Benzodiazepines. Introduction. . GABA, the Principal Inhibitory Transmitter in the Brain. Introductory article

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1 Hanns Möhler, ETH and University of Zurich, Zurich, Switzerland are a group of drugs with wide application as tranquillizers, hypnotics, muscle relaxants and anticonvulsants. They act by enhancing g-aminobutyric acid (GABA)ergic synaptic inhibition in the brain. Introduction An overall balance between excitatory and inhibitory neurotransmission is a prerequisite for normal brain function. A severe excess of either component is not compatible with life. It is therefore important to implement a balancing system that operates throughout the brain (Figure 1). This is accomplished by regulation of the membrane potential. Excitation of a neuron is based on the depolarization of its plasma membrane. This process is initiated by excitatory neurotransmitters, in particular by glutamate, which triggers the influx of cations (Na +,Ca 2+ ). Inhibitory neurotransmitters counterbalance the neuronal excitation, mainly by hyperpolarizing the membrane potential. This is accomplished largely by the neurotransmitter g-aminobutyric acid (GABA), which triggers an influx of anions (Cl 2 ). Hyperpolarization of the cell membrane makes it less likely that an excitatory depolarizing current will reach the threshold potential for the initiation of an action potential. Benzodiazepine drugs act by enhancing the GABAergic inhibitory neurotransmission in the central nervous system (CNS). GABA, the Principal Inhibitory Transmitter in the Brain GABA is the predominant inhibitory neurotransmitter in the mammalian CNS. Most GABAergic neurons are local, Figure 1 Excitatory and inhibitory neurotransmission. Ligand-gated ion channels, which are selectively permeable to particular cations or anions, mediate excitatory and inhibitory neurotransmission. The principal excitatory and inhibitory neurotransmitters are glutamate and g- aminobutyric acid (GABA), respectively.. Introduction Introductory article Article Contents. GABA, the Principal Inhibitory Transmitter in the Brain. Operation of a GABA Synapse. GABA Type A Receptors: Ligand-gated Chloride Channels. Enhancement of the Activity of GABA Type A Receptors by. Potency and Duration of Action. : Among the Most Frequently Prescribed Drugs. Tranquillizers. Hypnotics. Anticonvulsants. Side Effects. The Benzodiazepine Antagonist Flumazenil. Not All GABA Receptor Subtypes are Affected by. A Role for Corticotrophin-releasing Hormone in the Antistress Response of. Dependence Liability of. Barbiturates Distinct from doi: /npg.els shortcircuit interneurons that innervate neurons in the immediate vicinity. The GABA neurons themselves are activated by collaterals of excitatory afferents arriving from distant regions (forward inhibition) or from excitatory neurons in their vicinity (recurrent or feedback inhibition). Frequently, the GABA interneuron forms a feedback loop impinging on the same excitatory neuron by which it is activated. The physiological role of these GABAergic interneurons is obvious: they prevent overexcitation of neurons in response to strong excitatory inputs by limiting their firing rate and terminating burst activity. However, the inhibitory action of GABA at a synaptic contact is not necessarily translated into an overall reduced neuronal activity in a neuronal network because GABAergic neurons can themselves be the target of GABAergic inputs. This serial arrangement of GABAergic neurons can produce disinhibition of downstream neurons. Thus, a synaptic inhibition in particular synapses does not necessarily result in depression of complex CNS functions. The physiological action of GABA can be observed, for instance, at the onset of sleep. GABA neurons in the thalamus begin to inhibit the influx of signals, which would otherwise continue to reach the cerebral cortex, as is the case during wakefulness. The activation of thalamic GABA neurons underlies the emergence of spindle activity registered in the electroencephalogram at the beginning ENCYCLOPEDIA OF LIFE SCIENCES & 2005, John Wiley & Sons, Ltd. 1

