Clozapine normalizes prefrontal cortex dopamine transmission in monkeys subchronically exposed to phencyclidine.

The mechanism responsible for the therapeutic effects of the prototypical atypical antipsychotic drug, clozapine, is still not understood; however, there is persuasive evidence from in vivo studies in normal rodents and primates that the ability to elevate dopamine neurotransmission preferentially in the prefrontal cortex is a key component to the beneficial effects of clozapine in schizophrenia. Theoretically, such an effect of clozapine would counteract the deficient dopaminergic innervation of the prefrontal cortex that appears to be part of the pathophysiology of schizophrenia. We have previously shown that following repeated, intermittent administrations of phencyclidine to monkeys there is lowered prefrontal cortical dopamine transmission and impairment of cognitive performance that is dependent on the prefrontal cortex; these biochemical and behavioral changes therefore model certain aspects of schizophrenia. We now investigate the effects of clozapine on the dopamine projections to prefrontal cortex, nucleus accumbens, and striatum in control monkeys and in those withdrawn from repeated phencyclidine treatment, using a dose regimen of clozapine that ameliorates the cognitive deficits described in the primate phencyclidine (PCP) model. In normal monkeys, clozapine elevated dopamine turnover in all prefrontal cortical, but not subcortical, regions analyzed. In the primate PCP model, clozapine normalized dopamine (DA) turnover in the dorsolateral prefrontal cortex, prelimbic cortex, and cingulate cortex. Thus, the present data support the hypothesis that the therapeutic effects of clozapine in this primate model and perhaps in schizophrenia may be related at least in part to the restoration of DA tone in the prefrontal cortex.     

A comparison between some biochemical and behavioural effects produced by neuroleptics

Acute administration of five neuroleptics to rats produced a dose-dependent increase in brain homovanillic acid (HVA) which was of greater magnitude and of longer duration in the corpus striatum than in the tuberculum olfactorium. 4-p-Fluorophenyl-5-N(N'-o-methoxy-phenyl) piperazinoethyl-4-oxazolin-2-one (LR 511) appeared to be 5--10 times more potent than fluanisone and clozapine, as active as chlorpromazine (CPZ) but at least twentyfold less active than haloperidol. Time-course studies on dopamine turnover have indicated that LR 511 at a moderate pharmacological dose has some similarities with clozapine, e.g., a lower difference between striatal and limbic tissues. All the neuroleptics caused inhibition of the conditioned avoidance response and at their ED50S on this parameter (with the exception of clozapine) caused also an equal increment of the cerebral HVA level. At their ED50S on catalepsy, the neuroleptics evoked HVA changes which varied according to the drug tested. Thus, potent cataleptogenic agents caused either strong stimulation of the DA turnover in both brain structures (haloperidol and CPZ) or weak stimulation only in corpus striatum (fluanisone). On the other hand a poor cataleptogenic agent, such as LR 511, was accompanied by the highest HVA levels and also the non-cataleptogenic clozapine at the dose of 40 mg/kg increased the cerebral HVA levels. It is suggested that the acceleration of DA turnover in animals is not an essential prerequisite for predicting the antipsychotic activity in man and that the preferential reactivity of dopaminergic limbic receptors is not necessarily linked to a better ratio of antipsychotic versus extrapyramidal effects in man.     

The effects of kainic acid lesions on dopaminergic responses to haloperidol and clozapine

The antipsychotic drugs haloperidol and clozapine have the common action of increasing dopamine metabolism in the striatum (nucleus accumbens, caudate-putamen) of the rat. Intracerebroventricular administration of kainic acid (KA) produces neuronal loss in limbic-cortical brain regions which project directly or indirectly to the striatum. In the present study, dopamine metabolism in subregions of the striatum was examined in rats with KA lesions after acute and chronic haloperidol or clozapine administration. The main findings was that the elevating effect of acute haloperidol treatment on the dopamine metabolite, DOPAC, was blocked in the nucleus accumbens shell and diminished in medial and laterodorsal caudate-putamen of the KA-lesioned rats. In addition, the elevating effects of both acute and chronic haloperidol treatment on dopamine turnover were attenuated in the laterodorsal caudate-putamen of KA-lesioned rats. The levels of dopamine, DOPAC, and HVA after chronic clozapine treatment were greater in KA-lesioned than control rats. These results indicate that dopaminergic responses to haloperidol may be diminished by limbic-cortical neuropathology, while such pathology does not significantly alter dopaminergic responses to clozapine. Received: 30 September 1996 / Final version: 6 March 1997     

Fatty acid derivatives of clozapine: prolonged antidopaminergic activity of docosahexaenoylclozapine in the rat.

