Effect of chronic administration of alprazolam and adinazolam on clonidine- or apomorphine-induced aggression in laboratory rodents

The activity of chronic (3 weeks) treatment with the triazolobenzodiazepines, alprazolam and adinazolam, on clonidine- and apomorphine-induced aggression were studied. Adinazolam, like desipramine, potentiated aggression induced by clonidine while diazepam and alprazolam completely abolished it. In the apomorphine-induced aggression, adinazolam suppressed both aggressivity and stereotypy, while diazepam slightly potentiated it. Alprazolam did not modify the effect of aggression induced by apomorphine. On the whole, while adinazolam seemed to develop an activity closer to that of a classical antidepressant like desipramine, alprazolam appeared to be more similar to the benzodiazepines on clonidine-induced aggression in mice. Compared to desipramine and diazepam, adinazolam left these two effects induced by apomorphine almost unchanged. The experiments performed showed differences between the profiles of action of the two triazolobenzodiazepines studied.     

Pharmacological profile of the antidepressant adinazolam, a triazolobenzodiazepine

Adinazolam, which is a 1-dimethylaminomethyl triazolobenzodiazepine, is an effective anxiolytic agent as defined by suppression of stress-induced increases in plasma corticosteroids. Adinazolam is also an effective antagonist of pentylenetetrazole. The 1-dimethylaminoethyl triazolo analog, U-43,465F, was inactive in the stressed rat test and only weakly active against pentylenetetrazole. Adinazolam and U-43,465F have been previously shown to have antidepressant activity in classical screening tests. They have also been found to potentiate the effect of norepinephrine and this is consistent with the activity of the known antidepressants; U-43,465F was found to be equieffective to imipramine in this test. Adinazolam was also effective; however, the magnitude of the potentiation was not as great. The uptake of norepinephrine was only weakly affected by either compound. Potentiation or uptake of serotonin were not significantly-altered pharmacological factors. Receptor binding studies were negative except at the benzodiazepine receptor. Chronic treatment with adinazolam did not decrease the number of beta-adrenergic receptors in the cerebral cortex of the rat, in contrast to the positive effect of imipramine. The discovery of triazolobenzodiazepines with antidepressant activity is of special interest. These agents will hopefully have lower toxicity than the tricyclic antidepressants and thus possess a more favourable therapeutic index. This would be advantageous in the treatment of depression.     

U 43,465F: a benzodiazepine with antidepressant activity? Interaction with Ro 15-1788 and d,1-propranolol

U-43,465F (8-chloro-1-[2-(dimethylamino)ethyl]-6-phenyl-4H-s-triazolo [4,3-a][1,4]benzodiazepine p-toluene sulfonate) showed two fold activity at 32 mg/kg i.p.: antipunishment activity inhibited by the benzodiazepine receptor antagonist Ro 15-1788, and an antidepressant-like reversal of apomorphine-induced hypothermia, antagonized by the beta-adrenergic receptor blocker d,1-propranolol. U-43,465F hence produced effects not mediated by benzodiazepine receptors and these effects might be related to antidepressant activity.     

Comparison of the spectrum of cognitive effects of alprazolam and adinazolam after single doses in healthy subjects

Single doses of alprazolam (0, 0.5, 1.5 mg) or adinazolam mesylate sustained release tablets (SR) (0, 15, 45 mg) were administered to separate groups of 12 healthy men in a crossover design. Psychomotor performance was assessed by digit symbol substitution (DSST), and memory was assessed using a test battery which reflects various aspects of memory, including attention/working memory, explicit memory (recall of categorically related words), semantic memory (fragmented picture recognition, generation of category exemplars), and implicit memory (time saved in resolving fragmented pictures on the second exposure). Maximal psychomotor performance and memory decrements for the highest active doses were significantly different from placebo for all tasks at some time after dosing. The maximum decrement in DSST was not significantly different between drugs at the high dose (P=0.288). Maximum attention/working memory decrements were significantly different between the high doses of the active compounds (P=0.031), and the difference in maximum category recall decrement was marginally significant (P=0.067). Access to knowledge memory was not significantly altered by these drugs; these results are similar to those obtained for other benzodiazepines. Both drugs exhibited slight effects on implicit memory. The results suggest that the sedative and memory effects of these triazolobenzodiazepines may not be closely related and suggest that adinazolam has a somewhat different spectrum of cognitive effects relative to alprazolam.     

