Loxapine and clozapine decrease serotonin (S2) but do not elevate dopamine (D2) receptor numbers in the rat brain

Chronic administration of loxapine or clozapine in rats for 4 weeks or 10 weeks did not produce enhancement of striatal dopamine receptor density. However, there was a marked reduction (50-60%) of cortical serotonin receptor density associated with clozapine or loxapine administration. Acute doses of clozapine or loxapine produced the same potent effect. The possibility that these two antipsychotic drugs act via the serotonin system in the brain is proposed.     

Antipsychotic drugs affect glucose uptake and the expression of glucose transporters in PC12 cells.

1. Adherence of the PC12 cell line to poly-l-lysine (PLL) on tissue culture dishes stimulated glucose transport into the cells. Fluphenazine, chlorpromazine, clozapine and haloperidol inhibited glucose uptake in this system after a short (30 min) preincubation with drug. The IC50's for this effect were typically in the range of 5-40 microM. 2. Following longer exposures of the drugs (24 hr), there was a significant increase (approximately 3-fold) in the cellular levels of the glucose transporter (GLUT) isoforms, GLUT1 and GLUT3. 3. Long-term incubation (48 hr), especially with the phenothiazine drugs, was accompanied by a marked reduction in cell growth and proliferation. The rank ordering of the potencies of the drugs was essentially the same for these various effects: fluphenazine > chlorpromazine > clozapine approximately haloperidol. 4. It is suggested that the effects on glucose transport reported here may complicate the interpretation of positron emission tomography (PET) studies that rely on the uptake of radiolabeled glucose analogs to measure the physiological response to these drugs.     

Uptake of clozapine into HL-60 promyelocytic leukaemia cells

The atypical antipsychotic, clozapine, exerts superior efficacy in therapy-resistant schizophrenia, but unfortunately induces agranulocytosis with an incidence of 0.8 - 1 %. In this study, we investigated the cellular uptake of clozapine into human promyelocytic leukaemia HL-60 cells using HPLC with electrochemical detection. On incubation with 1.25 to 40 microM clozapine for 30 min, a saturable, energy- and temperature-dependent uptake process takes place (K m = 18.8 microM, k cat = 1.36 nmol/5 min/mg protein at 37 degrees C). This suggests membrane passage of clozapine by a carrier mechanism. 10 microM Indatraline, an inhibitor of dopamine, noradrenaline and serotonin (5-HT) reuptake, but not the selective 5-HT reuptake inhibitor fluvoxamine, markedly reduced the transport of clozapine by 62 %, whereas addition of 10 mM glucose to the incubation medium increased intracellular clozapine concentrations by 28 %. Since cyclosporine A, vinblastine or verapamil up to a final concentration of 10 microM did not alter the intracellular accumulation of clozapine, an involvement of P-glycoprotein seems to be unlikely. In summary, clozapine uptake into HL-60 cells meets criteria of an active unidirectional transport. Its molecular correlates remain to be established.     

Bromocriptine and clozapine regulate dopamine 2 receptor gene expression in the mouse striatum

In a previous study, we showed that the psychoactive drug caffeine alters the expression of the dopamine 2 receptor (D2R) gene in vitro and in vivo. Here, we report that acute administration of antipsychotic and anti-parkinsonian drugs also regulate D2R gene expression in PC12 cells and in the mouse striatum. Treatment of PC12 cells with the atypical antipsychotic and specific 5-HT antagonist clozapine (60 microM) reduced D2R/luciferase reporter expression by 46% after 24 h. However, male and female mice treated with a clinical dose of clozapine (10 mg/kg) showed no changes in striatal D2R mRNA expression when assayed by quantitative RT-PCR. Treatment of PC12 cells with the specific D2R agonist anti-parkinsonian drug, bromocriptine mesylate (BCM; 5 microM) also resulted in decreased D2R/luciferase reporter activity (27%). In contrast to clozapine, a clinical dose of BCM (16 mg/kg) led to a 21% decrease and a 45% increase in striatal D2R mRNA expression in male and female mice, respectively, after 24 h. Coadministration of clozapine and BCM in PC12 cells resulted in a synergistic decrease in D2R/luciferase reporter expression (68%), and coadministration of these drugs in vivo led to decreases in striatal D2R mRNA expression in both male and female mice (45% and 22%, respectively). Collectively, these results indicate that clozapine, BCM, or a combination of these drugs have differential effects on dopamine receptor gene expression and might also affect striatal physiology in a sexually dimorphic manner.     

