Calming the brain benzodiazepines and related drugs from laboratory to clinic 9781000102154, 1000102157, 9781000119329, 1000119327, 9781000144468, 1000144461, 9781003076407, 1003076408

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Calming the Brain: Benzodiazepines and related drugs from laboratory to clinic

ADAM DOBLE

PhD

Consultant in Medical Research,

Fresnes, France

IAN L MARTIN

PhD

Professor of Pharmacology,

Pharmacy,

School of Life and Health Sciences,

Aston University, Birmingham, UK

DAVID NUn OM

MRCP FRCpsych

Professor of Psychopharmacology,

Psychopharmacology Unit,

School of Medical Sciences,

University of Bristol, UK

0 cfll

CRC Press Taylor & Francis Group Boca Ratan London New York

CRC Press is an imprint of the Taylor & Francis Group, an informa business

e 2003 Martin Dunitz CRC Press is an imprint of Taylor &: Franeis Group. an Informa business First published in the United Kingdom in 2004 by Martin Dunitz, an imprint of the Taylor & Francis Group, 11 New Fetter Lane, London EC4P 4EE Tel: +44 (0) 20 7583 9855 Fax: +44 (0) 20 7842 2298 E-mail: [email protected] Website: http://www.dunitz.co.uk All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior permission of the publisher or in accordance with the provisions of the Copyright, Designs and Patents Act 1988 or under the terms of any licence permitting limited copying issued by the Copyright Licensing Agency, 90 Tottenham Court Road, London W1POLP. Although every effort has been made to ensure that all owners of copyright material have been acknowledged in this publication, we would be glad to acknowledge in subsequent reprints or editions any omissions brought to our attention. A CIP record for this book is available from the British Library.

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Contents

Preface.. ..................... . .... .. . . . .. . .. .. .... ................ ..... ..........

v

1

The story of modem tranquilliser drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1

2

Interaction of the benzodiazepines with the GABA A receptor. . . . . . . . . . . . . . . . . . . . . . . .

15

3

Structural characterisation of the GABA A receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

43

4

Functional characterisation of the GABA A receptor ................................

69

5

Structures of benzodiazepine recognition site ligands ............ ... ...............

85

6

Behavioural pharmacology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

99

7

Pharmacokinetic determinants of clinical activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

131

8

Benzodiazepine drugs in sleep disorders ..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

145

9

Benzodiazepines as anxiolytics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

155

10

Benzodiazepines: Anticonvulsant and other clinical uses. . . . . . . . . . . . . . . . . . . . . . . . . . .

167

11

Conclusions. .. . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . .. . . . .

177

Index................ ..... ...... .. . .... ........... ... ................... ..........

181

Preface

Many neurological and psychiatric disorders seem to involve an excessive activation 10,000

7.0 ±0.3 1.2 ±0.3 2.4 ± 0.5 0.4±0.1 0.5 ± 0 .04 490 ± 120 >15,000

>10,000 >10,000

107 ± 26 5.0± 0.9 >10,000

90±20 5 .1±0.8 >10,000

The K, values (nM) are taken from Hadingham et al (1992. 1993a.b). The data for triazolam. bretazenil and CL 218.872 were obtained from receptors which contained the 1J1 subunit In place of the 1J2 subunit. with the exception that all studies conducted with a4 and a6 subunit containing receptors are taken from Wisden et al (1991)

50 CALMING THE BRAIN

Table 3.2 Effect of ligand affinity

~

subunlt lsoform on

al~Y'Y2

~1

~2

~3

Aunitrazepam 11.5 ± 1.6 8.0±0.2 22.4±2.5 0.81 ± 0.07 1.01 ± 0.06 0.92 ±0. 04 Aumazenil Rc 154513 CL 218,872 Zclpidem The

Figure 3.4 Venn diagram representing benzodiazepine recognition site pharmacology of a(1-6)~2'Y2 recombinant receptors. Within Box 1 all receptors exhibit a high affinity for muscimol and Ro 15-4513. Box 2 contains receptors with a high affinity for diazepam and the classical benzodiazepines. Box 3 delineates those receptors with a low affinity for diazepam and the classical benzodiazepines. The BZ1 phenotype is found in Box 4 while the BZ2 phenotype is found in Box 5. The affinity for zolpidem is markedly reduced within Box 7 compared to Box 6.

recognise the classical benzodiazepine agonists with high affinity (Wieland et al, 1992; Korpi et al, 1993; Duncalfe et ai, 1996). ~

subunlt class

The particular p subunit isoform appears to have little influence on the characteristics of the benzodiazepine recognition site in recombinant receptors from the series aX~Y"Y2 (Table 3.2) (Wisden and Seeburg, 1992; Hadingham et ai, 1993b). Transient expression of a1~1'Y2, a1~2'Y2 or a1~3'Y2 receptors in HEK 293 cells demon­ strated that the particular ~ subunit isoform did not modify the increase in GABA-activated cur­ rents produced by 10 JLM diazepam (Puia et al, 1991). In oocytes the exchange of ~1 for ~2 sub­ units, in combination with a1 and 'Y2 subunit expression, resulted in a decrease in the appar­ ent affinity for GABA but a marked increase in

10.0±0.6 10.4±0.5 8. 9±0.9 290± 32 220±37 301±28 59.6 ± 20.1 64.1 ± 7.5 112± 17

Kt values (nM) were taken from

Hadlngham et al (1993b)

the potentiation of the GABA-activated current by 1 JLM diazepam (Sigel et aI, 1990). While complete dose-response curves were not assem­ bled in these experiments it is clear that allosteric effects of the benzodiazepines may be modulated by the particular ~ subunit isoform but perhaps as a consequence of their primary interaction with the natural agonist GABA. However, the ~ subunits have important determinants for agonist recognition. It is sug­ gested that they are the principal site of photo­ incorporation of muscimol (Deng et aI, 1986; Casalotti et al, 1986), and mutagenic studies have identified specific amino acid residues involved in agonist binding (Smith and 01sen, 1994; Amin and Weiss, 1993; Siegel et al, 1992; Boileau et al, 1999; Newell et aI, 2000). It is also clear that recognition of the anticonvulsant loreclazole dif­ ferentiates between GABAA receptor subtypes depending on their ~ subunit isoform composi­ tion (Wafford et al, 1994; Wingrove et aI, 1994). 'Y subunlt class All three 'Y subunit isoforms, when expressed together with a and ~ subunits, confer benzodi­ azepine recognition properties on the expressed receptors (Table 3.3) (Pritchett et aI, 1989a; Knoflach et al, 1991; Ymer et aI, 1990). In combi­ nation with an a and a ~ subunit, the 'Y1 subunit produces a marked decrease in affinity for the antagonist flumazenil and the inverse agonist

STRUCTURAL CHARACTERISATION OF THE GABAA RECEPTOR 51

Table 3.3 Effect of "y sUbunlt Isoform on ligand affinity alj32'Vx 'Vi

'(2

Aunitrazepam

20.0 ± 1.6 8 .0 ± 0.2

Aumazenil

>10,000

'V 3

67 ±4

1.01 ± 0.06 0 .9 ± 0.3

Ro 154513

10.4 ± 0.5

2.8±0.9

CL 218,872

220± 37

8 .0 ± 1.0

ZOlpidem

59.6 ± 20.1 >10,000

The K, values (nM) were taken from Ymer et al (1990) for the "(1 containing receptors; Hadingham et al (1993b) for the "(2 containing receptors and Lliddens et al (1994) for the "(3 containing receptors

DMCM in comparison to 'Y2 containing combi­ nations (Ymer et aI, 1990). In contrast, a1p2'Y3 shows a marked decrease in benzodiazepine agonist affinity, relative to that of an a1p2'Y2 receptor, while both combinations have similar affinities for antagonists and inverse agonists (Herb et aI, 1992). Interestingly, receptors con­ taining 'Y3 displayed much greater differences in affinities for certain benzodiazepine agonists (eg midazolam versus zolpidem) than did the cor­ responding 'Y2 containing combination (Herb et aI, 1992), and exhibited particularly high affini­ ties for CL 218,872 (Liiddens et aI, 1994). While the -y2 subunit isoform is the one most ubiquitously expressed, if replaced with 'Yl in many a~'Y combinations, it results in agonist activity of both DMCM and j3CCM (Puia et aI, 1991; Wafford et aI, 1993). These particular recombinant experiments provide an explana­ tion of the data obtained in cultured astrocytes, where it had previously been shown that both these compounds function as agonists (Bormann and Kettenmann, 1988) since these cells have since been shown to express the 'Yl subunit to higher levels than those of other 'Y subunits (Bovolin et aI, 1992).

& subunit class Until recently, only limited information was available concerning the functional importance of

the B subunit in the GABA A receptor subtypes in which it is expressed. This subunit can form homo-oligomers in HEK 293 cells that display GABA-gated channels with small currents that are sensitive to picrotoxin, bicuculline and pen­ tobarbital (Shivers et aI, 1989). If the B subunit replaces the 'Y2 subunit in recombinant receptors, high affinity benzodiazepine binding sites are lost (Shivers et al, 1989). However, it is clear that the B subunit is expressed together with a4 and a p subunit in the rat thalamus where the receptors exhibit recognition sites for muscimol but not the benzodiazepines (Sur et al, 1999). Interestingly, in transgenic mice in which the a6 subunit gene is disrupted by homologous recombination, the & subunit protein is lost from the cell surface recep­ tors suggesting a specific association between a6 and &subunits (Jones et al, 1997). p subunlt class

Two p subunits have been identified in man (Cutting et al, 1991, 1992). Their expression was originally thought to be restricted to the retinal bipolar cells but recent evidence suggests that this is not the case, and they have now been located in various brain regions, albeit in relatively low densities (Enz and Cutting, 1998, 1999). While functional GABA A receptors are hetero-oligomeric the p subunits form homo-oligomers or pseudo-homo-oligomers (containing both pI and p2) that display large GABA-gated currents (Cutting et aI, 1991; Shimada et aI, 1992; Enz and Cutting, 1999). These receptors display an unusual pharma­ cology that is not altered by co-expression with a, p or 'Y subunits. While the expressed receptors are picrotoxin-sensitive, they are insensitive to barbiturates, benzodiazepines, bicuculline and baclofen (Cutting et al, 1991; Shimada et al, 1992). It is likely that the receptor forms pseudo-homo­ oligomers in the retina in vivo as elimination of the pI subunit, using a knock-out strategy, elimi­ nated the expression of these receptors in the mouse retina (McCall et al, 2(02); in other brain regions it is believed that responses from this receptor subtype are mediated by p2 homomers. These receptors are commonly referred to as GABAc rather than GABAA (Shimada et al, 1992),

52 CALMING THE BRAIN

although there remains some discussion about this nomenclature (Woodward et aI, 1993; Bamard et aI, 1998; Bormann,2000). Other subunit classes

The 1T subunit is most closely related to the ~ (37%), & (35%) and p (33%) subunits. Studies with this subunit are limited but it does appear to express together with both a5~3 and a5~3'Y2 recombinants to form receptors with distinct biophysical and pharmacological properties. Only the recombinant containing the 'Y2 subunit is sensitive to the benzodiazepine, diazepam (Neelands and Macdonald, 1999). The 8 subunit is most closely related to the 'Y subunits with between 38% and 43% amino acid identity. Functional recombinant receptors appear to form when the 8 subunit is expressed together with al and ~3 subunits to produce both spon­ taneously active and GABA-activated currents with a conductance of about 24 pS. The expressed receptor responds to GABA in a concentration-a5 = a4>al>a3>a2 (Ebert et aI, 1994; see also Hevers, Liiddens, 1998). In the same study it was shown that receptors con­ taining the (33 subunit isoform are more sensi­ tive to GABA than those containing 131 or (32. Similarly, in those receptors containing the 'Y3 subunit, GABA is more potent than those con­ taining 'Yl or 'Y2 (Ebert et aI, 1994). It is clear that while the absolute requirement for agonist activation is the expression of a(3 subunits, not only does the 'Y subunit exert some control on the functionality but the precise subunit isoform composition is an important factor in the response of the receptor subtype to the natural agonist. Receptors containing the & subunit, which may substitute for a 'Y subunit, are insensitive to the benzodiazepines but can exhibit a 50-fold decrease in ECso compared to those containing 'Y2 (Saxena and Macdonald, 1996). There is limited information on the characteristics of receptors containing the E subunit, which, like the & subunit may also replace a 'Y subunit in functional receptors (Whiting et aI, 1997; Neelands et aI, 1999). Partial agonlsts

While the subunit composition of particular GABA A receptor subtypes appears to be of minor significance to the high affinity binding sites for many of the synthetic agonists, marked variations in their ECso and efficacies are appar­

ent in functional studies (Ebert et aI, 1997). THIP, for example, proves to be a partial agonist at receptors containing al'Y2 and any of the 13 subunit isoforms, but is a full agonist at a2(33'Y2, a5(33'Y2 and a6(33'Y2 receptors. P4S, however, exhibits a distinct profile being a full agonist at a2(33'Y2 and a5(33'Y2 receptors but a relatively weak partial agonist at those recep­ tors containing aI, a3 or a6 subunit isoforms. Interestingly, THIP exhibits a greater efficacy than GABA at a4(33& containing receptors (Brown et aI, 2002). Channel-gatlng kinetics and function

While the subunit composition of GABA A receptors clearly defines agonist potency, they also have a marked effect on the gating charac­ teristics of the integral ion channel. Initial com­ parisons of a 1(32'Y2 and a3(32'Y2 receptors demonstrated the reduced potency of GABA at the a3 containing receptors by about one order of magnitude, in line with previous studies (Verdoom, 1994). It was also clear that the pres­ ence of the a3 subunit slowed activation, desen­ sitisation and deactivation on termination of agonist application (Gingrich et aI, 1995). One of the most pronounced differences in gating results from the replacement of the 'Y2 subunit in al133'Y2L receptors with the & subunit. The receptors containing the &subunit activate about four times more slowly and desensitise to only some 56% during the 4 second agonist application compared to over 90% for the 'Y2L-containing receptors (Haas and Macdonald, 1999). It appears that the extracellu­ lar residues of TMl play an important part in defining the desensitisation of these two recep­ tor subtypes (Bianchi et aI, 2001). Tonic and phaslc Inhibition

It has become increasingly clear that not all GABAergic inhibition is mediated through tight synaptic contacts. There is now a substantial body of evidence that extrasynaptic receptors may well outnumber those located within synapses (Mody, 2001). The synaptic recep­ tors experience brief exposures to very high

72 CALMING THE BRAIN

concentrations of GABA, approaching 1 mM, consequential to its release from presynaptic sites (phasic inhibition) while, in the case of the extrasynaptic receptors, the agOnist concentra­ tion will be lower and its presence extended as it diffuses from its release site (tonic inhibition). It is suggested that receptors containing the ,,/2 subunit, important in anchoring receptors to the cytoskeleton (Essrich et aI, 1998; Wang et aI, 1999; Kneussel et al, 2000), are probably located within the synapse while those containing 8 sub­ units are extrasynaptic (Nusser et al, 1995, 1998). The rapid kinetic properties of a1133,,/2L recep­ tors, at their synaptic locations, suggest that they would exhibit larger peak currents with a rapid desensitisation and prolonged current decay while (11331) receptors extrasynaptica1ly located would produce smaller peak currents and desen­ sitise slowly on prolonged agonist exposure (Haas and Macdonald, 1999). This differential inhibition proves to have some interesting properties. Recent studies carried out in hippocampal slice preparations demonstrated that the tonic inhibition was increased over 300% by the introduction of an inhibitor of GAT-I, the GABA transporter, but this failed to modulate phasic transmission. On the contrary, application of zolpidem increased phasic inhibition by 60% but failed to modulate tonic inhibition, due to the presence of a I) subunit in the extrasynaptic receptors (Nusser and Mody, 2002). It is thus clear that the subunit composition of receptor subtypes may provide a subtlety of response to agonist expo­ sure, and thus neuronal inhibition, that is yet to be fully explored. BARBITURATES, NEUROACTIVE STEROIDS AND ANAESTHETICS

The barbiturates, neuroactive steroids, together with both gaseous and intravenous anaesthet­ ics, share a number of functional characteristics. All facilitate GABA-mediated transmission but, unlike the benzodiazepines, their actions do not appear to be markedly receptor sub type­ dependent (Lambert et al, 2001; Thompson and Wafford, 2001). However, these agents may also induce channel opening in the absence of

the agonist, although there is evidence that this action is dependent on the subunit composition of the receptor subtype, where this has been investigated (see eg Adkins et al, 2(01). Studies with these compounds have relied on func­ tional characterisation; no sites have been iden­ tified by radioligand binding techniques although indirect biochemical studies have been used (Peters et aI, 1988; Kirkness and Turner, 1988). A detailed discussion of the mutagenic studies conducted with these agents in recent years has been amply reviewed recently (Lambert et aI, 2001; Thompson and Wafford, 2(01). However, it is interesting that the focus of these studies has been the extracellular seg­ ments of the first three hydrophobic domains within the a and 13 subunits (Carlson et al, 2000; Belelli et al, 1997, 1999a). Whether this location should be considered an allosteric site or simply a direct channel-gating modulation will undoubtedly yield to further investigation (Belelli et aI, 1999b). It is perhaps also interesting that these agents, in contrast to the benzodiazepines, have no requirement for the "/ subunits and are active at both a4 and a6 containing receptors (Schofield et al,1987; Puia et al, 1990; Wafford et aI, 1996). There is increasing evidence that the presence of a4 and a6 subunits, or the lack of a ,,/2 subunit, within particular receptor subtypes, indicates that the receptors are located extrasy­ naptically. It is also clear that the extrasynaptic receptors, which are responsible for tonic inhi­ bition, are responsible for much greater charge transfer than the synaptically located receptors. It is perhaps tempting to speculate that these agents may differ in their overt effects from the benzodiazepines as a consequence of their activity on tonic inhibition where the benzodi­ azepines are inactive (see above). ORIGINS OF FUNCTIONAL HETEROGENEITY OF BENZODIAZEPINES

The initial studies of functional heterogeneity can be traced back to work carried out by the team at Lederle who had described CL 218,872, one of the first non-benzodiazepines discovered

FUNCfIONAL CHARACfERlSAnON OF GABAA RECEPTORS 73

to interact with the benzodiazepine site on the GABA A receptors. They had observed that this triazolopyridazine, which exhibited a higher affinity for the BZ1 receptor subtype, produced anxiolytic and anticonvulsant actions at doses considerably below those that caused sedation and myorelaxation. The authors argued that it was the ligand preference for the BZ1 receptor subtype that provided the preferential anxioly­ sis over sedation, concluding that it was the BZ2 subtype that was responsible for the seda­ tive and muscle relaxant activity (Squires et ai, 1979; Lippa et aI, 1979). However, later behav­ ioural studies provided evidence that CL 218,872 was able to antagonise the loss of right­ ing reflex induced by diazepam, suggesting that it was a partial agonist (Gee et aI, 1983). In subsequent electrophysiological experiments this proved to be the case, the compound was a partial agonist in primary cultured neurons from both chick and frog (Farb et aI, 1984; Yakushiji et aI, 1989). It was not until the early 1990s that the differential subtype efficacy of CL 218,872 became apparent. Methodological considerations

In the majority of cases efficacy determinations have been carried out in receptors ectopically expressed in Xenopus oocytes using a 2-electrode voltage clamp. The system is robust but the size of the oocytes, around 2 mm diameter, pre­ cludes the determination of rapid kinetics. Thus, the peak responses can be compromised by desensitisation, particularly in receptor sub­ types in which desensitisation is rapid. Generally, radioligand binding studies have not been conducted with oocytes, although it is possible (Chang and Weiss, 1999). Alternatively, mammalian cell expression systems have been used where rapid application is readily achieved and affinity determinations can be easily carried out with radioligand binding methods on the cell population. In the case of the modulatory sites of the GABA A receptor care must be taken in the com­ parison of the efficacy determinations from dif­ ferent laboratories. Some investigators choose to determine efficacy at the EClO for GABA acti­

vation in which case the maximum effect of the positive allosteric modulators is numerically large. However, others carry out the determina­ tions at GABA ECso, where both negative and positive modulation can be quantified with facility. Clearly, measurement of efficacy at multiple receptor subtypes, with a single com­ pound, also requires the determination of agonist concentration-response curves for each receptor subtype. These procedures are time consuming and rigorous comparisons are rarely possible with the data that are currently avail­ able in the public domain. Efficacy: Importance of the subunlt Isoforms

The concept of efficacy at the benzodiazepine site is confounded, to an extent, by the lack of a natural agonist. It has been common practice to assume that the classical benzodiazepines, such as diazepam and flunitrazepam, are full agonists and comparisons are thus made against this criterion. Under these conditions only one compound, triazolam, has thus far been reported to have an efficacy greater than diazepam and only at a1 subunit containing receptors (Ducic et aI, 1993). However, this observation is not universally supported (Wafford et aI, 1993a). Clonazepam is one of the few classical benzodiazepine ligands that was originally reported to exhibit partial agonism (Chan and Farb, 1985). While this appears to be the case in rat cortex using maximal concentrations of clonazepam, the decreased efficacy appears to amount to only 70% of that found for the full agonists at recep­ tors containing ~ny2 together with a2, a3 or a5 subunits; at the a1 containing receptor clon­ azepam exhibits similar efficacy to diazepam (Puia et aI, 1991). By construction of concentration-response curves rather more useful data can be obtained. In recombinant receptors comprising 13112 together with either a1 or a3 subunits, the partial agonist FG 8205 exhibits an efficacy about 60% of that of flunitrazepam at both receptor suhtypes while CL 218,872 displays 50% efficacy at the a1 containing receptors that is reduced to 30% in those that contain a3.

74 CALMING THE BRAIN

Zolpidem appears to be a full agonist at a2 containing receptors and retains 80% and 90% efficacy at a 1 and a3 containing receptors, respectively (Wafford et aI, 1993a,b). Interes­ tingly, the 1 subunit has a significant effect not only on the ECso values but also on the efficacy of partial agonists. In a2J31 combinations the efficacy of zolpidem at receptors that also contain 11 is only 30% but it is a full agonist at those containing the 12 subunit. In contrast, CL 218,872 proves to be a full agonist at a2J3111 but displays an efficacy only 60% or that of fluni­ trazepam a tal J3112 receptors (Wafford et aI, 1993b). Similar studies have been carried out with the distinct J3 subunit isoforms, although these do not appear to play a major role in determining efficacy with the benzodiazepine ligands, the barbiturates or the neurosteroids (Hadingham et al, 1993). The j3-carboline series have also been studied with regard to their efficacy at distinct receptor subtypes. Abecarnil is a full agonist at a1 and a3 containing receptors but a partial agonist at those which comprise a2 or as in combination with 12 and a J3 subunit (Knoflach et al, 1993). However, while DMCM and j3CCM exhibit clear inverse agonist activity at a1J3112 recep­ tors, at a 1J31 11 receptors they are efficacious agonists (Puia et aI, 1991). Interestingly, DMCM displays inverse agonist activity at a4j3112 receptors, although with rather less efficacy than at a1 containing receptors, while the partial agonists bretazenil and FG 8205 also retain their efficacy at this receptor subtype indicating that certain benzodiazepine site ligands will recognize a4 containing subtypes (Puia et al, 1992; Wafford et al, 1996). The relationship between recognition and receptor subtype is quite distinct from that between efficacy and receptor subtype. However, it is clear that residues previously identified as important in recognition may play a role in the definition of efficacy. An example of this can be seen in the characterisation of mutants of rat a1H101 (Dunn et al, 1999). Two of the mutants, when expressed with J3212, failed to recognise flunitrazepam (K and E) while others (F, Y and Q) had little effect on the ability of this ligand to potentiate GABA responses. The effects on recog­

nition of flumazenil and Ro 15-4513 were signifi­ cant but at the mutant H101K both ligands exhib­ ited partial agonist properties. The evidence for the relationship between ligand, receptor subtype recognition and efficacy continues to emerge but complete pictures are difficult to assemble from information that is in the public arena. It is not possible within the con­ fines of this text to provide more than examples of the progress that continues to be made in this area. However, it seems clear from the clinic that agents currently used as hypnoties have either a preferential affinity or efficacy for a 1 subunit containing receptors, such as zolpidem and zale­ pIon. Preclinical data also provides evidence for receptor subtype selectivity with regard to func­ tion. SL 651498, which exhibits full agonist activ­ ity at a2 and a3 subunit containing receptors but is a partial agonist at those containing the a1 subunit, is effective in behavioural tests that predict anxiolytic activity but fails to impair motor coordination or memory, tempts the con­ clusion that it is the a21 a3 containing receptors which may be targeted for non-sedative anxi­ olyties (Griebel et al, 2(01). The targeting of anxi­ olytic activity to the a21 a3 containing receptors is also exemplified in the compound L 838417, which has a high affinity for aI, a2, a3 and as containing receptors but does not recognise those containing a4 or a6. However, unlike the classical benzodiazepines it exhibits zero efficacy at a 1 containing receptors being a partial agonist at other GABA A receptors which it recognises (McKeman et al,2000). A similar approach has been used in attempts to develop cognition enhancers that display inverse agonist activity at as containing recep­ tors. Mice with targeted disruption of the as subunit gene exhibit significantly enhanced spatial learning (Collinson et aI, 2002) suggest­ ing that inverse agonists at as containing recep­ tors may well prove useful cognition enhancers that lack proconvulsant or kindling effects seen with non-selective inverse agonists (see, for example, Chambers et aI, 2(02). In this context, it is interesting that GABA receptors containing the as subunit are only marginally reduced in Alzheimer disease patients (Howell et aI, 2000). This discussion will be expanded in Chapter 6.

FUNCfIONAL CHARACTERISAnON OF GABA A RECEPTORS 75

GENETIC MANIPULATION

The 'knock-out' mouse

Attempts have been made to identify the func­ tion of distinct receptor subtypes by the genera­ tion of mouse lines in which the genes encoding specific GABA receptor subunit isoforms have been inactivated, the so-called 'knock-out' mouse. In these animals the expression of spe­ cific gene products may be suppressed and the effect on the behaviour of the animal evaluated. The first of these studies involved the tar­ geted disruption of the -y2 gene (Gunther et aI, 1995), this subunit being present in around 60% of all GABA A receptors expressed in the brain. Mice, homozygous in the deleted gene, die peri­ natally, probably because the -y2 subunit is important in synaptic clustering of the receptor (Essrich et aI, 1998). However, the heterozy­ gotes survive until adulthood and breed. These animals are less sensitive to the benzodi­ azepines than their unaffected littermates and show a behavioural syndrome of hypervigi­ lance and anxiety. However, the -y2 subunit can exist in a long form, 'Y2L, or the short form, -y2S, in which a 24 base pair exon is deleted, result­ ing in the expression of a protein with 8 fewer amino acids in the intracellular loop between M3 and M4 (see Chapter 3). In animals in which this exon is deleted from the genome the animals appear normal but treatment with either zolpidem or midazolam prolongs their sleep time, an effect not seen when the animals are exposed to etomidate or pentobarbital (Homanics et aI, 1999; Quinlan et aI, 2000). The discovery of the impact of deletion of the Gabr~-3 gene encoding the ~3 subunit arose quite serendipitously from genetic studies of known mouse mutations causing albinism and cleft palate. The pink-eyed locus on mouse chromosome 7 is homologous to the region of human chromosome 15 that carries the gene for Angelman syndrome, a hereditary neurobehav­ ioural disorder. The pink-eyed cleft palate mutation - peep) - is characterised by hypopig­ mentation and deft palate. Since they cannot wean, peep) mice generally die shortly after birth. The survivors show tremor and gait dis­ turbances. It appears that in this mutation, the

Gabry-3, Gabra-S and

Gabr~-3 genes as well as the p locus are deleted (Nakatsu et aI, 1993). The appearance of deft palate appears to be caused by deletion of the Gabr~-3 gene (Culiat et aI, 1993, 1995). Interestingly, in human neonates, cleft palate has been described as an occasional teratological consequence of the use of certain benzodiazepines by pregnant woman (Safra and Oakley, 1975). This finding implies that GABA A receptors containing the ~3 subunit may have a previously unsuspected non-neuronal role in directing palate formation during embryogenesis. Palate formation can be restored in peep) mice by introducing a Gabr~-3 transgene (Culiat et aI, 1995). Targeted disruption of the gabr~-3 gene pro­ duces an original neurological phenotype, in addition to deft palate. Although these mice have difficulty suckling, due to the cleft palate, if successfully weaned they are quite viable. Such mice are hyperactive, show poor motor coordination and abnormal reflexes, and have spontaneous seizures (Homanics et aI, 1997a). Hippocampal neurons and dorsal root ganglion cells from these transgenic mice show attenu­ ated responses to GABA. Furthermore, results of pharmacological experiments with loreda­ zole are consistent with the notion that the GABA A receptors now contain the ~2 subunit in place of the ~3 subunit (Krasowski et aI, 1998). Lowered sensitivity of hippocampal neurons may well explain the lowered convulsant thresholds in these animals. These mice also show a reduced sensitivity to a number of anaesthetic agents including halothane, etomi­ date, enflurane and midazolam (Quinlan et aI, 1998). No difference in ethanol sensitivity was noted. Deletions of either Gabry-3 alone or Gabry-3 and Gabra-S do not cause cleft palate, nor seem deleterious to survival (Culiat et aI, 1994). Adult mice with these deletions are neurologi­ cally normal, suggesting that either receptors containing a5 or -y3 subunits do not fulfil a vital role in brain function, or other subunits can take the place of the missing a5 and 'Y3 to main­ tain normal functioning. Targeted disruption of the Gabra-6 gene, which is expressed uniquely in the granule cell

76 CALMING THE BRAIN

population of the cerebellum, has also been per­ formed (Homanics et aI, 1997b; Jones et aI, 1997). Cerebellar development and function are apparently normal, and these mice are viable and can reproduce. Nonetheless, there is com­ plete absence of expression of benzodiazepine­ insensitive GABAA receptors in the cerebellum where only half the number of GABAA recep­ tors are expressed compared to wild type animals. Interestingly, in these animals the & subunit appears to be selectively degraded. While & sub unit mRNA levels in the granule cells of the cerebellum were present at wild type levels, immunological methods provided evidence of post-translational loss of the mature protein (Nusser et aI, 1999). Pharmacological studies with Ro 15-4513, which recognises receptors containing a6 and/ or &subunits had suggested that these receptors may be responsible for mediating some of the central nervous system depressant effects of alcohol (Ticku and Kulkarni, 1988; Liiddens et aI, 1990). This hypothesis received further support when it was demonstrated that an 'alcohol-resistant' rat strain developed in Finland carried a point mutation (Rl00Q) in its a6 subunit (Hellevuo et aI, 1987; Korpi and Seeburg, 1993). It was therefore quite surprising to observe that targeted deletion of the Gabra-6 gene does not lead to any changes in the sensi­ tivity of mice to alcohol, or in the development of tolerance to alcohol (Homanics et al, 1998). The a6/& containing GABA A receptor does not thus appear to play a crucial role in brain func­ tion or in the effects of central nervous system depressant drugs. However, mice deficient in the &subunit, generated using similar technol­ ogy, have been shown to exhibit a selective attenuation of responses to the neuroactive steroids (Mihalek et aI, 1999; Vicini et al, 2002). Clearly, the 'knock out' approach is powerful but not particularly subtle. The means by which these animals are engineered may not only compromise expression of other neigh­ bouring genes but also lead to adaptive changes during development. For example, not only does the a6 'knock out' suffer a decreased expression of the & subunit in the cerebellar granule cells, but also there is an increase in the

expression of the 133 subunit (Nusser et aI, 1999) and up-regulation of the potassium channel TASK-1 (Brickley et aI, 2001). In view of the abundance of receptors containing the al and 132 subtypes it is perhaps surprising that mice lacking either of these subunits survive despite a reduction to less than half of the normal GABA A receptor population (Sur et aI, 2001). Here again, subsequent generations indi­ cate that subunit plasticity comes to the rescue (Kralic et aI, 2002). The interpretation of such complex phenotypes is difficult. 'Knock-In' mice

The 'knock-in' strategy, by which a single amino acid cod on within a defined gene is replaced in vivo, provides a rather more restrained approach. GABAA receptors containing aI, a2, a3 or as subunits, together with 13 and 'V, recog­ nise the classical benzodiazepines but those containing a4 or a6 do not (see Chapter 3). The reason for this is that histidine, found at position 101 (or equivalent) of those a subunits that do recognise the benzodiazepines, is replaced by arginine in those that do not. By replacement of H101 with arginine in the benzodiazepine­ sensitive a subunits, benzodiazepine recogni­ tion can be ablated but the receptors exhibit the normal response to the natural agonist GABA (Kleingoor et ai, 1993; Wieland et aI, 1992; Rudolph et al, 1999). Thus, by introducing this molecular switch into the germ line, mutant animals may be engineered which express the aI, a2, a3 or a5 subunits normally but these mutated subunits no longer recognise the benzodiazepines. The technology, thus far, has provided infor­ mation concerning the relative importance of aI, a2 and a3 subunits in the pharmacological spectrum of benzodiazepine action (Rudolph et aI, 1999; McKernan et al, 2000; Low et aI, 2000). The GABA responses in the a1(HI01R) and a2(H101R) mutated animals appeared to be unaltered with respect to their wild-type rela­ tives (Rudolph et aI, 1999; Low et aI, 2000), the distribution of the mutated subunits matched that of their parent and the animals showed no overt phenotype.

FUNCTIONAL CHARACTERISAnON OF GABAA RECEPTORS 17

Those mice expressing the 0.1HI0IR were not sedated by diazepam, as measured by locomo­ tor activity, neither were they fully protected against pentylenetetrazole-induced convulsions by the drug. Interestingly, however, although the anticonvulsant activity of diazepam was not totally compromised in these animals, the anti­ convulsant effects of zolpidem were completely lost (Crestani et ai, 2000). It was also clear that the anterograde amnesia, associated with diazepam exposure in these animals, was lost as a consequence of this mutation. However, other overt effects of diazepam, such as anxiol­ ysis, muscle relaxation or ethanol potentiation, remained unaffected. In mice expressing 0.2HI0IR, diazepam­ induced anxiolytic activity, assessed using the elevated plus maze and light-dark box selection tests (discussed further in Chapter 6), was absent but this was not the case for those mice expressing 0.3HI0IR (Low et ai, 2000). Thus, targeting of agents to the 0.2 subunit containing receptors could be expected to provide relief from anxiety without the sedative side-effect profile that compromises the use of the cur­ rently available agents. This indeed appears to be the case (McKernan et ai, 2000). While it is clear that experiments of this type can be criticised (Sieghart, 2000), the insight that they provide is significant. There is now evidence both from molecular genetics and ligand-specific approaches that 0.1 containing receptors are responsible for the sedative effects and anterograde amnesia of the classical benzo­ diazepines while those that contain the 0.2 subtype mediate their anxiolytic properties (Mohler et al, 2001; Rudolph et ai, 2001). IMMUTABILITY OF EXPRESSION PATTERNS AS IT PERTAINS TO FUNCTION

One of the principal concerns about the use of the benzodiazepines, particularly over exten­ ded periods, has been their propensity to produce tolerance and, in a significant percent­ age of the population, dependence that mani­ fests itself as withdrawal when the medication is stopped (see Chapters 6 and 10 for discussion). The molecular events, which underlie these phe­

nomena, remain poorly understood, but over the past decade significant progress has been made. Tolerance

One of the early suggestions was that tolerance developed to the benzodiazepines as a conse­ quence of receptor down-regulation. Most of the early work was carried out using radioli­ gand binding studies and, although a number of different drugs and treatment paradigms were used, it was generally found that no change in the binding capacity occurred (Gallager et al, 1984; Miller et aI, 1989). There were some reports of receptor down-regulation but in a number of these very high benzodi­ azepine doses were used (Rosenberg and Chiu, 1979,1981). Interestingly, in some of the early chronic treatment studies, it was noted that the func­ tionality of the benzodiazepine site was lost: in exposed animals chloride flux measurements, carried out in vitro, suggested that the benzodi­ azepines were no longer able to augment the response to GABA, and in binding assays GABA failed to increase the affinity of the ben­ zodiazepine binding site for agonists in tissue recovered from chronically treated animals (Gallager et ai, 1984; Marley and Gallager, 1989). Subsequent studies, in which either primary cultured cells (Roca et ai, 1990; Hu and Ticku, 1994a,b; Prasad and Reynolds, 1992) or cells engineered to express specific GABA A receptor subtypes, were exposed to benzodi­ azepines in vitro (Klein et ai, 1994, 1995; Primus et aI, 1996; Wong et aI, 1994) again showed a loss of functional coupling between the GABA and benzodiazepine sites. In those studies in which it was investigated, no change in either the GABA or benzodiazepine binding site capacity was observed and the coupling between the sites could be recovered by with­ drawal of the benzodiazepine from the culture medium. The loss of coupling in vitro proved to be rapid, of the order of hours, and thus seemed to be an unattractive hypothesis to explain tolerance that occurs as a consequence of long-term exposure to the benzodiazepines. Indeed, later studies in vivo showed that the

78 CALMING THE BRAIN

uncoupling phenomenon was somewhat tran­ sient in nature. A single injection of diazepam to animals resulted in a loss of the GABA­

hypothesis attractive. First, it would appear that cessation of exposure allows the subunit isoform expression patterns to return to normal.

benzodiazepine coupling between 4 and 12

Second, both partial agonists and other benzo­

hours after the injection, but 24 hours later, pre­ sumably when the diazepam had been essen­ tially cleared from the animal by metabolism, the coupling between the sites had returned to control levels (Holt et aI, 1999). In these studies, chronic treatment of the animals with diazepam, daily by injection for 14 days, affected neither the time course of appearance nor disappear­ ance of the uncoupling between the sites. However, it is undoubtedly an important obser­ vation and may well be the prelude to other molecular events, which are now often referred to as 'subunit isoform switching'. Tolerance and dependence

The appearance of tolerance to some of the overt effects of the benzodiazepines is relatively slow, the sedative effects decline after a few days while the anticonvulsant effects are compro­ mised in man only after several months (see Chapter 6 for further discussion). Dependence also occurs on a similar time scale and many groups accumulated evidence that, over this time frame of benzodiazepine exposure, the expression patterns of a number of GABA A receptor subunit isoforms changed. The studies were initially difficult to interpret for the various groups used different experimental par­ adigms, studied distinct sets of GABAA receptor subunit isoforms or carried out their work in different brain regions (eg Heninger et aI, 1990; Kang and Miller, 1991; O'Donovan et aI, 1992; Primus and Gallager, 1992; Impagnatiello et aI, 1996; Holt et aI, 1996). However, it now seems clear that chronic treat­ ment of experimental animals with agonist benzodiazepine ligands modulates the expres­ sion of different subunit isoforms, changes that are brain region-specific and time-dependent. Much of the work relied on the quantification of steady state mRNA levels but in several cases changes in protein levels have now been shown to reflect these. Several additional pieces of information make the subunit isoform switching

diazepine site ligands, which appear to be less prone to cause tolerance and dependence, do not induce marked changes in expression (Holt et aI, 1996; Impagnatiello et al, 1996; Holt et al, 1997a). Finally, different benzodiazepines seem to have a commonality of action, for example, the a4 subunit, which renders GABAA receptors insensitive to the benzodiazepines, is increased by chronic exposure. However, while these studies provide correlations between subunit expression and the temporal dependence of tol­ erance development it will undoubtedly become clear that additional changes occur as a conse­ quence of exposure to these agents. The mechanisms that are responsible for this modulation of subunit expression are not well defined. It is clear that in the case of ,,(2, chronic treatment of animals with diazepam leads to a change in gene transcription, which occurs in a brain region-specific manner reflecting the observed change in steady state mRNA levels (Holt et al, 1997b). There have been no reports on the effect of benzodiazepines on mRNA degradation. Studies in primary cultured neurons have also demonstrated changes in receptor subunit isoform expression as a conse­ quence of benzodiazepine exposure and, further, that the changes are dependent on phosphorylation mechanisms (Johnston et aI, 1998; Brown and Bristow, 1996; Johnston and Bristow, 1998). This is interesting for it has been reported that GABAA receptor subunit cycling between intracellular compartments and the cell membrane is dependent on phosphorylation (Connolly et al, 1999; Kittler et al, 2000, 2(02), as is the association of the receptor with clathrin, important in receptor internalisation (Tehrani and Barnes, 1991, 1993). The observation that internalised GABAA receptors, which are associ­ ated with clathrin, appear to be uncoupled from the benzodiazepine sites (Tehrani et aI, 1997), brings the story temptingly back to the begin­ ning of this discussion. Although the validity of the plot must be viewed with some circumspec­ tion at this time, its development in the future

FUNCTIONAL CHARACTERISA nON OF GABAA RECEPTORS 79

will undoubtedly provide valuable guidance to our understanding of the complex disease states in which these drugs are used. REFERENCES

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FUNCTIONAL CHARACTERISAnON OF GABA A RECEPTORS 81

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82 CALMING THE BRAIN

Krasowski MD, Rick CE, Harrison NL, et al. A deficit of functional GABA(A) receptors in neurons of beta 3 subunit knockout mice. Neurosci Lett 1998; 240: 81-84. Lambert JJ, Belelli D, Hamey se, et al. Modulation of native and recombinant GABA A receptors by endogenous and synthetic neuroactive steroids. Brain Res Rev 2001; 37: 68-80. Uppa A, Coupet I, Greenblatt EN, et al. A synthetic non-benzodiazepine ligand for benzodiazepine receptors: a probe for investigating neuronal sub­ strates of anxiety. Pharmacol Biochem Behav 1979; 11: 99-106. Low K, Crestani F, Keist R, et al. Molecular and neu­ ronal substrate for the selective attenuation of anxiety. Science 2000; 290: 131-134. Liiddens H, Pritchett DB, Kohler M, et al. Cerebellar GABAA receptor selective for behavioural alcohol antagonist. Nature 1990; 346: 648-651. Marley RI, Gallager DW. Chronic diazepam treat­ ment produces regionally specific changes in GABA-stimulated chloride influx. Eur , Pharmacol 1989;159:217-223. McKeman RM, Rosahl TW, Reynolds OS, et al. Sedative but not anxiolytic properties of the benzo­ diazepines are mediated by the GABAA receptor a1 subtype. Nature Neurosci 2000; 3: 587-592. Mihalek RM, Banerjee PK, Korpi ER, et al. Attenuated sensitivity to neuroactive steroids in 'Y-amino­ butyrate type A receptor delta subunit knockout mice. Proc Natl Acad Sd USA 1999; 96: 12905-12910. Miller LG, Roy RB, Weill Cl. Chronic clonazepam administration decreases gamma-aminobutyric acidA receptor function in cultured cortical neurons. Mol Phannacol1989; 36: 796--802. Mody I. Distinguishing between GABAA receptors responsible for tonic and phasic conductances. Neurochem Res 2001; 26: 907-913. M6hler H, Crestan F, Rudolph U. GABAA-receptor subtypes: a new pharmacology. Curr Opin PharmacoI2001;1:22-25. Nakatsu Y, Tyndale RF, Delorey TM, et al. A cluster of three GABA A receptor subunit genes is deleted in a neurological mutant of the mouse p locus. Nature 1993; 364: 448-450. Neelands TR, Fisher IL, Bianchi M, Macdonald RL. Spontaneous and 'Y-aminobutyric acid (GABA)­ activated GABA A receptor channels formed by E-subunit containing isoforms. Mol Pharmacoll999; 56: 168-178. Newell IG, Dunn SMI. Functional consequences of the loss of high affinity agonist binding to gamma ­ aminobutyric acid type A receptors: Implications

for receptor desensitization. , Bioi Chem 2002; 277: 21423-21430. Newell IG, Davies M, Bateson AN, Dunn SMI. Tyrosine 62 of the 'Y-aminobutyric acid type A receptor 132 subunit is an important determinant of high affinity agonist binding. , Bioi Chem 2000; 275: 14198-14204. Nusser Z, Mody I. Selective modulation of tonic and phasic inhibitions in dentate gyrus granule cells. , Neuraphysiol 2002; 87: 2624-2628. Nusser Z, Roberts ID, Baude A, et al. Relative densi­ ties of synaptic and extrasynaptic GABA A recep­ tors on cerebellar granule cells as determined by a quantitative immunogold method.' Neurosci 1995; 15: 2958-2960. Nusser Z, Sieghart W, Somogyi P. Segregation of dif­ ferent GABAA receptors to synaptic and extrasy­ naptic membranes of cerebellar granule cells. ,Neurosci 1998; 18: 1693-1703. Nusser Z, Ahmad Z, Tretter V, et al. Alterations in the expression of GABA A receptor subunits in cerebellar granule cells after disruption of the a6 subunit gene. Eur' Neurosci 1999; 11: 1685-1697. O'Donovan MC, Buckland PR, Spurlock G, et al. Bidirectional changes in the levels of messenger RNAs encoding 'Y-aminobutyric acid A receptor alpha subunits after flurazepam treatment. Eur , PharmacoI1992;226:335-341. Olsen RW, Bergman MO, Van Ness PC, et al. 'Y-Aminobutyric acid receptor binding in mam­ malian brain: heterogeneity of binding sites. Mol Pharmacol 1981; 19: 217-227. Olsen RW, Yang I, King RG, et al. Benzodiazepine receptor function: V. barbiturate and benzodi­ azepine modulation of GABA receptor binding and function. Life Sci 1986; 39: 1969-1976. Peters lA, Kirkness EF, Callachan H, et al. Modulation of the GABA A receptor by depressant barbiturates and pregnane steroids. Br , Pharmacol 1988;94:1257-1269. Prasad A, Reynolds IN. Uncoupling of GABA­ benzodiazepine receptors in chick cerebral cortical neurons requires co-activation of both receptor sites. Brain Res 1992; 591: 327-331. Primus RI, Gallager DW. GABA A receptor subunit mRNA levels are differentially influenced by chronic FG 7142 and diazepam exposure. Eur' PharmacoI1992;226:21-28. Primus RJ, Yu I, xU J, et al. Allosteric uncoupling after chronic benzodiazepine exposure of recombinant gamma-aminobutyric acid (A) receptors expressed in Sf9 cells: ligand efficacy and subtype selectivity. ,Pharmacol Exp Ther 1996; 276: 882--890.

FUNCTIONAL CHARACTERISAnON OF GABAA RECEPTORS 83

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Sigel E, Baur R, Trube G, et al. The effect of subunit composition of rat brain GABAA receptors on channel function. Neuron 1990; 5: 703-711. Sigel E, Baur R, Kellenberger 5, Malherbe P. Point mutations affecting antagonist and agonist depen­ dent gating of GABAA receptor channels. EMBO J 1992;11:2017-2023. Smith GB, Olsen RW. Identification of [3H]muscimol photoaffinity substrate in bovine 'Y-aminobutyric acid A receptor a subunit. J Bioi Chem 1994; 269:20380-20387. Squires RF, Benson 01, Braestrup C, et al. Some properties of brain specific benzodiazepine recep­ tors: new evidence for multiple receptors. Pharmacol Biochem Behav 1979; 10: 825-830. Sur C, Wafford KA, Reynolds OS, et al. Loss of major GABAA receptor subtype in brain is not lethal in mice. J Neurosci 2001; 21: 3409-3418. Tehrani MH, Bames EM. Agonist dependent inter­ nalization of -y-aminobutyric acid A/benzodi­ azepine receptors in chick cortical neurons. J Neurochem 1991; 57: 1307-1312. Tehrani MH, Bames EM. Identification of GABAA/benzodiazepine receptors on clathrin­ coated vesicles from rat brain. J Neurochem 1993; 60: 1755--1761. Tehrani MH, Baumgartner BJ, Bames EM. CIathrin­ coated vesicles from bovine brain contain uncou­ pled GABA A receptors. Brain Res 1997; 776: 195--203. Thompson 5-A, Wafford K. Mechanism of action of general anaesthetics - new information from mole­ cular pharmacology. Curr Opin Pharmacol 2001; 1: 7S--83. Ticku MK, Kulkami SK. Molecular interactions of ethanol with GABAergic system and potential of Ro 15-4513 as an ethanol antagonist. Pharmacol Biochem Behav 1988; 30: 501-510. Tretter V, Ehya N, Fuchs K, Sieghart W. Stoichiometry and assembly of recombinant GABA A receptor subtypes. J Neurosci 1997; 17: 272S--2737. Verdoom TA. Formation of heteromeric gamma­ aminobutyric acid type A receptors containing two different alpha subunits. Mol Pharmacol 1994; 45: 475--480. Verdoom TA, Oraguhn A, Ymer S, et al. Functional properties of recombinant GABA A receptors depend upon subunit composition. Neuron 1990; 4: 919-928. Vicini S, Losi G, Homanics G. GABA A receptor 8 subunit deletion prevents neurosteroid modula­ tion of inhibitory synaptic currents in cerebellar neurons. Neuropharmacology 2002; 43: 646-tiSO.

84 CALMING THE BRAIN

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5

Structures of benzodiazepine recognition

site ligands

In the mid 1950s pharmacologists at Hoffmann­ La Roche instituted a search for compounds to replace the then popular minor tranquilliser, meprobamate. The in vivo pharmacological screening procedures available at that time, and reported in some detail by Randall (Randall and Kappell, 1973), proved both sensitive and discriminatory. A collaboration between this group and a team of medicinal chemists, headed by Leo Sternbach (Sternbach, 1979), uncovered a spectrum of pharmacological activity which was to transform the treatment of anxiety over a period of more than forty years. The first compound to emanate from this work was chlordiazepoxide, first marketed in 1960 under the trade name Librium (Figure 5.1). Inevitably, attempts were made to refine the activity profile of this initial market lead. Using

essentially the same suite of in vivo screening procedures they revealed the primary struc­ tural features now associated with the benzodi­ azepine minor tranquillisers. Interestingly, reading the older reports, it was clear that several of the compounds produced by Sternbach's team may well have revealed the full spectrum of activity that we associate with compounds acting at the benzodiazepine site today, for several appeared to be inactive or convulsant. These were natu­ rally discarded as of little therapeutic value, although we would now perhaps be able to associate them with antagonist or inverse agonist activity at the site. Over the subsequent years, many of these screening methods were replaced by more sophisticated in vivo (Kyburz, 1986) and in vitro (Haefely, 1988) techniques. The development of radioligand binding assays proved invaluable for later studies of structure-activity relation­ ships at the benzodiazepine recognition site (Fryer, 1990). Not only did these circumvent the pharmacokinetic and metabolic complications that contaminated the interpretation of the earlier in vivo studies but also they revealed novel chemical structures that appeared to interact with the same recognition site on the GABA A receptor as the classical benzodi­ azepines. Several of these have subsequently

spawned the subtype-specific agents that are Figure 5.1.

Chlordiazepoxide.

currently appearing in the patent literature. However, before we delve into this speculative area we must review the original structure­

86 CALMING THE BRAIN

activity relationships as, in many cases, they underpin subsequent refinements. CLASSICAL 1,4-BENZODIAZEPINES

The early studies of Sternbach and his col­ leagues indicated that the presence of the seven-membered amino-lactam ring was essen­ tial for activity, although limited substitution was allowed at positions 1,3,7 and 2' (Figure 5.2) (Stembach et ai, 1968). Electronegative substitution of the A ring at position 7 yields high affinity ligands, with the nitro group being the most effective substituent and fluorine the least. Cl substitution at posi­ tions 6, 8, or 9 in the A ring has a detrimental effect on affinity with substitution at position 6 being particularly disadvantageous. Halogen substitution at 2' leads to compounds with increased affinity, compared to their congeners with no substitution in the A ring, but only marginally increases the affinity of those com­ pounds with electronegative substituents at position 7. The A ring may be replaced by certain heterocydes with a small reduction in affinity. Position 1 is relatively accommodating to substitution. Aliphatic amines and alcohols reduce the affinity, relative to hydrogen or methyl substituents, although the effect is rela­ tively small. However, the introduction of

bulky aliphatic substituents or acids results in a marked reduction in affinity. The carbonyl function at position 2 is im­ portant for binding activity; its removal results in a decrease in affinity by two orders of magni­ tude. Saturation of the 4,5 double bond results in complete loss of in vitro activity. The S-phenyl substituent, ring C, may be saturated, or replaced with a heterocycle; both produce some reduction in binding activity. A detailed review of the structural modifications and the consequences of these on the pharmacological properties of the compounds is available (Haefely et al, 1985). The majority of the benzodiazepines have no chiral centre; however, the seven-membered ring B can adopt one of two energetically preferred boat conformations; the molecule is thus termed pro-chiral. The methylene group at the 3-position of a l,4-benzodiazepine ring is known to be either above or below the plane of the fused benzene ring, imparting conformational chirality to the molecule. It was originally thought that this puckering of the seven-membered ring placed the carbonyl oxygen atom on the same side of the molecule as the methylene carbon atom (Fryer, 1983). It was later shown, after examining many X-ray structures, that this proton-accepting oxygen atom can exist either above or below the plane of the A ring to an extent of approximately ± o.sA. In order to eluci­ date the stereospecificity of the receptor inter­ action several enantiomeric pairs with a chiral centre at position 3 have been prepared (Haefely et ai, 1985). The introduction of a methyl sub­ stituent at this position leads to stabilisation of the conformation of the seven-membered ring. Both nuclear magnetic resonance (Sunjic et al, 1979) and X-ray crystallographic studies (Blount et al, 1983) are consistent with the suggestion that the conformation of ring B, responsible for high affinity interaction with the receptor, is that shown for diazepam in Figure 5.3. 1 ,2-ANNELATED 1,4-BENZODIAZEPINES

FIgure 5,2

The 1,4-benzodiazepinones.

The condensation of both triazolo- and imidazo­ rings to the 1,2 bond of the classical benzo­ diazepine nucleus (the 'a' face) has resulted in the synthesis of a novel and interesting series

STRUCTURES OF BENZODIAZEPINE RECOGNITION SITE LIGANDS 87

f3-CARBOLlNES

Figure 5.3

The active conformation of diazepam,

of high affinity ligands (Figure 5.4) for this recep­ tor. The substitutions within this nucleus appear to exhibit structure activity relation-ships distinct from those of the classical1,4-benzodiazepines. The 1,2-annelation of triazolo- or imidazo­ rings to classical 1,4-benzodiazepines generally results in an increase in affinity only for those compounds with a relatively low affinity in the classical series; little increase in affinity is found for the high affinity 1,4-benzodiazepines. Substitution at positions 8 and 2' appears to be much less important for activity than equivalent substitutions in the classical 1,4­ benzodiazepines (Haefely et aI, 1985). Within the imidazobenzodiazepine series those com­ pounds lacking substitution at position 3, or with an amide in this position display agonist activity. However, ester substitution at this position can lead to antagonist activity.

Figure 5.4

, Th e 1.2-annelated benzod'lazeplnes,

The [3-carbolines (Figure 5.5 and Table 5.1) rep­ resent a structurally distinct class of ligands for the benzodiazepine recognition site, many of which exhibit high affinity. The [3-carbolines are able to evoke the whole gamut of behavioural responses and represen­ tatives can be found within this class of full agonists, antagonists and inverse agonists. However, the first member of this class to be investigated was the partial inverse agonist ethyl f3-carboline-3-carboxylate, [3CCE. Inverse agonists with greater intrinsic efficacy were subsequently described, [3CCM and DMCM, partial and full inverse agonists, respectively (Corda et aI, 1983; Ninan et aI, 1982; Dorow et aI, 1983; File et aI, 1982; Braestrup et aI, 1982). Increasing the bulk of the aliphatic ester at the 3-position decreased the efficacy of these com­ pounds and [3CCPr, together with its t-butyl analogue were found to be essentially devoid of intrinsic activity, they were antagonists. Substitution in the A ring revealed some interesting structure function relationships. ZK 93426 proved to be an antagonist (see Figure 5.5) (Jensen et al., 1983, 1984; Shanon et aI, 1984; Stephens et aI, 1985) but further aromatic sub­ stitution in this ring provided the first agonists in this series with ZK 91296, a partial agonist (Stephens et aI, 1985; Petersen et aI, 1984), and

88 CALMING THE BRAIN

Ring substitutions on the p.carboIlne skeleton

Table &.1

Compound

R1

R3

R4

R,

Ra

RT

I3CCM

H

C02CH 3

H

H

H

H

C02C2 Hs

H

H

H

H OCH 3

H

~CCE

DMCM

H

C0 2CH 3

C2HS

H

OCHa

I3CCPr

H

CO2 ''C 3 H7

H

H

H

H

t-butyl analogue

H

C02 fC 4 Hg

H

H

H

H

ZK 93426 ZK 91296 ZK 93426

H

C02C2 Hs

CH 3

OiC 3H7

H

H

H

C0 2C2 Hs

CH 20CH a

OCH 2 Ph

H

H

H

C0 2C2 Hs

CH 2OCH 3

H

OCH 2 Ph

H

Abecamil

H

C02iC 3 H7

CH 2OCH 3

H

OCH 2Ph

H

ZK 93423, a full agonist (stephens et al, 1984, 1985). Both compounds exhibit similar pharma­ cological characteristics to the agonist benzodi­ azepines (Step hens et al, 1984, 1985; Meldrum et al, 1983; Klockgether et al, 1985; Mereu et aI, 1987; Giorgi et al, 1987). Abecarnil (ZK 112-119) also exhibits agonist activity although develop­ ment of this compound is now at an end (stephens et al, 1990). Abecarnil has a high affinity for the benzo­ diazepine receptor and possesses anxiolytic and anticonvulsant properties; it is more potent than diazepam in most rodent tests of anxiolytic activity and in reducing locomotor activity in mice and rats. In other tests (motor coordination, potentiation of the effects of ethanol and hexo­ barbital on motor performance) it is less potent than diazepam. Its affinity is increased in vitro by

Figure 5.5

The ~arbolines.

GABA, although the effect is less pronounced than that found for diazepam, and the com­ pound also antagonises the effects of diazepam on the righting reflex, both observations compat­ ible with its classification as a partial agonist (stephens et aI, 1990). However, the ability of abecarnil to distinguish between distinct classes of the GABA A receptor (BZI versus BZ2) may well explain some of its differential pharmaco­ logical effects compared to those of the classical benzodiazepines (Haefely et al, 1990). However, it is also clear that this compound displays dif­ ferential efficacy at distinct subtypes of GABAA receptor being a full agonist at cll and a3 con­ taining receptors and a partial agonist at those receptors containing a2 or as (Knoflach et aI, 1993). Perhaps even more interesting is the asso­ ciation of decreased propensity of this com­ pound to produce tolerance and difficulties in withdrawal (Loscher, 1993), an effect which may be related to its apparent inability to modulate GABA A receptor subunit steady state mRNA levels on chronic treatment (Holt et aI, 1996). Reviewing the structural characteristics of the above compounds it would appear that substitu­ tion of larger alkyl groups in the ester function at position 3 increases agonist activity of the com­ pound, as does the introduction of hydrophobic substituents at positions 5 or 6. To an extent the generality of this statement is compromised as methoxy substitution at positions 6 and 7, in

STRUCTURES OF BENZODIAZEPINE RECOGNmON SITE LIGANDS 89

Figure 5.6

The cyclopyrrolones.

DMCM, results in efficacy movement in the reverse direction, that of inverse agonist.

ring fused in the isoindolic moiety and by a bicyclic substituent at the isoindole nitrogen.

CYCLOPYRROLONES

TRIAZOLOPYRIDAZINES

The first compound of this series to be synthe­ sised and described was zopiclone, which displayed an IC so of about 30 nM versus PH]­ diazepam in rat cortex (Blanchard et aI, 1979). Later, more potent cyclopyrrolone compounds, including suriclone (Blanchard et aI, 1983), suproclone (Garret et aI, 1984), RP 59037 (Doble et aI, 1993) and DN 2327 (Wada and Fukuda, 1991) were synthesised. Figure 5.6 shows the chemical structures of zopiclone, suriclone and suproclone. It is clear from structure-activity studies within this series that the methyl-4 piperazinyl carbonyl group is essential to activ­ ity, while a chlorine group in the para position of the pyridine ring results in an increase of activ­ ity and a decrease of toxicity. Other substituents, -CN, -OCH3 or -N02, at this position do not improve activity. The ring fused in the isoindolic moiety can be benzene (RP 25519) or a hetero­ cyclic ring (zopiclone). Suriclone, which exhibits high affinity, is characterised by a heterocyclic

Synthesis, pharmacology and structure-activity data have been reported for the series of 1,2,4­ triazolo[4,3-b]pyridazines, a class of compound with activity in tests predictive of anxiolytic activity (Albright et aI, 1981). Figure 5.7 shows CL 218,872, the most widely studied member of the series. The most active derivatives are the unsubsti­ tuted phenyl compound (with a methyl at posi­ tion 3), p-fluoro, m-fluoro and m-trifluoromethyl analogues (with and without a methyl group at position 3). Decreased activity, or no activity, results from introduction of ethyl, propyl, phenyl, benzyl, chloromethyl groups in the C-3 position. Introduction of methyl groups at the C-7 and C-8 positions decreased or eliminated activity. [3H]-diazepam binding affinities for most compounds ranged from about 10-7 to 10-5 M. The most potent compound of the origi­ nal series, CL 218,872, is active in most in vivo tests predictive of anxiolytic activity (Lippa et al,

90 CALMING THE BRAIN

FIgure 5.7

The structures of CL 218,872 and zaleplon.

FIgure 5.8

The phenylquinolines.

1979; Sepinwall et aI, 1985; Budhram et al, 1986; Gardner, 1986; Guy and Gardner, 1985; Pellow and File, 1986; Gardner and Budhram, 1987). The most recently introduced hypnotic, zale­ pIon, is a pyrazolopyrimidine analogue derived from this series which displays subtype selectivity (preferring BZ1 subtype receptors) and exhibits an elimination half-life of one hour (Figure 5.7) (Sanger et aI, 1995; Beer et al, 1994, 1997; Hurst and Noble, 1999). PHENYLQUINOLlNES

The phenylquinoline derivatives, PK 8165 and PK 9084 (Figure 5.8) exhibit an atypical phar­ macological profile. While these compounds

increase punished responding in the rat con­ flict procedures, they are not anticonvulsant (Pellow and File, 1986; Gardner and Budhram, 1987; Keane et aI, 1984; Benavides et aI, 1984) and do not produce ataxia or sedation at doses 5-20 times higher than those effective in the conflict test. They selectively inhibit PH]­ diazepam binding in vitro (Budhram et aI, 1986; Le Fur et aI, 1981; Pellow, 1985), although Keane (Keane et aI, 1984) has sug­ gested that they have little direct effect on the benzodiazepine sites in vivo. Their pharmaco­ logical effects appear to be complex and may well be mediated by other mechanisms associ­ ated with the GABA A receptor complex (Simmonds, 1985).

STRUCTURES OF BENZODIAZEPINE RECOGNITION SITE LIGANDS 91

Figure 5.9

The pyrazoloquinolinones.

PYRAZOLOQUINOLlNONES

IMIDAZOPYRIDINES

The pyrazoloquinolinones, CGS 8216, CGS 9895 and CGS 9896 (Figure 5.9) all exhibit high affinity for the benzodiazepine recogni­ tion site in vitro. CGS 8216 has been classified as an antagonist (Boast et aI, 1983); the substi­ tution of p-methoxy or p-CI substituents on the 2-phenyl ring produces compounds, CGS 9895 and CGS 9896, respectively, with some agonist activity. CGS 9896 is active in most animal models of anxiety (Sepinwall, 1985; Gardner and Budhram, 1987; Goldberg et aI, 1983; Yokoyama et aI, 1982; Patel et aI, 1983; Bennet and Petrack, 1984; File and Pellow, 1986; Gardner and James, 1987) and in some anticonvulsant tests (Sepinwall, 1985; Pellow, 1985; Gardner and Budhram, 1987; Goldberg et aI, 1983; Yokoyama et al, 1982; Patel et aI, 1983; Bennet and Petrack, 1984; File and Pellow, 1986; Gardner and James, 1987). However, its antagonism of the anticonvul­ sant effects of diazepam is consistent with the suggestion that this compound is a partial agonist (Bernard et aI, 1985; Morel et aI, 1986; Brown et aI, 1984); CGS 9895 is an even weaker partial agonist than CGS 9896 (Yokoyama et aI, 1982; Brown et aI, 1984; Katzman and Shannon, 1985).

The imidazopyridines, zolpidem and alp idem (Figure 5.10), exhibit high affinity for the benzo­ diazepine receptor. Zolpidem is a hypnotic drug with rapid onset and short duration of action; it also displays anticonvulsant, myorelaxant and anticonflict activities (Arbilla et aI, 1985; Deporteere et aI, 1986; Nicholson and Pascoe, 1986; Benavides et aI, 1988), although at higher doses than those required for sedation. Early radioligand binding data indicated that zolpidem preferentially bound to the BZ1 recep­ tor subtype (ArbiUa et aI, 1985; Niddam et aI, 1987) while later recombinant studies have shown that it has an extremely low affinity for those receptors containing an a5 subunit isoform (Pritchett and Seeburg, 1990), although in some regions of the brain, such as the cerebral cortex, a5 containing receptors retain a high affinity for zolpidem suggesting that it is perhaps co­ expressed with another a subunit in these cases (Mertens et al, 1993). Alpidem shows a similar behavioural profile to that of zolpidem (ie seda­ tive dose 48 h)

0.5-1 h 1-1.5 h lh 1-1.5 h 3h 2-3 h 2h lh 0.5-4 h

8h

10-20 h

10 h

16-48 h

39 h

5-8 h

1-4 h

lh

1.2-1.7 h 0.5-4 h 2h 2h lh lh

4h

32-47 h (biphasic)

0 .5-2 h 1 .5 h 1.5h

77h

65 h

2.2-3 h 4-6 h (desmethyldiazepam)

5-15 h 30-150 h (desmethyldiazepam) -

-

- -

- - -

This table shows the time of peak plasma concentrations (T..... ) and elimination half-lives (T1 "2) of several important hypnotic and anxiolytlc drugs. Data are taken from the appropriate French or British product prescribing information

ual effects are particularly troublesome for active individuals who need to be alert and reactive in the workplace during the day. The most widely used hypnotic benzodiazepines today have elimination half-lives of 8 hours or less (Table 7.2). A too rapid rate of elimination, on the other hand, can be a handicap for a hypnotic benzo­ diazepine, since the hypnotic effect will wane during the night and not protect the individual

against nocturnal awakenings. For this reason, the choice of hypnotic benzodiazepine needs to take into account whether symptoms relate predominantly to difficulties falling asleep, or difficulties maintaining sleep later in the night, and a drug with an elimination half-life appro­ priate for the individual used. The difference between sleep induction and sleep maintenance is exploited by zaleplon, the most recent hypnotic drug, which has an extremely short

136 CALMING THE BRAIN

Figure 7.4 Theoretical pharmacokinetic profile of a hypnotic benzodiazepine. The shaded zone indicates the range of plasma levels over which a sedative­ hypnotic effect is observed. Plasma levels need to rise rapidly after ingestion to ensure a rapid onset of hypnotic action, and then to fall over the night, so that only subthreshold drug levels are present the next morning.

elimination half-life of one hour (Figure 7.5). Zaleplon is promoted as a sleep-inducer, rather than a hypnotic, and it has been suggested that this hypnotic can be taken during the night to help falling back to sleep after a nocturnal awakening without running the risk of persis­ tence of sedative effects the next day. This is the only hypnotic drug that has this pharmacoki­ netic profile and that could be expected to be free of next-day effects after administration in the small hours of the night. METABOLISM

The vast majority of benzodiazepines are metabolised in the liver by Type I oxidation involving cytochrome P450 enzymes (Chouinard et aI, 1999). N1-Dealkylation is a particularly common route of metabolism, occurring for the vast majority of benzodiazepines substituted in this position. For the triazolobenzodiazepines, in which this route is not available, the principal metabolic pathway is a-hydroxylation of the triazolo ring by CYP3A subtypes, fol­ lowed by scission of the benzodiazepine ring, as well as by 4-hydroxylation (Gorski et aI, 1994, 1999; Perloff et aI, 2000). Zopiclone is

Figure 7.5 Plasma levels of zopiclone (7.5 mg). zolpidem (20 mg) and zaleplon (10 mg) in healthy volunteers after oral administration of a single dose. Data are taken from Gaillot et ai, 1983, and Drover et ai, 2000.

transformed into three metabolites (by demethylation, decarboxylation and N-oxida­ tion) by CYP2C8 and CYr3A4 (Becquemont et aI, 1999), as is zolpidem (by demethylation at the 5- and 4'-positions, and by ring hydroxyla­ tion), principally by CYP3A4 (Pichard et aI, 1995). Zaleplon has a single major metabolite, 5­ oxozaleplon, formed by aldehyde oxidase (Lake et aI, 2002). On the other hand, a few benzodiazepines are metabolised primarily by glucuronidation (Type 11 metabolism). These include oxazepam and temazepam (Figure 7.6). The enzymes responsible for these conjugation reactions are of high capacity, with the consequence that metabolic rates are relatively insensitive to changes in hepatic function. For this reason, benzodiazepines, such as oxazepam, are more suitable for use in patients with hepatic insuffi­ ciency, and, particularly, for the acute detoxifi­ cation of alcohol dependent subjects who often have impaired hepatic function. Another benefit associated with Type 11 metabolism is a reduced liability for drug interactions.

PHARMACOKINETIC DETERMINANTS OF CLINICAL ACTIVITY 137

Figure 7.6 Metabolism of temazepam and oxazepam.

Unlike barbiturates, benzodiazepines do not induce cytochrome P450 metabolising enzymes, and this is one of their principal advantages over the earlier class of tranquilliser drugs. However, they can, at least in principle, inhibit these enzymes, and this is a source for potential drug interactions. Moreover, other drugs that inhibit cytochrome P450 enzyme sub types can interfere with the metabolism of benzodiazepines and increase their plasma levels. In the case of hypnotic benzodiazepines (or zopiclone, zolpidem and zaleplon), drug could then persist in the circulation for longer than normal, with the consequence of daytime sedation. For anxiolytics, the increased plasma levels can also lead to unwanted sedation. This issue of drug interactions which increase the sedative effects of benzodiazepines is particu­ larly relevant, since the principal cytochrome P450 subtype involved in the metabolism of most benzodiazepines (and of the z-hypnotics) is CYP3A4, the major xenobiotic drug­ metabolising enzyme in humans, with a large number of inhibitors known amongst com­ monly prescribed drugs. Given that Nl-dealkylation is a major meta­ bolic pathway for benzodiazepines, and that many clinically important benzodiazepines are NI-substituted, exposure to circulating N­ dealkyl metabolites is high. This has important clinical implications, since these metabolites are both biologically active and slowly eliminated from the organism. One such metabolite is desmethyldiazepam. This is produced from many benzodiazepines, most importantly diazepam, but also halazepam, prazepam, oxa­ zolam and others. Indeed, desmethyldiazepam (or nordazepam) is marketed as an anxiolytic in its own right in a number of countries, includ­ ing Germany. Desmethyldiazepam probably

contributes significantly to the biological activ­ ity of diazepam and other precursors, particu­ larly after chronic administration, and at later times after single administration. The pharma­ cokinetic profiles of diazepam and desmethyl­ diazepam after single administration are illustrated in Figure 7.7. It can be seen that plasma levels of the metabolite actually over­ take those of the parent drug during the second day after administration, and remain elevated for over a week, when all traces of diazepam in the organism have essentially disappeared. Desmethyldiazepam is itself slowly metabolised to oxazepam, which is then, as we have seen, rapidly removed from the organism by glu­ curonoconjugation and renal excretion. The formation and persistence of desmethyl­ diazepam in the organism has several impor­ tant repercussions in clinical practice. The most important of these is accumulation of bioactive

Figure 7.7 Plasma levels of diazepam and desmethyldiazepam in a healthy volunteer following administration of a Single oral dose of diazepam (5 mg). Data are adapted from Divoll et ai, 1983.

138 CALMING THE BRAIN

material in the organism, which will be dis­ cussed in more detail below. In addition, if treatment needs to be stopped in patients for any reason, several days will go by after the last dose of medication before the system is cleared of bioactive material, and drug effects may persist for a long time. This is particularly important in cases of diazepam overdose, where a significant period of time may be required to re-establish normal arousal and attention levels, due to the persistence of high plasma concentrations of desmethyldiazepam. On the other hand, when patients are being discontinued from long-term benzodiazepine treatment, the persistence and slow elimination of desmethyldiazepam provides a natural taper, and thus reduces the chances of the emergence of a withdrawal syndrome. This probably helps explain why the emergence of rebound insomnia and anxiety after stopping diazepam is less prominent than for benzodi­ azepines without long-lasting active metabo­ lites, such as triazolam. Desalkylflurazepam is another important active metabolite formed both directly from flu­ razepam, to whose biological activity it con­ tributes the major part (Greenblatt et aI, 1981 b), and indirectly from quazepam (Figure 7.8). Quazepam is oxidised to 2-oxoquazepam, which is then dealkylated to desalkylflurazepam

(Zampaglione et aI, 1985). Both 2-oxoquazepam and desalkylflurazepam are considerably more active than the parent drug and probably con­ tribute to its sedative activity. PRODRUGS

Certain benzodiazepines are converted by hepatic metabolism into other species that are responsible for exerting their biological effects. An example is prazepam, which, after absorp­ tion in the gastrointestinal tract, is converted by first-pass metabolism in the liver to desmethyl­ diazepam before it even reaches the general circulation (Smith et aI, 1979). Another some­ what different example is clorazepate. This benzodiazepine carries a charged carboxylate group at position 3, and as such will neither bind to the GABAA receptor nor penetrate the CNS. Decarboxylation of clorazepate by hepatic cytochrome P450 enzymes leads to desmethyl­ diazepam, which is brain-permeable and will activate central benzodiazepine binding sites, thus providing the desired clinical effect (Figure 7.9) . Ethyl loflazepate is thought to behave very similarly, acting following decar­ boxylation to desalkylflurazepam (Chambon et aI, 1985). Oxazolam is another benzodiazepine devoid of activity at the benzodiazepine binding site that is metabolised in vivo to Figure 7.8 Metabolism of lIurazepam and quazepam.

PHARMACOKlNETIC DETERMINANTS OF CLINICAL ACTIVITY 139

Figure 7.9 Metabolism of diazepam, clorazepate, prazepam and oxazolam.

desmethyldiazepam (Nakatsuka et aI, 1985). These different prodrugs all generate the same active principal, desmethyldiazepam. However, the rate at which desmethyldiazepam is gener­ ated varies between the different agents (Ochs et aI, 1984), and this may influence the time­ course of the biological response. Such pro­ drugs are not suitable for use as hypnotics for two reasons. First, the need for prior conversion to desmethyldiazepam will introduce a lag into the onset of clinical activity, which may be quite variable between subjects due to inter­ individual differences in oxidative metabolism. Second, the long elimination half-life of desmethyldiazepam leads to persistence of high circulating levels of bioactive material, and thus of sedative effects, during the daytime. ACCUMULATION

A Significant problem occurs with the forma­ tion of active metabolites that are eliminated only slowly from the organism. When benzodi­ azepines with such metabolites are used chroni­ cally, the latter will accumulate in the organism and continue to exert their pharmacological effects when the parent drugs have been elimi­ nated. Thus, the organism will be exposed to increasing amounts of biologically active mater­

ial, increasing the risk of appearance of side­ effects, in particular sedation. In the case of hypnotic benzodiazepines, the persistence of biologically active metabolites in the daytime following dosing the previous evening is partic­ ularly problematic, since these cause daytime sedation, performance deficits and the feeling of muzzy headedness known as 'benzodi­ azepine hangover'. Desalkylflurazepam is an example of an active metabolite that is eliminated from the body very slowly. As described above, this metabolite is produced in high quantities after administration of both flurazepam and quazepam and is probably responsible for a considerable part of the biological activity of these two hypnotic benzodiazepines. This metabolite persists in the body well after the parent benzodiazepines have been eliminated, due to its higher plasma elimination half-life (Table 7.3; Kaplan et aI, 1973; Greenblatt et aI, 1981b; Zampaglione et aI, 1985). Accumulation of desalkylflurazepam has been detected after chronic dosing of both these hypnotics (Kaplan et aI, 1973; Chung et aI, 1984). Another example of an accumulating metabolite is desmethyl­ diazepam (see above). The later generation of benzodiazepines, such as triazolam, and alprazolam, as well as zopi­

140 CALMING THE BRAIN

Table 7.3 Comparison of plasma elimination

half-llves for flurazepam, quazepam and their

metabolltes after administration of a single

dose to young healthy males

Benzodlazeplne

11/2

Aurazepam

Very short

Quazepam 2-Oxoquazepam

39 h

Desalkylflurazepam

70-75 h; 74 h

40 h

Data are taken from Zampaglione et al. 1985. and Greenblatt et al. 1981b

clone and zolpidem are not significantly trans­ formed to active metabolites. In consequence, their use is not associated with accumulation of sedative drug in the organism. AGE

The effects of benzodiazepines can change with the age of the patient to whom they are admin­ istered. This is a consequence of changes in hepatic function with ageing and also of the sensitivity of the CNS to such drugs. The principal hepatic factor influencing responses to benzodiazepines is the decline in oxidative capacity of cytochrome P4S0 enzymes with age. As we have seen, most benzodiazepines are inactivated principally by such metabolic processes (Type I metabolism). This means that, with ageing, levels of active

drug in the general circulation will persist longer in elderly patients than those younger (Figure 7.10; Greenblatt et al, 1989). For many

commonly used benzodiazepines, plasma half­

lives are twice as long in the elderly as they are in the young