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Differences between cyclopyrrolones (suriclone and zopiclone) and benzodiazepines bincling to rat hippocampus photolabelled membranes

The intravenous general anaesthetic propofol (2,6-di-isopropylphenol) is frequently combined with a benzodiazepine. There are clinical reports of synergism between these two agents for induction of general anaesthesia. To investigate a possible mechanism of this synergistic interaction between propofol and benzodiazepines, the effect of propofol and flurazepam on GABA_A receptor function was examined in Xenopus occytes expressing human recombinant α₁β₂γ_2L and α₂β₂γ_2L receptor constructs. Potentiation of GABA receptor-activated current by low (1–10 μM) concentrations of propofol together with flurazepam (0.25–0.5 μM) was significantly greater than predicted by an additive response. Isobolographic analysis indicated a strong synergistic interaction between propofol and flurazepam at either of the receptor constructs examined. In contrast, the cyclopyrrolone derivative zopiclone, which produced a similar facilitation of GABA receptor-activated current compared to flurazepam, produced a less than additive potentiation when combined with propofol. Flurazepam significantly decreased the EC₅₀ concentration of propofol for potentiation of GABA responses. Thus, flurazepam, in addition to facilitating GABA receptor activity on its own, also increases the apparent affinity of the GABA_A receptor complex to propofol, resulting in a greater than expected potentiation by the combination of propofol plus flurazepam.

Zopiclone is a new short half-life cyclopyrrolone hypnotic agent acting at the GABA-benzodiazepine receptor complex. In order to characterize its pharmacological profile, the effects of 7.5 mg zopiclone on nocturnal melatonin secretion were investigated under polysomnographic control in 11 healthy subjects following acute and subchronic administration as well as after abrupt discontinuation of the drug. No effect of zopiclone on the melatonin plasma levels could be observed. Regarding both total melatonin production and the temporal pattern of melatonin secretion during the night, there was no difference between placebo baseline condition, acute and subchronic administration, and discontinuation. In contrast, the sleep EEG data demonstrated the hypnotic efficacy of zopiclone under acute administration and indicated a rebound insomnia after abrupt discontinuation. Moreover, alterations of sleep architecture were found under treatment as well as after discontinuation. Whereas, with regard to sleep EEG parameters, zopiclone appears to be comparable with some short-acting benzodiazepines, a discrepancy between the missing effect of zopiclone on pineal function and the suppressing influence of benzodiazepines known from the literature becomes obvious. The fact that zopiclone does not interfere with nocturnal melatonin secretion at pharmacologically active doses as indicated by alterations in sleep EEG parameters might possibly point to a pharmacodynamic difference between the two drug classes.

Polysomnographic examinations (PSG) were performed on 6 male schizophrenic outpatients who were being treated with benzodiazepine (BZD) hypnotics in combination with neuroleptics and 6 healthy male volunteers. In schizophrenic subjects, zopiclone (ZPC), 15 mg/day, was substituted for the BZD hypnotics, and PSGs were recorded again during ZPC therapy. All-night sleep stage scoring was carried out by visual analysis, and computerized period-amplitude analysis of sleep EEG was also performed. The schizophrenics showed marked reduction in the amount of slow-wave sleep (SWS) and in the number of delta half-waves during all-night sleep, especially those with higher amplitude, as compared to the normals. The number of delta half-waves in the patients was markedly reduced during the first sleep cycle. The average amplitude of delta half-waves during all-night sleep in the schizophrenics was significantly lower than that in the normals. The half-wave count of total delta waves in the schizophrenics was higher during treatment with ZPC than with BZDs, although no significant differences were observed in the amount of SWS between the two treatments. Soundness of sleep in the subjective sleep assessment was better evaluated during treatment with ZPC than BZDs. These results suggest that reduction of SWS in schizophrenia may be attributable mainly to the decrease in the number of delta waves with higher amplitude and that ZPC may induce deeper sleep in schizophrenics than BZDs.

