Clozapine Exposure Research Articles

T50 - Longitudinal Epigenetic Analysis Of Clozapine Use In Treatment-Resistant Schizophrenia: Data From The Crestar Consortium

Background Approximately one-third of patients with schizophrenia are considered treatment-resistant. For these patients, the atypical antipsychotic drug clozapine is recommended as the only evidence based treatment available. However, there is still significant variability in treatment-response. Animal studies have demonstrated that clozapine influences histone modification and DNA methylation, and a recent EWAS study in humans identified multiple differentially methylated positions (DMPs) and differentially methylated regions (DMRs) in clozapine-exposed samples. We used a longitudinal, within-participant design to conduct genome-wide analysis of DNA methylation changes in treatment resistant patients over 6 months of clozapine use. Methods We recruited 20 participants with a diagnosis of treatment-resistant schizophrenia, before they were prescribed clozapine. We then collected whole-blood samples at baseline and follow-up (6 weeks, 12 weeks and 6 months after clozapine start date), alongside clinical assessments. We quantified DNA methylation at ~ 480,000 sites across the genome using the Illumina 450 K HumanMethylation array and following pre-processing, normalization and quality control, an epigenome-wide association study was performed comparing DNA methylation at each time point. Results Preliminary data demonstrates an overall reduction in DNA methylation in the 6 months of clozapine use, with multiple individual CpG sites showing changes in DNA methylation that were found to be significantly associated with length of time exposed to clozapine. During analysis of the first 10 patients, the most significant CpG site was located in the gene body of CRB1; CRB1 is expressed exclusively in the eye, and the central nervous system, and has been previously associated with Lever’s congenital amaurosis and retinitis pigmentosa. The findings and analysis for all 20 participants will be presented at the meeting. Discussion This is the first study to identify longitudinal epigenetic changes following clozapine exposure in human subjects, replicating findings in animal studies of decreased methylation. Recruitment is ongoing and further analysis will look at whether epigenetic changes are associated with treatment-response/adverse reactions. Ultimately, these data will help us understand the mechanisms involved in clozapine, potentially providing biomarkers to predict clozapine response.

Clozapine-induced agranulocytosis: Evidence for an immune-mediated mechanism from a patient-specific in-vitro approach

Use of the atypical antipsychotic clozapine (CZP) is compromised by the risk of potentially fatal agranulocytosis/granulocytopenia (CIAG). To address this, we have established a simple, personalized cell culture-based strategy to identify CIAG-susceptible patients, hypothesizing that an immunogenic and possibly haptene-based mechanism underlies CIAG pathophysiology.To detect a putative haptene-induced response to CZP in vitro exposure, a traditional lymphocyte stimulation assay was adapted and applied to patient-specific peripheral blood-derived mononuclear cells (PBMC). 6 patients with a history of CIAG, 6 patients under CZP treatment (without CIAG) and 12 matched healthy controls were studied.In vitro CZP exposure, even at strikingly low levels, resulted in significantly increased proliferation rates only in CIAG patients' PBMC. Other parameters including cell viability and mitogen-induced proliferation were also affected by in vitro CZP exposure, yet there was no significant difference between the groups.This personalized approach is a starting point for further investigations into a putative haptene-based mechanism underlying CIAG development, and may facilitate the future development of predictive testing.

Aspiration pneumonia associated with clozapine

Clozapine is effective in managing patients with treatment‐resistant schizophrenia. Clinicians who use this drug are generally well aware of the need to monitor patients for infrequent but well‐recognized and potentially life‐threatening adverse effects, such as agranulocytosis. Clozapine also has more common adverse effects that are generally considered to entail less medical risk, such as sialorrhea (excessive salivation). Some reports suggest a risk of aspiration pneumonia related to clozapine‐induced sialorrhea.1,2,3 A clinical report recently described an occurrence of aspiration pneumonia as a result of clozapine exposure.4

NeuN+ neuronal nuclei in non-human primate prefrontal cortex and subcortical white matter after clozapine exposure

