Investigation of the development of tolerance to the actions of zolpidem and midazolam

It is well established that tolerance can develop very rapidly to the behaviour-suppressing effects of benzodiazepines. In previous studies, however, the depressant action of zolpidem, a novel hypnotic acting at the benzodiazepine receptor, on operant behaviour in rats was maintained after many injections. An experiment was carried out, therefore, to compare the effects of acute and chronic administration of zolpidem and midazolam. Rats were trained to press a lever in standard operant test chambers to obtain 45 mg food pellets on an FR 10 schedule. Dose-response curves were then established, before, immediately after and 4 weeks after the daily administration of midazolam (3.0 mg/kg s.c.) or zolpidem (1.0 mg/kg) for 10 days. In confirmation of previous work, marked tolerance developed to the response-rate-decreasing effect of midazolam, the dose-response curve being shifted to the right by a factor of 6 and also flattened. No significant dissipation of this tolerance occurred during a period of 4-6 weeks. In contrast, repeated administration of zolpidem produced only a small degree of tolerance, the dose-response curve being shifted by a factor of two. There was little evidence for cross tolerance between the two drugs, zolpidem-treated rats being sensitive to a dose of midazolam and midazolam-treated rats sensitive to a dose of zolpidem. Although the explanation for the development of tolerance to midazolam is unknown, these results suggest that the mechanisms of action of midazolam and zolpidem in reducing response rates are different.     

Comparison of the effects of zaleplon,zolpidem, and triazolam at various GABA(A) receptor subtypes

The pyrazolopyrimidine zaleplon is a hypnotic agent that acts at the benzodiazepine recognition site of GABA(A) receptors. Zaleplon, like the hypnotic agent zolpidem but unlike classical benzodiazepines, exhibits preferential affinity for type I benzodiazepine (BZ(1)/omega(1)) receptors in binding assays. The modulatory action of zaleplon at GABA(A) receptors has now been compared with those of zolpidem and the triazolobenzodiazepine triazolam. Zaleplon potentiated GABA-evoked Cl(-) currents in Xenopus oocytes expressing human GABA(A) receptor subunits with a potency that was higher at alpha1beta2gamma2 receptors than at alpha2- or alpha3-containing receptors. Zolpidem, but not triazolam, also exhibited selectivity for alpha1-containing receptors. However, the potency of zaleplon at these various receptors was one-third to one-half that of zolpidem. Zaleplon and zolpidem also differed in their actions at receptors containing the alpha5 or gamma3 subunit. Zaleplon, zolpidem, and triazolam exhibited similar patterns of efficacy among the different receptor subtypes. The affinities of zaleplon for [(3)H]flunitrazepam or t-[(35)S]butylbicyclophosphorothionate ([(35)S]TBPS) binding sites in rat brain membranes were lower than those of zolpidem or triazolam. Furthermore, zaleplon, unlike zolpidem, exhibited virtually no affinity for the peripheral type of benzodiazepine receptor.     

Specificity within the GABAA receptor supramolecular complex: a consideration of the new omega 1-receptor selective imidazopyridine hypnotic zolpidem

The relative contribution of different recognition sites within the GABAA receptor supramolecular complex (GRSC) to the pharmacological effects of anxiolytic and hypnotic drugs is unknown. The development of the omega 1 (ex BZ1) specific hypnotic zolpidem allows a more direct approach to the problem. In contrast to many benzodiazepine hypnotic/anxiolytics (e.g., flunitrazepam, diazepam), zolpidem shows a specificity for GABAergic function, e.g., selectively reversing isoniazide-induced seizures. Furthermore, zolpidem produces a highly specific hypnotic action as compared to myorelaxant or amnesic effects (ratio of ED50's greater than 4.0 for zolpidem; less than 1 for flunitrazepam). Zolpidem exerts its action within the GRSC as it enhances 35S-TBPS binding, as do mixed omega 1/omega 2 compounds or GABA agonists. Both the in vivo and in vitro actions of zolpidem are reversed by flumazenil and the enhanced 35S-TBPS binding is also bicuculline-sensitive. Thus, omega 1 recognition site stimulation (e.g., by zolpidem) is sufficient to produce potent pharmacological effects and modulation of the GABAA receptor-gated chloride ionophore.     