Differential effects of clozapine and haloperidol on ketamine-induced brain metabolic activation

Subanesthetic doses of N-methyl- -aspartate (NMDA) receptor antagonists such as ketamine and phencyclidine precipitate psychotic symptoms in schizophrenic patients. In addition, these drugs induce a constellation of behavioral effects in healthy individuals that resemble positive, negative, and cognitive symptoms of schizophrenia. Such findings have led to the hypothesis that decreases in function mediated by NMDA receptors may be a predisposing, or even causative, factor in schizophrenia. The present study examined the effects of the representative atypical (clozapine) and typical (haloperidol) antipsychotic drugs on ketamine- induced increases in -2-deoxyglucose (2-DG) uptake in the rat brain. As previously demonstrated, administration of subanesthetic doses of ketamine increased 2-DG uptake in specific brain regions, including medial prefrontal cortex, retrosplenial cortex, hippocampus, nucleus accumbens, basolateral amygdala, and anterior ventral thalamic nucleus. Pretreatment of rats with 5 or 10 mg/kg clozapine alone produced minimal or no change in 2-DG uptake, yet clozapine completely blocked ketamine-induced changes in 2-DG uptake in all brain regions studied. In striking contrast, a dose of haloperidol (0.5 mg/kg) that produces a substantial cataleptic response, potentiated, rather than blocked, ketamine-induced activation of 2-DG uptake. These results demonstrate, in a model with potential relevance to schizophrenia, a striking neurobiological difference between the actions of prototypical typical and atypical antipsychotic drugs. The dramatic blockade by clozapine of ketamine-induced brain metabolic activation suggests that antagonism of the consequences of reduced NMDA receptor function could contribute to the superior therapeutic effects of this atypical antipsychotic agent. The results also suggest that this model of ketamine-induced alterations in 2-DG uptake may be extremely useful for understanding the complex neural mechanisms of atypical antipsychotic drug action.     

Nonketotic hyperglycemia associated with loxapine and amoxapine: case report

A case of nonketotic hyperglycemic coma associated with the recent introduction of loxapine is presented. Drug discontinuation led to a return of normal fasting blood glucose. Later challenge with amoxapine was also associated with acute hyperglycemia. Their common metabolite, 7-OH amoxapine, is implicated.     

Inhibition of system A-mediated glycine transport in cortical synaptosomes by therapeutic concentrations of clozapine: implications for mechanisms of action

Clozapine is an atypical antipsychotic with particular efficacy in schizophrenia, possibly related to potentiation of brain N-methyl-D-aspartate receptor (NMDAR) -mediated neurotransmission. NMDARs are regulated in vivo by glycine, which is regulated in turn by glycine transporters. The present study investigates transport processes regulating glycine uptake into rat brain synaptosomes, along with effects of clozapine on synaptosomal glycine transport. Amino-acid uptake of amino acids was assessed in rat brain P2 synaptosomal preparations using a radiotransport assay. Synaptosomal glycine transport was inhibited by a series of amino acids and by the selective System A antagonist MeAIB (2-methyl-aminoisobutyric acid). Clozapine inhibited transport of both glycine and MeAIB, but not other amino acids, at concentrations associated with preferential clinical response (0.5-1 microg/ml). By contrast, other antipsychotics studied were ineffective. The novel glycine transport inhibitor N[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl]sarcosine (NFPS) produced biphasic inhibition of [(3)H]glycine transport, with IC(50) values of approximately 25 nM and 25 microM, respectively. NFPS inhibition of [(3)H]MeAIB was monophasic with a single IC(50) value of 31 microM. Clozapine significantly inhibited [(3)H]glycine binding even in the presence of 100 nM NFPS. In conclusion, this study suggests first that System A transporters, or a subset thereof, may play a critical role in regulation of synaptic glycine levels and by extension of NMDA receptor regulation, and second that System A antagonism may contribute to the differential clinical efficacy of clozapine compared with other typical or atypical antipsychotics.     