2 of sleep. The contribution of GABAergic inhibition is also apparent in the pathophysiology of certain CNS disorders. In epilepsy, a reduced inhibitory GABAergic influence is thought to facilitate the synchronized discharge pattern of neurons that is typical of epileptogenic activity. Operation of a GABA Synapse When a GABA neuron is activated by an action potential, GABA is released from the presynaptic terminal into the synaptic cleft and induces an immediate and short-term inhibitory response in the postsynaptic neuron by activating postsynaptic GABA A receptors (Figure 2). These receptors constitute ligand-gated ion channels and therefore provide a response within milliseconds. GABA can also induce an inhibitory response with a slow onset by activating postsynaptic GABA B receptors. The latter operate via an enzymatic pathway (G proteins) to open K + channels. In this case, hyperpolarization is achieved through an outflow of K + ions. GABA B receptors can also be located presynaptically on the terminal of the GABA neuron, where they mediate a self-limiting feedback response of synaptic GABA by inhibiting the release of GABA from the terminal (a calcium-dependent mechanism). As GABA B receptors are also present on terminals of excitatory neurons, diffusion of synaptically released GABA to neighbouring glutamate neurons also curtails the release of excitatory amino acids. Thus, GABA B receptors mediate inhibition with a slow onset either postsynaptically (opening of K channel) or presynaptically (inhibition of the release of glutamate). However, the major inhibitory response of GABA is mediated via postsynaptic GABA A receptors. It is this response that is enhanced by benzodiazepine drugs. The action of GABA in the synaptic cleft is terminated by GABA transporters, through which GABA is taken up into the terminal from which it was originally released. GABA Type A Receptors: Ligand-gated Chloride Channels GABA A receptors are membrane proteins made up of five subunits (molecular sizes kda), which form a central pore with a selectivity for chloride ions (Figure 2). In each subunit, the amino (N)-terminal part of the polypeptide chain is followed by four transmembrane regions, of which the second transmembrane domain is thought to line the channel. The carboxy (C)-terminal part is again located extracellularly. Although the pentameric basic design of GABA A receptors is maintained throughout the brain, multiple receptor variants are generated in different populations of neurons by the expression and assembly of different types of subunits encoded by a repertoire of 17 genes (a1 6, b1 3, g1 3, d, r1 3, e) and splice variants thereof. However, a majority of GABA A receptors consist of an a subunit variant, a b subunit variant and the g2 subunit. Different populations of neurons are characterized by specific GABA A receptor subtypes. Binding of GABA to GABA A receptors causes the ion channel to open, thus permitting the influx of chloride ions within milliseconds. The influx process consists of bursts of channel openings interrupted by closed intervals. The dissociation of GABA from the receptor terminates the operation of the ion channel. Although several amino acid residues have been identified to be critical for GABA binding, the molecular anatomy of the gating process by which GABA opens the channel is presently not understood. Enhancement of the Activity of GABA Type A Receptors by Figure 2 g-aminobutyric acid (GABA)ergic synapse. After being released from the nerve terminal, the neurotransmitter GABA activates GABA A receptors located in the postsynaptic membrane. They constitute GABAgated chloride channels (Cl 2 ) whose performance is enhanced in the presence of a benzodiazepine (e.g. diazepam). The action of GABA is terminated by GABA transporters (GT), by which the neurotransmitter is taken up into the nerve terminal. The term benzodiazepine refers to the chemical structure of a heterocyclic ring system that has come to denote the respective class of drugs (prototype diazepam) in which the two N atoms are mostly located in position 1 and 4 (1,4- benzodiazepines) (Figures 3 and 4). These drugs have found wide therapeutic application as tranquillizers, hypnotics and anticonvulsants (see below). They share a common mechanism of action by binding to a common modulatory site located on GABA A receptors, which is termed the benzodiazepine site. The ligands of the benzodiazepine site include not only benzodiazepines but also chemically 2

3 Potency and Duration of Action Figure 3 Diazepam, the prototypical benzodiazepine drug. The chemical structure of diazepam, a 1,4-benzodiazepine, and the pharmacological spectrum of benzodiazepine drugs. Therapeutic indications and chemical structures of various other benzodiazepine drugs are given in Tables 1 and 2, and in Figure 4. different drugs (e.g. zopiclone and zolpidem). The basic mechanism of action of benzodiazepines also applies to nonbenzodiazepine ligands of the benzodiazepine site. Binding of a benzodiazepine to the benzodiazepine site of a GABA A receptor enhances GABAergic inhibition by increasing the opening frequency of the GABA-gated ion channel. This effect is equivalent to an increase in the affinity of GABA for the receptor, which is apparent in the shift of the GABA dose response curve to the left in the presence of a constant concentration of a benzodiazepine (Figure 5). In view of the wide distribution of GABA A receptors in the brain, it is somewhat surprising that these drugs exert selective actions, such as anxiolytic activity. An explanation