Pharmacological profiles of benzodiazepinergic hypnotics and correlations with receptor subtypes]

We examined the behavioral pharmacological properties of six benzodiazepine (omega) receptor ligands including brotizoram, nitrazepam, quazepam, rilmazafone, zolpidem and zopiclone and the binding of these drugs with omega receptor subtypes. Behavioral tests were performed at the time of the maximal effects induced by each drug following its oral administration to mice. All of these drugs dose-dependently induced impairment of motor coordination as rotarod performance and potentiation of thiopental-induced anesthesia as hypnotic effect. The hypnotic effects of rilmazafone, whose major metabolites were bound to both omega1 and omega2 receptors with high affinity, and omega1 selective quazepam were about 20 times more effective than the induction of motor impairments when compared with ED50 values. However, there was no difference between the ED50 values of omega1 selective zolpidem alone in these two tests. An antianxiety efficacy of zolpidem was relatively weak unlike that of other drugs in the elevated plus-maze. It has been reported that omega2, but not omega1, receptors are associated with motor impairment and anxiolytic effect. The weak anxiolytic effect of zolpidem supports the previous hypothesis. However, the strong motor incoordination of zolpidem suggests that not only omega2 but also omega1 receptors are related to motor impairment unlike the previous hypothesis.     

Interaction of the hypnotic lormetazepam with central benzodiazepine receptor subtypes omega 1, omega 2 and omega 3]

To clarify the action of lormetazepam, 3-hydroxybenzodiazepine, on the benzodiazepine (BZ) receptor subtypes, effects of lormetazepam on motor performance in the traction test and hexobarbital-induced loss of righting reflex in mice and the binding to BZ receptor subtypes were investigated in comparison with those of other BZ hypnotics. Lormetazepam prolonged the duration of hexobarbital-induced loss of righting reflex. The minimal effective dose (1 mg/kg, p.o.) was higher than that of flunitrazepam, lower than those of diazepam and zopiclone, and the same as those of triazolam and brotizolam. Lormetazepam showed the ataxic effect at 10 mg/kg, p.o., but the separation between its effective doses for the hypnotic and ataxic effects was the largest among the hypnotics tested. In the displacement study on [3H]flumazenil binding to cerebellar and spinal cord membranes, lormetazepam bound with a higher affinity to omega 1 receptor (Ki = 10 nM) than to omega 2 receptor (Ki = 29 nM). The GABA-ratios of lormetazepam to omega 1 and omega 2 receptors were 3.9 and 4.0, respectively; and they were higher and lower than those of flunitrazepam to omega 1 and omega 2 receptors, respectively. In the displacement study on [3H] Ro5-4864 binding to kidney membranes, lormetazepam bound with a lower affinity to the omega 3 receptor (Ki = 213 nM) than flunitrazepam. Thus, lormetazepam was suggested to be a potent hypnotic with weaker ataxic effects than other BZ hypnotics, which may be due to its selective and potent agonistic action on central omega 1 receptors.     

Effects of zolpidem,a new imidazopyridine hypnotic,on the acquisition of conditioned fear in mice

Benzodiazepines and other compounds which act at benzodiazepine binding sites have been shown previously to attenuate the acquisition of conditioned fear in rodents when administered before the acquisition session, an effect which may parallel the disruption of human memory produced by anxiolytics and sedatives. Such an action is usually, but not invariably, produced by doses which have direct behavioural depressant effects. The present study was carried out to extend previous work by investigating the effects of the hypnotic benzodiazepine triazolam and the nonbenzodiazepines zolpidem and CL 218,872 on the acquisition of learned fear in mice. All these drugs reduced locomotor activity shortly after injection. They also produced disruptions of the acquisition of learned fear. Triazolam exerted behavioural effects similar to those found previously with other benzodiazepines, the doses which disrupted the acquisition of conditioned fear being similar to, or lower than, the doses which depressed locomotion. In contrast, the results indicated that zolpidem was more potent at reducing locomotion than at interfering with fear conditioning, a result which may reflect the preferential sedative action of zolpidem.     

