Neuroactive steroids acting at GABAA receptors

S.08 GABAA receptor modulation: recent pharmacological findings and clinical promise DAS, and to this GABHS-triggered autoimmune response? A numberof investigations are ongoing to explore these questions, and these studies will be presented and discussed in terms of the larger question of a role of neuroimmunologic dysfunction in various neuropsychiatric disorders. References Osler W (1894), OnChorea andChoreiform Affections. Philadelphia: HKLewis. Swedo SE (1994), Sydenham's chorea: a model for childhood autoimmune neuropsychiatric disorders. JAMA 272: 1788-1791. Swedo SE,Leonard HL, Schapiro MB et al. (1993), Sydenham's chorea: physical andpsychological symptoms of St. Vitus' dance. Pediatrics 91:706-713.

S.08 GABAA receptor modulation: recent pharmacological findings and clinical promise

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18.08.01 Genetics andfunction of GABAA-receptor subtypes

H. Mohler,J.-M. Fritschy,D. Benke, 1. Benson, B. Luscher. Institute of Pharmacology; ETHand University of Zurich, Switzerland Receptor subtypes provide operational flexibility in signal transduction, display differential regulationand allow cell-type specificdrug-targeting. Based on a repertoire of subunits encoded by at least 15 genes (a 1-6, f3l-3, yl-3, 8, yl-2) GABAA-receptors are prime examples of receptor heterogeneity in the CNS. GABAA-receptor subtypes appear to be tailor-madefor the respective neuronal circuits [l--4J. Pharmacology of GABA A -receptor subtypes: The development of novel ligands acting at the benzodiazepine (BZ) site of GABAA-receptors aims to retain the therapeutic effectiveness of classical benzodiazepines while reducing unwanted side effects such as tolerance, dependence liability, memory impairment, and ataxia [1-3]. Two strategies are being followed to achieve this goal: 1) reduction of efficacy (partial agonists) and 2) targeting of particular receptor subtypes (selectiveagonists). Partial agonists, typified by bretazenil and imidazenil,display high affinityfor most if not all receptors but act with reduced efficacy compared to classical full agonists. Other ligands display efficacies that vary depending on the GABAA receptor subtype. For example, abecarnil acts as a partial agonist on recombinant receptors containing a2 and a5 subunits but acts as full agonist on receptors containing the a 1 and a3 subunits. The second strategy concerns ligands with subtype-selective affinity. The hypnotic zolpidem shows high affinity for receptors containing the a 1 subunit while displaying lower affinity for receptors containing the a2 and a3 subunits and even lower affinity for most receptors containing the a5 subunit. Thus, favourable profiles of novel BZ site ligands can be achieved either by reducing the efficacy of the ligand at all receptors (bretazenil, imidazenil) or by avoidinga full activation of certainreceptor subtypes (abecarnil, zolpidem). The findings concerning abecarnil and zolpidem are of particular interest with regard to receptor heterogeneity. Receptors containing a2 and a5 subunits might be expected to be located in neuronal circuits which are involved in mediating unwanted rather than desirable drug effects. Indeed, it is notable that receptors containing the a2 subunit are strongly expressed in brain areas mediating reward (e.g., nucleus accumbens), while receptors containing the a5-subunit are concentrated in certain areas linked to memory functions (e.g. hippocampus). In the future, it should be possible to determine the pharmacological relevance of certain GABAA -receptor subtypes by generating mutant mice that contain pharmacologically inactive receptor subtypes. Such animals will help to determine the contribution of particular receptors to the pharmacological spectrum of BZ site ligands. Targeted mutations of subunits: Gene targeting of particular subunitsis being used to determine the physiological and pharmacological function of GABAA-receptor subtypes in vivo. As an initial step an attempt was made to generate BZ-insensitive mice by targeting the y2 subunit gene [5]. In y2-subunit deficient mice, GABAA-receptors continued to be formed from a and f3 subunits in practically unaltered number and distribution. The y2 subunit is thereforenot requiredfor subunitassembly and transport. The GABA response however failed to be potentiated by

