This paper discusses some of the pharmacological and neurochemical properties of clozapine, and the special attributes that differentiate clozapine from the classical neuroleptics. The question as to whether or not clozapine blocks DA-receptors - a crucial point in regard to the dopamine hypothesis of schizophrenia - has received particular attention. Neurochemical, neuropharmacological, and endocrinological evidence is presented which speaks against a DA-receptor blockade by clozapine in pharmacologically relevant doses. These findings are difficult to reconcile with the dopamine hypothesis which proposes a direct relationship between antipsychotic effect and DA-receptor blockade.
Die tierexperimentellen Befunde mit Clozapin, im besonderen derjenigen Eigenschaften, in denen sich Clozapin von den klassischen Neuroleptika unterscheidet, werden diskutiert. Die Frage, ob Clozapin zentrale dopaminerge Systeme beeinflußt, ist in bezug auf die Dopamin-Theorie der Schizophrenie von besonderer Bedeutung. Neurochemische, neuropharmakologische und endokrinologische Untersuchungen zeigen jedoch, daß dieses Präparat in pharmakologisch relevanten Dosen keine Blockade der Dopaminrezeptoren bewirkt. Diese Befunde sind mit der Dopamin-Theorie der Schizophrenie, welche eine direkte Beziehung zwischen antipsychotischer Wirkung und Dopaminrezeptoren-Blockade postuliert, schwer zu vereinbaren.
In the selection of possible antipsychotic agents from the numerous compounds synthesized by the chemist, psychopharmacologists depend largely on screening procedures that were developed on the basis of pharmacological or biochemical properties of pre-existing drugs with proven therapeutic efficacy. It is clear that such an approach will not lead to new types of drugs, but will only produce agents that resemble to a greater or lesser degree the drug with whose help the screening tests were developed. Hence, novel psychotropic drugs are still identified only by careful analysis of their action in man. An example in recent years is provided by clozapine, a drug which proved to have a potent antischizophrenic activity, and yet which, according to the current criteria, could hardly have been classified by the pharmacologists as a neuroleptic agent. It is the purpose of this paper to discuss some of the pharmacological and neurochemical properties of clozapine and the special attributes that differentiate clozapine from classical neuroleptics such as haloperidol and chlorpromazine. Early pharmacological and clinical observations revealed that chlorpromazine is an effective antiemetic drug (1) which causes a Parkinson-like syndrome in patients (2). These observations led to the development of laboratory tests which still enjoy wide-spread popularity for the selection of compounds with possible antischizophrenic activity, namely the measurement of cataleptogenic activity and protection against apomorphine- or amphetamine-induced stereotypies (3). Further, chlorpromazine and other neuroleptics were shown to accelerate the metabolism of dopamine (DA) in the brain, an effect attributed to feedback activation of presynaptic dopaminergic neurones due to blockade of striatal DA-receptors (4, 5, 6). Thus emerged the DA-hypothesis of schizophrenia, which was to explain both the extrapyramidal effects and the antischizophrenic activity of neuroleptic drugs (2, 7, 8, 9). According to this hypothesis, blockade of DA-receptors in animals results in protection against apomorphineinduced stereotypies and emesis, and, as a result of attenuation of striatal transmission, in the
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Recent observations suggest, however, that there may be no causal relationship between extrapyramidal activity of neuroleptics and their antipsychotic action in man. When potent neuroleptics such as loxapine or haloperidol were administered to rats for several days, tolerance developed to the extrapyramidal manifestations of these drugs, i.e., cataleptogenic behaviour, apomorphine antagonism and the changes in striatal DA metabolism (12). It is interesting to note that a number of reports (see 13 for a review) stress that anti-Parkinson medication, often needed initially together with neuroleptic therapy, can be discontinued within a few weeks without the re-appearance of extrapyramidal symptoms. However, no tolerance develops to the antipsychotic activity of the neuroleptic drugs. The tolerance to the extrapyramidal effects in animals has been shown to be due to alterations in the properties of DA-receptors, with a reduced sensitivity toward the effects of neuroleptics and an increased sensitivity toward dopaminergic agents (14, 15, 16, 17), so that it would seem that long-term treatment with neuroleptic agents brings about new functional equilibria of the striatal system. These findings challenge the view expressed by the DA-hypothesis that the DA-receptor blockade per se is the mechanism by which neuroleptics mediate, antischizophrenic activity, and suggest that the therapeutic effect of these drugs could equally well be due to functional alterations of the kind seen after chronic administration. Furthermore, the question is posed whether any meaningful concept for the explanation of the therapeutic actions of neuroleptics can be deduced from data obtained after single administration of these drugs, when it is known that neuroleptic therapy usually lasts for weeks or even months before the desired therapeutic effect is achieved. Further doubts of the relevance of the DA-hypothesis arose when early reports about the antipsychotic efficacy of clozapine (18, 19, 20) were confirmed in different countries in trials involving large numbers of patients ( 2 1 , 22). If the usual pharmacological criteria for selecting antipsychotic drugs are applied, clozapine would not have qualified as a potential neuroleptic (23). Clozapine is non-cataleptogenic, provides no protection against apomorphine stereotypies, inhibits conditioned avoidance behaviour only in doses causing severe sedation, and in man causes few, if any, parkinsonoid effects. It follows that the drug can hardly be classified as a neuroleptic according to the accepted pharmacological definition of the term (24) (Table 1).