Zolpidem for insomnia

INTRODUCTION: The imidazopyridine derivative zolpidem , which acts as a benzodiazepine (BZ) receptor agonist, is the most widely prescribed hypnotic drug in the US. AREAS COVERED: This review addresses the neuroreceptor properties of zolpidem; clinical pharmacokinetics, pharmacodynamics and drug interactions; efficacy as a hypnotic; adverse effects; tolerance, dependence and withdrawal; relation to motor vehicle accidents and complex sleep behaviors; and new dosage forms. EXPERT OPINION: Approved doses of zolpidem (10 mg for adults, 5 mg for the elderly) are consistently effective in reducing sleep latency and consequently increasing sleep duration in patients with insomnia. However, favorable effects on sleep maintenance are observed less consistently. Residual daytime effects are unlikely with recommended doses, and provided that at least 8 h elapse prior to arising. Hypnotic efficacy is maintained with repeated nightly use, and the risk of rebound insomnia is low. Dependence and abuse of zolpidem are no more likely to occur than with typical benzodiazepines. Newly available novel dosage forms of zolpidem have increased therapeutic options for patients with insomnia variants such as sleep maintenance insomnia and middle-of-the-night awakening.     

The interaction between zolpidem and beta-CMC: a clue to the identification of receptor sites involved in the sedative effect of zolpidem

The interaction of beta-CMC, an amino beta-carboline recently described as a selective antagonist of the sedative effect of diazepam, with zolpidem, an imidazopyridine hypnotic, which like beta-CMC binds preferentially to the omega 1 (BZ-1) site of the GABA benzodiazepine chloride channel receptor complex, was investigated. In mice, beta-CMC antagonized the effect of zolpidem against isoniazid-induced convulsions without affecting its activity against convulsions induced by pentylenetetrazole or electroshock. beta-CMC also antagonized the decrease in locomotor activity and the impairment in muscle strength provoked by zolpidem. In rats trained to discriminate zolpidem, beta-CMC antagonized both the interoceptive stimulus and the decrease in the rate of lever pressing produced by zolpidem. This selective antagonism, inhibition of effects of zolpidem exerted by low doses (locomotor activity and isoniazid-induced convulsions) as well as effects produced by high doses (muscle strength) but not those provoked by intermediate doses (pentylenetetrazole and electroshock-induced convulsions), could not be explained by a receptor occupancy hypothesis. These results suggest that the anticonvulsant and sedative effects of zolpidem do not involve the same receptor subtype and that the hypnoselective properties of zolpidem may be linked to its selectivity for the omega 1 (BZ-1) site of the GABAA receptor.     

Differences in pharmacological profiles of a new generation of benzodiazepine and non-benzodiazepine hypnotics

The hypnotics, quazepam (a benzodiazepine), brotizolam (a thienotriazolodiazepine), zopiclone (a cyclopyrrolone) and zolpidem (an imidazopyridine) have a common ability to bind to the benzodiazepine recognition site (omega receptor) within the GABAA receptor. For this reason we compared their pharmacological profiles in mice. All compounds shared anticonvulsant and central depressant effects. However, the sedative activity of zolpidem appeared at much lower doses than did the anticonvulsant and myorelaxant effects but the opposite was observed with the other hypnotics. In contrast to brotizolam, quazepam and zopiclone, zolpidem did not increase food intake in mice placed in a novel environment, indicating that this drug lacks disinhibitory activity. Moreover the efficacy of zolpidem at the GABAA receptor, as indicated by its activity against convulsions induced by the GABA synthesis inhibitor, isoniazid, was much greater than that of other hypnotics. These results suggest that the hypnoselective properties observed with zolpidem might be related to a high selectivity for the omega 1 recognition site of the GABAA receptor coupled with a very high intrinsic activity.     

