Mice lacking Gad2 show altered behavioral effects of ethanol, flurazepam and gabaxadol.

Gamma-aminobutyric acid (GABA) is synthesized in brain by two isoforms of glutamic acid decarboxylase (Gad), Gad1 and Gad2. Gad1 provides most of the GABA in brain, but Gad2 can be rapidly activated in times of high GABA demand. Mice lacking Gad2 are viable whereas deletion of Gad1 is lethal. We produced null mutant mice for Gad2 on three different genetic backgrounds: predominantly C57BL/6J and one or two generations of backcrossing to 129S1/SvimJ (129N1, 129N2). We used these mice to determine if actions of alcohol are regulated by synthesis of GABA from this isoform. We also studied behavioral responses to a benzodiazepine (flurazepam) and a GABAA receptor agonist (gabaxadol). Deletion of Gad2 increased ethanol palatability and intake and slightly reduced the severity of ethanol-induced withdrawal, but these effects depended strongly on genetic background. Mutant mice on the 129N2 background showed the above three ethanol behavioral phenotypes, but the C57BL/6J inbred background did not show any of these phenotypes. Effects on ethanol consumption also depended on the test as the mutation did not alter consumption in limited access models. Deletion of Gad2 reduced the effect of flurazepam on motor incoordination and increased the effect of extrasynaptic GABAA receptor agonist gabaxadol without changing the duration of loss of righting reflex produced by these drugs. These results are consistent with earlier proposals that deletion of Gad2 (on 129N2 background) reduces synaptic GABA but also suggest changes in extrasynaptic receptor function.

Metabolism of two new benzodiazepine-type anti-leishmanial agents in rat hepatocytes and hepatic microsomes and their interaction with glutathione in macrophages.

OBJECTIVES: To measure the metabolism and toxicity of 7-chloro-4-(cyclohexylmethyl)-1-methyl-3,4-dihydro-1H-1,4-benzodiazepine-2,5-dione (BNZ-1) and 4-cyclohexylmethyl-1-methyl-3,4-dihydro-1H-1,4-benzodiazepine-2,5-dione (BNZ-2), two new benzodiazepine analogues found to be effective against Leishmania amastigotes in vitro. METHODS: The metabolism of BNZ-1 and -2 was investigated in isolated rat hepatocytes and rat liver microsomes. The toxicity of the compounds was assessed in a murine macrophage cell line by determining cell viability and reduced glutathione (GSH) content. The metabolism and toxicity of flurazepam was assessed for comparison. KEY FINDINGS: BNZ-1 and BNZ-2 underwent similar metabolic transformations by the liver systems, forming N-demethylated and hydroxylated metabolites, with subsequent O-glucuronidation. Flurazepam and both analogue compounds depleted macrophage GSH levels without affecting cell viability at the concentrations used (up to 100 microM), but only flurazepam inhibited glutathione reductase activity, indicating that it is acting by a different mechanism. CONCLUSIONS: The exact mechanism responsible for GSH depletion is unknown at present. Further experiments are needed to fully understand the effects of BNZs on the parasite GSH analogue, trypanothione, which may be a direct or indirect target for these agents. Pharmacokinetic evaluation of these compounds is required to further progress their development as potential new treatments for leishmaniasis.

Nitrendipine decreases benzodiazepine withdrawal seizures but not the development of benzodiazepine tolerance or withdrawal signs.

1. The effects of the calcium channel blocking agent, nitrendipine, were studied on seizures in mice produced during withdrawal from chronic benzodiazepine treatment and on the development of tolerance to benzodiazepines. 2. Nitrendipine produced a dose-dependent decrease in seizure incidence, when seizures were produced by the partial inverse agonist FG7142 during withdrawal from seven days treatment with flurazepam. 3. Nitrendipine did not raise the seizure thresholds in naïve mice to the full inverse agonist methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM), or to the gamma-aminobutyric acid (GABA) antagonist, bicuculline. 4. When given concurrently with flurazepam for seven days, nitrendipine did not affect the incidence of seizures during flurazepam withdrawal. 5. When given concurrently with the benzodiazepines, nitrendipine did not prevent the development of tolerance to midazolam general anaesthesia or tolerance to the ataxic actions of flurazepam or midazolam. 6. Chronic treatment with flurazepam for seven days did not affect the Kd or Bmax of [3H]-nimodipine binding in mouse whole brain or cerebral cortex. 7. These results with benzodiazepines are partially in contrast with those for ethanol, where nitrendipine not only decreased ethanol withdrawal seizures when given acutely, but also prevented the development of tolerance and withdrawal signs when given concurrently with ethanol. However, they do confirm the selectivity of nitrendipine for withdrawal-induced seizures.