Comparative analysis of acute and chronic administration of haloperidol and clozapine using [3H] 2-deoxyglucose metabolic mapping

In an effort to compare and contrast the mechanisms of action of typical and atypical antipsychotic drugs, [3H] 2-deoxyglucose metabolic mapping was employed following acute and chronic administration of haloperidol (1 mg/kg i.p. acute and 0.5 mg/kg i.p. chronic) and clozapine (20 mg/kg i.p., both acute and chronic). Optical density ratios (ODR) were measured in 62 brain structures. An overall decrease in ODR was observed in many of the regions analyzed. Acute haloperidol elicited significant decreases, particularly in the thalamus and hippocampus. Acute clozapine decreased glucose uptake in the caudate putamen, hippocampus, central gray, locus coreleus, and the thalamus. In both chronically treated haloperidol and clozapine animals, significant decreases in ODR were seen in the thalamus and hippocampal areas most dramatically, with other changes in the superior colliculus, retrospenial cortex, and the cerebellum. Clozapine caused significant effects in 32 nuclei acutely and only 19 nuclei chronically. Haloperidol caused significant effects in 23 nuclei acutely and 15 nuclei chronically. The pattern of change induced by haloperidol and clozapine were remarkably similar when considering their pharmacology is somewhat different. Both antipsychotics elicited fewer significant changes upon chronic administration.     

Effects of typical and atypical antipsychotics on human glycine transporters

Augmentation strategy in the treatment of schizophrenia with the NMDA receptor co-agonist glycine has demonstrated significant improvement in patient symptoms. Interestingly, the therapeutic efficacy of glycine was more consistent among patients that were not co-administered clozapine suggesting that clozapine modulates glycine levels in brain. Since cerebral glycine concentration in the vicinity of NMDA receptors is thought to be controlled by the glia expressed glycine transporter type 1 (GlyT1), the effects of several typical and atypical antipsychotics on glycine uptake were examined in human placenta choriocarcinoma (JAR) cells expressing human GlyT1a. The selectivity of these compounds was investigated by measuring their inhibitory potency at the closely related glycine transporter type 2 (GlyT2). Typical antipsychotics haloperidol, thioridazine and chlorpromazine non-selectively inhibited [(14)C]glycine uptake mediated by GlyT1a and GlyT2 with potency of 9-21 microM. The atypical antipsychotic, clozapine antagonized glycine transport by human GlyT1a with an IC(50) of 100 microM and was weaker at recombinant GlyT2. Its main metabolites, N-desmethylclozapine and clozapine N-oxide were very weak inhibitors at all glycine transporters. Similarly, olanzapine did not potently block GlyT1a- and GlyT2-mediated uptake. Detailed kinetic analysis of hGlyT1a in the presence and absence of haloperidol and clozapine revealed that both drugs were not competitive inhibitors of glycine uptake. Data also indicated that these compounds did not interact with the Na(+) and Cl(-) sites of hGlyT1a. Our results have revealed the existence of an inhibitory interaction between some antipsychotics and hGlyT1a and raise the possibility that these drugs could interact with GlyT1 function at therapeutic doses.     

Cytotoxicity of conventional and atypical antipsychotic drugs in relation to glucose metabolism

The goal of these studies was to analyze the cytotoxicity of both the conventional and atypical antipsychotic drugs in relation to their effects on glucose metabolism. The drugs were evaluated for their effects on the viability of PC12 cells, which are an established model of neuronal cells in culture. In general, the conventional drugs, such as chlorpromazine, fluphenazine and pimozide, were more toxic than the atypical drugs, including clozapine, quetiapine and risperidone. Olanzapine was unique in that it stimulated cell proliferation in this system. There was a good correlation between the cytotoxicity of a drug and its ability to block glucose transport, although there were some exceptions to this trend. Conventional antipsychotics also affected the expression of glucose transporter proteins in whole cell extracts and at the cell surface. Overall, the data support the notion that many of the antipsychotic drugs associated with the development of movement disorders in patients are cytotoxic for cultured cells.     