A series of benzodiazepine receptor ligands with different chemical structures were evaluated for their affinities at diazepam-sensitive and diazepam-insensitive binding sites for [³H]Ro 15-4513 (ethyl-8-azido-5,60dihydro-5-methyl-6-oxo-4H-imidazo-[1,5a][1,4]benzodiazepine-3-carboxylate) in cerebellar GABA_A receptors. Rats of Wistar strain and of alcohol-sensitive (ANT) and alcohol-insensitive (AT) lines were used. The ANT rats possess a single point mutation in their GABA_A receptor α6 subunit, which makes their diazepam-insensitive sites sensitive to benzodiazepine agonists, unlike those of AT and Wistar rats. All compounds evaluated displayed high-affinity binding to diazepam-sensitive sites (K_i < 50 nM). In contrast, a wider range of affinities were observed at diazepam-insensitive sites which depended upon the basic structure and substitutions. The 7- and 8-halogen substituted imidazobenzodiazepines and 12-halogen substituted diimidazoquinazolines displayed the highest affinities (K_i < 15 nM), while intermediate to low affinities (100 < K_i < 4000 nM) were displayed by imidazoquinazolines, thienopyrimidines, one oxoimidazoquinoxaline, and some cyclopyrrolones. The imidazoquinoxalines evaluated displayed the lowest affinity (K_i > 10000 nM). The oxoimidazoquinoxaline, 6-chloro-3-(5-cyclopropyl-1,2,4-oxadiazol-3-yl)-4,5-dihydro-5-isopropyl-4-oxo-imidazol[1,5-a]quinoxaline (NNC 14-0578) and suriclone represent the first benzodiazepine receptor full agonists to bind with relatively high affinity (K_i ∼ 100 nM) to diazepam-insensitive sites. The 5 position substituted methoxybenzyl, dimethylallyl, and 4-fluorobenzyl oxoimidazoquinoxaline analogs demonstrated a 58–336-fold higher affinity for ANT than AT diazepam-insensitive sites. Classical benzodiazepines having a 5-phenyl substituent have demonstrated a similar preference for ANT sites, suggesting that all these structures bind to diazepam-insensitive sites in the same orientation. The other compounds evaluated demonstrated only a more modest selectivity (1–12-fold), indicating different structural requirements for binding to mutant ANT and wild-type AT and Wistar receptors. These results expand the range of ligands which display high affinity for diazepam-insensitive sites. Such compounds shoudl be helpful in determining intrinsic actions of high-affinity ligands at these sites and in assessing the contribution of these sites in enhanced sedative sensitivity of cerebellar function in the ANT rats.

The mechanism of action of the cyclopyrrolone hypnotic drug zopiclone involves allosteric modulation of the GABAA receptor. Zopiclone displaces the binding of [³H]-flunitrazepam with an affinity of 28 nM, and enhances the binding of the channel blocker [³⁵S]-TBPS. The binding of zopiclone, unlike that of hypnotic benzodiazepines, is not facilitated by GABA. Zopiclone does not distinguish between GABAA receptors containing different α-subunits (BZ₁ and BZ₂ phenotype). Studies with protein-modifying agents (eg diethylpyrocarbonate) and photoaffinity labelling suggest that cyclopyrrolones bind to a domain on the GABAA receptor different from the benzodiazepine binding domain. The consequence of this interaction with the GABAA receptor is to potentiate responses to GABA, as can be demonstrated by electrophysiological methods. Subchronic treatment of mice with high doses of zopiclone does not produce the changes in sensitivity of the GABAA receptor that are observed with hypnotic benzodiazepines.

The effects of the cyclopyrrolones suriclone and RP 59037 on body temperature were investigated in male TO mice. The full agonist suriclone (3, 10, 30 mg/kg i.p.) produced significant hypothermia which was inhibited by concurrent administration of benzodiazepine receptor antagonists of both benzodiazepine (flumazenil; 10 mg/kg i.p.) and β-carboline (ZK 93426; 3 mg/kg i.p.) structure. The response to suriclone (10 mg/kg i.p.) was also attenuated by benzodiazepine (Ro 17-1812; 10 mg/kg i.p.) and β-carboline (ZK 91296; 30 mg/kg i.p.) partial agonists — which have no effect on body temperature per se. In contrast with these compounds, the cyclopyrrolone partial agonist RP 59037 (10, 30 mg/kg i.p.) produced significant hypothermia itself (although it was much less efficacious in this respect than the full agonist) and at a dose of 30 mg/kg failed to block the decrease in body temperature induced by suriclone (10 mg/kg i.p.). Thus suriclone acts as a full agonist at benzodiazepine receptors in the body temperature paradigm. RP 59037 possesses some partial agonist properties in this model, however, it appears to have greater intrinsic activity than other partial agonists tested previously.