Evaluation of experimental combined toxicity by use of dose-frequency curves: comparison with theoretical additivity as well as independence.

Dose-frequency curves of toxic effects of a substance A were evaluated in the absence and in the presence of a fixed dose of a second substance B. Data were fitted by the curve-fitting program ALLFIT. Observed combined frequencies of A + B were compared statistically with the expected frequencies of additivity and (or) independence by the phi 2-square goodness-of-fit test. The theoretical dose-frequency curves expected for an additive response were obtained by a solely graphical procedure and the theoretical curves for independent effects were calculated from the effects of B and A at certain doses. In rotarod tests with trained mice, the combined deteriorating effect of ethanol and benzodiazepines were significantly over-additive. However, their lethal interaction appeared underadditive in mice. The lethal underadditive interaction of ethanol and phencyclidine (PCP) can be ascribed largely to independent actions of these compounds. Loss of righting reflex was additively enhanced by PCP, whereas PCP overadditively enhanced the effect of ethanol. The insecticidal action of the cholinesterase inhibitors malathion and parathion appeared additive and significantly different from independent interaction. A comparison of results from dose-response curves with isoboles showed good agreement. The method appears as an attractive alternative or as a complementary procedure to the isobolographic analysis. Combination experiments as described can be carried out and evaluated rather simply, with a minimum of expenditure and a maximum of information.

Carcinogenicity study of doxefazepam administered in the diet to Sprague-Dawley rats.

Groups of 50 male and 50 female Sprague-Dawley rats were given food containing sufficient doxefazepam, a benzodiazepine derivative, to ensure intakes of 0, 3, 10, or 30 mg/kg/day. These dosages respectively correspond to 2, 20, and 60 times the mean daily hypnotic dose level of an adult man. Rats were treated for 104 weeks and then euthanized. An extensive autopsy was performed on those animals that died intercurrently and on euthanized animals. The chronic administration of doxefazepam did not influence the survival of the rats. No treatment-related changes in clinical signs and body weight gains occurred and malignant tumor rates were similar in controls and treated animals. A significant linear trend in the incidence of hepatocellular neoplasms, primarily benign, was observed in the female treated groups. This higher incidence was not associated to a higher occurrence of focal hyperplasia or other preneoplastic lesions in treated rats. The brain, a target organ for the pharmacological activity of doxefazepam, was carefully examined to search for microscopic foci of proliferative cells. A total of 12 and 6 malignant gliomas were observed in male and female rats, respectively; only two were noticed at autopsy. These tumors were mainly of the oligodendroglioma type commonly found in aged rats. Their incidence was slightly higher in treated rats, but results were not of statistical significance. The overall evaluation of the present study indicates that doxefazepam is noncarcinogenic in rats. However, the increase in liver adenomas found here as well as in previous bioassays with similar drugs and the lack of reliable historical data on the incidence of brain tumors in benzodiazepine-treated rodents suggest that additional experimental and epidemiological studies should be undertaken to exhaustively assess the toxic potential of this widely used class of drugs.

Toxicological evaluations of the benzodiazepine doxefazepam.