Second-generation antipsychotic drugs, olanzapine, quetiapine, and clozapine enhance neurite outgrowth in PC12 cells via P13K/AKT, ERK, and pertussis toxin-sensitive pathways

Second-generation antipsychotic drugs, olanzapine, quetiapine, and clozapine, were found to enhance neurite outgrowth induced by nerve growth factor (NGF) in PC12 cells. These drugs increased the number of cells bearing neurites, the length of primary neurites, and the size of the cell body of NGF-differentiated PC12 cells. In addition, the drugs induced sprouting of neurite-like processes in PC12 cells in the absence of NGF. Olanzapine, quetiapine, and clozapine enhanced the phosphorylation of Akt and ERK in combination with NGF, and specific inhibitors of these pathways attenuated these effects. Pretreatment of cells overnight with pertussis toxin had no effect on NGF-induced differentiation but significantly decreased the effects of the antipsychotic drugs on neurite outgrowth, suggesting that Gi/Go-coupled receptors are involved in the response to drug. A better understanding of the mechanisms underlying the effects of the second-generation drugs might suggest new therapeutic targets for enhancement of neurite outgrowth.     

Glucose transport in primary cultured neurons

In this study, we have examined glucose uptake and its regulation by insulin in primary cultured neurons. Glucose transport was assessed by measuring the initial rate of uptake of 3H-2-deoxyglucose, a glucose analog that is transported and phosphorylated but not further metabolized. The uptake of 2-deoxyglucose was saturable; measurements of the intracellular concentration of 2-deoxyglucose and 2-deoxyglucose-6-phosphate revealed that hexokinase activity rather than membrane transport is the rate-limiting step for glucose uptake. Insulin had no effect on 2-deoxyglucose uptake at low (0.2 mM) or high (20 mM) concentrations of substrate. The order of potency of other hexoses to competitively inhibit the accumulation of 2-deoxyglucose was D-glucose (0.2 mM) = D-mannose (0.2 mM) greater than 3-0-methylglucose (9 mM) greater than D-galactose (90 mM). Cytochalasin B was a potent inhibitor of 2-deoxyglucose uptake (IC50 = 500 nM) and phloretin was more potent than ploridzin in inhibiting uptake. The structure of glucose transporters was examined by photoaffinity labeling using 3H-cytochalasin B and by immunologic detection using antibodies raised against the human erythrocyte transporter. 3H-cytochalasin B labeled two proteins of 55 kDa and 43 kDa and the antibody recognized primarily a 43 kDa protein. The subcellular distribution of glucose transporters, estimated by measuring the number of specific cytochalasin B binding sites in subfractions of neuronal homogenates, showed 3.62 pmol/mg protein in the 11,000g pellet and 1.34 pmol/mg protein in the 200,000g pellet. In conclusion: 1) Neuronal glucose transport is not acutely regulated by insulin. 2) The kinetics of 2-deoxyglucose uptake into neurons are determined largely by hexokinase activity rather than membrane transport. 3) The apparent molecular weight of neuronal glucose transporters is similar to transporters in other tissues. 4) The number of glucose transporters per milligram of protein is relatively low in neurons compared to other tissues.     

Second-generation antipsychotic drugs, olanzapine, quetiapine, and clozapine enhance neurite outgrowth in PC12 cells via P13K/AKT, ERK, and pertussis toxin-sensitive pathways

Second-generation antipsychotic drugs, olanzapine, quetiapine, and clozapine, were found to enhance neurite outgrowth induced by nerve growth factor (NGF) in PC12 cells. These drugs increased the number of cells bearing neurites, the length of primary neurites, and the size of the cell body of NGF-differentiated PC12 cells. In addition, the drugs induced sprouting of neurite-like processes in PC12 cells in the absence of NGF. Olanzapine, quetiapine, and clozapine enhanced the phosphorylation of Akt and ERK in combination with NGF, and specific inhibitors of these pathways attenuated these effects. Pretreatment of cells overnight with pertussis toxin had no effect on NGF-induced differentiation but significantly decreased the effects of the antipsychotic drugs on neurite outgrowth, suggesting that G_i/G_o-coupled receptors are involved in the response to drug. A better understanding of the mechanisms underlying the effects of the second-generation drugs might suggest new therapeutic targets for enhancement of neurite outgrowth.     

A neurochemical basis for the antipsychotic activity of loxapine: interactions with dopamine D1, D2, D4 and serotonin 5-HT2 receptor subtypes.