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flunitrazepam while pentobarbital remained active. In addition, the single channel conductance was drastically reduced (from 30 pS in wildtype to 11 pS in y2-/- mice). Although embryonal development appeared to be unimpaired, the life span of the homozygous mutant mice was reduced [5]. Most likely,the y2-deficient GABAA-receptors do not provide the precise control of GABAergic tone required in inhibitory synaptic transmission in order to control neuronal excitability. The timepoint by which most GABAergic synapses become operative coincides with the maximal life-span of the mice. The lack of GABAA-reeeptor regulation by a postnatally relevant endogenous BZ-site ligand might contribute to this phenotype. Animal model of neophobia: In heterozygous mutant mice, the expression of the y2 subunit was reduced, resulting in an alteration of a small population of GABAA-receptors (about 20%) as demonstrated biochemically. The animals developed normally and reached adulthood. Behaviourally, the heterozygous mice were characterizedby an increased reactivity to aversive stimuli. They exhibited neophobia in a free-choice exploratory paradigm and showed novelty-dependent hypolocomotionin an openfield test. These behavioural alterations appear to be selective since locomotion, memory function (Morris-water maze) and neuronal plasticity (LTP) were unaltered. Thus, emotional reactivity to novelty appearsto be selectively increasedby an impairmentof GABAA-receptor function. The heterozygous y2"subunit mutant mice may serve as an animal model of neophobia. References [I] Mohler, H., Knollach, E, Paysan, J., Motejlek, K., Benke, D., Luscher, B., and Fritschy, J.M. (1995) Heterogeneity of GABAA receptors: cell-specific expression, pharmacology and regulation. Neurochem. Research. 20,631-636 [2] Mohler, H.• Benke, D.• Benson. J., Liischer, B. and Fritscby, J.M. (1995). GABAA receptor subtypes in vivo: cellular localization, pharmacology and regulation. In: GABAA receptors andanxiety: from neurobiology to treatment Biggio, G.•Sanna, E., Serra, M.andCosta, E. eds., Raven Press, N.Y. pp.41-56 [3] Mohler, H.•Fritschy, J.-M., Benke, D., Benson, J., Rudolph, U. andLiischer, B. (1996) GABAA-receptor subtypes: pharmacological significance andmutational analysis in vivo. In: GABA Receptors, Transporters and Metabolism. C. Tanaka, N.G. Bowery eds., Birkhauser, Basel, pp 157-112 [4] Mohler, H., Fritschy, J.M., Luscher, B., Rudolph, U., Benson, J. and Benke, D.TheGABAA-receptors: from subunits todiverse functions In: IonChannels Vol. 4, T. Narahashi ed.,Plenum Press, N.Y. (inpress) [5] GUnther, U., Benson, J., Benke, D., Fritschy, J.M., Reyes, G., Knoflach, P, Crestani E, Aguzzi, A., Arigoni, M., Lang, Y., Bliithmann, H., Mohler, H. andLiischer B. (1995) Benzodiazepine-insensitive mice generated by targeted disruption of they2-subunit gene of GABAAreceptors. Proc. Nat!. Acad. Sci. USA, 92,7749-7753

I8.08.021 Neuroactive steroids acting at GABAA receptors Kelvin W. Gee. Department of Pharmacology, College of Medicine, University of California at Irvine, Irvine, CA92717, USA A class of neuroactive steroids, both endogenously/naturally occurring and synthetic, has been identified with specific and potent effects as allosteric modulators of the y-aminobutyric acid (GABAA) receptor in the central nervous system. These steroids are quite remarkable in that they are devoidof classic steroidhormone activityand yet they are among the most potent and specificmodulatorsof the GABAA receptor currently known. In view of these unique characteristics they have been named epalons. Epalons are defined as a unique class of synthetic and naturally occurring pregnane steroids with high specificity for a novel allosteric modulatorysit on the GABAA receptor complex (GRC). Their ability to allosterically potentiate GABA action at the GABAA receptor with high potency and specificity provides the basis and rationale for their potential use as CNS therapeutic agents. The first of the synthetic to enter clinical trials for an indication in which the positive modulation of GABAA receptors maybe useful is ganaxalone (3a-hydroxy-3f3-methyl-5a-pregnan-20-one). It is currently in phaseII clinicaltrials for thetreatmentof epilepsy.Preclinically, ganaxalone is effective against pentylenetetrazol-, (+)bicuculline-, picrotoxin-, t-butylbicyclophosphorothionate-, and aminophylline-induced seizures in rodents at non-toxic doses. It is also a potent inhibitorof kindled seizures in the rat. Ganaxalone has completed phase I clinical trials in which safety, tolerability and pharmacokinetics following single and multiple doses wereevaluated in over 100healthy volunteers [6]. This epalon was