1-(2-Hydroxyethyl)-3-hydroxyl-7-chloro-1,3-dihydro-5-(O-fluorophenyl)-2H - 1,4-benzodiazepin-2-one (doxefazepam, SAS 643, Doxans) was investigated in a series of toxicological studies. Oral LD50 values were greater than 2000 mg/kg in mice, rats and dogs, while endoperitoneal LD50 values were 746 and 544 mg/kg in the mice and rats, respectively, and greater than 1000 mg/kg in the dogs. Subacute and chronic studies in rats and dogs evidenced a transient ataxia after administration of the test compound, which was dose-dependent in the subacute experiment, and occurred only at the highest dose in the chronic studies. No pathological findings were registered at necropsy or in microscopic observations, except an increase of liver weight at the highest dosage in the chronic study in the rat. Doxefazepam did not exert any teratogenic effects in rats and rabbits. Moreover in rats it did not alter the reproductive performance. The mutagenic studies did not reveal any mutagenic potential. In the cancerogenicity study in rats doxefazepam did not show positive carcinogenic potential.

Interaction between flurazepam and ethanol.

The interaction of a representative benzodiazepine, flurazepam, and ethanol has been assessed in ICR albino mice. Tests done included loss of "rotarod" performance, light sedation, deep sedation, loss of righting reflex, anesthesia, and lethality. LD50 and ED50s were plotted as isobolograms (plots of equieffective dose combinations). Data for anaesthesia and lethality showed little or no interaction between the two drugs. In contrast, the four motor and behaviour tests showed synergism, especially with higher doses of ethanol (over 2 g/kg) for righting reflex, and (over 1.5 g/kg) for deep sedation. Synergism occurred over all doses for light sedation and rotarod performance. It is expected that concurrent use of benzodiazepines and ethanol can result in a significantly higher accident risk in humans, but little additional risk of death from simple overdose.

Preclinical evaluation of behavioral deficits following hypnotic drug treatment.

Most hypnotic medications result in performance decrements, or "hangover effects," the morning after bedtime ingestion. However, the severity of hangover effects with new compounds is difficult to predict prior to clinical trials. This paper describes a preclinical procedure that is predictive of human hangover effects following bedtime ingestion of hypnotic medication. Four cebus monkeys were shaped to discriminate between two levers such that rapid responses following the cue light maximized the probability of reinforcement. Subsequently, performance was evaluated one hr (immediate condition) and 8 3/4 hr (delayed condition) after the administration of flurazepam, triazolam, pentobarbital and placebo. All drugs caused dose-dependent performance decrements in both conditions. Significantly higher doses of triazolam were required to impair performance during the delayed condition as compared to the immediate condition, but no such differences occurred with either flurazepam or pentobarbital. These findings parallel human data which indicate that sleep-inducing doses of flurazepam and many barbiturates, but not triazolam, cause performance decrements the morning after bedtime ingestion.

Central excitatory actions of flurazepam.

Toxic actions of flurazepam (FZP) were studied in cats, mice and rats. High doses caused an apparent central excitation, most clearly seen as clonic convulsions, superimposed on general depression. Following a lethal dose, death was always associated with convulsions. Comparing the relative sensitivity to central depression and excitation revealed that rats were least likely to have convulsions at doses that did not first cause loss of consciousness, while cats most clearly showed marked central excitatory actions. Signs of FZP toxocity in cats included excessive salivation, extreme apprehensive behavior, retching, muscle tremors and convulsions. An interaction between FZP and pentylenetetrazol (PTZ) was shown by pretreating mice with FZP before PTZ challenge. As a function of dose, FZP first protected against convulsions and death. At higher doses, however, convulsions again emerged. These doses of FZP were lower than those that would alone cause convulsions. These results may be relevant to the use of FZP in clinical situations in which there is increased neural excitability, such as epilepsy or sedative-hypnotic drug withdrawal.

Sedative-hypnotic properties of a new benzodiazepine in comparison with flurazepam. Pharmacological and clinical findings.

The sedative-hypnotic effects of a new benzodiazepine, 1-(2-hydroxyethyl)-3-hydroxy-7-chloro-1,3-dihydro-5-(o-fluorophenyl)-2H-1,4-benzodiazepin-2-one (SAS 643), were compared with those of flurazepam in mice and rats as well as in a double-blind clinical trial. It was found that SAS 643 has a potency 2--4 times greater than that of flurazepam while it is about one half less toxic than the latter drug. The results of the clinical trial confirm the greater activity of SAS 643 and indicate that the new benzodiazepine causes a significantly less amount of hangover than flurazepam.