Stable amides of clozapine derived from fatty acids prominent in cerebral tissue might enhance the central activity of clozapine and reduce its exposure to peripheral tissues. Such derivatives might enhance the safety of this unique drug, which is the only agent with securely established superior antipsychotic effectiveness, but with a risk of potentially lethal systemic toxicity. Amide derivatives of clozapine were prepared from structurally varied fatty acid chlorides and evaluated for ability to inhibit behavioral arousal in rat induced by dopamine agonist apomorphine and to induce catalepsy. Their duration-of-action and potency were compared to free clozapine, and concentrations of clozapine were assayed in brain and blood. Selected agents were also evaluated for affinity at dopamine receptors and other potential drug-target sites. Clozapine-N-amides of linoleic, myristic, oleic, and palmitic acids had moderate initial central depressant activity but by 6 h, failed to inhibit arousal induced by apomorphine. However, the docosahexaenoic acid (DHA) derivative was orally bioavailable, 10-times more potent (ED(50) 5.0 micromol/kg) than clozapine itself, and very long-acting (>/= 24 h) against apomorphine, and did not induce catalepsy. DHA itself was inactive behaviorally. Clozapine showed expected dopamine receptor affinities, but DHA-clozapine was inactive at these and other potential target sites. After systemic administration of DHA-clozapine, serum levels of free clozapine were very low, and brain concentrations somewhat lower than after administering clozapine. DHA-clozapine is a long-acting central depressant with powerful and prolonged antidopaminergic activity after oral administration or injection without inducing catalepsy, and it markedly reduced peripheral exposure to free clozapine. It lacked the receptor-affinities shown by clozapine, suggesting that DHA-clozapine may be a precursor of free, pharmacologically active clozapine. Such agents may represent potential antipsychotic drugs with improved central/peripheral distribution, and possibly enhanced safety.     

Central levels of noradrenaline, 3-methoxy-4-hydroxyphenylethyleneglycol and cyclic AMP in the rat after activation of Locus coeruleus neurons: Influence of single and repeated neuroleptic treatment

The influence of single and repeated neuroleptic treatment on noradrenaline (NA) metabolism in two areas of the central nervous system with different neuronal organizations (forebrain and spinal cord) was studied. The doses of the neuroleptics studied were chosen because of their maximum effects on turnover of dopamine in various brain areas.The endogenous levels of 3-methoxy-4-hydroxy-phenylethyleneglycol (MOPEG) in the forebrain and spinal cord of the rat were markedly increased by single intraperitoneal doses of clozapine (15 mg/kg), haloperidol (1.0 mg/kg) or chlorpromazine (2.0 mg/kg). The levels of noradrenaline (NA) in the hippocampus were decreased by a single dose of clozapine and haloperidol. Fluphenazine (0.1 mg/kg) and sulpiride (40 mg/kg) caused only slight increases of MOPEG in the forebrain and none in the spinal cord. Following repeated treatment with either clozapine or haloperidol tolerance to the stimulatory effects on NA turnover developed more rapidly in the spinal cord than in the forebrain (within 4 and 15 days respectively). After 4 days of repeated treatment the initial decrease in hippocampal NA levels had disappeared.Unilateral electrical stimulation of the locus coeruleus (LC) after a single dose of either clozapine or haloperidol induced smaller reductions of hippocampal NA (ipsilateral versus contralateral) than in saline treated control animals. In subchronically clozapine or haloperidol treated rats, LC-stimulation induced an ipsilateral decrease of NA similar to that in controls. The levels of MOPEG after LC-stimulation were elevated compared to untreated stimulated rats both in the ipsilateral and contralateral forebrain.Neither single nor repeated treatment with clozapine or haloperidol altered basal cyclic AMP levels or inhibited the cyclic AMP response to LC-stimulation.This study is evidence: (1) that neuroleptics decrease NA by release of the amine from a rapidly releasable pool (2) that even when the influence of subchronic neuroleptic treatment on cerebral NA metabolism has ceased, such treatment has a lasting influence on NA-neurons (3) that in vivo the formation of cyclic AMP is not influenced by neuroleptic treatment.     