is offered by the activity-dependent and selflimiting nature of benzodiazepine actions. The drug-induced modulation of the GABA A receptor is effective only in those synapses where receptors are activated by GABA. by themselves cannot activate the receptor in the absence of GABA. However, even within the group of GABA-activated synapses, the intensity of benzodiazepine action is not uniform. In synapses that contain a low concentration of GABA, the enhancement of GABA A receptors by benzodiazepines is much more pronounced than in synapses where GABA A receptors are almost maximally activated by high concentrations of GABA. Thus, while benzodiazepines enhance a weak GABA response, they do not enhance a GABA response beyond its physiological maximum. This self-limiting feature may explain why the enhancement of GABA transmission, even by high doses of benzodiazepines, is not life threatening (unless combined with other sedatives). This property distinguishes benzodiazepines from drugs such as barbiturates and propofol, which act at different sites of GABA A receptors (see below). More than a dozen benzodiazepine derivatives are in clinical use as are some nonbenzodiazepine ligands that act at the same site (Tables 1 and 2). Depending on their chemical structure, individual benzodiazepine drugs can differ strikingly in their potency (half-maximal dose to achieve a therapeutic effect). The potency is largely determined by the affinity of the drug for the benzodiazepine site, as illustrated for muscle relaxant activity in Figure 6. The higher the affinity of a particular benzodiazepine, the lower the dose required for its half-maximal effect. In addition, the duration of action can differ strikingly among different benzodiazepines (Tables 1 and 2). Their half-life is largely determined by the rate of metabolic degradation of the parent drug. In some cases, long-acting metabolites are generated (e.g. desmethyldiazepam) and contribute to the duration of action. Differences in half-life are relevant for the clinical indication. A short-acting hypnotic might be indicated in the case of failure to fall asleep, whereas a longer-acting hypnotic will be chosen to extend continued sleep during the entire night. : Among the Most Frequently Prescribed Drugs Since the introduction of the first benzodiazepine drugs chlordiazepoxide (Librium) and diazepam (Valium) benzodiazepines have remained among the most frequently prescribed drugs in medicine. Well-established therapeutic uses in psychiatry include the treatment of anxiety disorders and insomnia (Tables 1 and 2). In other areas of medicine, benzodiazepines are used for the induction of anaesthesia, relaxation of muscles, suppression of seizures and to calm anxiety and agitation that may be associated with illness. The popularity of these drugs derives from their high therapeutic:toxicity ratio; compared with other sedative drugs, they are effective and relatively safe when used according to the guidelines (see below). Tranquillizers are used as tranquillizers to alleviate anxiety states, in particular generalized anxiety disorders and panic anxiety attacks, but also in cases where anxiety accompanies other psychiatric disorders (e.g. depression) (Table 1). The anxiolytic effect is likely to involve mainly limbic structures in the brain. In general, anxiolytic effects are achieved at low doses of benzodiazepine, suggesting that modulation of only a small proportion of GABA A receptors is required to achieve an anxiolytic effect. In case of stress responses, benzodiazepines also dampen the endocrine response (see below). 3

4 Figure 4 Hypnotics Chemical structures of benzodiazepine drugs. The reduction of the sensitivity and reactivity to external stimuli is the basis for the use of benzodiazepines as hypnotics in the treatment of sleep disorders (Table 2). Short-acting benzodiazepines are chosen when the induction of sleep is disturbed. with a longer half-life are preferred when rewakening during the night is to be prevented. The sedative properties of benzodiazepines are also used as premedication before the induction of anaesthesia. 4

5 Figure 5 Enhancement of the g-aminobutyric acid (GABA) response by a benzodiazepine. In the presence of a benzodiazepine (e.g. diazepam), the dose response curve of GABA is shifted to the left. The action of benzodiazepines is activity dependent and self-limiting: in the absence of GABA, the benzodiazepine has no potentiating effect (bottom left part of the curves); when the GABA response is maximal, it is not further enhanced by the benzodiazepine (top right). Table 1 Benzodiazepine anxiolytic drugs Generic name Daily adult dosage (mg) Half-life a Chlordiazepoxide Long Diazepam 4 40 Long Clorazepate Long Prazepam Long Bromazepam Medium Clonazepam Medium Alprazolam Medium Lorazepam 2 6 Medium Oxazepam Short a Long, 24 h or