Inhibition of the binding and the behavioral effects of thyrotropin-releasing hormone (TRH) by the triazolobenzodiazepines

The abilities of 4 triazolobenzodiazepines, adinazolam, alprazolam, estazolam and triazolam, to inhibit thyrotropin-releasing hormone (TRH) receptor binding and to antagonize the narcoleptic effects of TRH were examined. The IC50 values for inhibition of 3H-3-methyl-His-2-TRH (MeTRH) binding ranged from 19 microM to 477 microM, and the Hill coefficient from 0.53 to 0.98. Similar ranges of values were obtained from benzodiazepines of other structural classes. Thus, the inhibition of TRH receptor binding by the triazolobenzodiazepines is similar to that produced by other types of benzodiazepines. Furthermore, the triazolobenzodiazepine, alprazolam, antagonized the narcoleptic effect of TRH. However, this action is not necessarily linked to its inhibition of TRH receptor binding since alprazolam also inhibited the narcoleptic effect of amphetamine.     

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.     

Kinetics and dynamics of intravenous adinazolam, N-desmethyl adinazolam, and alprazolam in healthy volunteers

The pharmacokinetics and pharmacodynamics of adinazolam mesylate (10 mg), N-desmethyl adinazolam mesylate (NDMAD, 10 mg), and alprazolam (1 mg) were investigated in 9 healthy male subjects in a randomized, blinded, single-dose, 4-way crossover study. All drugs were intravenously infused over 30 minutes. Plasma adinazolam, NDMAD, and alprazolam concentrations, electroencephalographic (EEG) activity in the beta (12-30 Hz) range, performance on the Digit Symbol Substitution Test (DSST), and subjective measures of mood and sedation were monitored for 12 to 24 hours. Mean pharmacokinetic parameters for adinazolam, NDMAD, and alprazolam, respectively, were as follows: volume of distribution (L), 106, 100, and 77; elimination half-life (hours), 2.9, 2.8, and 14.6; and clearance (mL/min), 444, 321, and 84. More than 80% of the total infused adinazolam dose was converted to systemically appearing NDMAD. All 3 benzodiazepine agonists significantly increased beta EEG activity, with alprazolam showing the strongest agonist activity and adinazolam showing the weakest activity. Alprazolam and NDMAD significantly decreased DSST performance, whereas adinazolam had no effect relative to placebo. Adinazolam, NDMAD, and alprazolam all produced significant observer-rated sedation. Plots of EEG effect versus plasma alprazolam concentration demonstrated counterclockwise hysteresis, consistent with an effect site delay. This was incorporated into a kinetic-dynamic model in which hypothetical effect site concentration was related to pharmacodynamic EEG effect via the sigmoid E(max) model, yielding an effect site equilibration half-life of 4.8 minutes. The exponential effect model described NDMAD pharmacokinetics and EEG pharmacodynamics. The relation of both alprazolam and NDMAD plasma concentrations to DSST performance could be described by a modified exponential model. Pharmacokinetic-dynamic modeling was not possible for adinazolam, as the data did not conform to any known concentration-effect model. Collectively, these results indicate that the benzodiazepine-like effects occurring after adinazolam administration are mediated by mainly NDMAD.     

The open field as a paradigm to measure the effects of drugs on anxiety-like behaviors: a review

The open field is a very popular animal model of anxiety-like behavior. An overview of the literature on the action elicited by effective or putative anxiolytics in animal subjected to this procedure indicates that classical treatments such as benzodiazepine receptor full agonists or 5-HT(1A) receptor full or partial agonists elicit an anxiolytic-like effect in this procedure in most cases (approximately 2/3). However, compounds (triazolobenzodiazepines such as adinazolam and alprazolam, selective serotonin reuptake inhibitors) that have a different spectrum of therapeutic efficacy in anxiety disorders such as panic attacks, generalized anxiety disorder or obsessive-compulsive disorder were poorly effective as anxiolytics in the open field test, suggesting that this paradigm may not model features of anxiety disorders. The procedure is also relevant for the study of compounds endowed with anxiogenic effects, as such effects were detected after treatments with benzodiazepine receptor inverse agonists or with corticotropin releasing factor (CRF) receptor agonists.     