Table 1 Pharmacological data of clozapine, haloperidol and loxapine Aporaorphine antagonism Catalepsy Conditioned avoidance response Inhibition of locomotor activity Mydriasis Oxotremorine antagonism Electrographic arousal inhibition: - Reticular stimulation - Arecoline stimulation For methods see (23)
Clozapine
Haloperidol
Rat Rat
ED 50 (mg/kg sc): ED 50 (mg/kg sc):
inactive inactive
0.14 0.30
0.07 0.10
Rat
ED 50 (mg/kg po):
20.0
0.38
0.06
Mouse Mouse Mouse
ED 50 (mg/kg po): ED 200 (mg/kg po): ED 50 (mg/kg po):
2.5 8.0 9.0
0.30 inactive > 20
ED 150 (mg/kg iv): Block (mg/kg iv):
1.5 1.1
inactive inactive
Rabbit
Loxapine
0.04 inactive 16.0 3.2 4.5
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development of catalepsy. In man, production of psychotic experiences is inhibited, and a Parkinson-like syndrome develops (2, 10). Extrapyramidal effects were considered indispensable for an antipsychotic agent, and indeed, some clinicians (11) considered the therapeutic dose level of a neuroleptic to be reached only when extrapyramidal effects appeared.
Clozapine and the Dopamine Hypothesis of Schizophrenia
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Although as judged by the usual pharmacological criteria clozapine cannot be regarded as a neuroleptic drug, various authors, using different experimental designs, concluded that clozapine may still be a DA-receptor blocker, the consequences of DA-receptor blockade being masked by the pronounced anticholinergic and/or muscle-relaxing properties of the drug (8, 9, 2 5 , 29, 30). For this reason experiments were designed specifically t o answer the question whether or not clozapine, in pharmacologically relevant doses, blocks DA-receptors. Three independent experimental approaches were chosen: The first approach is based on the fact that rats treated chronically with classical neuroleptics develop a tolerance to the extrapyramidal effects, as shown by the progressively diminishing HVA-increases in the corpora striata after repeated administration of the drugs (12, 28). Cross tolerance occurs between neuroleptics which may be quite unrelated structurally, as, e.g. haloperidol and loxapine (14). In striking contrast to these observations, repeated administration of high doses of clozapine caused no tolerance to the HVA-increasing action (28), nor did pretreatment with clozapine affect the HVA increase of a challenging dose of haloperidol or loxapine (14) (Table 2). Table 2 Homovanillic acid content of rat striatum after repeated doses of loxapine, chlorpromazine, haloperidol or clozapine, and challenged with a single dose of haloperidol or loxapine Pretreatment days 1 - 6 (mg/kg po) Water Haloperidol Loxapine Chlorpromazine Clozapine Water Loxapine Haloperidol Chlorpromazine Clozapine
( 3) ( 2) (32) (80) ( 2) ( 3) (32) (80)
Challenging dose on day 7 (mg/kg po)
N
HVA-content Mg/g S.D.
Haloperidol Haloperidol Haloperidol Haloperidol Haloperidol Loxapine Loxapine Loxapine Loxapine Loxapine
10 5 6 4 5 10 9 5 5 5
3.06 1.57 0.95 1.95 2.57 3.17 0.88 1.91 2.41 3.06
(3) (3) (3) (3) (3) (2) (2) (2) (2) (2)
a
0.38 0.05* 0.08* 0.18* 0.64 0.58b 0.13* 0.08* 0.24* 0.15
Rats were killed 3 h after the day 7 injection. N represents the number of determinations, each performed on the pooled homogenates of the striata of 5 rats. HVA-content in rats treated solely with water was 0.52 jug/g 0.05 (S.D., N = 15). * p < 0.001 compared to groups a and b respectively.
In a second experimental design, use was made of the fact that rats treated chronically with classical neuroleptics exhibit hypersensitivity t o the actions of apomorphine (15). This effect is best quantified by measuring the turning behaviour in rats with unilateral striatal lesions (14). The number of turns elicited after a standard dose of apomorphine serves as a measure for striatal DA-receptor sensitivity to apomorphine. It was shown that pretreatment with haloperidol for one week strongly augmented the turning response to apomorphine, which was given 27 h after the last dose of haloperidol. This result suggests DA-receptor hypersensitivity due t o prolonged
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Biochemical evidence shows that clozapine, in common with the classical neuroleptics, affects the dopaminergic system in the striatum ( 2 5 , 2 6 ) . However, its mechanism of action differs considerably from that of the classical neuroleptics. The latter drugs accelerate the turnover of DA in the striatum, increase the levels of the DA-metabolites homovanillic acid (HVA) and dihydroxyphenylacetic acid (DOPAC), and decrease the content of DA. In contrast, clozapine increases striatal HVA and DOPAC only in doses which exceed by a factor of between 100 and 1000 those of the classical neuroleptics, and it increases, instead of decreases the striatal DA-content (27, 28),
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Table 3 Apomorphine-induced circling in rats with unilateral striatal lesions
Haloperidol Haloperidol Clozapine Clozapine Clozapine Clozapine
Length of treatment in days
Daily dose mg/kg po
Total no. of turns (% of Control)
6 6 6 6 13 20
0.5 3.0 20.0 80.0 80.0 80.0
146* 179* 107 104 122 126
interaction of the neuroleptic with the DA-receptors. In marked contrast t o these findings, repeated administration of high doses of clozapine did not significantly increase the sensitivity of the lesioned rats to apomorphine (14) (Table 3). The third experimental approach involved a DA-system structurally and functionally distinct from that of the corpus striatum. Clozapine was compared with classical neuroleptics in respect of the action on prolactin secretion. The concentration of prolactin in the blood depends in part on the rate of its secretion, which is controlled by a hypothalamic inhibitory factor, the availability of which in turn is regulated by DA mechanisms. After administration of dopaminergic agents the blood concentration of prolactin is reduced, afjter DA-receptor blockers such as chlorpromazine it is elevated (for discussion see 31). It was shown that potent neuroleptics like clothiapine and pimozide dramatically increase the prolactin level in the serum, while clozapine even at very high doses caused only a slight increase in the prolactin level (Table 4). a
Table 4 Serum prolactin in ovariectomized rats >
D
0
Treatment Controls Clozapine Pimozide Clothiapine
Dosage mg/kg po
Serum prolactin 1 hr after administration 2 hrs after administration N ng/ml SD N ng/ml SD
80 40 20 1 1
6 6 6 6 6 5
5.4 17.9 12.2 10.7 72.7 94.0
3.1 12.2* 8.0 7.4 16.2*** 56.3**
6 6 6 6 6 5
5.1 16.3 7.1 4.7 79.7 71.4
3.0 12,9 8.5 6.2 14.1*** 16.0***
a
Courtesy of Dr. W. Ruch and Dr. P. Marbach, Med. Biol. Forschung, Sandoz AG, Basel * p < 0.05; ** p < 0.01; *** p < 0.001 compared to controls (t-test) We wish to thank the NIAMD rat pituitary hormone distribution program for radioimunoassay materials. The values are expressed in terms of NIAMD-Rat Prolactin-RP-1
b
c
The results of these experiments, viz., the failure of clozapine t o induce homologous tolerance and cross-tolerance with respect to the increase in striatal HVA-content, its failure t o induce hypersensitivity of striatal DA-receptors, and its negligible effect on prolactin secretion, confirm that clozapine does not act on central dopaminergic transmission by the same mechanism (DAreceptor blockade) as the classical neuroleptics. The findings are in full agreement with the observations that clozapine is non-cataleptogenic and provides no protection against apomorphine- or amphetamine-induced stereotypies in experimental animals, and that it causes virtually no extrapyramidal effects in humans. In view of the potent antischizophrenic properties of clozapine it would appear that DA-receptor blockade is not essential for antipsychotic activity in man.
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Aporaorphine (0.4 mg/kg sc) was administered 27 h after the last application of the test compound. * p < 0.001
However, other workers have argued that clozapine does not differ fundamentally from other neuroleptics with respect to its mechanism of action on striatal dopaminergic transmission (8, 9, 25, 2 9 , 3 0 ) . It has been suggested that the increased level of HVA in the striatum is evidence for DA-receptor blockade, although in the case of clozapine this blockade is of the "surmountable t y p e " (25). It must be remembered, however, that DA-receptor blockade is not the only method of increasing striatal HVA-levels. Catecholamine depleting agents such as reserpine and tetrabenazine, stimulants such as pyrovalerone and methylphenidate, cholinergic agents such as oxotremorine, and inhibitors of acid transport systems such as probenecid all lead to an increase in striatal HVA content (6, 32, 33, 48). Later it was shown that clozapine, at a dose of 50 mg/kg intravenously, markedly increased the output of acetylcholine from the caudate nucleus of the cat (30). This increase was postulated to be due to DA-receptor blockade, the cholinergic neurones in the striatum being under an inhibitory dopaminergic influence. However, it must be borne in mind that at this near-toxic dose (the LD 50 in rats is approximately 60 mg/kg iv [47]) the anticholinergic component of clozapine may play a considerable role, and it has been shown repeatedly that anticholinergic drugs increase the output of acetylcholine from the caudate nucleus (for references see 34). In recent years evidence has been presented that DA-sensitive adenylate cyclase may be part of or identical to the central "DA-receptor" (8, 35). Low concentrations of DA or of apomorphine stimulate adenylate cyclase activity. The stimulatory effect of DA was blocked by low concentrations of haloperidol or chlorpromazine (35), and also by clozapine (8, 36, 37), giving rise to the suggestion that clozapine too has potent DA-receptor blocking properties. However, pimozide (8, 36) and haloperidol (8), both of which are regarded as powerful DA-receptor blocking agents, are much weaker inhibitors of the DA-sensitive adenylate cyclase reaction than clozapine, so that a direct relationship between the effect on the adenylate cyclase reaction and DA-receptor blockade seems doubtful. Some investigators suggested that clozapine may be a neuroleptic of the classical type, but that the marked anticholinergic activity of the drug prevents the occurrence of extrapyramidal effects (9, 29, 38). This idea is not accepted by all workers (25, 28), and there are several factors which are difficult to reconcile with the hypothesis. Firstly, one of the more prominent side-effects of clozapine therapy is hypersalivation, which can be treated successfully with anticholinergic agents. Secondly, addition of clozapine to the treatment regime of patients receiving depot neuroleptics does not result in a significant reduction of extrapyramidal disturbances (39). In rats, the increase in striatal HVA-content induced by haloperidol can be attenuated by pretreatment with atropine, but not with clozapine (32). Of course, it may be argued that the anticholinergic effect of clozapine exactly balances the proposed DA-receptor blocking activity, rendering the last two points invalid. However, if this were the case, then administration of clozapine should result in the appearance of catalepsy and in protection against apomorphine-induced stereotypies when given in conjunction with a centrally-acting cholinergic agent. Further, if the anticholinergic component of clozapine prevents the development of DA-receptor hypersensitivity after repeated administration of the drug, then DA-receptor hypersensitivity should fail to develop after repeated administration of haloperidol in combination with an anticholinergic drug. Recent work has shown this not to be so. Clozapine still fails to induce catalepsy or to protect against apomorphine when given together with physostigmine, and haloperidol still induces DA-receptor hypersensitivity when given in conjunction with atropine (40). These findings do not support the suggestion that the anticholinergic component of clozapine is masking blockade of DA-receptors. In recent critical appraisals of the role of clozapine in psychiatry, it was observed that the most valuable contribution of this drug to medicine may be its demonstration that the capacity to provoke extrapyramidal effects, as postulated by the DA-hypothesis of schizophrenia, is not a prerequisite of an effective antipsychotic agent ( 4 1 , 42). In fact, as was pointed out by Stille and
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Clozapine and the Dopamine Hypothesis of Schizophrenia
Hippius (41), the DA-hypothesis of schizophrenia has proved to be a useful concept for the rationalisation of the mode of action of neuroleptics, but at the same time a hindrance in the development of new types of antipsychotic drugs. Excessive attention was paid to the effects of drugs on striatal dopaminergic transmission, with the tendency to lose sight of the fact that the nigro-neostriatal fibres are only part of a larger functional system which includes at least cholinergic and GABA-ergic pathways (43). Disturbances of dopaminergic transmission at the level of the DAreceptors, particularly after repeated administration of neuroleptic drugs, may conceivably alter the functional characteristics of the entire system, resulting in alterations in the sleep pattern (44), in extrapyramidal manifestations, and possibly in the therapeutic effect in schizophrenia. However, it is equally conceivable that similar alterations of the functional state of the system could be induced by drugs that do not act primarily on dopaminergic transmission, but on other sites of the circuit. More recently the limbic system has come under scrutiny from both the clinical (45) and pharmacological view, but again the pharmacological investigations seem t o have concentrated on the dopaminergic aspects of the system (8, 38, 46). The remarks made concerning the multiplicity of transmitters in the striatal system apply equally well to the limbic system. However, it remains to be shown explicitly that there is a cause-effect relationship between the action of neuroleptics on central DA transmission and their antipsychotic efficacy. The question as to whether and t o what extent other transmitter systems are involved cannot be ignored.
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20 Berzewski, H, H. Helmchen, H. Hippius, H. Hoffmann, S. Kanowski: Das klinische Wirkungsspektxum eines neuen Dibenzodiazepinderivates W-108/HF-1854: 8-Chlor-l l-(4'-methyl)-piperazino-5-dibenzo[b,e][l,4]diazepin. ArzneimittelForsch. 19 (1969) 495 21 Angst, J., D. Benfe, P. Berner, H. Heimann, H. Helmchen, H. Hippius: Das klinische Wirkungsbild von Clozapin (Untersuchung mit dem AMP-System) Pharmakopsychiat. 4 (1971) 201 22 Fischer-Comelssen, K., U. Femer, H. Steiner: Multifokale Psychopharmakaprüfung. Arzneimittel-Forsch. 24 (1974) 1006 23 Stille, G., H. Lauener, E. Eichenberger: The pharmacology of 8-chloro-ll-(4'-methyl-l-piperazinyl)-5H-dibenzo[b,e][l,4]diazepine (clozapine). II Farmaco, Ed. Prat. 26 (1971) 603 24 Van Rossum, J.M., P.A.J. Janssen, J.R. Boissier, L. Julou, D.M. Loew, I. Malier-Nielsen, I. Münkvad, A. Randrup, G. Stille, D.H. Tedeschi: Pharmacology. In: Modern Problems of Pharmacopsychiatry, The Neuroleptics. Vol. 5. Eds. D.P. Bobon, P.A.J. Janssen, J. Bobon (Karger, Basel) 1970, pp. 23-70 25 Bartholini, G., W. Haefeli, M. Jalfre, H.H. Keller, A. Pletscher: Effects of clozapine on cerebral catecholaminergic neurone systems. Brit. J. Pharmacol. 46 (1972) 736 26 Sedvall, G., H. Nybäck: Effect of clozapine and some other antipsychotic agents on synthesis and turnover of dopamine formed from MCtyrosine in mouse brain. Joint E.B.B.S.-I.C.F.P.B. Workshop Meeting, Jerusalem 1972 27 Bürki, H.R., W. Ruch, H. Asper, M. Baggiolini, G. Stille: Pharmakologische und neurochemische Wirkungen von Clozapin: neue Gesichtspunkte in der medikamentösen Behandlung der Schizophrenie. Schweiz, med. Wschr. 103 (1973) 1716 28 Bürki, H.R., W. Ruch, H. Asper, M. Baggiolini, G. Stille: Effect of single and repeated administration of clozapine on the metabolism of dopamine and noradrenaline in the brain of the rat. Europ. J. Pharmacol. 27 (1974) 180 29 Miller, R.J., CR. Hiley: Antimuscarinic properties of neuroleptics and drug-induced parkinsonism. Nature (Lond.) 248 (1974) 596 30 Stadler, H, K.G. Lloyd, G. Bartholini: Dopaminergic inhibition of striatal cholinergic neurons: synergistic blocking action of -butyrolactone and neuroleptic drugs. Naunyn-Schmiedeberg's Arch. Pharmak. exp. Path. 283 (1974) 129 31 Foumier, P.J.R., P.D. Desjardins, H.G. Friesen: Dr. H.R. Bürki, Dr. A.C. Sayers, Research Institute, Wander Ltd., P.O. Box 2747, CH-3001 Berne/Switzerland
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Clozapine and the Dopamine Hypothesis of Schizophrenia
Die tierexperimentellen Befunde mit Clozapin, im besonderen derjenigen Eigenschaften, in denen sich Clozapin von den klassischen Neuroleptika unterscheidet, werden diskutiert. Die Frage, ob Clozapin zentrale dopaminerge Systeme beeinflußt, ist in bezug auf die Dopamin-Theorie der Schizophrenie von besonderer Bedeutung. Neurochemische, neuropharmakologische und endokrinologische Untersuchungen zeigen jedoch, daß dieses Präparat in pharmakologisch relevanten Dosen keine Blockade der Dopaminrezeptoren bewirkt. Diese Befunde sind mit der Dopamin-Theorie der Schizophrenie, welche eine direkte Beziehung zwischen antipsychotischer Wirkung und Dopaminrezeptoren-Blockade postuliert, schwer zu vereinbaren.
In the selection of possible antipsychotic agents from the numerous compounds synthesized by the chemist, psychopharmacologists depend largely on screening procedures that were developed on the basis of pharmacological or biochemical properties of pre-existing drugs with proven therapeutic efficacy. It is clear that such an approach will not lead to new types of drugs, but will only produce agents that resemble to a greater or lesser degree the drug with whose help the screening tests were developed. Hence, novel psychotropic drugs are still identified only by careful analysis of their action in man. An example in recent years is provided by clozapine, a drug which proved to have a potent antischizophrenic activity, and yet which, according to the current criteria, could hardly have been classified by the pharmacologists as a neuroleptic agent. It is the purpose of this paper to discuss some of the pharmacological and neurochemical properties of clozapine and the special attributes that differentiate clozapine from classical neuroleptics such as haloperidol and chlorpromazine. Early pharmacological and clinical observations revealed that chlorpromazine is an effective antiemetic drug (1) which causes a Parkinson-like syndrome in patients (2). These observations led to the development of laboratory tests which still enjoy wide-spread popularity for the selection of compounds with possible antischizophrenic activity, namely the measurement of cataleptogenic activity and protection against apomorphine- or amphetamine-induced stereotypies (3). Further, chlorpromazine and other neuroleptics were shown to accelerate the metabolism of dopamine (DA) in the brain, an effect attributed to feedback activation of presynaptic dopaminergic neurones due to blockade of striatal DA-receptors (4, 5, 6). Thus emerged the DA-hypothesis of schizophrenia, which was to explain both the extrapyramidal effects and the antischizophrenic activity of neuroleptic drugs (2, 7, 8, 9). According to this hypothesis, blockade of DA-receptors in animals results in protection against apomorphineinduced stereotypies and emesis, and, as a result of attenuation of striatal transmission, in the
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Recent observations suggest, however, that there may be no causal relationship between extrapyramidal activity of neuroleptics and their antipsychotic action in man. When potent neuroleptics such as loxapine or haloperidol were administered to rats for several days, tolerance developed to the extrapyramidal manifestations of these drugs, i.e., cataleptogenic behaviour, apomorphine antagonism and the changes in striatal DA metabolism (12). It is interesting to note that a number of reports (see 13 for a review) stress that anti-Parkinson medication, often needed initially together with neuroleptic therapy, can be discontinued within a few weeks without the re-appearance of extrapyramidal symptoms. However, no tolerance develops to the antipsychotic activity of the neuroleptic drugs. The tolerance to the extrapyramidal effects in animals has been shown to be due to alterations in the properties of DA-receptors, with a reduced sensitivity toward the effects of neuroleptics and an increased sensitivity toward dopaminergic agents (14, 15, 16, 17), so that it would seem that long-term treatment with neuroleptic agents brings about new functional equilibria of the striatal system. These findings challenge the view expressed by the DA-hypothesis that the DA-receptor blockade per se is the mechanism by which neuroleptics mediate, antischizophrenic activity, and suggest that the therapeutic effect of these drugs could equally well be due to functional alterations of the kind seen after chronic administration. Furthermore, the question is posed whether any meaningful concept for the explanation of the therapeutic actions of neuroleptics can be deduced from data obtained after single administration of these drugs, when it is known that neuroleptic therapy usually lasts for weeks or even months before the desired therapeutic effect is achieved. Further doubts of the relevance of the DA-hypothesis arose when early reports about the antipsychotic efficacy of clozapine (18, 19, 20) were confirmed in different countries in trials involving large numbers of patients ( 2 1 , 22). If the usual pharmacological criteria for selecting antipsychotic drugs are applied, clozapine would not have qualified as a potential neuroleptic (23). Clozapine is non-cataleptogenic, provides no protection against apomorphine stereotypies, inhibits conditioned avoidance behaviour only in doses causing severe sedation, and in man causes few, if any, parkinsonoid effects. It follows that the drug can hardly be classified as a neuroleptic according to the accepted pharmacological definition of the term (24) (Table 1).
Table 1 Pharmacological data of clozapine, haloperidol and loxapine Aporaorphine antagonism Catalepsy Conditioned avoidance response Inhibition of locomotor activity Mydriasis Oxotremorine antagonism Electrographic arousal inhibition: - Reticular stimulation - Arecoline stimulation For methods see (23)
Clozapine
Haloperidol
Rat Rat
ED 50 (mg/kg sc): ED 50 (mg/kg sc):
inactive inactive
0.14 0.30
0.07 0.10
Rat
ED 50 (mg/kg po):
20.0
0.38
0.06
Mouse Mouse Mouse
ED 50 (mg/kg po): ED 200 (mg/kg po): ED 50 (mg/kg po):
2.5 8.0 9.0
0.30 inactive > 20
ED 150 (mg/kg iv): Block (mg/kg iv):
1.5 1.1
inactive inactive
Rabbit
Loxapine
0.04 inactive 16.0 3.2 4.5
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development of catalepsy. In man, production of psychotic experiences is inhibited, and a Parkinson-like syndrome develops (2, 10). Extrapyramidal effects were considered indispensable for an antipsychotic agent, and indeed, some clinicians (11) considered the therapeutic dose level of a neuroleptic to be reached only when extrapyramidal effects appeared.
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Although as judged by the usual pharmacological criteria clozapine cannot be regarded as a neuroleptic drug, various authors, using different experimental designs, concluded that clozapine may still be a DA-receptor blocker, the consequences of DA-receptor blockade being masked by the pronounced anticholinergic and/or muscle-relaxing properties of the drug (8, 9, 2 5 , 29, 30). For this reason experiments were designed specifically t o answer the question whether or not clozapine, in pharmacologically relevant doses, blocks DA-receptors. Three independent experimental approaches were chosen: The first approach is based on the fact that rats treated chronically with classical neuroleptics develop a tolerance to the extrapyramidal effects, as shown by the progressively diminishing HVA-increases in the corpora striata after repeated administration of the drugs (12, 28). Cross tolerance occurs between neuroleptics which may be quite unrelated structurally, as, e.g. haloperidol and loxapine (14). In striking contrast to these observations, repeated administration of high doses of clozapine caused no tolerance to the HVA-increasing action (28), nor did pretreatment with clozapine affect the HVA increase of a challenging dose of haloperidol or loxapine (14) (Table 2). Table 2 Homovanillic acid content of rat striatum after repeated doses of loxapine, chlorpromazine, haloperidol or clozapine, and challenged with a single dose of haloperidol or loxapine Pretreatment days 1 - 6 (mg/kg po) Water Haloperidol Loxapine Chlorpromazine Clozapine Water Loxapine Haloperidol Chlorpromazine Clozapine
( 3) ( 2) (32) (80) ( 2) ( 3) (32) (80)
Challenging dose on day 7 (mg/kg po)
N
HVA-content Mg/g S.D.
Haloperidol Haloperidol Haloperidol Haloperidol Haloperidol Loxapine Loxapine Loxapine Loxapine Loxapine
10 5 6 4 5 10 9 5 5 5
3.06 1.57 0.95 1.95 2.57 3.17 0.88 1.91 2.41 3.06
(3) (3) (3) (3) (3) (2) (2) (2) (2) (2)
a
0.38 0.05* 0.08* 0.18* 0.64 0.58b 0.13* 0.08* 0.24* 0.15
Rats were killed 3 h after the day 7 injection. N represents the number of determinations, each performed on the pooled homogenates of the striata of 5 rats. HVA-content in rats treated solely with water was 0.52 jug/g 0.05 (S.D., N = 15). * p < 0.001 compared to groups a and b respectively.
In a second experimental design, use was made of the fact that rats treated chronically with classical neuroleptics exhibit hypersensitivity t o the actions of apomorphine (15). This effect is best quantified by measuring the turning behaviour in rats with unilateral striatal lesions (14). The number of turns elicited after a standard dose of apomorphine serves as a measure for striatal DA-receptor sensitivity to apomorphine. It was shown that pretreatment with haloperidol for one week strongly augmented the turning response to apomorphine, which was given 27 h after the last dose of haloperidol. This result suggests DA-receptor hypersensitivity due t o prolonged
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Biochemical evidence shows that clozapine, in common with the classical neuroleptics, affects the dopaminergic system in the striatum ( 2 5 , 2 6 ) . However, its mechanism of action differs considerably from that of the classical neuroleptics. The latter drugs accelerate the turnover of DA in the striatum, increase the levels of the DA-metabolites homovanillic acid (HVA) and dihydroxyphenylacetic acid (DOPAC), and decrease the content of DA. In contrast, clozapine increases striatal HVA and DOPAC only in doses which exceed by a factor of between 100 and 1000 those of the classical neuroleptics, and it increases, instead of decreases the striatal DA-content (27, 28),
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Table 3 Apomorphine-induced circling in rats with unilateral striatal lesions
Haloperidol Haloperidol Clozapine Clozapine Clozapine Clozapine
Length of treatment in days
Daily dose mg/kg po
Total no. of turns (% of Control)
6 6 6 6 13 20
0.5 3.0 20.0 80.0 80.0 80.0
146* 179* 107 104 122 126
interaction of the neuroleptic with the DA-receptors. In marked contrast t o these findings, repeated administration of high doses of clozapine did not significantly increase the sensitivity of the lesioned rats to apomorphine (14) (Table 3). The third experimental approach involved a DA-system structurally and functionally distinct from that of the corpus striatum. Clozapine was compared with classical neuroleptics in respect of the action on prolactin secretion. The concentration of prolactin in the blood depends in part on the rate of its secretion, which is controlled by a hypothalamic inhibitory factor, the availability of which in turn is regulated by DA mechanisms. After administration of dopaminergic agents the blood concentration of prolactin is reduced, afjter DA-receptor blockers such as chlorpromazine it is elevated (for discussion see 31). It was shown that potent neuroleptics like clothiapine and pimozide dramatically increase the prolactin level in the serum, while clozapine even at very high doses caused only a slight increase in the prolactin level (Table 4). a
Table 4 Serum prolactin in ovariectomized rats >
D
0
Treatment Controls Clozapine Pimozide Clothiapine
Dosage mg/kg po
Serum prolactin 1 hr after administration 2 hrs after administration N ng/ml SD N ng/ml SD
80 40 20 1 1
6 6 6 6 6 5
5.4 17.9 12.2 10.7 72.7 94.0
3.1 12.2* 8.0 7.4 16.2*** 56.3**
6 6 6 6 6 5
5.1 16.3 7.1 4.7 79.7 71.4
3.0 12,9 8.5 6.2 14.1*** 16.0***
a
Courtesy of Dr. W. Ruch and Dr. P. Marbach, Med. Biol. Forschung, Sandoz AG, Basel * p < 0.05; ** p < 0.01; *** p < 0.001 compared to controls (t-test) We wish to thank the NIAMD rat pituitary hormone distribution program for radioimunoassay materials. The values are expressed in terms of NIAMD-Rat Prolactin-RP-1
b
c
The results of these experiments, viz., the failure of clozapine t o induce homologous tolerance and cross-tolerance with respect to the increase in striatal HVA-content, its failure t o induce hypersensitivity of striatal DA-receptors, and its negligible effect on prolactin secretion, confirm that clozapine does not act on central dopaminergic transmission by the same mechanism (DAreceptor blockade) as the classical neuroleptics. The findings are in full agreement with the observations that clozapine is non-cataleptogenic and provides no protection against apomorphine- or amphetamine-induced stereotypies in experimental animals, and that it causes virtually no extrapyramidal effects in humans. In view of the potent antischizophrenic properties of clozapine it would appear that DA-receptor blockade is not essential for antipsychotic activity in man.
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Aporaorphine (0.4 mg/kg sc) was administered 27 h after the last application of the test compound. * p < 0.001
However, other workers have argued that clozapine does not differ fundamentally from other neuroleptics with respect to its mechanism of action on striatal dopaminergic transmission (8, 9, 25, 2 9 , 3 0 ) . It has been suggested that the increased level of HVA in the striatum is evidence for DA-receptor blockade, although in the case of clozapine this blockade is of the "surmountable t y p e " (25). It must be remembered, however, that DA-receptor blockade is not the only method of increasing striatal HVA-levels. Catecholamine depleting agents such as reserpine and tetrabenazine, stimulants such as pyrovalerone and methylphenidate, cholinergic agents such as oxotremorine, and inhibitors of acid transport systems such as probenecid all lead to an increase in striatal HVA content (6, 32, 33, 48). Later it was shown that clozapine, at a dose of 50 mg/kg intravenously, markedly increased the output of acetylcholine from the caudate nucleus of the cat (30). This increase was postulated to be due to DA-receptor blockade, the cholinergic neurones in the striatum being under an inhibitory dopaminergic influence. However, it must be borne in mind that at this near-toxic dose (the LD 50 in rats is approximately 60 mg/kg iv [47]) the anticholinergic component of clozapine may play a considerable role, and it has been shown repeatedly that anticholinergic drugs increase the output of acetylcholine from the caudate nucleus (for references see 34). In recent years evidence has been presented that DA-sensitive adenylate cyclase may be part of or identical to the central "DA-receptor" (8, 35). Low concentrations of DA or of apomorphine stimulate adenylate cyclase activity. The stimulatory effect of DA was blocked by low concentrations of haloperidol or chlorpromazine (35), and also by clozapine (8, 36, 37), giving rise to the suggestion that clozapine too has potent DA-receptor blocking properties. However, pimozide (8, 36) and haloperidol (8), both of which are regarded as powerful DA-receptor blocking agents, are much weaker inhibitors of the DA-sensitive adenylate cyclase reaction than clozapine, so that a direct relationship between the effect on the adenylate cyclase reaction and DA-receptor blockade seems doubtful. Some investigators suggested that clozapine may be a neuroleptic of the classical type, but that the marked anticholinergic activity of the drug prevents the occurrence of extrapyramidal effects (9, 29, 38). This idea is not accepted by all workers (25, 28), and there are several factors which are difficult to reconcile with the hypothesis. Firstly, one of the more prominent side-effects of clozapine therapy is hypersalivation, which can be treated successfully with anticholinergic agents. Secondly, addition of clozapine to the treatment regime of patients receiving depot neuroleptics does not result in a significant reduction of extrapyramidal disturbances (39). In rats, the increase in striatal HVA-content induced by haloperidol can be attenuated by pretreatment with atropine, but not with clozapine (32). Of course, it may be argued that the anticholinergic effect of clozapine exactly balances the proposed DA-receptor blocking activity, rendering the last two points invalid. However, if this were the case, then administration of clozapine should result in the appearance of catalepsy and in protection against apomorphine-induced stereotypies when given in conjunction with a centrally-acting cholinergic agent. Further, if the anticholinergic component of clozapine prevents the development of DA-receptor hypersensitivity after repeated administration of the drug, then DA-receptor hypersensitivity should fail to develop after repeated administration of haloperidol in combination with an anticholinergic drug. Recent work has shown this not to be so. Clozapine still fails to induce catalepsy or to protect against apomorphine when given together with physostigmine, and haloperidol still induces DA-receptor hypersensitivity when given in conjunction with atropine (40). These findings do not support the suggestion that the anticholinergic component of clozapine is masking blockade of DA-receptors. In recent critical appraisals of the role of clozapine in psychiatry, it was observed that the most valuable contribution of this drug to medicine may be its demonstration that the capacity to provoke extrapyramidal effects, as postulated by the DA-hypothesis of schizophrenia, is not a prerequisite of an effective antipsychotic agent ( 4 1 , 42). In fact, as was pointed out by Stille and
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Clozapine and the Dopamine Hypothesis of Schizophrenia
Hippius (41), the DA-hypothesis of schizophrenia has proved to be a useful concept for the rationalisation of the mode of action of neuroleptics, but at the same time a hindrance in the development of new types of antipsychotic drugs. Excessive attention was paid to the effects of drugs on striatal dopaminergic transmission, with the tendency to lose sight of the fact that the nigro-neostriatal fibres are only part of a larger functional system which includes at least cholinergic and GABA-ergic pathways (43). Disturbances of dopaminergic transmission at the level of the DAreceptors, particularly after repeated administration of neuroleptic drugs, may conceivably alter the functional characteristics of the entire system, resulting in alterations in the sleep pattern (44), in extrapyramidal manifestations, and possibly in the therapeutic effect in schizophrenia. However, it is equally conceivable that similar alterations of the functional state of the system could be induced by drugs that do not act primarily on dopaminergic transmission, but on other sites of the circuit. More recently the limbic system has come under scrutiny from both the clinical (45) and pharmacological view, but again the pharmacological investigations seem t o have concentrated on the dopaminergic aspects of the system (8, 38, 46). The remarks made concerning the multiplicity of transmitters in the striatal system apply equally well to the limbic system. However, it remains to be shown explicitly that there is a cause-effect relationship between the action of neuroleptics on central DA transmission and their antipsychotic efficacy. The question as to whether and t o what extent other transmitter systems are involved cannot be ignored.
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Clozapine and the Dopamine Hypothesis of Schizophrenia