more; medium, h; short, 10 h or less. Clonazepam is not approved by the US Food and Drug Administration for the treatment of anxiety or panic. Drug registration may vary between countries. Table 2 Insomnia: commonly prescribed drugs acting at the benzodiazepine site Generic name Daily adult dose (mg) Half-life a Flurazepam Long Estazolam 1 2 Medium Quazepam Medium Temazepam Medium Zopiclone b 7.5 Medium Midazolam Short Triazolam Short Zolpidem b 2 6 Short a Long, 20 h or more; medium, 5 20 h; short; h. Drug registration may vary between countries. b Zopiclone and zolpidem are chemically not benzodiazepines, but act via the benzodiazepine site of GABA type A receptors like the benzodiazepine drugs. Figure 6 Structure activity relationship of the benzodiazepines. The pharmacological potency of benzodiazepines correlates closely with their affinity to the benzodiazepine-binding site. This is exemplified for the muscle relaxant activity of various benzodiazepines. Their affinity was determined by competition for the binding site with [ 3 H]diazepam. The numbers refer to benzodiazepines not in clinical use. Ed min, minimum effective dose. Anticonvulsants The anticonvulsant action of benzodiazepines is explained by the GABAergic inhibition of neuronal responsiveness to excitatory inputs. This concerns, in particular, the synchronous discharge of excitatory neurons and the spread of focal epileptogenic activity to other brain areas. Tolerance to the anticonvulsant effect limits the usefulness of some benzodiazepines for the chronic treatment of epilepsy (clonazepam), but benzodiazepines are the drugs of choice in the life-threatening emergency of status epilepticus (lorazepam or diazepam). Side Effects The side effects of acutely administered benzodiazepines include daytime drowsiness, potentiation of the sedative effects of ethanol, anterograde amnesia and ataxia. These side effects are related to the dose and to individual susceptibility. They are mediated via the benzodiazepine site of GABA A receptors, as they can be antagonized by flumazenil (see below). Chronic administration can lead to tolerance to certain benzodiazepine effects and to physical dependence (see below). The Benzodiazepine Antagonist Flumazenil The clinically used tranquillizers, hypnotics and anticonvulsants that act at the benzodiazepine site are pharmacologically classified as agonists (high-efficacy ligands). The benzodiazepine flumazenil (Figure 4) acts as an antagonist. Although it binds with high affinity to all 5

6 benzodiazepine sites of GABA A receptors, it is practically devoid of any pharmacological activity when given alone. However, by occupying the benzodiazepine site, flumazenil efficiently blocks the action of benzodiazepine agonists in a competitive manner. Flumazenil can therefore be used to terminate the sedative action of benzodiazepines, for instance after surgery when a benzodiazepine was given before anaesthesia. In addition, flumazenil acts as an antidote in cases of benzodiazepine overdosage. Not All GABA Receptor Subtypes are Affected by The GABA A receptors in the brain that are responsive to the classical benzodiazepines are characterized by particular subunit variants (a1 3, a5, b2, b3 and g2). Their benzodiazepine sites are located on the respective a subunits (a1 3 and a5), characterized molecularly by a conserved histidine residue in a critical position. In other a subunits (a4, a6), the histidine residue is replaced by an arginine, which renders the respective GABA A receptors insensitive to diazepam. The latter receptors are located mainly in the cerebellum and, to a small degree, in the thalamus. The physiological significance of GABA A receptors that are either sensitive or insensitive to diazepam is not understood, in particular as there is presently no evidence for an endogenous ligand acting at the benzodiazepine site in the brain. Recently, progress was made in attributing the pharmacological spectrum of benzodiazepines to defined receptor subtypes. GABA A receptors containing the a1 subunit were found to mediate the sedative, amnestic and, to a large degree, anticonvulsant activity. The anxiolytic action was attributed to the population of a2 receptors, while a5 receptors were involved in the formation of spatial and temporal memory. Thus, in the future, subtype-specific benzodiazepines may provide a new generation of anxiolytic drugs, which are expected to display fewer side effects than the drugs in current clinical use. A Role for Corticotrophin-releasing Hormone in the Antistress Response of In response to stressful and aversive stimuli, including the anticipation of threatening events, corticotrophin-releasing hormone (CRH) is released from the hypothalamus and acts on the anterior pituitary to release adrenocorticotrophic hormone (ACTH), which in turn acts on the adrenal cortex to