Anxiolytic activity on locus coeruleus-mediated suppression of muricidal aggression

The evidence suggests that stimulation of brain noradrenergic neurons plays an inhibitory role in rat mouse-killing (muricidal) aggression. Anxiolytic benzodiazepines inhibit locus coeruleus activity and previous data showed that chlordiazepoxide was capable of antagonizing the locus coeruleus-mediated suppression of muricidal aggression. The present experiments showed that this effect is common to new anxiolytic triazolobenzodiazepines and to other non-benzodiazepine derivatives with anxiolytic activity. In this framework, 10 mg/kg of buspirone, of 1-pyrimidine-piperazine and of MJ-13805 proved to be as active as 2.5 mg/kg of alprazolam and as 5 mg/kg of chlordiazepoxide in inhibiting the locus coeruleus-mediated suppression of muricidal aggression.     

A re-evaluation of the role of alpha 2-adrenoceptors in the anxiogenic effects of yohimbine,using the selective antagonist delequamine in the rat.

1. The acute behavioural effects of the alpha2-adrenoceptor antagonists, yohimbine, idazoxan and delequamine (RS-15385-197) were compared in two tests of exploratory behaviour in the rat, operated in tandem. These were the elevated X-maze test (5 min) and a modified holeboard test (12 min), which comprised a holeboard arena with a small roof in one corner as a ''refuge''. Rats were first placed into this corner, thus enabling measurements of initial emergence latency and the number of forays. The experiments were always done with a concomitant vehicle control group, with 10-12 rats per group, and with the treatment blinded. 2. In order to validate the tests, the effects of representatives of four classes of psychoactive agents were examined, viz. picrotoxin (anxiogenic), chlordiazepoxide (anxiolytic), (+)-amphetamine (stimulant) and diphenhydramine (sedative). The modified holeboard tended to be more sensitive than the measurement of total arm entries in the elevated X-maze at detecting drug effects on exploratory behaviour, but unlike the X-maze it could not clearly identify each class of agent. Thus, picrotoxin (5 mg kg(-1), i.p.) reduced total arm entries and open arm exploration in the X-maze (P<0.02) and suppressed most measures of activity in the holeboard (P<0.05); chlordiazepoxide (7.5 mg kg(-1), i.p.) increased total arm entries and open arm exploration (P<0.02) in the X-maze, without clear-cut effects in the holeboard; (+)-amphetamine (1 mg kg(-1), i.p.) had no significant effects in the X-maze, but increased most holeboard activities (P<0.05), and diphenhydramine (30 mg kg(-1), i.p.) reduced total arm entries in the X-maze (P<0.002) and hole exploration in the holeboard (P<0.05).     

Benzodiazepines reduce gastric ulcers induced in rats by stress.

1 The sedative and antiulcer effects of chlordiazepoxide (5 to 50 mg/kg) and lorazepam (0.25 to 2.5 mg/kg) were investigated in the rat. 2 Sedation was measured by recording locomotor activity in a holeboard. Ulceration of the glandular stomach was induced by a 2 h period of restrain at 4 degrees C. 3 Acutely, both drugs produced significant sedation at all doses; high doses only (chlordiazepoxide 10 and 50 mg/kg; lorazepam 2.5 mg/kg) produced a significant reduction in ulcer formation. 4 With chronic treatment, after 5 and 10 days administration of chlordiazepoxide (50 mg/kg), tolerance to sedation was observed without a similar change in antiulcer action. 5 Cimetidine (20 mg/kg) and atropine (0.2 mg/kg) decreased ulcer formation without causing sedation. 6 The antisecretory profile of chlordiazepoxide (2 x 10(-4) M), in the rat isolated gastric mucosa, resembled that of atropine (10(-7) M) rather than cimetidine (10(-5) M). 7 These observations suggest that the antiulcer effect of benzodiazepines probably results from a combination of sedative, anxiolytic and antisecretory actions.     

Double-blind comparison of alprazolam and adinazolam for panic and phobic disorders

Subjects primarily suffering from DSM-III panic disorders (agoraphobia with panic attacks, 13 subjects; panic disorder, one subject) were tested with two drug treatments after a baseline was established using single-blind placebo capsules three times daily for 1 week. Double-blind, 4-week crossover treatments were given with alprazolam, 0.5-6.0 mg/day, and adinazolam mesylate, 10-120 mg/day. Mean final doses in mg/day were 3.1 for alprazolam and 95.5 for adinazolam mesylate. Both were broadly effective in comparison with the baseline condition. Measures included self-rated symptoms and global impressions, physician-rated global impressions, and two forms of challenges, agoraphobic and noradrenergic. The two active drugs were highly similar in overall efficacy across the sample, but alprazolam was favored globally in six subjects, and adinazolam was favored globally in another six subjects. Only two subjects obtained maximal improvement ratings without side effects with both drugs. No diagnostic or demographic factor correlated with the differential in responses to the two active treatments. No clinically significant laboratory abnormalities occurred with either drug.     