Loxapine is a typical neuroleptic that shows great structural and functional homology to the atypical antipsychotic clozapine. Chronic loxapine treatment is usually associated with extrapyramidal symptoms (EPS), whereas clozapine treatment is not. Conversely, loxapine does not produce the agranulocytosis that often results from protracted clozapine treatment. Earlier studies of loxapine have usually implicated D2 receptor blockade as the cause of the tardive dyskinesia that occurs with chronic treatment. More recently, loxapine's ability to potentiate serotonergic neurotransmission has also been implicated. In this study, the pharmacological affinities of loxapine for the dopamine D1, D2, D4, as well as serotonin-2 (5-HT2) and NMDA receptor subtypes, were investigated through direct radioreceptor assays. The findings indicate that loxapine displays an extremely strong binding affinity for dopamine D4 and serotonin 5-HT2 receptors, which suggests that both serotonergic and dopaminergic mechanisms contribute to the antipsychotic drug action and EPS associated with loxapine in the treatment of schizophrenia.     

The ability of atypical antipsychotic drugs vs. haloperidol to protect PC12 cells against MPP+-induced apoptosis

The present study examined the effects of the atypical antipsychotic drugs clozapine, olanzapine, quetiapine and risperidone, on N-methyl-4-phenylpyridinium ion-induced apoptosis and DNA damage in PC12 cells, and explored the molecular mechanisms underlying these effects. Haloperidol, a typical antipsychotic drug, was used for comparison. Exposure of PC12 cells to 50 micro m N-methyl-4-phenylpyridinium ion for 24 h resulted in a 35-45% loss of cells in culture. Pretreatment with the aforementioned atypical antipsychotic drugs significantly reduced the N-methyl-4-phenylpyridinium ion-induced cell loss, whereas haloperidol (10-100 micro m) did not have this protective effect. Hoechst 33258 staining revealed the apoptotic nuclear features of the N-methyl-4-phenylpyridinium ion-induced cell death, and showed that the atypical antipsychotic drugs, but not haloperidol, effectively prevented PC12 cells from this N-methyl-4-phenylpyridinium ion-induced apoptosis. DNA fragmentation assays further confirmed the N-methyl-4-phenylpyridinium ion-induced nuclear fragmentation. Pretreatment with the atypical antipsychotic drugs completely prevented this nuclear fragmentation, whereas haloperidol only partially prevented it. In vitro oligonucleotide assays indicated an activation of a specific glycosylase that recognizes and cleaves bases (at the 8-hydroxyl-2-deoxyguanine site) that were damaged by N-methyl-4-phenylpyridinium ion. Pretreatment with the atypical antipsychotic drugs more effectively attenuated this N-methyl-4-phenylpyridinium ion-induced activation than did haloperidol. Northern blot analyses showed that the atypical antipsychotic drugs, but not haloperidol, blocked the N-methyl-4-phenylpyridinium ion-induced substantial increase of copper/zinc superoxide dismutase mRNA in PC12 cells. Atypical antipsychotic drugs slightly up-regulated the expression of copper/zinc superoxide dismutase mRNA, whereas haloperidol strongly increased the expression of copper/zinc superoxide dismutase mRNA. These data may account for the different therapeutic effects and side-effect profiles of typical and atypical antipsychotic drugs in schizophrenia.     

Transport of 2-deoxy-d-glucose by dissociated brain cells

The characteristics of glucose transport into dissociated cells from rat brain were determined using [1,2-³H]2-deoxyglucose as substrate. The rate of net uptake exhibited biphasic saturation kinetics with increasing substrate concentration; two values each for K_m (8.85 and 1.05 mM) and V_max (20.41 ± 5.99nmol/min/mg protein) were obtained, indicating the presence of two transport systems. d-glucose competed with [1,2-³H]2-deoxyglucose as shown by increasing degrees of inhibition of uptake of labeled substrate with increasing concentrations of d-glucose. The presence of an accelerative exchange mechanism was demonstrated by enhanced rates of uptake of labeled substrate by cells pre-loaded with high levels of unlabeled 2-deoxyglucose. Transport was inhibited by cytochalasin B, phloretin and phloridzin in a manner suggesting that the system is sodium-independent. Transport was also inhibited by sodium cyanide, potassium cyanide and dinitrophenol, but not by sodium arsenite or ouabain. Insulin status of the animals had no effect on the rate of transport of this substrate. Net transport was significantly lower in neonatal (4-day-old) rats than in either older sucklings (14–16-day-old) or adult animals; no significant difference between the latter two groups was observed. These findings demonstrate that two carrier-mediated systems for glucose transport are present on the membranes of these mixed brain cells suggesting that the kinetic characteristics of glucose transport may differ between neurons and glial cells. The age change in transport rate may reflect age-associated glial cell proliferation and/or an age-dependent increase in the number of transporters per cell in one brain cell type.     