Different effects of L-type and T-type calcium channel blockers on the hypnotic potency of triazolam and zolpidem in rats.

We examined the effects of an L-type Ca2+ channel blocker, nilvadipine (0.5 and 2.0 mg/kg), and that of a T-type Ca2+ channel blocker, flunarizine (10.0 and 40.0 mg/kg), on the hypnotic potency of both a benzodiazepine (BZ)-hypnotic, triazolam (1.0 mg/kg), and a non-BZ hypnotic, zolpidem (20.0 mg/kg), in rats. The polysomnogram was recorded for 6 h after administration of the vehicle solution alone, or after one of the Ca2+ channel blockers, with or without one of the hypnotics. Both Ca2+ channel blockers prolonged the increased total time of non-rapid eye movement (non-REM) sleep induced by either hypnotic. In the case of triazolam, however, the non-REM sleep-enhancing effect induced by nilvadipine was greater than that induced by flunarizine. These findings indicate that the hypnotic action of triazolam is potentiated more strongly by an L-type Ca2+ channel blocker than by a T-type Ca2+ channel blocker.     

125I]iodoclonazepam, a specific high affinity radioligand for the identification of BZ1 and BZ2 sites in rat brain.

Binding of the radioligand [125I]iodoclonazepam to three different areas of rat brain (cerebellum, hippocampus and striatum) has been characterised. In all three regions binding is rapid, saturable and of high affinity (cerebellum Bmax = 1.49 +/- 0.3 pmol/mg of protein, Kd = 0.39 +/- 0.06 nM; hippocampus Bmax = 1.5 +/- 0.14 pmol/mg of protein, Kd = 0.38 +/- 0.6 nM and striatum Bmax = 0.53 +/- 0.1 pmol/mg of protein, Kd = 0.34 +/- 0.03 nm, n = 3). In all regions only one population of sites was apparent. However, competition for [125I]iodoclonazepam sites by a series of benzodiazepine agonists and antagonists showed some regional differences. The BZ1 selective compounds zolpidem and CL218,872 showed a 4.3-fold and 5.2-fold selectivity for cerebellar binding sites respectively. We conclude that [125I]iodoclonazepam is a novel, high affinity ligand which recognises both BZ1 and BZ2 classes of receptor and should be a useful addition to the panel of benzodiazepine ligands currently available.     

Benzodiazepine-induced intestinal motor disturbances in rats: mediation by omega 2 (BZ2) sites on capsaicin-sensitive afferent neurones.

1. The central and peripheral effects of the omega (benzodiazepine) site ligands, clonazepam, alpidem, zolpidem, triazolam, flumazenil, ethyl beta carboline-3-carboxylate (beta-CCE) and N-methyl beta carboline-3-carboxylate (beta-CCM) on intestinal myoelectrical activity were evaluated in conscious rats, chronically fitted with Nichrome electrodes implanted on the duodenum and jejunum. The localization of the omega (benzodiazepine) receptors involved in these effects was evaluated by use of systemic and perivagal capsaicin treatments. 2. When administered intraperitoneally (i.p.) the omega site inverse agonists beta-CCE and beta-CCM, and the omega site antagonist flumazenil, did not affect the duodeno-jejunal motility. Alpidem and zolpidem, two selective omega 1 site agonists induced an inhibition of migrating myoelectric complexes (MMCs) only at a high dose (5 mg kg-1). In contrast, clonazepam (a mixed omega 1/omega 2 agonist) and triazolam (a preferential omega 2 site agonist) disrupted the MMC-pattern at doses as low as 0.05 mg kg-1, the effect of trizolam being of much longer duration than that of clonazepam. None of these drugs altered MMC-pattern when administered centrally (i.c.v.). 3. Administered i.p. or i.c.v. prior to triazolam, alpidem blocked the effect of triazolam on duodenojejunal spike activity. Administered i.p. prior to triazolam, flumazenil suppressed the triazolam-induced MMC-disruption. Previous systemic but not perivagal capsaicin treatment suppressed the effects of clonazepam on MMCs. 4. It is concluded that omega-site agonists but not, antagonist or inverse agonists, administered systemically induced intestinal motor disturbances which may be linked to activation of omega 2 (BZ2) sites located on nonvagal capsaicin-sensitive afferent neurones.     