Caffeine-induced anxiogenesis: the role of adenosine, benzodiazepine and noradrenergic receptors

The purpose of this study was to determine the mechanism by which caffeine increases anxiety. Rats were tested in the social interaction test of anxiety after administration of caffeine (20 or 40 mg/kg) alone or in combination with various compounds. In order to investigate the role of adenosine receptors, caffeine was given in combination with 2-chloroadenosine (0.1 and 1 mg/kg). To investigate the role of benzodiazepine receptors, chlordiazepoxide (5 mg/kg), a benzodiazepine antagonist, flumazenil (RO 15-1788, 1 and 10 mg/kg) and a triazolobenzodiazepine U-43,465 (32 mg/kg) were used. Finally, an alpha 2-receptor agonist, clonidine (0.1 and 0.025 mg/kg) and a beta-adrenoceptor antagonist, DL-propranolol (5 mg/kg), were used to study the role of noradrenergic systems in the effects of caffeine. Caffeine (20 and 40 mg/kg) reduced the time spent in social interaction and this effect was antagonized by chlordiazepoxide, U-43,465 and DL-propranolol, but not by flumazenil, 2-chloroadenosine or clonidine. It was therefore concluded that the anxiogenic effect of caffeine was unlikely to be due to its effects at adenosine or benzodiazepine receptors. It is suggested that the reversal of caffeine's effects by chordiazepoxide may have been "functional," i.e., merely a cancellation of two opposite effects. It is discussed whether the reversal of caffeine's effects by propranolol and U-43,465 are functional, or reflect a noradrenergic site of action.     

Anxiolytic effect of wogonin,a benzodiazepine receptor ligand isolated from Scutellaria baicalensis Georgi

The search for novel anxiolytics devoid of undesirable side-effects typical of classical benzodiazepines (BDZs) has been intense, and flavonoids, as a relative new class of ligands, have been shown to possess anxiolytic effects in vivo. The present study evaluated the pharmacological properties of a naturally occurring monoflavonoid, 5,7-dihydroxy-8-methoxyflavone or wogonin. The affinity (K(i)) of wogonin for the benzodiazepine site (BZD-S) on the gamma-aminobutyric acid(A) (GABA(A)) receptor complex was 0.92 microM. Using electrophysiological techniques, we showed that wogonin enhanced the GABA-activated current in rat dorsal root ganglion neurons, and in Xenopus laevis oocytes expressing recombinant rat GABA(A) receptors, the enhancement was partially reversed by the co-application of a 1 microM concentration of the BZD-S antagonist anexate (Ro15-1788). Acute toxicity and behavioral effects were examined in mice. Acute lethal activity was low, with an LD(50) of 3.9 g/kg. Oral administration of wogonin (7.5 to 30 mg/kg) elicited an anxiolytic response that was similar to that elicited by diazepam in the elevated plus-maze; a dose-dependent increase in open arm entries and time spent in open arms was observed. More importantly, its anxiolytic effect was blocked by the co-administration of Ro15-1788. In the holeboard test, not only did wogonin-treated mice experience an increased number of head-dips but they also spent more time at it, showing no signs of sedation. Furthermore, wogonin did not cause myorelaxant effects in the horizontal wire test. Taken together, these data suggest that wogonin exerts its anxiolytic effect through positive allosteric modulation of the GABA(A) receptor complex via interaction at the BZD-S. Its anxiolytic effect was not accompanied by sedative and myorelaxant side-effects typical of BDZs.     

Synthesis of substituted 3-anilino-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepine-2-ones and their evaluation as cholecystokinin-ligands

3-Amino-1,4-benzodiazepines as well as chemically related diverse amines were prepared from oxazepam and subsequently screened on the cholecystokinin receptor in a radiolabel binding assay. Oxazepam 2 was activated via its 3-chloro-1,4-benzodiazepine intermediate 3 and was reacted with a large series of aliphatic and aromatic amines. The substituted 3-anilino-1,4-benzodiazepine structure was identified as lead structure in a diverse series of 3-amino-1,4-benzodiazepines 4-38 and the full SAR (structure-activity relationship) optimisation provided 3-anilinobenzodiazepines 16-38 with CCK1 receptor selectivity to CCK2. The compounds 18, 24, 28 and 33 have shown affinities at the CCK1 receptor of 11, 10, 11 and 9 nM, respectively. These equipotent CCK1 ligands were fully evaluated in behaviour pharmacological essays. An antidepressant effect was identified in the tail suspension- and the Porsolt swimming-test. The ED50 values for 24 and 28 were determined in these assays as 0.46 and 0.49 mg/kg. The mixed antagonist 37 showed in addition to the antidepressant effects anxiolytic properties.     

