetetrazol from saline had this cue antagonized by the benzodiazepine, clorazepate. Ro 15-1788 reversed the antagonism of the pentylenetetrazol cue produced by clorazepate. Similarly, Ro 15-1788 blocked the anti-conflict effect of clorazepate. Rats trained to discriminate clorazepate from saline, however, generalized this cue to Ro 15-1788. These results demonstrate that Ro 15-1788 is not a pure benzodiazepine antagonist, but has partial agonist properties.