Activity of the chloro- and triazolo-benzodiazepines. Behavioural studies in the monkey (Macaca mulatta)

The effects of 1,4-benzodiazepines with various heterocyclic ring structures (triazolo, triazino and imidazo), and of 1,4-benzodiazepines with chlorine substitution in the phenyl ring (4'- chlorodiazepam , 2'- chlorodiazepam and 2'- chloronordiazepam ) were studied on spatial-delayed alternation and delayed matching-to-sample tasks in the monkey. Within the dose range 0.25-0.75 mg kg-1, the triazolo compounds had marked activity, decreasing the number of correct responses and increasing total response time. The triazino was less potent and the imidazo compounds were the least likely to disrupt performance. Smaller doses (0.005, 0.01 and 0.05 mg kg-1) of triazolam did not impair performance on the tasks, and larger doses of imidazo (1.0, 3.0 and 7.5 mg kg-1) were without effect on delayed alternation. Within the dose range 1.0-10.0 mg kg-1, 4'- chlorodiazepam was without effect and only the largest dose of diazepam and 2'- chlorodiazepam impaired performance. 2'- Chloronordiazepam decreased the number of correct responses at all doses (1.0, 3.0 and 10.0 mg kg-1) and with the largest dose, the total response time was increased. The studies suggest that the effects of a drug on higher nervous function in the monkey cannot easily be predicted from standard pharmacological tests in other animals or other species of monkey.     

Flurazepam hydrochloride and its principal metabolites: Behavioural studies in the monkey (Macaca mulatta)

Effects of flurazepam and its two principal metabolites, hydroxyethylflurazepam and desalkylflurazepam (0.5, 1.0, 3.0 and 10.0 mg/kg), were studied on a delayed differentiation task in the monkey. Flurazepam (10.0 mg/kg) and hydroxyethylflurazepam (3.0 and 10.0 mg/kg) decreased the number of correct responses and increased response latency; effects persisted to 4 h with the highest dose. Marked impairment of performance was observed with all doses of desalkylflurazepam and effects of 3.0 and 10.0 mg/kg persisted to 24 h. Evidence of a disinhibitory effect of desalkylflurazepam was demonstrated. The study would point to a consideration of both metabolites in the hypnotic effect and in the impairment of performance observed after acute ingestion of flurazepam in man.     

Behavioural responses to diazepam of drug-naive and experienced monkeys (Macaca mulatta)

Effects of 5 mg/kg diazepam given once a week for several weeks to drug-naive and drug-experienced monkeys were studied on a spatial delayed-alternation task. In both groups response latency was increased and the number of correct responses was reduced. The effects were consistent over weeks in the experienced monkeys, but in the naive monkeys there were greater initial effects on response latency and on repeat errors, and these lessened over 2–3 weeks. Thereafter, effects resembled those observed in the experienced group. Results are discussed in terms of the possible factors involved in the development and persistence of tolerance to benzodiazepines.     

Behavioural sequelae of methaqualone in man and in the monkey (Macaca mulatta).

1 Residual effects in man of methaqualone hydrochloride (400 mg) were studied by adaptive tracking and by reaction time. Performance was measured at 10 h, 13 h, 16 h, 19 h and 34 h after the overnight ingestion of the drug. There was no evidence of impaired performance on adaptive tracking from 10 h to 19 h, but enhanced performance (P = 0.001) was observed 34 h after ingestion. With reaction time an increase (P = 0.01) was observed 10 h and a decrease (P = 0.05) was observed 19 h after ingestion. 2 Effects in the monkey (Macaca mulatta) of methaqualone (20 and 30 mg/kg body weight) were studied by a delayed matching task in which total response time was measured. No consistent effects on matching behaviour or on total response time were observed 2 h after intraperitoneal injection. 3 The studies suggest that methaqualone hydrochloride may be a valuable hypnotic for occasional use by persons involved in skilled activity.     

Comparative studies with thieno- and benzodiazepines: spatial delayed alternation behaviour in the monkey (Macaca mulatta)

The effects of three benzodiazepines, temazepam, nordiazepam and clobazam, and two triazolo-1,4-thienodiazepines, brotizolam and WE 973, were studied on spatial delayed alternation in the monkey (Macaca mulatto). Experiments were carried out 1 hr after the intraperitoneal injection of doses ranging from 0.5 to 3.0 mg.kg^?1 body weight. No behavioural effects were observed with temazepam and clobazam. Nordiazepam reduced the number of correct responses, and the thienodiazepines reduced the number of correct responses, and increased total response time. In further experiments with doses ranging from 5.0 to 25.0 mg.kg^?1 body weight there was still no effect with clobazam, but temazepam reduced the number of correct responses and increased total response time. There would appear to be differential effects of diazepines on behaviour. The triazolo-1,4-thienodiazepines are particularly potent compared with the benzodiazepines, and the question arises whether they possess specific behavioural activity.     

Teratologic evaluation of metaproterenol in the rhesus monkey (Macaca mulatta)

In order to further evaluate the teratologic potential of metaproterenol (Alupent), an experiment was conducted in timed pregnant rhesus monkeys (Macaca mulatta). Pregnancy was established by bioassay of serum for chorionic gonadotropin according to the method of Tullner and Hertz using the uterine weight of immature mice. Metaproterenol as a 1 mg/ml solution in water was administered by stomach tube at a dose of 5 mg/kg (approximately 3 times the human dose) for 11 consecutive days beginning on days 18–23 of gestation in a total of 12 animals. At approximately day 100 of gestation, the fetus was removed from each monkey by cesarean section. Each fetus was examined for extermal abnormalities, the viscera for any deviations from normal, and the skeletons, after clearing and staining with Alizarin Red S, for any structural deviations. No changes were observed that could be attributed to the administration of metaproterenol. Four concurrently untreated pregnant animals were included.     

Fluorescence--TLC densitometric determination of diazepam and other 1,4-benzodiazepines in serum.

A sensitive fluorescence--TLC densitometric procedure was developed for the specific determination of diazepam (I) and its two metabolites, desmethyldiazepam (II) and oxazepam (III), in serum. After extraction from serum with benzene, the compounds were separated by TLC and converted with a sulfuric acid spray to greenish-yellow fluorescence spots with Rf values of 0.72, 0.43, and 0.17, respectively. Quantitation of the TLC plate was accomplished by scanning with a densitometer at 380 nm. The sensitivities of the assay were 18 (I), 6 (II), and 5 (III) ng/ml of serum. This procedure was also was applicable to other 1,4-benzodiazepines in biological fluids.     

Immediate effects on human performance of a 1,5-genzodiazepine (clobazam) compared with the 1,4-benzodiazepines, chlordiazepoxide hydrochloride and diazepam.

1 The immediate effects on human performance of the 1,5-benzodiazepine, clobazam (20 mg), and the 1,4-benzodiazepines, chlordiazepoxide hydrochloride (20 mg) and diazepam (10 mg), were studied by adaptive tracking and measurement of reaction time. Each drug was ingested at 09.00 h and performance was measured at 09 h 30 min (0.5 h), 11 h 30 min (2.5 h), 14 h 30 min (5.5 h) and 18 h 30 min (9.5 h after ingestion). 2 With diazepam decrements in performance on adaptive tracking were observed at 0.5 h and 2.5 h and performance was enhanced at 9.5 h after ingestion. With clobazam performance at individual times did not differ significantly from control, but there was evidence of an improvement in performance during the day. There was no evidence of impaired performance on adaptive tracking after chlordiazepoxide hydrochloride. 3 Reaction time was slowed at 0.5 h and 2.5 h after diazepam and chlordiazepoxide hydrochloride. A decrease in reaction time was observed at 9.5 h after diazepam. No changes in reaction time were observed after clobazam. 4 The subjects as a group differentiated correctly between performance decrements on adaptive tracking after diazepam and the absence of performance decrements after clobazam and chlordiazepoxide hydrochloride. The persistence of the decrement in performance after diazepam was accurately assessed. 5 It is evident that the nature and persistence of impaired performance and the ability to appreciate impaired performance vary considerably between the benzodiazepines, and that the choice of a benzodiazepine should include careful consideration of performance sequelae.     

Lead effects on neurobehavioral development in the neonatal rhesus monkey (Macaca mulatta)

Effects of lead exposure on behavioral development during the first month of postnatal life were examined in rhesus monkeys using a multi-item assessment scale developed for the evaluation of neonatal rhesus monkeys. Lead was administered daily beginning at day 8 postpartum at levels that produced blood lead levels of about 20 μg/dl by week 4 (n = 48); controls were treated identically but given vehicle only (n = 24). All monkeys were tested once a week for the first 4 weeks postpartum. The first principal component explained a substantial portion of the variance and was relatively consistent across ages for both groups. Analyses of the individual items and of both conceptually derived and empirically defined summary scores yielded no significant effects of lead. Furthermore, there were no systematic relationships between blood lead level and performance on the test. Correlation coefficients indicated more similarity across age for control monkeys than for lead-exposed monkeys suggesting that continuity of development, as measured by this test, was disrupted by lead. The relationship between outcome on these early assessments and later behavior will be explored in subsequent studies of these monkeys.     

Metabolism of 14C-labeled doxylamine succinate (Bendectin) in the rhesus monkey (Macaca mulatta)

The time-course of the metabolic fate of [14C]doxylamine was determined after the p.o. administration of 13 mg/kg doxylamine succinate as Bendectin plus [14C]doxylamine succinate to the rhesus monkey. Urine and plasma samples were analyzed by reversed-phase high performance liquid chromatography (HPLC), chemical derivatization, and mass spectrometry. The cumulative 48-hr urinary metabolic profile contained 81% of the administered radiolabeled dose and consisted of at least six radiolabeled peaks. They were peak 1: unknown polar metabolites (8% of dose); peak 2: 2-[1-phenyl-1-(2-pyridinyl)ethoxy] acetic acid, 1-[1-phenyl-1(2-pyridinyl)ethoxy] methanol, and another minor metabolite(s) (31%); peak 3: doxylamine-N-oxide (1%); peak 4a: N,N-didesmethyldoxylamine (17%); peak 4b: doxylamine (4%); and peak 5: N-desmethyldoxylamine (20%). The plasma metabolic profile was the same as the urinary profile except for the absence of doxylamine-N-oxide. The maximum plasma concentrations and elapsed time to attain these concentrations were as follows. Peak 1: 540 ng/mL, 4 hr; peak 2: 1700 ng/mL, 1 hr; peak 4a: 430 ng/mL, 4 hr; peak 4b: 930 ng/mL, 2 hr; and peak 5: 790 ng/mL, 2 hr. These data suggest that in the monkey, doxylamine metabolism follows at least four pathways: a minor pathway to the N-oxide; a minor pathway to unknown polar metabolites; a major pathway to mono- and didesmethyldoxylamine via successive N-demethylation; and a major pathway to side-chain cleavage products (peak 2) via direct side-chain oxidation and/or deamination.     

Toxicity and disposition of 2,3,4,7,8-pentachlorodibenzofuran (4PeCDF) in the rhesus monkey (Macaca mulatta)

The toxicity and disposition of 2,3,4,7,8-pentachlorodibenzofuran (4PeCDF), a ubiquitous and acutely toxic environmental contaminant, was examined in three adult male Rhesus monkeys administered a single iv dose of 34 micrograms (0.1 mumol)/kg. Within 20 min, 4PeCDF was eliminated from the blood and was distributed to the liver, skin, adipose, and muscle tissues. Excretion occurred primarily via the feces with a minimum whole body half-life approximately 38 days. Within 7-14 days after administration, the packed cell volume and serum triglyceride and bile acid concentrations were significantly increased while serum cholesterol, protein, and albumin concentrations were decreased relative to pretreatment levels. Thyroid hormone levels were also altered with an increase in TSH and a decrease in T3 and T4 concentrations. After 28 days, two monkeys began exhibiting alopecia, hyperkeratinization of the toe and finger nails, facial chloracne-like lesions, and loss of body weight. They subsequently died 40 and 48 days after treatment. Similar symptoms of toxicity were observed in the third animal 58 days after 4PeCDF administration, but this animal appeared to fully recover and was administered 4PeCDF orally and [3H]1,2,3,7,8-pentachloro-dibenzofuran (1PeCDF) dermally 238 days after the initial iv dose. In this animal, approximately 2% of an oral dose of [14C]-4PeCDF was absorbed from the stomach and small intestine in 6 hr and was distributed mainly to the muscle and skin and less than 99% of a dermal dose of 1PeCDF remained at the site of application. Pathological findings in the monkeys that died indicated hyperplastic and metaplastic changes in the gastric mucosa, the Meibomian glands of the eyelid, and the ceruminous glands of the ear. Regression of these lesions was present in the surviving animal. Therefore, 4PeCDF produces dioxin-like toxicity in the monkey similar to that reported for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and in the same dose range.     

β-Adrenoceptor antagonists: studies on behaviour (delayed differentiation) in the monkey (Macaca mulatta)

1 Activity of six β-adrenoceptor antagonists was studied on behavioural activity (delayed differentiation) in the monkey (Macaca mulatta). The drugs, three relatively lipophilic antagonists (propranolol, oxprenolol and metoprolol), and three relatively hydrophilic antagonists (acebutolol, atenolol and sotalol), were given by intraperitoneal injection (5 to 30 mg/kg).

2 With atenolol (25 to 30 mg/kg), total response time was increased, but there was no effect on the number of correct responses. With acebutolol (25 to 30 mg/kg), the number of correct responses was reduced, but there was no effect on total response time. With metoprolol (25 to 30 mg/kg), there was an increase in total response time and a decrease in the number of correct responses, and correct responses were decreased 4 h after injection over the whole dose range (5 to 30 mg/kg).