Specific effects of benzodiazepines and tricyclic antidepressants in panic disorder: comparisons of clomipramine with alprazolam SR and adinazolam SR

In order to compare the efficacy and safety of tricyclic antidepressants and benzodiazepines in panic disorder, with or without agoraphobia, two studies were carried out comparing clomipramine with alprazolam sustained release (SR) or with adinazolam SR. Two hundred and fifty‐seven patients received alprazolam SR (2–6 mg/day given in two divided daily doses) or clomipramine (50–150 mg/day given in two divided daily doses) for 12 weeks in a single‐blind, randomised, multicentre study and 347 patients received adinazolam SR (30–90 mg/day given in two divided daily doses) or clomipramine (50–150 mg/day given in two divided daily doses) for 24 weeks in a double‐blind, randomised, multicentre study. Both benzodiazepines showed an earlier onset of therapeutic efficacy than clomipramine. At the end of the treatment periods, however, clomipramine was equally as effective as alprazolam SR and more effective than adinazolam SR. Withdrawal problems were also somewhat less common with clomipramine than with alprazolam SR and adinazolam SR. Both benzodiazepines were clearly better tolerated than clomipramine. The rate of premature withdrawal was also notably higher with clomipramine than with alprazolam SR. In conclusion, the benzodiazepines alprazolam and adinazolam SR are better tolerated than the tricyclic antidepressant clomipramine in the treatment of panic disorder, but have no advantages in terms of efficacy. Copyright © 1999 John Wiley & Sons, Ltd.     

Adinazolam pharmacokinetics and behavioral effects following administration of 20–60 mg oral doses of its mesylate salt in healthy volunteers

The pharmacokinetics and pharmacodynamics of adinazolam were studied in 15 normal, healthy, non-obse volunteers. Placebo capsules and capsules containing 20, 40, and 60 mg adinazolam mesylate were administered as single oral doses in a randomized, 4-way crossover design. Plasma concentrations of adinazolam and mono-N-desmethyladinazolam (NDMAD) were determined by HPLC. Psychomotor performance and memory tests were performed and the degree of sedation assessed at designated times following drug administration. Adinazolam and NDMAD pharmacokinetics were linear throughout the dosage range studied. The ratio of NDMAD to adinazolam area under the curve was approximately 4:1. Dose-related decrements in psychomotor performance and memory were observed up to 8h after dosing (P<0.025 in all cases). Psychomotor performance decrements correlated more closely with NDMAD plasma concentrations than with adinazolam concentrations. These results suggest that NDMAD is responsible for a significant degree of the sedative and psychomotor effects observed after the administration of adinazolam.     

Influence of dosing regimen on alprazolam and metabolite serum concentrations and tolerance to sedative and psychomotor effects

The relationships between alprazolam and metabolite concentrations and CNS effects were determined in a double-blind placebo controlled four-way crossover trial in 16 normal male volunteers. Active drug treatments consisted of 4-day regimens of 4 mg alprazolam PO daily as 2 mg bid., 1 mg qid, and 0.25 mg each hour. On days 1 and 4, the kinetics, sedative and psychomotor effects were evaluated. Plasma concentrations of the 4- and α-hydroxy metabolites of alprazolam were less than 10% of unchanged alprazolam levels on both days. Accumulation of these metabolites and alprazolam was dependent on alprazolam half-life (11.6 h). Acute and chronic tolerance to the sedative and psychomotor effects was observed with all active drug treatments. All alprazolam treatments resulted in significantly greater sedation than placebo on days 1 and 4. However, on day 4, sedation was 16–36% less than observed on day 1, despite plasma concentrations 1.4–2.76 times the day 1 concentrations. Sedation from alprazolam was reduced in each successive study phase, suggesting a tolerance which was sustained during the 10-day washout between phases. By day 4, psychomotor performance was not different from placebo, indicating more complete development of tolerance than occurred for the sedative effect. Sedation and psychomotor impairment on day 1 were greatest with 2 mg alprazolam bid. During the initiation of therapy, the patient will likely experience less sedation and psychomotor impairment with smaller, more frequent doses. Since tolerance develops to these effects, the advantage of more frequent dosing regimen dissipates by the 4th day.     