Discriminative stimulus effects of drugs acting at GABA(A) receptors: differential profiles and receptor selectivity.

The GABA(A) receptor complex contains a number of binding sites at which a variety of psychotropic drugs, including benzodiazepines, barbiturates, and some neurosteroids, act to potentiate or inhibit the effect of the transmitter. Many studies have reported that these drugs can produce discriminative stimulus actions, but the cueing effects of compounds acting at different sites to enhance the effects of GABA are not identical. The discriminative stimulus effects of benzodiazepines have been analyzed in detail, and there is also a great deal of information available on the effects of nonbenzodiazepine compounds acting at BZ(omega) recognition sites, which form part of the GABA(A) receptor complex. Of particular interest are compounds with selectivity for the BZ1(omega1) receptor subtype including zolpidem, zaleplon, and CI 218,872. BZ1(omega1)-selective drugs substitute for the discriminative stimulus produced by chlordiazepoxide only partially and at sedative doses. This is consistent with the view that sedative effects of BZ(omega) receptor agonists are mediated by the BZ1(omega1) receptor subtype, whereas the discriminative stimulus produced by chlordiazepoxide may be produced by activity at the BZ2(omega2) subtype. Analysis of this hypothesis is complicated by the variety of levels of intrinsic activity shown by different drugs.     

Gaboxadol -- a different hypnotic profile with no tolerance to sleep EEG and sedative effects after repeated daily dosing

Gaboxadol, a selective extra synaptic GABA(A) receptor agonist, has been in clinical development for the treatment of insomnia. Development of tolerance to therapeutic effects (e.g. hypnotic and anticonvulsant and sedative) and withdrawal symptoms (e.g. REM sleep rebound and reduced seizure threshold) upon treatment discontinuation is reported for GABA(A) receptor allosteric modulators acting via the benzodiazepine binding site, e.g. zolpidem and indiplon. We conducted a head to head comparison in rats of the hypnotic (sleep EEG after 21 daily doses and 24 and 48 h after the last dose) and seizure threshold modifying (bicuculline assay 24 h after 28 daily doses) effects of gaboxadol and benzodiazepine ligands. Furthermore, we investigated in further details a previously reported apparent rapid development of tolerance to gaboxadol's effects in a rat rotarod motor coordination assay and related this effect to CNS exposure levels and in vitro potency at extra synaptic GABA(A) receptors. Sleep EEG studies demonstrated lack of tolerance and withdrawal effects after 28 daily doses with gaboxadol, whereas zolpidem produced both tolerance and withdrawal effects under a similar dosing regimen. Daily dosing with gaboxadol, zolpidem or indiplon for 28 days and acute discontinuation of treatment left the threshold to bicuculline-induced seizures unchanged. The rapidly attenuated effect of repeated gaboxadol dosing was confirmed in the rotarod model. However, re-challenge of gaboxadol insensitive animals with gaboxadol produced a maximum response, ruling out that receptor desensitisation accounts for these effects. By comparing CNS exposure at rotarod responses and concentration response relation at cloned GABA(A) receptors expressed in Xenopus oocytes it appears that the decline in response in the rotarod model coincides with the steep part of the concentration response curve for gaboxadol at extra synaptic GABA(A) receptors. In conclusion, rat sleep EEG repeated dose studies of gaboxadol confirm a hypnotic-like profile and no withdrawal effects, whereas tolerance and withdrawal effects were shown with zolpidem. Withdrawal from gaboxadol, zolpidem and indiplon did not affect the seizure threshold to bicuculline. Gaboxadol's apparent rapid development of tolerance in the rotarod assay appears to be kinetically determined.     