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5.08 GABAA receptor modulation: recent pharmacological findings and clinical promise

found to be safe and well-tolerated after single (up to 1500 mg) and proportional increase in the AUC and Cmax values with increasing dose. Safety and tolerability have been unremarkable. Ganaxalone is currently beingevaluated for safety, tolerability and potentialefficacy in paediatric, adolescent and adult patients exhibiting partial or generalized seizures refractory to conventional anti-epileptic drugs. In summary, it is too early to tell whether the epalons will prove to be clinicallyuseful in the treatmentof CNS disorders (i.e., anxiety, epilepsy and insomnia) amenable to positive modulation of GABAA receptors. However, their mechanisms of action and efficacy in well-established animal models provide a sound theoretical basis for predicting their clinical utility in the aforementioned indications.

I8 .08.031 with Phannacology of benzodiazepine receptor ligands different intrinsic efficacy D.N. Stephens. Laboratory of Experimental Psychology. University of Sussex. Falmer; Brighton ON] 9QG, UK Drugs from the benzodiazepine substance class are the standard treatments for anxietydisorders, and are effective and safe drugs, witha rapid onset of action. Nevertheless, their broadactionas modulators of GABAA receptor function, and the importance of GABA as the brain's main inhibitory transmitter, means that benzodiazepines exert effects on many brain functionsapart from emotionalsystems. Benzodiazepines therefore give rise to a range of side effects, including sedation, reduced muscle tone, amnesia, and more general impairments of cognition. Their action at GABAAreceptorsalso accounts for their ability to potentiate the CNS depressant effects of ethanol and barbiturates. Lastly, clinicalexperience indicates that dependence occursfollowing long term treatment withbenzodiazepines, and this aspect of benzodiazepine pharmacology has led to increasing concern among prescribers. For this reason, much effort has been expended in identifying novel approaches to the therapy of anxiety disorders. One approach to a novel therapy with the therapeutic efficacy of benzodiazepines, but with less dependence potential. side effects, and interactions with CNS depressants is to identify substances with partial or selectiveagonist actionsat benzodiazepine receptors. Partial agonists: Partialagonistsat benzodiazepine receptors show less efficacy than full agonists in potentiating GABA's effects at GABAA receptors. At the singlecell levelthis is reflected by partial agonists showing a lower maximal enhancement of GABA-induced currents than full agonists, This phenomenon (differences in efficacy) mustbe distinguished from differences in potency. wherebyat a givendose, one drug may show less effectsthan anotheractingat the same receptors, but at optimaldoses of each drug the maximal effect is the same. Since their efficacy is less than that of full agonists, in order to exert the same degreeof potentiation of GABAergic inhibition of neuronal activity, partial agonists need to occupy a greater proportion of the available receptors. In other words, in order to achieve any given pharmacological effect, partial agonists require to occupy a higher proportion of receptors within the relevant neuronal system. In general, anxiolytic effects of benzodiazepines occur at lower doses than their sedative effects.For full agonists, the steepness of the dose effect relationship results in only a poor separation of these effects. Partial agonists, on the other hand, show shallower dose effect relationships, allowing a greater separation of the wanted and unwanted actions of the drug. For this reason, they may retain the ability to induce anxiolytic effects (albeit at higher fractional receptor occupancies than full agonists) while losing the ability to induce sedation or ataxia, effects whichgenerally require higherfractional receptor occupancies than anxiolytic effects(e.g. Jones et al, 1993). Nevertheless, this modelis based on certainassumptions which are not proven.It assumes,for instance, that the degreeof GABAergic activation required to treat anxiety is a constant, and that partial agonists are able to achieve this level of GABA potentiation. If, however, more severe cases of anxiety require more extensive activation of the GABA system, then it seems possible that partialagonists may be less effective than full agonistsin their ability to reduceanxietysymptoms. Selective agonists: GABAA receptors exist in a large number of subtypes, many of which are sensitive to the modulatory effects of benzodiazepines. These subtypes are distributed heterogenously in the CNS, suggesting that different subtypes may be involved in different effects