Effect of neuroleptics on endogenous norepinephrine in rat brain

The neuroleptic drugs clozapine, thioridazine and methiothepin, but not chlorpromazine, markedly reduced the norepinephrine (NE) levels in the brain of rats kept normothermic. Clonidine prevented the NE lowering effect of clozapine. In animals with sectioned spinal cords, clozapine decreased the NE levels only in the segment cranial to the lesion whereas in normal animals the drug induced a similar NE decrease in the cranial and caudal part of the spinal cord. After pretreatment with the dopamine-β-hydroxylase inhibitor FLA 63 in a dose which did not markedly diminish the cerebral NE, chlorpromazine caused a small but significant decrease of this amine.It is concluded that, due to blockade of NE receptors and a subsequent feed-back activation of NE neurones, clozapine, thioridazine and methiothepin increase the release of NE to such a degree that the amine loss cannot be fully compensated by synthesis possibly as a consequence of an insufficient rate of β-hydroxylation of dopamine. In contrast, in the case of chlorpromazine which seems to be a relatively weak NE-receptor blocking agent, this compensation is still possible.     

Regional changes in the concentrations of cerebral monoamines and their metabolites after ethanolamine-O-sulphate induced elevation of brain gamma-aminobutyric acid concentrations.

The effect of ethanolamine-O-sulphate induced elevation of cerebral GABA concentrations on monoamine and their metabolites levels has been studied in various regions of the rat brain. Increased GABA concentrations were associated with a decrease in turnover of dopamine in limbic regions: striatal dopamine was not significantly affected. An increased turnover of 5-hydroxytryptamine was also observed in other brain areas. Increased cerebral GABA concentrations had no effect on regional noradrenaline turnover. The possible sites of interaction between the neurotransmitters are discussed.     

Effects of clozapine on ketamine-induced linguopharyngeal events in rats

We tested the effects of clozapine (0.02-20 mg/kg i.p.) on ketamine-induced linguopharyngeal events in rats anesthetized with i.m. injections of ketamine hydrochloride (100 mg/kg) and mounted on a stereotaxic with the tip of the tongue tied to a force displacement transducer monitoring tongue protrusions, retrusions and swallows. Reduction began at the 0.04 mg/kg dose. At 4.8 mg/kg there was total suppression of events. At 20 mg/kg, suppression lasted for 1 h. Notably clozapine doses causing total suppression of events in our model were much lower than those usually reported to alter dopamine turnover.     

Effects of a Single Administration of Clozapine on Mouse Brain Catecholamines

Abstract The effects of clozapine on the accumulation and disappearance of ¹⁴C-labelled catecholamines formed from ¹⁴C-tyrosine, and on the endogenous levels of catecholamines have been studied in mouse brain. Rather low doses of clozapine increased noradrenaline accumulation and disappearance (from 2.5 and 0.16 mg/kg, respectively), whereas dopamine accumulation was only increased from 10 mg/kg and disappearance after 80 mg/kg. However, in the dosage interval of 1.25 to 5 mg/kg dopamine disappearance was decreased. Endogenous noradrenaline levels were decreased from 10 mg/kg in contrast to the dopamine levels which remained unchanged, except in the dosage interval 2.5 to 10 mg/kg, where they were elevated, at the same time at which dopamine disappearance was decreased. It is concluded that clozapine differs biochemically from the typical neuroleptics by changing the metabolism of noradrenaline in doses much lower than those influencing dopamine metabolism. It is suggested that this property together with the ability of clozapine to decrease dopamine disappearance in a certain dose range, could contribute to the unique psychopharmacological action of clozapine.     