Novel benzodiazepine receptor ligands: palatable food intake following zolpidem, CGS 17867A, or Ro23-0364, in the rat

The potent imidazopyridine hypnotic, zolpidem, binds to central benzodiazepine receptors and has predominantly sedative properties, as determine in animal models. In tests of palatable food consumption in nondeprived male rats, the present results indicate that zolpidem (0.3-3.0 mg/kg) had no effect on food intake. Its lack of effect contrasts sharply with other benzodiazepine agonists which strongly stimulate palatable food intake. Two other novel compounds, both of which bind to benzodiazepine receptors, and which have reduced propensity to induce sedative effects, increased palatable food consumption, although in differing ways. The imidazobenzodiazepine Ro23-0364 (0.3-10.0 mg/kg) dose-dependently increased feeding in the standard procedure, but failed to stimulate food intake in presatiated animals. The pyrazoloquinoline CGS 17867A (1.0-30.0 mg/kg) increased food intake in both test procedures, although the dose-effect relationship was nonmonotonic. Taken together, the data indicate a probable separation between hyperphagic and sedating effects of benzodiazepine receptor agonists. If zolpidem's sedative effect is linked to an action at a receptor subtype (benzodiazepine Type 1 or omega 1), then the hyperphagic effect of benzodiazepines may depend more on the alternative subtype.     

Tolerance,cross-tolerance and dependence measured by operant responding in rats treated with triazolam via osmotic pumps

Previous research has found that drugs with affinity for (benzodiazepine) sites differ in their abilities to produce tolerance and dependence. The present study therefore investigated the effects of ligands of (BZ) sites in rats that had been rendered tolerant to a benzodiazepine. Two experiments were carried out in separate groups of rats. Behavioral changes induced by chronic infusion of triazolam (3 mg/kg/day, SC, for 14 days) via osmotic pumps were studied in animals trained on a fixed ratio 10 schedule of food presentation. Control animals were implanted with pumps containing the vehicle. Test drugs were administered IP using cumulative dosing. In one experiment triazolam decreased response rates on days 1, 2 and 3 after implantation of the pumps and tolerance developed to this depressant effect. In the other experiment, vehicle and triazolam treated rats differed in their responding during chronic infusion but differences were not statistically significant on any particular day. Flumazenil (3.0–30 mg/kg) greatly decreased rates of responding on day 11 in triazolam treated rats. This effect may represent a precipitated withdrawal syndrome. However, no withdrawal effects on operant performance were observed upon pump removal. Chronic infusion of triazolam did not affect the sensitivity of rats to alpidem on day 11 (10–100 mg/kg) whereas it abolished the stimulant effect of bretazenil (0.1–1.0 mg/kg). Chronic triazolam treatment produced tolerance to the depressant effects of triazolam (1.0–3.0 mg/kg), lorazepam (0.3–3.0 mg/kg) and zopiclone (10 mg/kg) but no tolerance to those of CL 218,872 (3.0–30 mg/kg) and zolpidem (0.3–3.0 mg/kg) when tested 3–14 days after pump removal. Differences between compounds highlighted with this model are in agreement with previous observations that these agents possess different pharmacological profiles and different potentials to induce tolerance and dependence.     

GABA(A) receptor alpha-1 subunit deletion alters receptor subtype assembly,pharmacological and behavioral responses to benzodiazepines and zolpidem

