Driven by chemistry but increasingly guided by pharmacology and the clinical sciences, drug research has contributed more to the progress of medicine during the past century than any other scientific factor. The advent of molecular biology and, in particular, of genomic sciences is having a deep impact on drug discovery. Recombinant proteins and monoclonal antibodies have greatly enriched our therapeutic armamentarium. Genome sciences, combined with bioinformatic tools, allow us to dissect the genetic basis of multifactorial diseases and to determine the most suitable points of attack for future medicines, thereby increasing the number of treatment options. The dramatic increase in the complexity of drug research is enforcing changes in the institutional basis of this interdisciplinary endeavor. The biotech industry is establishing itself as the discovery arm of the pharmaceutical industry. In bridging the gap between academia and large pharmaceutical companies, the biotech firms have been effective instruments of technology transfer.

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Drug Discovery: A Historical Perspective

The current studies further investigated the effects, in animal models of anxiety, of novel putative anxiolytic and anxiogenic compounds believed to induce their effects by actions at the GABA-benzodiazepine receptor complex. It was expected that the results would also provide further validation for a novel test of anxiety based on the ratio of open to closed arm entries in an elevated plus maze in the rat. The novel putative anxiolytics CL 218,872 (10–20 mg/kg) and tracazolate (5 mg/kg) significantly elevated the percentage of time spent on the open arms of an elevated plus-maze, consistent with their anxiolytic activity in several other animal tests. Also consistent with results from other animal tests, no anxiolytic activity was observed for the phenylquinoline PK 8165 (10–25 mg/kg), the 3,4-benzodiazepine tofisopam (25–50 mg/kg), or buspirone (0.5–20 mg/kg). The benzodiazepine receptor inverse agonists FG 7142 (1–5 mg/kg) and CGS 8216 (3–10 mg/kg) had anxiogenic activity in this test, as did the atypical benzodiazepine Ro 5-4864 (1–5 mg/kg). Interestingly, however, the benzodiazepine receptor antagonists Ro 15-1788 (10–20 mg/kg) and ZK 93426 (5–10 mg/kg) had no anxiogenic activity in this test.

Anxiolytic and anxiogenic drug effects on exploratory activity in an elevated plus-maze: a novel test of anxiety in the rat

National High Blood Pressure Education Program.

NEUROTRANSMISSION effected by GABA (γ-aminobutyric acid) is predominantly mediated by a gated chloride channel intrinsic to the GABAA receptor. This heterooligomeric receptor1 exists in most inhibitory synapses in the vertebrate central nervous system (CNS) and can be regulated by clinically important compounds such as benzodiazepines and barbiturates2. The primary structures of GABAA receptor α- and β-subunits have been deduced from cloned complementary DNAs3,4. Co-expression of these subunits in heterologous systems generates receptors which display much of the pharmacology of their neural counterparts, including potentiation by barbiturates3–5. Conspicuously, however, they lack binding sites for, and consistent electrophysiological responses to, benzodiazepines4,5. We now report the isolation of a cloned cDNA encoding a new GABAA receptor subunit, termed γ2, which shares approximately 40% sequence identity with α-and β-subunits and whose messenger RNA is prominently localized in neuronal subpopulations throughout the CNS. Importantly, coexpression of the γ2 subunit with α1 and β1 subunits produces GABAA receptors displaying high-affinity binding for central benzodiazepine receptor ligands.

Importance of a novel GABAA receptor subunit for benzodiazepine pharmacology.

This article does not aim to review in detail the properties of γ-aminobutyric acidA(GABAA)breceptors, because recent accounts of that topic are available. In this same journal, a review of the binding properties and pharmacology of these receptors has been published ([Sieghart, 1995][1]). Other

https://pharmrev.aspetjournals.org/content/pharmrev/50/2/291.full.pdf

International Union of Pharmacology. XV. Subtypes of gamma-aminobutyric acidA receptors: classification on the basis of subunit structure and receptor function.