Subcortical dopamine and serotonin turnover during acute and subchronic administration of typical and atypical neuroleptics

The effects of acute (1 day) and subchronic (28 days) treatment with three atypical antipsychotic drugs [clozapine, (±)-sulpiride and (–)-3-PPP] on dopamine and serotonin turnover in both the nucleus accumbens (NA) and corpus striatum (CS) of rodents was compared to haloperidol and saline treatment. The equivalent doses of all drugs were determined based upon their ability to compete in vivo for³H-spiperone binding in the NA and CS. All three atypical drugs, compared to haloperidol, produced preferential elevations of dopamine turnover in the NA. Further, the development of tolerance to this effect was more apparent for the three atypical drugs than for haloperidol. Surprisingly, all three atypical drugs, but not haloperidol, produced changes in serotonin turnover, despite the fact that (±)-sulpiride and (–)-3-PPP have no known direct effects on brain serotonin systems. All three atypical drugs produced acute increases in serotonin turnover in both the NA and CS, followed by later decreases.     

Effects of amitriptyline and nortriptyline on cerebral activity of the CDF-1 mouse strain

1. Equal dose regimens of amitriptyline, a tertiary amine tricyclic antidepressant, were more potent than nortriptyline, a secondary amine derivative, in suppressing CDF-1 mouse locomotor activity. 2. A suggestive increase in dopamine turnover rate in mouse cerebral cortex and striatal brain regions was apparent by amitriptyline but not nortriptyline. 3. A suggestive increase in serotonin turnover in mouse cerebellum and striatum was determined for nortriptyline. 4. Both antidepressants increased cerebral cortex, midbrain and cerebellum serotonin levels from saline control. 5. Increases of regional brain dopamine by amitriptyline and serotonin by nortriptyline concurrent with reuptake blockade of the respective serotonin and dopamine may contribute to their differential extrapyramidal and sedating side effects.     

DIFFERENTIAL BLOCKADE OF OCTOPAMINE AND DOPAMINE RECEPTORS BY ANALOGUES OF CLOZAPINE AND METOCLOPRAMIDE

1. Sulpiride, but not procainamide, antagonizes the excitatory effects of (±)-octopamine receptors in the Tapes ventricle. Neither compound attenuates dopamine excitation. 2. Clozapine will attenuate the effects of (±)-octopamine and (-)-α-methyl octopamine at the octopamine receptor but not the excitatory effects of dopamine at dopamine receptors. 3. Clozapine is more potent than its 2-positional isomer HF 2046 in attenuating octopamine excitation. However, HF 2046, unlike clozapine, will attenuate the excitatory effects of dopamine. 4. These data indicate that replacement of the 8-chloro substituent in the clozapine nucleus with a 2-chloro substituent decreases the ability of the compound to blockade octopamine receptors. However, the 2-chloro-substituted compound (HF 2046) now has the added ability to blockade excitatory dopamine receptors. 5. The greater potency of clozapine than HF 2046 as an octopamine antagonist suggests that it is the 8-chloro-substituted aromatic ring of clozapine which overlaps the aromatic site usually occupied by the octopamine aromatic ring.     

Discriminative stimulus properties of N-desmethylclozapine, the major active metabolite of the atypical antipsychotic clozapine, in C57BL/6 mice

N-desmethylclozapine (NDMC) is the major active metabolite of the atypical antipsychotic drug clozapine and may contribute to the therapeutic efficacy of clozapine. Although they share many pharmacological features, it is noteworthy that NDMC is a partial dopamine D2 and cholinergic muscarinic M1/M4 agonist, whereas clozapine is a weak dopamine D2 receptor inverse agonist/antagonist and a nonselective muscarinic antagonist. To better understand the in-vivo pharmacological mechanisms of these drugs, male C57BL/6NHsd-wild-type mice were trained to discriminate 10.0 mg/kg NDMC from vehicle in a two-lever drug discrimination procedure for food reward. It was found that the parent drug clozapine fully substituted for NDMC, whereas the typical antipsychotic drug haloperidol (dopamine D2 antagonist) and the atypical antipsychotic drug aripiprazole (D2 partial agonist) did not substitute for NDMC. These results demonstrated that clozapine and its major metabolite NDMC share in-vivo behavioral properties (i.e. discriminative stimulus properties) that are likely due to shared pharmacological mechanisms that differ from other antipsychotic drugs. The discriminative stimulus properties of NDMC probably reflect a compound cue similar to that of its parent drug clozapine due to its diverse binding profile.     