Potentiation of GABA(A) receptor activation through allosteric benzodiazepine (BZ) sites produces the anxiolytic, anticonvulsant and sedative/hypnotic effects of BZs. Using a mouse line lacking alpha1 subunit expression, we investigated the contribution of the alpha1 subunit to GABA(A) receptor pharmacology, function and related behaviors in response to BZ site agonists. Competitive [(3)H]flunitrazepam binding experiments using the Type I BZ site agonist, zolpidem, and the Type I and II BZ site non-specific agonist, diazepam, demonstrated the complete loss of Type I BZ binding sites in alpha1(-/-) mice and a compensatory increase in Type II BZ binding sites (41+/-6%, P<0.002). Chloride uptake analysis in alpha1(-/-) mice revealed an increase (108+/-10%, P<0.001) in the efficacy (E(max)) of flunitrazepam while the EC(50) of zolpidem was increased 495+/-26% (alpha1(+/+): 184+/-56 nM; alpha1(-/-): 1096+/-279 nM, P<0.01). An anxiolytic effect of diazepam was detected in both alpha1(+/+) and alpha1(-/-) mice as measured on the elevated plus maze; however, alpha1(-/-) mice exhibited a greater percentage of open arm entries and percentage of open arm time following 0.6 mg/kg diazepam. Furthermore, alpha1(-/-) mice were more sensitive to the motor impairing/sedative effects of diazepam (1-10 mg/kg) as measured by locomotor activity in the open field. Knockout mice were insensitive to the anticonvulsant effect of diazepam (1-15 mg/kg, P<0.001). The hypnotic effect of zolpidem (60 mg/kg) was reduced by 66% (P<0.001) in alpha1(-/-) mice as measured by loss of righting reflex while the effect of diazepam (33 mg/kg) was increased 57% in alpha1(-/-) mice (P<0.05). These studies demonstrate that compensatory adaptations in GABA(A) receptor subunit expression result in subunit substitution and assembly of functional receptors. Such adaptations reveal important relationships between subunit expression, receptor function and behavioral responses.     

Biochemical Characterization of the Effects of the Benzodiazepine,Midazolam, on Mitochondrial Electron Transfer

Abstract: Midazolam, a water soluble benzodiazepine used as a preanaesthetic and hypnotic drug, showed a concentration-related (0.1-0.75 mM) depressant effect on both Adenosine 5′-diphosphate (ADP)-induced oxygen consumption and oxidative phosphorylation of rat liver mitochondria if the substrate was oxidized at different steps in the oxidation chain, but not when the substrate was ascorbate plus tetramethyl-p-phenylenediamine (complex IV). Furthermore, midazolam did not affect citrate synthase activity, but inhibited the 2,4 dinitrophenol (DNP)-uncoupled mitochondrial respiration. This result shows that midazolam primarily acts as a mitochondrial electron transport inhibitor. This inhibition is mainly due to the fact that midazolam decreases NADH ubiquinone reductase (complex I) and ubiquinol cytochrome c reductase (complex III) activities, but it also inhibits complex II activity. Spectrophotometric measurements of redox states of rat skeletal muscle mitochondria cytochromes show a decrease in the reduction of aa₃ and c+c₁ cytochromes in the presence of the benzodiazepine. Midazolam significantly decreased the reduced ubiquinone/total ubiquinone ratio (evaluated by means of HPLC and electrochemical detection) in rat liver mitochondria in both P-hydroxybutyrate and succinate. Ubisemiquinone may be the redox component affected by midazolam, whether or not bound to the iron-sulfur proteins present in all three mitochondrial complexes. These effects of midazolam, not necessarily related to the preanaesthetic and hypnotic action are probably mediated via mitochondrial benzodiazepine receptors.     

The pharmacology and mechanisms of action of new generation, non-benzodiazepine hypnotic agents

The new generation hypnotic drugs, zolpidem, zopiclone and zaleplon, are at least as efficacious in the clinic as benzodiazepines and may offer advantages in terms of safety. These drugs act through the BZ binding sites associated with GABAA receptors, but show some differences from benzodiazepines in pharmacological effects and mechanisms of action. Of particular interest is the finding that zolpidem shows a wide separation between doses producing sedative effects and those giving rise to other behavioural actions, and induces less tolerance and dependence than benzodiazepines. Zolpidem also demonstrates selectivity for GABAA receptors containing alpha1 subunits. Recent studies using genetically modified mice have confirmed that receptors containing alpha1 subunits play a particularly important role in mediating sedative activity, thus providing an explanation for the pharmacological profile of zolpidem.