of benzodiazepines. Selective agonists, by addressing only particular subtypes of benzodiazepine receptor, can therefore be expected to give rise to a selective behavioural profile, and, depending on the particular selectivity of a drug, may possess anxiolytic effects in the absence of interactions with eNS systemsgiving rise to unwanted effects. Although it is not yet known which (or even whether) particular subtypes are responsible for the various effects of benzodiazepines, it is notable that theimidazopyridine, zolpidem,a hypnoticwith little anxiolyticactivity in animalmodels, showsnegligible affinity for GABAA receptorscontaining as subunits, while compounds with lower affinities for receptors containing a3 subunits may have little ataxic activity (Turski and Stephens, 1993). although it is not clear whethersubtype selectivity would enable a novel drug to avoid dependence potential, it seems reasonablethat the fewer receptors occupied during chronic treatment, the fewer and less severe the withdrawal symptoms will be. Several reports suggest that subtype-selective compounds suchas zolpidemmay possessless potential to inducedependence. Mixed partial agonist/subtype selective profiles.: The existence of multiple subtypes of GABAA receptorallowsthe existenceof drugs with different efficacies at different subtypes. Abecarnil is a compound from the p-carboline chemical class which combines receptor subtype selectivity and partial agonism: i.e., abecamil, in contrast to benzodiazepines, shows differential affinity for certain GABAA receptor subtypes, while at the same time displaying full agonist efficacy at some receptors, and partialagonist at others. The binding properties were tested with isoreceptors containing 3 different a-subunits together with b2g2 subunits, expressed in a human kidneycell line, HEK 293. In contrast to diazepam, abecarnil differentiated between the 3 isoreceptors in regard to binding affinity. The IC50 towards the al-containing isoreceptor was 29-fold higher than towards the a3-containing one, with an intermediate value for the a5b2g2 isoreceptor. In oocytes, abecamil as well as diazepam produced no effect on their own when applied to the superfusate. In oocytesexpressingaxb2g2 combinations, abecamil resembled diazepam in its ability to potentiate GABA-induced currentsin al -, or a3-containing receptors,but therewasa clear difference betweenthe two compoundsin the maximalpotentiation of GABA-induced currents in oocytes expressing as!lzgz combinations. This mixedprofile at the receptorlevelfindsits reflection in a behavioural profilewhichis differentiated from the typicalprofileof benzodiazepines in that in animal models abecarnil shows potent anxiolytic and anticonvulsant activity, with lower potency in causing in particular, muscle relaxant and ataxic effects. Following chronic administration, withdrawal of the compound givesrise to few and weak withdrawal symptoms. Relationship of anxioiytic effect to fractional receptor occupancies.: An indication of the relative efficacy of drugs acting as full and partial agonists at a receptor site can be obtained from the level of receptor occupancy which they require to achieve a particular pharmacological effect, i.e., full agonistsrequire to occupy relatively fewer receptors than partialagonists, In orderto obtaininformation on the efficacy of abecarnil in comparison to standard benzodiazepines, and to the partialagonistbretazenil , data obtained in vivo for the displacement of 3H-benzodiazepine binding to brain receptors in mice and rats by doses of abecamil and benzodiazepines wasused to calculate the fractional receptoroccupancies achieved by the same compounds over dose ranges in several tests of anxiolytic activity. Thus, the relative potencies of the compounds could be compared at doses which gave rise to equivalent numbers of ligandreceptor complexes. In terms of fractional receptor occupancy, abecarnil was as potent as diazepam and alprazolam in the plus maze test, and these three drugs all significantly increased time spent in the open arms of the maze, as well as the numbers of entries into the open arms, at doses giving rise to approximately 30% receptor occupancy defined by 3H-lormetazepam labelled receptors. The benzodiazepine receptorpartial agonist, bretazenil was ineffective at doses giving rise to up to 89% receptor occupancy. These data suggest that abecarnil possessesan efficacy in this test as high as alprazolarn and diazepam (Jones et al, 1993). Similaranalyses for the 4-plate test of anxiolytic activityin mice, reveal that abecarnil, like alprazolam and diazepam, induced antipunishment effects at doses giving rise to approximately 20% receptor occupancies. In this test, the partial agonist, bretazenil also gave rise to a significant antipunishment activity at about 20% receptor occupancy (Jones et al, 1993).