Trough oestradiol levels associated with cognitive impairment in post-menopausal women after 10 years of oestradiol implants

RATIONALE: Clozapine is a unique antipsychotic with very low propensity to cause motor side effects. In contrast to most other antipsychotics that block more than 70% of dopamine D(2) receptors at therapeutic doses, clozapine occupies less than 70%. Furthermore, even at maximum occupancy, 70% is not exceeded. Several mechanisms have been proposed as explanations for this low D(2) receptor occupancy, but clear evidence is limited. OBJECTIVES: In patient studies the data are limited by the dose-range that can be safely used; therefore, the aims of this study were to examine the maximum occupancy of dopamine D(2) receptors with up to 5.0 mg/kg of bolus injection of clozapine to non-human primates and to measure the time course of occupancy. METHODS: PET examination with [(11)C]raclopride was performed to measure the dopamine D(2) receptor occupancy in the striatum of two monkeys after the bolus injection of 0.2-5.0 mg/kg clozapine. [(11)C]raclopride was injected sequentially to follow the time course of occupancy up to 7 h after the clozapine injection. RESULTS: Dopamine D(2) receptor occupancy reached up to 83% after 5.0 mg/kg clozapine injection. Occupancy decreased with a half-life of 7.22 h after 5.0 mg/kg clozapine and 5.25 h after 1.0 and 2.0 mg/kg clozapine. CONCLUSIONS:Clozapine could occupy a high proportion of dopamine D(2) receptors. The time course of occupancy was relatively fast, with a half-life of several hours.     

Hypophysectomy does not prevent increased cerebral dopamine turnover following sulpiride administration

Hypophysectomy is claimed to prevent increased forebrain dopamine turnover produced by administration of sulpiride. We have measured the increase in dopamine metabolite concentrations caused by sulpiride following surgical removal of the pituitary. In saline-treated control animals and in hypophysectomized rats 1 week or 1 month following surgery, administration of sulpiride caused marked elevations of striatal, nucleus accumbens and tuberculum olfactorium, homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC) concentrations which were maximal 4-8 h following drug administration. The maximal increases in nucleus accumbens and tuberculum olfactorium were generally comparable in control and hypophysectomized animals, except for a greater increase in HVA levels in the nucleus accumbens 1 month following hypophysectomy. However, maximal increases in HVA and DOPAC in striatum were more pronounced in hypophysectomized rats 1 week or 1 month following surgery compared with control animals. At 30 min following sulpiride administration only inconsistent changes in dopamine turnover were observed in both control and hypophysectomized rats. Hypophysectomy does not prevent sulpiride from increasing forebrain dopamine turnover suggesting this is due to a direct interaction with cerebral dopamine receptors.     

Increase of 3-methoxy-4-hydroxyphenylethylene glycol in rat brain by neuroleptic drugs

The potency of various neuroleptic drugs in increasing the content of endogenous 3-methoxy-4-hydroxyphenyl-ethylene glycol (MOPEG) in rat brain decreased in the order methiothepin, haloperidol, clozapine, thioridazine, chlorpromazine, pimozide. The neuroleptics, except pimozide and chlorpromazine, also caused a slight to moderate diminution of the endogenous cerebral noradrenaline (NA).Based on these and earlier findings it is concluded that (a) changes in brain NA turnover induced by neuroleptics can be estimated, in a relatively simple way, by measuring cerebral MOPEG; (b) these drugs markedly differ in their ability to activate noradrenergic neurons; and (c) the activation of noradrenergic neurons by neuroleptics does not seem to parallel that of dopaminergic neurons.     

Effects of acute and chronic clozapine on dopamine release and metabolism in the striatum and nucleus accumbens of conscious rats.

1. The effect of single and repeated (once daily for 23 days) oral doses of 20 and 60 mg kg-1 clozapine on dopamine release and metabolism were studied by intracerebral dialysis in the striatum and nucleus accumbens of conscious rats. 2. The basal output of dopamine, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the striatum and nucleus accumbens of rats given clozapine 20 or 60 mg kg-1 chronically, measured one day after the last drug dose, was not significantly different from that of vehicle-treated animals. 3. Challenge doses of 20 or 60 mg kg-1 clozapine produced similar increases in dopamine levels in the striatum and nucleus accumbens of animals which had received vehicle or clozapine 20 or 60 mg kg-1 once daily for 23 days, except that 1 h after administration 60 mg kg-1 clozapine had a greater effect in the nucleus accumbens. 4. In animals treated chronically with clozapine 20 and 60 mg kg-1 or vehicle, DOPAC levels in the striatum and nucleus accumbens were increased to the same extent by challenge doses of clozapine (20 or 60 mg kg-1). In animals treated chronically with clozapine, a challenge dose of 60 mg kg-1 had significantly greater effect on HVA only in the nucleus accumbens. 5. When DOPAC and HVA were measured post mortem in the striatum and nucleus accumbens 2 h after various oral doses of clozapine, it was found that 10 mg kg-1 significantly increased dopamine metabolites only in the nucleus accumbens whereas 100 mg kg-1 had this effect in both regions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bupivacaine induces apoptosis via mitochondria and p38 MAPK dependent pathways

N-desmethylclozapine (NDMC or norclozapine) is the major active metabolite of the antipsychotic clozapine in humans. The activity of NDMC differs from clozapine at a number of neurotransmitter receptors, probably influencing the pharmacological effects of clozapine treatment. Here, we tested the properties of NDMC in comparison with clozapine at recombinant human dopamine D(2) and serotonin 5-HT(1A) receptors, using a panel of functional assays implicating diverse signalling pathways. At dopamine D(2) receptors, NDMC as well as clozapine did not display agonist activity in measures of G protein activation by [(35)S]GTPγS binding and in the sensitive Extracellular Signal-Regulated Kinase 1/2 (ERK1/2) phosphorylation assay. In contrast, there were weak partial agonist actions of NDMC (but not of clozapine) for dopamine D(2)-dependent activation of Ca(2+) liberation via coexpressed chimeric Gα(q/o) proteins and for G protein-coupled inward rectifier potassium channel (GIRK) current induction in Xenopus oocytes. Intriguingly, GIRK currents induced by NDMC via dopamine D(2) receptors showed a rapid and transient time course, strikingly different from currents recorded with other receptor agonists. At serotonin 5-HT(1A) receptors, NDMC was a more efficacious partial agonist than clozapine for [(35)S]GTPγS binding, ERK1/2 phosphorylation and GIRK activation. Respective low and moderate partial agonist properties of NDMC at dopamine D(2) and serotonin 5-HT(1A) receptors thus differentiate the metabolite from its parent drug and may contribute to the overall effects of clozapine pharmacotherapy.     

Anti-cataleptic effects of clozapine, but not olanzapine and quetiapine, on SCH 23390- or raclopride-induced catalepsy in rats.

The present study investigated potential anti-cataleptic properties of the prototype atypical antipsychotic clozapine and two newly developed atypical antipsychotics, olanzapine and quetiapine, which are structurally related and display similar pharmacological profiles to clozapine. Clozapine (2.5 mg kg(-1), s.c.), but not olanzapine (2.0 mg kg(-1), s.c.) and quetiapine (20.0 mg kg(-1), s.c.), blocked catalepsy induced either by the dopamine D(1/5) receptor antagonist SCH 23390 (50.0 microg kg(-1), s.c) or the selective dopamine D(2/3) receptor antagonist raclopride (4.0 mg kg(-1), s.c.). Such findings are consistent with the beneficial effects of clozapine in the management of drug-induced psychosis in parkinsonian patients, and suggest that neither olanzapine nor quetiapine may be a safe alternative to clozapine in this field. Furthermore, the results indicate that clozapine has a unique pharmacological profile that distinguishes it from olanzapine and quetiapine. The mechanisms underlying anti-cataleptic or anti-parkinsonian properties of clozapine are unclear but may be related to dopamine D(1) receptor agonism of clozapine.