3 Some animals failed to respond or complete the task with 30 mg/kg oxprenolol, 25 mg/kg sotalol and 20 mg/kg propranolol. With 25 mg/kg oxprenolol, the total response time was increased and the number of correct responses was decreased. With 5-20 mg/kg sotalol, total response time was increased, but there was no effect on the number of correct responses. With 15 mg/kg of (±)-propranolol and its isomers, there were increases in total response time and decreases in correct responses.

4 The studies suggest that lipophilic antagonists, such as propranolol, oxprenolol and metoprolol, are likely to have, at least, effects on the central nervous system, while hydrophilic antagonists may modify the peripheral nervous system. In the dose-ranges studied, propranolol had the greatest, and atenolol and acebutolol had the least effects. Atenolol and acebutolol may prove to be particularly useful in man when disturbances of the nervous system are to be avoided.

     

Effect of d-amphetamine and diazepam on the greeting behavior of rhesus monkeys (Macaca mulatta)

The greeting behavior of rhesus monkeys (Macaca mulatta) was evaluated in a pharmacological test. It was observed in pairs of juvenile subjects reunited after a separation of two days. The observational measures were the duration of greeting behavior (social grooming, social play, and huddling) and the frequency of presentations, mounts and solicitations. d-Amphetamine (0.2 and 0.1 mg X kg-1) was found to shorten the duration of greeting behavior and increase the frequency of presentations and mounts. Diazepam (1 and 0.5 mg X kg-1) was found to prolong the duration of greeting behavior.     

Kinetics and mechanisms of hydrolysis of 1,4-benzodiazepines II: oxazepam and diazepam.

The hydrolysis kinetics of oxazepam and diazepam leading to a benzophenone product and a glycine derivative were quantified from pH 1 to 11. For oxazepam, two intermediates were isolated and identified, indicating a parallel consecutive reaction mechanism. The hydrolysis occurred uncatalyzed and demonstrated acid-base catalysis for both reaction steps. One intermediate was observed by TLC for diazepam hydrolysis. This intermediate, resulting from breakage of the azomethine linkage, was different than the major intermediate isolated for oxazepam hydrolytic degradation (amide hydrolysis preferred). Stability parameters involving rate constant-temperature dependence are reported.     

Pharmacokinetics of diazepam and nordiazepam in the cat

The cat has been used extensively as an experimental model for studying the pharmacology of compounds that exhibit CNS activity including diazepam and nordiazepam. However, since little is known about the distribution and elimination of diazepam in this species, the pharmacokinetics of diazepam and nordiazepam were studied in the cat following intravenous doses of 5, 10, and 20 mg/kg of diazepam and 5 and 10 mg/kg of nordiazepam. The disappearance of diazepam and nordiazepam from blood was fitted with classical equations. Theoretical and trapezoidal areas under the curve (AUCth and AUCtr) were calculated. The volumes of distribution (Vd beta) were calculated as model-independent parameters for diazepam and nordiazepam. Intrinsic hepatic clearance, extraction ratio, and tissue binding parameters were also calculated for diazepam. From the observed data, it is apparent that the blood concentrations and the resulting areas under the curves are proportional to the dose of diazepam administered and that the pharmacokinetics of diazepam were linear over the dose range studied. In addition, nordiazepam formed after diazepam administration appeared to be proportional to the dose of diazepam administered. The terminal elimination rate constant of nordiazepam remained constant over the dose range studied. It appears that both diazepam and nordiazepam are highly bound to tissue. The total body clearance of diazepam (4.72 +/- 2.45 mL/min/kg) is approximately six times that of nordiazepam (0.85 +/- 0.25 mL/min/kg). Approximately 50% of an administered dose of diazepam was biotransformed to nordiazepam in the cat.     

Quinazolines and 1,4-benzodiazepines. 92. Conformational recognition of the receptor by 1,4-benzodiazepines

We propose that the conformation of 1,4-benzodiazepines that is recognized by the binding site on the benzodiazepine receptor complex is one in which the planes formed by the fused benzene ring and the methylene group (and the two adjoining atoms) of the diazepine ring are in the R configuration. The derivation of this conformation was based on comparisons of computer-generated 3-dimensional structures obtained from single-crystal X-ray data for diazepam, (R)- and (S)-1,3-dimethyl-5-(2-fluorophenyl)-7-nitro-1,3-dihydro-2H-1,4-benzodiazepin-2- one, and the structurally rigid ethyl (S)-7-chloro-11,12,13,13 alpha-tetrahydro-9-oxo-9H-imidazo[1,5-alpha] pyrrolo[2,1-d][1,4]benzodiazepine-1-carboxylate. The affinity of ligands for the benzodiazepine binding site was determined using the [3H]-diazepam binding assay.