GABA(A) receptor physiology and its relationship to the mechanism of action of the 1,5-benzodiazepine clobazam

Clobazam was initially developed in the early 1970s as a nonsedative anxiolytic agent, and is currently available as adjunctive therapy for epilepsy and anxiety disorders in more than 100 countries. In October 2011, clobazam (Onfi?; Lundbeck Inc., Deerfield, IL, USA) was approved by the US FDA for use as adjunctive therapy for the treatment of seizures associated with Lennox-Gastaut syndrome in patients aged 2 years and older. It is a long-acting 1,5-benzodiazepine whose structure distinguishes it from the classic 1,4-benzodiazepines, such as diazepam, lorazepam and clonazepam. Clobazam is well absorbed, with peak concentrations occurring linearly 1-4 hours after administration. Both clobazam and its active metabolite, N-desmethylclobazam, are metabolized in the liver via the cytochrome P450 pathway. The mean half-life of N-desmethylclobazam (67.5 hours) is nearly double the mean half-life of clobazam (37.5 hours). Clobazam was synthesized with the anticipation that its distinct chemical structure would provide greater efficacy with fewer benzodiazepine-associated adverse effects. Frequently reported adverse effects of clobazam therapy include dizziness, sedation, drowsiness and ataxia. Evidence gathered from approximately 50 epilepsy clinical trials in adults and children indicated that the sedative effects observed with clobazam treatment were less severe than those reported with 1,4-benzodiazepines. In several studies of healthy volunteers and patients with anxiety, clobazam appeared to enhance participants' performance in cognitive tests, further distinguishing it from the 1,4-benzodiazepines. The anxiolytic and anticonvulsant effects of clobazam are associated with allosteric activation of the ligand-gated GABA(A) receptor. GABA(A) receptors are found extensively throughout the CNS, occurring synaptically and extrasynaptically. GABA(A) receptors are composed of five protein subunits, two copies of a single type of α subunit, two copies of one type of β subunit and a γ subunit. This arrangement results in a diverse assortment of receptor subtypes. As benzodiazepine pharmacology is influenced by differences in affinity for particular GABA(A) subtypes, characterizing the selectivity of different benzodiazepines is a promising avenue for establishing appropriate use of these agents in neurological disorders. Molecular techniques have significantly advanced since the inception of clobazam as a clinical agent, adding to the understanding of the GABA(A) receptor, its subunits and benzodiazepine pharmacology. Transgenic mouse models have been particularly useful in this regard. Comparative studies between transgenic and wild-type mice have further defined relationships between GABA(A) receptor composition and drug effects. From such studies, we have learned that sedating and amnesic effects are mediated by the GABA(A) α(1) subunit, α(2) receptors mediate anxiolytic effects, α subunits are involved with anticonvulsant activity, α(5) may be implicated in learning and memory, and β(3) subunit deficiency decreases GABA inhibition. Despite progress in determining the role of various subunits to specific benzodiazepine pharmacological actions, the precise mechanism of action of clobazam, and more importantly, how that mechanism of action translates into clinical consequences (i.e. efficacy, tolerability and safety) remain unknown. Testing clobazam and 1,4-benzodiazepines using a range of recombinant GABA(A) receptor subtypes would hopefully elucidate the subunits involved and strengthen our understanding of clobazam and its mechanism of action.     

Synthesis and evaluation of N1-substituted-3-propyl-1,4-benzodiazepine-2-ones as cholecystokinin (CCK2) receptor ligands

A novel synthetic approach towards N1-alkylated 3-propyl-1,4-benzodiazepines was developed in five synthetic steps from 2-amino-4-chlorobenzophenone, in which the N-oxide 4 served as a key intermediate. The structure-activity relationship optimization of this 3-propyl-1,4-benzodiazepine template was carried out on the N1-position by selective alkylation reactions and resulted in a ligand with an improved affinity on the cholecystokinin (CCK2) receptor. The N-allyl-3-propyl-benzodiazepine 6d displayed an affinity towards the CCK2 (CCK-B) receptor of 170 nM in a radiolabelled receptor-binding assay. The anxiolytic activity of this allyl-3-propyl-1,4-benzodiazepine 6d was subsequently determined in in-vivo psychotropic assays. This novel ligand had ED50 values of 4.7 and 5.2 mg kg(-1) in the black and white box test and the x-maze, respectively, and no significant sedation/muscle relaxation was observed.