Benzodiazepines produce most, if not all, of their numerous effects on the central nervous system (CNS) primarily by increasing the function of those chemical synapses that use gamma-amino butyric acid (GABA) as transmitter. This specific enhancing effect on GABAergic synaptic inhibition is initiated by the interaction of benzodiazepines with membrane proteins of certain central neurones, to which drugs of this chemical class bind with high affinity and specificity. The molecular processes triggered by the interaction of these drugs with central benzodiazepine receptors, and which result in facilitation of GABAergic transmission, are still incompletely understood. Theoretically, benzodiazepines could mimic the effect of hypothetical endogenous ligands for the benzodiazepine receptors, although there is no convincing evidence for their existence; in vitro studies indicate that benzodiazepines might compete with a modulatory peptide which is present in the supramolecular assembly formed by GABA receptor, chloride ionophore and benzodiazepine receptor and which reduces the affinity of the GABA receptor for its physiological ligand. The mechanisms of action of benzodiazepines at the molecular level are likely to be better understood following our recent discovery of benzodiazepine derivatives, whose unique pharmacological activity is to prevent or abolish in a highly selective manner at the receptor level all the characteristic centrally mediated effects of active benzodiazepines. Here, we describe the main properties of a representative of this novel class of specific benzodiazepine antagonists.

Selective antagonists of benzodiazepines.

The effects of several benzodiazepines on a variety of nervous activities known or presumed to depend on GABA are presented and compared with those of agents that deplete or increase the level of endogenous GABA: antagonism of various convulsant agents in mice, enhancement of presynaptic inhibition in the spinal cord and the cuneate nucleus of cats, decrease of the spontaneous firing rate of cerebellar Purkinje cells in cats and rats, antagonism of bicuculine-induced depression of the strio-nigral-evoked potential in the cat, potentiation of haloperidol-induced catalepsy in rats, GABA-mimetic actions on drug-induced PGO-waves in cats and on eserine-induced circling in guinea pigs. Diazepam slightly increased the GABA level in the cat spinal cord and in the total brain of mice and rats; this increase does not seem to be due to an increase of GABA synthesis. It is concluded that benzodiazepines probably enhance presynaptic inhibition at all levels of the neuraxis and that this effect requires not only the presence of GABA but is also dependent on an activity of GABA-ergic neurons. Benzodiazepines also appear to enhance postsynaptic inhibition where this is mediated by GABA. Many actions of benzodiazepines can be tentatively explained by a stimulus-bound enhancement of GABA effects.

Possible involvement of GABA in the central actions of benzodiazepines.

In neurological and behavioral studies in mice, rats, dogs and squirrel monkeys, the imidazobenzodiazepinone Ro 15-1788 acted as a potent benzodiazepine antagonist. The antagonistic activity was both preventive and curative and seen at doses at which no intrinsic effects were detected. It was highly selective in that it acted against CNS effects induced by benzodiazepines but not against those produced by other depressants, such as phenobarbitone, meprobamate, ethanol, and valproate. The onset of action was rapid even after oral administration. Depending on the animal species studied, the antagonistic effects lasted from a few hours to 1 day. The acute and subacute toxicity of Ro 15-1788 was found to be very low. Benzodiazepine-like effects were not seen.

Benzodiazepine antagonist Ro 15-1788: Neurological and behavioral effects

The potent benzodiazepine receptor ligands β-carboline-3-carboxylic acid ethyl ester (β-CCM) and the corresponding methylester (β-CCM) administered i.v. depressed segmental dorsal root potentials in spinal cats, reversed the prolongation of dorsal root potentials by phenobarbitone, and abolished the depression of a motor performance task induced by phenobarbitone in mice; β-CCE enhanced the low-frequency facilitation of pyramidal population spikes in the hippocampus of anaesthetized rats. These effects of β-carbolines reflect a depression of GABAergic synaptic transmission and, thus, are diametrically opposed to the enhancing action of benzodiazepine tranquilizers. The specific benzodiazepine antagonist, Ro 15-1788, while not affecting dorsal root potentials, hippocampal population spikes or phenobarbitone-induced motor performance depression, abolished the effects of β-CCE on the three parameters and similar effects of β-CCM on the spinal cord and motor performance.

A three-state model of the benzodiazepine receptor explains the interactions between the benzodiazepine antagonist Ro 15-1788, benzodiazepine tranquilizers, beta-carbolines, and phenobarbitone.

R. Schaffner

Papers

Selective antagonists of benzodiazepines.

Possible involvement of GABA in the central actions of benzodiazepines.

Benzodiazepine antagonist Ro 15-1788: Neurological and behavioral effects

A three-state model of the benzodiazepine receptor explains the interactions between the benzodiazepine antagonist Ro 15-1788, benzodiazepine tranquilizers, beta-carbolines, and phenobarbitone.

General Pharmacology and Neuropharmacology of Benzodiazepine Derivatives