Tolerability and therapeutic effect of clozapine. A retrospective investigation of 216 patients treated with clozapine for up to 12 years

Two hundred and sixteen psychiatric patients (183 men and 33 women) hospitalized in Sct. Hans Hospital were treated with clozapine between 1971-1983. All had been treated previously with one or more neuroleptic(s) and had either failed to respond adequately, or their response was limited by side effects. Eighty-five patients were treated exclusively with clozapine, while the remaining 131 received additional medication, mainly other neuroleptic drugs. The mean clozapine dosage was 317 mg/day (range 50-1200), and the mean duration of treatment was 23/4 years (range 1/12-12). The tolerability to clozapine was determined by an evaluation of haematological changes, pronounced side effects and mortality. One patient treated with clozapine (8 months) and nitrofurantoin (8 days) developed a reversible granulocytopenia. One patient (treated with a combination of drugs) had clinically insignificant depression of the leucocytes and three of segmented granulocytes. Seven had a reduction in thrombocytes. Two patients developed cardiac insufficiency, and four epileptic seizures. None of the patients treated exclusively with clozapine developed neurological side effects. A global estimation of therapeutic effect revealed that clozapine alone or in combination with other neuroleptic drugs was significantly better than previous antipsychotic therapy, although 47-63% of the patients showed no change. It is concluded that clozapine is a potent antipsychotic drug offering particular advantages in the treatment of schizophrenic patients with a pronounced symptomatology and tendency towards developing extrapyramidal side effects. Caution is advised in patients with cardiac insufficiency and epilepsy. There appears to be a slight risk of granulocytopenia, and therefore the present monitoring of WBC should continue in order to prevent this reaction and to obtain more complete information regarding risk of granulocytopenia.     

Effects of amoxapine on serotonin uptake in human blood platelets of depressed patients and normal controls

The effect of amoxapine and imipramine on the serotonin (5-HT) uptake of blood platelets from depressed patients and normal controls was studied ex vivo or in vitro, respectively. Amoxapine was approximately one-tenth as potent as imipramine in inhibiting 5-HT uptake in blood platelets from normal controls in vitro. Both drugs inhibited 5-HT uptake in a competitive manner. However, ex vivo studies demonstrated that imipramine produced a mixed inhibition and amoxapine, a competitive inhibition of 5-HT uptake. There was no relationship between the change in the platelet affinity for 5-HT after treatment with amoxapine and clinical response to amoxapine.     

The expression of glutamate transporter GLT-1 in the rat cerebral cortex is down-regulated by the antipsychotic drug clozapine.

We show here that clozapine, a beneficial antipsychotic, down-regulates the expression of the glutamate transporter GLT-1 in the rat cerebral cortex, thereby reducing glutamate transport and raising extracellular glutamate levels. Clozapine treatment (25--35 mg kg(-1) day(-1) orally) reduced GLT-1 immunoreactivity in several brain regions after 3 weeks; this effect was most prominent after 9 weeks and most evident in the frontal cortex. GLT-1 protein levels were reduced in the cerebral cortex of treated rats compared with controls and were more severely affected in the anterior (71.9 +/- 4.5%) than in the posterior (53.2 +/- 15.4%) cortex. L-[(3)H]-glutamate uptake in Xenopus laevis oocytes injected with mRNA extracted from the anterior cerebral cortex of rats treated for 9 weeks was remarkably reduced (to 30.6 +/- 8.6%) as compared to controls. In addition, electrophysiological recordings from oocytes following application of glutamate revealed a strong reduction in glutamate uptake currents (46.3 +/- 10.2%) as compared to controls. Finally, clozapine treatment led to increases in both the mean basal (8.1 +/- 0.7 microM) and the KCl-evoked (28.7 +/- 7.7 microM) output of glutamate that were 3.1 and 3.5, respectively, higher than in control rats. These findings indicate that clozapine may potentiate glutamatergic synaptic transmission by regulating glutamate transport.