stimulate the synthesis and release of glucocorticoids. These steroids exert major feedback responses at the level of the pituitary and the brain. The release of CRH is controlled by several neurotransmitter pathways. Since GABA neurons inhibit the release of CRH, benzodiazepine drugs reduce the plasma cortisol concentration. In addition to its endocrine function, CRH acts as a neurotransmitter at extrahypothalamic sites such as the hippocampus, amygdala and neocortex. Intraventricular administration of CRH induces anxiety behaviour in animals, which can be reduced by benzodiazepines. Thus, enhancing the GABAergic control of the CRH system may contribute to the anxiolytic effect of benzodiazepines, particularly in conditions of stress. Dependence Liability of Chronic administration of benzodiazepines leads to adaptive changes in the brain that are yet ill understood. These changes manifest themselves as physical dependence withdrawal symptoms that become apparent upon discontinuation of the drug. These symptoms are related to the dose and duration of treatment and can include withdrawal anxiety, insomnia, convulsions and sensory hyperreactivity. They are frequently difficult to distinguish from the actual disease symptoms. To avoid withdrawal symptoms, chronic treatment with benzodiazepines is discontinued under gradual tapering of the dose over an extended period of time. Abuse or misuse implies the nontherapeutic use of benzodiazepines, generally in high doses, for the purpose of euphoria. Abusers of benzodiazepines constitute a small group of persons, frequently with a history of substance abuse. Treatment with benzodiazepines should therefore be avoided in patients with an earlier history of drug or alcohol addiction or abuse. As a general precaution, guidelines discourage the use of benzodiazepine hypnotics for more than 4 weeks and of benzodiazepine anxiolytics for more than 6 months. Experimental investigations of the adaptive changes induced by chronic benzodiazepine administration have focused mainly on changes in GABA A -receptor function. They include downregulation of the receptor, reduced sensitivity to GABA or an uncoupling of the receptor function from the allosteric modulation at the benzodiazepine site. Indeed, most of the drug discontinuation symptoms may be explained by reduced GABA A -receptor function. However, a deeper understanding of the neurobiology of dependence is still elusive. Barbiturates Distinct from The allosteric modulatory site by which barbiturates enhance the function of GABA A receptors differs from that of benzodiazepines. The allosteric modulation of the receptor plays an important role in the sedative action of barbiturates, 6

7 in particular at low doses. Barbiturates such as pentobarbitone increase the channel open time and prolong the duration of the bursts of channel openings. At higher doses, barbiturates exert anticonvulsant and anaesthetic actions, which are attributed to their ability to open the GABA A - receptor channel directly (i.e. in the absence of GABA). In this case, the physiological maximum of GABAergic inhibition is exceeded, as even those GABA A receptors are activated that were not gated by the endogenous neurotransmitter GABA. This ability to directly activate the receptor may explain why barbiturates but not benzodiazepines display anaesthetic activity. Barbiturates at higher doses also inhibit receptors for excitatory amino acids and affect voltage-gated channels. Their therapeutic:toxicity ratio is considerably lower than that of benzodiazepines. Further Reading Collinson N (2002) Enhanced learning and memory and altered GABAergic synaptic transmission in mice lacking the a 5 subunit of the GABA A receptor. Journal of Neuroscience 22: Crestani F, Keist R and Fritschy JM (2002) Trace fear conditioning involves hippocampal a 5 GABA A receptors. Proceedings of the National Academy of Sciences of the USA 99: Kupfer DJ and Reynolds CF (1997) Management of insomnia. New England Journal of Medicine 336: Lo w K, Crestani F and Keist R (2000) Molecular and neuronal substrate for the selective attenuation of anxiety. Science 290: McKernan RM, Rosahl TW and Reynolds DS (2000) Sedative but not anxiolytic properties of benzodiazepines are mediated by the GABA A receptor a 1 subtype. Nature Neuroscience 3: Mo hler H, Benke D, Benson J et al. (1997) Diversity in structure, pharmacology and regulation of GABA A -receptors. In: Enna SJ and Bowery NG (eds) The GABA Receptors pp Totowa, NJ: Humana Press. Mo hler H and Okada T (1977) Benzodiazepine receptor: demonstration in the central nervous system. Science 198: Rudolph U, Crestani F, Benke D et al. (1999) Benzodiazepine actions mediated by specific GABA A -receptor subtypes. Nature 401: Shader RJ and Greenblatt DJ (1993) Use of benzodiazepines in anxiety disorders. New England Journal of Medicine 328: