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The benzodiazepbmes: A critical review
Prop. NeumPsycJ~opharmacol8 Biol Psychiot. Printed in Great Britain. All right9 reserved
1983. Vol. 7, PP. 421-426 copyr@ht
THE BENZODIAZEPINES:
0
027~5846/83 $0.00 + 30 1983 Pergamon Press Ltd.
A CRITICAL REVJEW
IAN L. MARTIN MRC Neurochemical Pharmacology Unit Medical Research Council Centre, Cambridge, U.K.
(Final form, June 1983)
The benxodiaxepines were introduced into clinical practice over 20 years ago with the marketing of chlordiazepoxide (Librium). Since that time they have become the most frequently prescribed of all psychotropic drugs and in Canada 10% of the population receives a prescription for one of these compounds each year, while for those in hospital this proportion rises to 30% (Ban et al., 1981), figures which are not atypical for other developed countries. These drugs are used as anxiolytics, hypnotics, anticonvulsants and muscle relaxants and their popularity is due, at least in some considerable part, to their large therapeutic ratio and essential lack of disturbing peripheral side effects. The occurrence of withdrawal phenomena The these drugs led to several widespread prescription of has commentaries regarding their possible over-use or misuse (Lader, 1978; Marks, 1978) and these have stimulated some detailed investigations into the possible risks and advantages accruing from these medicines. It is clear that in the period between 1967/68 and 1977/78 there was a significant change in the prescribibg habits for psychotropic drugs in the U.K., with a marked increase in the number of multiple long term repeats (Marks, 1983), suggesting that perhaps the use of these drugs was becoming somewhat indiscriminate. Such evidence led to concern about possible dependence upon, and the development of withdrawal reactions from these compounds. Recently this evidence has been carefully reviewed (Marks, 1983). All bensodiazepines which have been available as therapeutic agents for some time are implicated, by literature citation, as able to produce withdrawal phenomena. It is clear, however, that the risk is extremely low. Only 962 cases were reported up to the middle of 1981 and analysis of this information indicated that when no additive factors, such as alcohol, were present, characteristics of withdrawal were seen only very rarely in those taking therapeutic levels of the benxodiaxepines continuously for less than 6 months. Subsequent to this time the risk increased considerably, perhaps to 10% at times greater than 1 year. There are, though, other predisposing factors which exacerbate the risk such as high doses, concomitant use of other sedatives or alcohol so-called and the "dependence prone" personality. It has been pointed out that true withdrawal phenomena are difficult to differentiate from rebound anxiety (Lapierre, 1981) but even with this proviso it is clear that a very small, though significant, level of risk in their use exists. However, when judged against the level of psychiatric morbidity in developed countries it has been suggested that the current use of tranquillisers may be no greater, and perhaps even less, than expected (Marks, 1982). It is, however, clear that the risk of withdrawal reactions increases considerably on long-term therapy and it would therefore appear sensible to limit treatment accordingly. There are other criteria which suggest such a 421
422
I.L. Martin
limitation. It has been shown that the benzodiazepines impair the ability of experimental animals to adjust to certain stressful situations (Davis et al., 1981; Gray, 1982). If this phenomenon extends to man it would provide a convincing argument in support of only short term therapy with these compounds. The decision has to be taken on individual cases and is naturally one of clinical judgement but there seems to be little merit in the Calvinistic ethic if the patient fails to function adequately without the appropriate drug therapy. Mechanism of
action
of
the benzodiazepines
The first clues as to the mechanism by which the benzodiazepines produced their effects came from the electrophysiological studies of Schmidt et al. These experiments showed that diazepam was able to increase (1967). presynaptic inhibition and enhance dorsal root potentials in the cat spinal cord, an effect which was later shown to be mediated by y-aminobutyric acid (GABA) (Pole Since that et al., 1974). time the the ability of benzodiazepines to facilitate GABA mediated transmission in many areas of the mammalian CNS has been demonstrated. This amino acid is thought to produce its effects on neuronal excitability via chloride ion channels in the cell membrane and Study and Barker (1981) have used cultured mouse spinal cord neurones to investigate the GABA-benzodiazepine interaction in more detail. They have been able to show that the benzodiazepines increase the probability of GABA stimulated chloride channel opening in these cells. The effect was distinct from that of the barbiturates which produced their main effects on channel open life-time. Therefore these two groups of drugs, with similar pharmacological profiles, both appear to cause an increase in the membrane chloride ion flux, but by distinct mechanisms. While this work has given very considerable information about the effects of these drugs at the cellular level, undoubtedly the greatest stimulus to this area of research was the discovery of specific, high-affinity binding sites for the benzodiazepines in the mammalian CNS (Squires and Braestrup, 1977; MOhler and Okada, 1977). The ability of various members of this 3EI-diazepam from these binding sites was shown to be series to displace highly correlated with the efficacy of these compounds in a number of This animal tests, thought to be predictive of pharmacological efficacy. led to the belief that these binding sites were in fact part of the pharmacological receptor through which the benzodiazepines exerted their effects. Further characterisation of these receptors suggested that although their density varied throughout the CNS the intrinsic properties of the site remained constant both in the rat (Squires and Braestrup, 1977; Mbhler and Okada, 1977) and human (Braestrup et al., 1977; Mbhler et al., 1978). It was also possible to show that the addition of GABA (Tallman et al., 1978; 1978; Martin and Candy, 1978; Earobath and Sperk, 1979) or Wastek et al., certain anions (Martin and Candy, 1978; Costa, Rodbard and Pert, 1979; 1981) to _in vitro binding assays were able to increase the Costa et al., affinity of this receptor for the benzodiazepines, implying some functional linkage between the GABA receptor system and that of the benzodiazepines. Such data was consistent with the idea that the benzodiazepine receptor possessed unique recognition sites for both the benzodiazepines and GABA together with the effector machinery linked to a chloride ionophore. other non-benzodiazepinoid However, it soon became apparent that certain from their binding compounds could displace 3H-diazepam or 3B-flunitrazepam sites (Squires et al., 1979). This, in itself, was of some interest though the nature of the displacement curves were complex and this was interpreted in terms of multiple benzodiazepine receptors. Similar data was later obtained for ethyl B-carboline-3-carboxylate (8-CCE) and the concept was extended to suggest that the sites be termed BZl, with a high affinity for B-CCE, and BZ2, with a lower affinity for this ligand. Both sites exhibited the same affinity for benzodiazepines and while the the cerebellum consisted of mainly the BE1 subtype, the two were present in approximately equal proportions in the hippocampus (Nielsen and Braestrup, 1980; Braestrup and Nielsen, 1980). This interpretation of the radioligand binding data can only be made if certain well defined criteria are met; it
Benzodiazepines: A critical review
423
has not yet been possible to verify these and the hypothesis of multiple benzodiazepine binding sites remains unproven (see Martin et al., 1983, for a more detailed review of this question). There is, however, additional evidence to suggest that the idea may have substance. Flunitrazepam can be used as a photoaffinity label for the benzodiazepine receptor, as originally shown by M8hler et al. (1980). The technique has been used by Sieghart and Karobath (1980) to label the benzodiazepine receptor from a number of different brain regions and subsequently to separate these modified proteins by SDS-PAGE. These results show that in the cerebellum a single band of labelled material can be identified while in the hippocampus at least two are apparent, a result in accord with the interpretation of radioligand binding experiments. However, although certain compounds do appear to differentiate between these putative receptor subtypes the degree of differentiation has so far been insufficient to ascertain whether or not they each mediate different aspects of the behavioural profile of these compounds. Although the preoise interpretation of the above data is a matter of some dispute it is clear that many complexities are apparent in the interaction of ligands with the benzodiazepine receptor, complexities which have yet to be adequately rationalised in any coherent hypothesis. However, it is the behavioural actions of a number of these compounds which have recently stimulated attempts to generate such hypotheses. In many aspects 8-CCE has behavioural actions which are diametrically opposed to those of the benzodiazepines; it is a proconvulsant and antagonises the anticonvulsant and sedative effects of the benzodiazepines in viva (Tenen and Hirsch, 1980; Oakley and Jones, 1980; Cowan et-al., The compound also aonears to block =8ll. the actions of the benzbdiazepines on a GABA receptoi-responsible for increasing the potassium stimulated release of glutamate from rat striatal tissue in vitro (Mitchell and Martin, 1980). This is not difficult to explain if B-CCEassumed to act as an antagonist at the benzodiazepine receptor. However, the situation appears to be rather more complex than this. Two compounds, Ro 15-1788 and CGS 8216 have recently become available which are able to reverse rapidly the actions of the benzodiazepines yet produce no overt behavioural actions in their own right (Bunkeler et al, 1981; Czernik et al., 1981, 1982). The compound Ro 15-1788, however, has been shown to reverse the effect of 8-CCE on seizure thresholds in vivo, its effects on the superior cervical ganglion in vitro (Nutt et al., 1982) and its actions on certain electrophysiological?eEs (Pole et al., 1982). This data can be rationalised if the benzodiazepine is envisaged as a protein with distinct recognition sites for the benzodiazepines and the 8carboline esters, each ligand acting as an agonist at its own site. Occupation of the benzodiazepine site with induces a the agonist conformational change in the receptor protein such that the GABA recognition site becomes more efficiently coupled to the effector machinery responsible for opening the chloride channel; while similar occupation of the 8-carboline site reduces the efficiency of this linkage. These two sites are obviously involved in a close collaboration and an antagonist, such as Ro 15-1788, could effectively block the actions of both types of ligand by occupation of one of these sites (Nutt et al., 1982; Pole et al., 1982). There is much to learn yet about the recognition properties of these ligands and such an hypothesis has yet to be adequately evaluated but it provides a stimulating basis for the design of further experiments. Structure activity relationships Although considerable strides have been made with our understanding of the means by which the benzodiazepines produce their effects, the desire to produce more efficacious compounds has continued and two different approaches have been used. Here attempts The first, though somewhat esoteric, holds much promise. of the to correlate the structural characteristics have been made benzodiazepines with their affinity at the benzodiazepine 'receptor as determined by their ability to displace 3H-flunitrazepam from its binding sites in rat brain membranes. The structural characteristics of a series
424
I.L. Martin
of 5-phenyl-1,4_benzodiazepines have been determined using X-ray crystallographic techniques. These data have been used as the basis of molecular orbital calculations to determine the distribution of the electronic charge in these molecules and a number of other parameters, such as dipole moment, which may be important in determining the initial recognition of these molecules by the benzodiazepine receptor. The information has then been used in multiple regression analysis to obtain clues about which particular structural parameters are important in determining the affinity of the receptor for these compounds, and thus the characteristics of the receptor recognition site (see Hamor and Martin, 1983, for a more detailed discussion of these data). It is important to realise that this approach can only allow tentative conclusions to be drawn at present as X-ray analyses are available for only 20 benzodiazepines and clearly the structural data on any compound has many more variables than this. The approach does, however, allow the generation of hypotheses which can then be tested by looking at further compounds and so the information becomes sequentially refined. The alternative approach, far more pragmatic in nature, is to modify the benzodiazepine structure chemically and then investigate the pharmacological characteristics of this analogue in whole animal experiments: an Using this protocol approach which has been so successful in the past. certain triazolobenzodiazepines have been shown to have not only anticonvulsant activity, so characteristic of the benzodiazepines, but also some properties typical of the classical anti-depressants (Hester et al., It would appear that the antidepressant profile, as judged by the 1980). ability of the compound to reverse apomorphine induced hypothermia, is not mediated through the benzodiazepine receptor system as it is not blocked by the benzodiazepine antagonist Ro 15-1788 (Thiebot et al., 1982) The Future Even though the benzodiazepines have been available to the clinician since 1960 there remain many intriguing questions unanswered about the means by which they produce their effects. Are different aspects of their pharmacological profile mediated by the same underlying neurochemical mechanism of action? If not, perhaps distinct receptor subpopulations are responsible for the separate parts of their spectrum of activity. Clearly many complexities exist in the interaction of these compounds with their receptors in the CNS and it is the further elucidation of these which may lead to the development of drugs with a more restricted, and thus more useful, therapeutic-profile. -
References BAN, T.A., BROWN, W.T., DA SIWA, T., GAGNON, M.A., LAMONT, C.T., LEMANN, Therapeutic H.E., LGWY, F.W., RUEDY, J. and SELLERS, E.M. (1981) monograph on anxiolytic-sedative drugs. Can. Pharmaceutical J. 114: 301308. BRAESTRUP, C., ALBRECETSEN, R. and SQUIRES, R.F. (1977) High densities of benzodiazepine receptors in human cortical areas. Nature 269: 702-704. Multiple benzodiazepx receptors. BRAESTRUP, C. and NIELSEN, M. (1980) Trends in Neurosciences 1: 301-303. COSTA, T., RODBARD, D. and PERT, C. (1979) Is the benzodiazepine receptor coupled to a chloride anion channel? Nature 277: 315-317. COSTA, T., RUSSELL, L., PERT, C.B. and RODBARDTD. (1981) Halide and yaminobutyric acid-induced enhancement of diazepam receptors in rat brain. Mol. Reversal by disulfonic stilbene blockers of anion channels. Pharmacol. 20: 470-476. (1981) Ethyl 8COWAN, P.J.,-EEN, A.R., NUTT, D.J. and MARTIN, I.L. carboline carboxylate lowers seizure threshold and antagonises flurazepam induced sedation in rats. Nature 290: 54-55.
Benzodiazepines:A criticalreview
425
CZERNIK, A.J., PETRACK, B., KALINSKY, H.J., PSYCHOYOS, S., CASH, W.D., LA., m.mIsH, D.E. and TSAI, C., RINEHART, R.K., GRANAT, F.R., LowaL, WADE, R. (1982) CGS 8216; receptor binding characteristics of a potent benzodiazepine antagonist. Life Sci. 30: 363-372. CZERNIK, A.J., PETRACK, B., TSAI, C., GRANAT, R.F., RINEHART, R.K., High affinity (1981) KALINSKY, J.H., LOVELL, R.A. and CASH, W.D. occupancy of benzodiazepine receptors by the novel antagonist CGS-8216. Pharmacologist 23: 160. (1981) BRBKES, S., GRAY, J.A. and RAWLINS, J.N.P. DAVIS, N.M., Chlordiazepoxide and resistance to punishment. Quart. J. Exp. Psychol., 33B: 227-239. GMT J.A. Personal communication. (1983) Structure-activity relationships in a HAMOR, T.A. and MARTIN, I.L. series of 5-phenyl-1,4_benzodiazepines. In: K-ray crystallography and drug action, ed. A.S. Horn, Oxford University Press, in press. (1980) lHESTER Jr., J.B., RUDZIK, A.D. and VON VOIGT LANDER, F. benzodiazepines with (Aminoalkyl)-6-amyl-4H-s-triazolo[4,3-a][l,4] antianxiety and antidepressant activity. J. Med. Chem. 23: 392. HUNKELER, W., MGHLER, H., PIERI, L., POLC, P., BONETTI,E.P., CUMIN, R., Selective antagonists of (1981) SCHAFFNER, R. and HAEFELY, W. benzodiazepines. Nature 290: 514-516. Stimulation of benzodiazepine receptor KAROBATH, M. and SPERK, G.7979) binding by y-aminobutyric acid. Proc. Natl. Acad. Sci., U.S.A. -76: 10041006. LADER, M. (1978) Benzodiazepines - The opium of the masses? Neuroscience 3: 159-165. Can. J. Psychiat. -26: Benzodiazepine withdrawal. LAFIERE, Y.D., (1981) 93-95. The benzodiazepines: Use, overuse, misuse, and abuse. MARKS, J. (1978) MTP, Lancaster Benzodiazepines and dependence. In: Benzodiazepiner (1982) MARKS, J. til behandling af angst, ed. O.J. Rafaelson, neuroleptika eller Scantican, Aarhus, Denmark, pp. 59-65. Int. The benzodiazepines for good or evil. (1983) MARKS, J. Pharmacopsychiat. in press. (1983) Multiple benzodiazepine MARTIN. 1.L.. BROWN. C.L. and DOBLE. A. receptors: -structures in the brain or structures in the-mind? A critical review. Life Sci., 32: 1925-1933. MARTIN, 1.L. and CANDY7J.M. (1978) Facilitation of benzodiazepine binding by sodium chloride and GABA. Neuropharmacology 17: 993-998. Ethyl Fcarboline-3-carboxylate MITCHELL, P.R. and MARTIN, I.L. (1980) Eur. antagonises the effect of diazepam on a functional GABA receptor. J. Pharmacol. 68: 513-514. Benzodiazepine MSHLER, H., BATXRSBY, M.K. and RICHARDS, J.G. (1980) identified and visualised in brain tissue by a receptor protein photoaffinity label. Proc. Natl. Acad. Sci., U.S.A. 77: 1666-1670. demonstration in MGHLER, H. and OKADA, T. (1977) Benzodiazepine recept= the central nervous system. Science 198: 849-851. Biochemical MGHLER, H., OKADA, T., ULRICH, J. and HEITZ, P. (1978) identification of the site of action of benzodiazepines in human brain by 3H-diazepam binding. Life. Sci. 22: 985-996. Ethyl $-carboline-3-carboxylate (1980) NIELSEN, M. and BRAESTRUP, C. shows differential benzodiazepine receptor interaction. Nature, Lond. 286: 606-607. NUX D.J., COWAN, P.J. and LITTLE, H.J. (1982) Unusual interactions of benzodiazepine receptor antagonists. Nature 295: 436-438. The pro-convulsant and diazepamOAKLEY, N.R. and JONES, B.J. (1980) reversing effects of ethyl 8-carboline-3-carboxylate. Eur. J. Pharmacol. 68: 381-382. POT, P., BONETTI, E.P., SCHAFFNER, R. and HAEFELY, W. (1982) A three state model of the benzodiazepine receptor explains the interactions benzodiazepine antagonist Ro the 15-1788, benzodiazepine between tranquillisers, 8-carbolines and phenobarbitone. Naunyn Schmiedeberg's Arch. Pharmacol. 321: 260-264.
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POLC, P., HSHLER, 8. and HAEFELY, W. (1974) The effect of diazepam on spinal cord activities: possible sites and mechanisms of action. NaunynSchmiedeberg's Arch. Pharmacol. 284: 319-337. SCHMIDT, R.F., VOGEL, E. and ZIMMERMAN, M. Die Wirkung von (1967) Diazepam auf die prasynaptische Hemmung und andere Rhckermarksreflexe. Naunyn Schmiedeberg's Arch. Pharmacol. 258: 69-82. SIEGHART, W. and KAROBATH, M. Molecular heterogeneity of (198r benzodiazepine receptors. Nature, Lond. 286: 285-287. SQUIRES, R.F., BENSON, D.I., BRAESTRUP, c., COUPET, J., KLEPNER, C.A., Some properties of brain specific MYERS, V. and BEER, B. (1979) receptors: evidence for multiple receptors. benzodiazepine New Pharmacol. Biochem. Behav. 10: 825-830. (1977) Benzodiazepine receptors in rat SQUIRES, R.F. and BRAESTRUP,C. brain. Nature 266: 732-734. STUDY, R.E. andBARKER, J.L. Diazepam and (-)pentobarbital: (1981) Fluctuation analysis reveals different mechanisms for potentiation of yaminobutyric acid responses in cultured central neurons. Proc. Natl. Acad. Sci., U.S.A. 78: 7180-7184. and GALLAGER, D.W. (1978) GABAergic modulation TALLWAN, J.F., THOMAS7J.W. of benzodiazepine binding site sensitivity. Nature 274: 383-385. (1980) Antagonism of dizpam activity by 8TENEN, S.S. and HIRSCH, J.D. carboline-3-carboxylic acid ethyl ester. nature 288: 609-610THIEBOT, M.-H, WARE, L., PUECH, A.J. and SIMON,r (1982) U43,465F: a benzodiazepine with antidepressant activity? Interaction with Ro15-1788 Eur. J. Pharmacol. 84: 103-106 (1982). and d,l-propranolol. The WASTER, G.J., SPETH, R.C., REISINE, T.D. and YAMAMURA, H.I. (1978) effect of y-aminobutyric acid on [3H] flunitrazepam binding in rat brain. Eur. J. Pharmacol. -50: 445-447. Inquiries and reprint requests should be addressed to: Dr I.L. Martin MRC Neurochemical Pharmacology Unit Medical Research Council Centre Medical School, Hills Road Cambridge CB2 248, U.K.
EditorialNote: Althoughthe formatof the paper does not followentirelythe standardsof the journal,the presentpaper is publishedbecauseof its importantscientificcontribution as well ae to avoid undue delay in the publicationof the "6th CCNP Proceedings".
1983. Vol. 7, PP. 421-426 copyr@ht
THE BENZODIAZEPINES:
0
027~5846/83 $0.00 + 30 1983 Pergamon Press Ltd.
A CRITICAL REVJEW
IAN L. MARTIN MRC Neurochemical Pharmacology Unit Medical Research Council Centre, Cambridge, U.K.
(Final form, June 1983)
The benxodiaxepines were introduced into clinical practice over 20 years ago with the marketing of chlordiazepoxide (Librium). Since that time they have become the most frequently prescribed of all psychotropic drugs and in Canada 10% of the population receives a prescription for one of these compounds each year, while for those in hospital this proportion rises to 30% (Ban et al., 1981), figures which are not atypical for other developed countries. These drugs are used as anxiolytics, hypnotics, anticonvulsants and muscle relaxants and their popularity is due, at least in some considerable part, to their large therapeutic ratio and essential lack of disturbing peripheral side effects. The occurrence of withdrawal phenomena The these drugs led to several widespread prescription of has commentaries regarding their possible over-use or misuse (Lader, 1978; Marks, 1978) and these have stimulated some detailed investigations into the possible risks and advantages accruing from these medicines. It is clear that in the period between 1967/68 and 1977/78 there was a significant change in the prescribibg habits for psychotropic drugs in the U.K., with a marked increase in the number of multiple long term repeats (Marks, 1983), suggesting that perhaps the use of these drugs was becoming somewhat indiscriminate. Such evidence led to concern about possible dependence upon, and the development of withdrawal reactions from these compounds. Recently this evidence has been carefully reviewed (Marks, 1983). All bensodiazepines which have been available as therapeutic agents for some time are implicated, by literature citation, as able to produce withdrawal phenomena. It is clear, however, that the risk is extremely low. Only 962 cases were reported up to the middle of 1981 and analysis of this information indicated that when no additive factors, such as alcohol, were present, characteristics of withdrawal were seen only very rarely in those taking therapeutic levels of the benxodiaxepines continuously for less than 6 months. Subsequent to this time the risk increased considerably, perhaps to 10% at times greater than 1 year. There are, though, other predisposing factors which exacerbate the risk such as high doses, concomitant use of other sedatives or alcohol so-called and the "dependence prone" personality. It has been pointed out that true withdrawal phenomena are difficult to differentiate from rebound anxiety (Lapierre, 1981) but even with this proviso it is clear that a very small, though significant, level of risk in their use exists. However, when judged against the level of psychiatric morbidity in developed countries it has been suggested that the current use of tranquillisers may be no greater, and perhaps even less, than expected (Marks, 1982). It is, however, clear that the risk of withdrawal reactions increases considerably on long-term therapy and it would therefore appear sensible to limit treatment accordingly. There are other criteria which suggest such a 421
422
I.L. Martin
limitation. It has been shown that the benzodiazepines impair the ability of experimental animals to adjust to certain stressful situations (Davis et al., 1981; Gray, 1982). If this phenomenon extends to man it would provide a convincing argument in support of only short term therapy with these compounds. The decision has to be taken on individual cases and is naturally one of clinical judgement but there seems to be little merit in the Calvinistic ethic if the patient fails to function adequately without the appropriate drug therapy. Mechanism of
action
of
the benzodiazepines
The first clues as to the mechanism by which the benzodiazepines produced their effects came from the electrophysiological studies of Schmidt et al. These experiments showed that diazepam was able to increase (1967). presynaptic inhibition and enhance dorsal root potentials in the cat spinal cord, an effect which was later shown to be mediated by y-aminobutyric acid (GABA) (Pole Since that et al., 1974). time the the ability of benzodiazepines to facilitate GABA mediated transmission in many areas of the mammalian CNS has been demonstrated. This amino acid is thought to produce its effects on neuronal excitability via chloride ion channels in the cell membrane and Study and Barker (1981) have used cultured mouse spinal cord neurones to investigate the GABA-benzodiazepine interaction in more detail. They have been able to show that the benzodiazepines increase the probability of GABA stimulated chloride channel opening in these cells. The effect was distinct from that of the barbiturates which produced their main effects on channel open life-time. Therefore these two groups of drugs, with similar pharmacological profiles, both appear to cause an increase in the membrane chloride ion flux, but by distinct mechanisms. While this work has given very considerable information about the effects of these drugs at the cellular level, undoubtedly the greatest stimulus to this area of research was the discovery of specific, high-affinity binding sites for the benzodiazepines in the mammalian CNS (Squires and Braestrup, 1977; MOhler and Okada, 1977). The ability of various members of this 3EI-diazepam from these binding sites was shown to be series to displace highly correlated with the efficacy of these compounds in a number of This animal tests, thought to be predictive of pharmacological efficacy. led to the belief that these binding sites were in fact part of the pharmacological receptor through which the benzodiazepines exerted their effects. Further characterisation of these receptors suggested that although their density varied throughout the CNS the intrinsic properties of the site remained constant both in the rat (Squires and Braestrup, 1977; Mbhler and Okada, 1977) and human (Braestrup et al., 1977; Mbhler et al., 1978). It was also possible to show that the addition of GABA (Tallman et al., 1978; 1978; Martin and Candy, 1978; Earobath and Sperk, 1979) or Wastek et al., certain anions (Martin and Candy, 1978; Costa, Rodbard and Pert, 1979; 1981) to _in vitro binding assays were able to increase the Costa et al., affinity of this receptor for the benzodiazepines, implying some functional linkage between the GABA receptor system and that of the benzodiazepines. Such data was consistent with the idea that the benzodiazepine receptor possessed unique recognition sites for both the benzodiazepines and GABA together with the effector machinery linked to a chloride ionophore. other non-benzodiazepinoid However, it soon became apparent that certain from their binding compounds could displace 3H-diazepam or 3B-flunitrazepam sites (Squires et al., 1979). This, in itself, was of some interest though the nature of the displacement curves were complex and this was interpreted in terms of multiple benzodiazepine receptors. Similar data was later obtained for ethyl B-carboline-3-carboxylate (8-CCE) and the concept was extended to suggest that the sites be termed BZl, with a high affinity for B-CCE, and BZ2, with a lower affinity for this ligand. Both sites exhibited the same affinity for benzodiazepines and while the the cerebellum consisted of mainly the BE1 subtype, the two were present in approximately equal proportions in the hippocampus (Nielsen and Braestrup, 1980; Braestrup and Nielsen, 1980). This interpretation of the radioligand binding data can only be made if certain well defined criteria are met; it
Benzodiazepines: A critical review
423
has not yet been possible to verify these and the hypothesis of multiple benzodiazepine binding sites remains unproven (see Martin et al., 1983, for a more detailed review of this question). There is, however, additional evidence to suggest that the idea may have substance. Flunitrazepam can be used as a photoaffinity label for the benzodiazepine receptor, as originally shown by M8hler et al. (1980). The technique has been used by Sieghart and Karobath (1980) to label the benzodiazepine receptor from a number of different brain regions and subsequently to separate these modified proteins by SDS-PAGE. These results show that in the cerebellum a single band of labelled material can be identified while in the hippocampus at least two are apparent, a result in accord with the interpretation of radioligand binding experiments. However, although certain compounds do appear to differentiate between these putative receptor subtypes the degree of differentiation has so far been insufficient to ascertain whether or not they each mediate different aspects of the behavioural profile of these compounds. Although the preoise interpretation of the above data is a matter of some dispute it is clear that many complexities are apparent in the interaction of ligands with the benzodiazepine receptor, complexities which have yet to be adequately rationalised in any coherent hypothesis. However, it is the behavioural actions of a number of these compounds which have recently stimulated attempts to generate such hypotheses. In many aspects 8-CCE has behavioural actions which are diametrically opposed to those of the benzodiazepines; it is a proconvulsant and antagonises the anticonvulsant and sedative effects of the benzodiazepines in viva (Tenen and Hirsch, 1980; Oakley and Jones, 1980; Cowan et-al., The compound also aonears to block =8ll. the actions of the benzbdiazepines on a GABA receptoi-responsible for increasing the potassium stimulated release of glutamate from rat striatal tissue in vitro (Mitchell and Martin, 1980). This is not difficult to explain if B-CCEassumed to act as an antagonist at the benzodiazepine receptor. However, the situation appears to be rather more complex than this. Two compounds, Ro 15-1788 and CGS 8216 have recently become available which are able to reverse rapidly the actions of the benzodiazepines yet produce no overt behavioural actions in their own right (Bunkeler et al, 1981; Czernik et al., 1981, 1982). The compound Ro 15-1788, however, has been shown to reverse the effect of 8-CCE on seizure thresholds in vivo, its effects on the superior cervical ganglion in vitro (Nutt et al., 1982) and its actions on certain electrophysiological?eEs (Pole et al., 1982). This data can be rationalised if the benzodiazepine is envisaged as a protein with distinct recognition sites for the benzodiazepines and the 8carboline esters, each ligand acting as an agonist at its own site. Occupation of the benzodiazepine site with induces a the agonist conformational change in the receptor protein such that the GABA recognition site becomes more efficiently coupled to the effector machinery responsible for opening the chloride channel; while similar occupation of the 8-carboline site reduces the efficiency of this linkage. These two sites are obviously involved in a close collaboration and an antagonist, such as Ro 15-1788, could effectively block the actions of both types of ligand by occupation of one of these sites (Nutt et al., 1982; Pole et al., 1982). There is much to learn yet about the recognition properties of these ligands and such an hypothesis has yet to be adequately evaluated but it provides a stimulating basis for the design of further experiments. Structure activity relationships Although considerable strides have been made with our understanding of the means by which the benzodiazepines produce their effects, the desire to produce more efficacious compounds has continued and two different approaches have been used. Here attempts The first, though somewhat esoteric, holds much promise. of the to correlate the structural characteristics have been made benzodiazepines with their affinity at the benzodiazepine 'receptor as determined by their ability to displace 3H-flunitrazepam from its binding sites in rat brain membranes. The structural characteristics of a series
424
I.L. Martin
of 5-phenyl-1,4_benzodiazepines have been determined using X-ray crystallographic techniques. These data have been used as the basis of molecular orbital calculations to determine the distribution of the electronic charge in these molecules and a number of other parameters, such as dipole moment, which may be important in determining the initial recognition of these molecules by the benzodiazepine receptor. The information has then been used in multiple regression analysis to obtain clues about which particular structural parameters are important in determining the affinity of the receptor for these compounds, and thus the characteristics of the receptor recognition site (see Hamor and Martin, 1983, for a more detailed discussion of these data). It is important to realise that this approach can only allow tentative conclusions to be drawn at present as X-ray analyses are available for only 20 benzodiazepines and clearly the structural data on any compound has many more variables than this. The approach does, however, allow the generation of hypotheses which can then be tested by looking at further compounds and so the information becomes sequentially refined. The alternative approach, far more pragmatic in nature, is to modify the benzodiazepine structure chemically and then investigate the pharmacological characteristics of this analogue in whole animal experiments: an Using this protocol approach which has been so successful in the past. certain triazolobenzodiazepines have been shown to have not only anticonvulsant activity, so characteristic of the benzodiazepines, but also some properties typical of the classical anti-depressants (Hester et al., It would appear that the antidepressant profile, as judged by the 1980). ability of the compound to reverse apomorphine induced hypothermia, is not mediated through the benzodiazepine receptor system as it is not blocked by the benzodiazepine antagonist Ro 15-1788 (Thiebot et al., 1982) The Future Even though the benzodiazepines have been available to the clinician since 1960 there remain many intriguing questions unanswered about the means by which they produce their effects. Are different aspects of their pharmacological profile mediated by the same underlying neurochemical mechanism of action? If not, perhaps distinct receptor subpopulations are responsible for the separate parts of their spectrum of activity. Clearly many complexities exist in the interaction of these compounds with their receptors in the CNS and it is the further elucidation of these which may lead to the development of drugs with a more restricted, and thus more useful, therapeutic-profile. -
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POLC, P., HSHLER, 8. and HAEFELY, W. (1974) The effect of diazepam on spinal cord activities: possible sites and mechanisms of action. NaunynSchmiedeberg's Arch. Pharmacol. 284: 319-337. SCHMIDT, R.F., VOGEL, E. and ZIMMERMAN, M. Die Wirkung von (1967) Diazepam auf die prasynaptische Hemmung und andere Rhckermarksreflexe. Naunyn Schmiedeberg's Arch. Pharmacol. 258: 69-82. SIEGHART, W. and KAROBATH, M. Molecular heterogeneity of (198r benzodiazepine receptors. Nature, Lond. 286: 285-287. SQUIRES, R.F., BENSON, D.I., BRAESTRUP, c., COUPET, J., KLEPNER, C.A., Some properties of brain specific MYERS, V. and BEER, B. (1979) receptors: evidence for multiple receptors. benzodiazepine New Pharmacol. Biochem. Behav. 10: 825-830. (1977) Benzodiazepine receptors in rat SQUIRES, R.F. and BRAESTRUP,C. brain. Nature 266: 732-734. STUDY, R.E. andBARKER, J.L. Diazepam and (-)pentobarbital: (1981) Fluctuation analysis reveals different mechanisms for potentiation of yaminobutyric acid responses in cultured central neurons. Proc. Natl. Acad. Sci., U.S.A. 78: 7180-7184. and GALLAGER, D.W. (1978) GABAergic modulation TALLWAN, J.F., THOMAS7J.W. of benzodiazepine binding site sensitivity. Nature 274: 383-385. (1980) Antagonism of dizpam activity by 8TENEN, S.S. and HIRSCH, J.D. carboline-3-carboxylic acid ethyl ester. nature 288: 609-610THIEBOT, M.-H, WARE, L., PUECH, A.J. and SIMON,r (1982) U43,465F: a benzodiazepine with antidepressant activity? Interaction with Ro15-1788 Eur. J. Pharmacol. 84: 103-106 (1982). and d,l-propranolol. The WASTER, G.J., SPETH, R.C., REISINE, T.D. and YAMAMURA, H.I. (1978) effect of y-aminobutyric acid on [3H] flunitrazepam binding in rat brain. Eur. J. Pharmacol. -50: 445-447. Inquiries and reprint requests should be addressed to: Dr I.L. Martin MRC Neurochemical Pharmacology Unit Medical Research Council Centre Medical School, Hills Road Cambridge CB2 248, U.K.
EditorialNote: Althoughthe formatof the paper does not followentirelythe standardsof the journal,the presentpaper is publishedbecauseof its importantscientificcontribution as well ae to avoid undue delay in the publicationof the "6th CCNP Proceedings".