Increased neuronal densities in subcortical white matter have been reported for some cases with schizophrenia. The underlying cellular and molecular mechanisms remain unresolved.We exposed 26 young adult macaque monkeys for 6months to either clozapine, haloperidol or placebo and measured by structural MRI frontal gray and white matter volumes before and after treatment, followed by observer-independent, flow-cytometry-based quantification of neuronal and non-neuronal nuclei and molecular fingerprinting of cell-type specific transcripts.After clozapine exposure, the proportion of nuclei expressing the neuronal marker NeuN increased by approximately 50% in subcortical white matter, in conjunction with a more subtle and non-significant increase in overlying gray matter. Numbers and proportions of nuclei expressing the oligodendrocyte lineage marker, OLIG2, and cell-type specific RNA expression patterns, were maintained after antipsychotic drug exposure. Frontal lobe gray and white matter volumes remained indistinguishable between antipsychotic-drug-exposed and control groups.Chronic clozapine exposure increases the proportion of NeuN+ nuclei in frontal subcortical white matter, without alterations in frontal lobe volumes or cell type-specific gene expression. Further exploration of neurochemical plasticity in non-human primate brain exposed to antipsychotic drugs is warranted.

Genome-wide association study of lymphoblast cell viability after clozapine exposure.

Clozapine is an antipsychotic drug with proven efficacy in treatment-resistant schizophrenia but also known to induce potentially lethal agranulocytosis (CIA) in 1% of patients. Genetic factors are likely to play a role in the molecular basis of CIA. We explored an in vitro system to study the genetic susceptibility of CIA. Cell viability was measured in 90 lymphoblast cell lines exposed to a series of increasing concentrations of clozapine for 48 hr. Quantitative trait measures of cell viability as well as area under the survival curve were used in a linear mixed model for genome-wide association analyses. The estimated heritability of clozapine-induced cell viability reduction in these cell lines is h2=0.76. No genome-wide significant association was observed after correction for multiple testing. Two independent loci with nominal evidence of association were observed at 30× clinical clozapine concentration: rs2709505 (P=1.41×10(-8)) in an intron of MDFIC and rs10457252 (P=1.79×10(-8)) located in a gene desert at chromosome 6q21. We identified one locus (rs1293970) near PRG4 that was consistently associated for all separate concentration analyses at P<5×10(-5). PRG4 encodes hemangiopoietin, a growth stimulator for hematopoietic stem cells. No evidence was observed for involvement of the MHC region. Our results demonstrate that clozapine-induced viability reduction in lymphoblast cell lines is a heritable, polygenic trait. Thus, in vitro models of CIA might be a useful tool for future discovery of genetic risk factors, although larger sample sizes will be required to unambiguously identify these loci.

Mean platelet volume in schizophrenia unaltered after 1 year of clozapine exposure

This study investigated the MPV of patients with major psychoses before and after 1year of clozapine exposure. Data were obtained from chart reviews of patients who were initiated on clozapine at the Centre of Addiction and Mental Health (CAMH) in Toronto. 100 patients were eligible for this study; 65 remained on clozapine after 1year. Prior to clozapine initiation, MPV was 10.79±0.91fL and there was no difference in MPV after 1year of clozapine exposure (p=0.777). We found high MPV in patients with schizophrenia and related psychoses, which was unaltered after 1year of clozapine treatment.

Adult response to olanzapine or clozapine treatment is altered by adolescent antipsychotic exposure: A preclinical test in the phencyclidine hyperlocomotion model

This study examined how repeated olanzapine (OLZ) or clozapine (CLZ) treatment in adolescence alters sensitivity to the same drug in adulthood in the phencyclidine (PCP) hyperlocomotion model. Male adolescent Sprague-Dawley rats (postnatal day (P) 44-48) were first treated with OLZ (1.0 or 2.0 mg/kg, subcutaneously (sc)) or CLZ (10.0 or 20.0 mg/kg, sc) and tested in the PCP (3.2 mg/kg, sc)-induced hyperlocomotion model for five consecutive days. Then a challenge test with OLZ (0.5 mg/kg) or CLZ (5.0 mg/kg) was administered either during adolescence (~P 51) or after the rats matured into adults (~P 76 and 91). During adolescence, repeated OLZ or CLZ treatment produced a persistent inhibition of PCP-induced hyperlocomotion across the five test days. In the challenge test during adolescence, rats previously treated with OLZ did not show a significantly stronger inhibition of PCP-induced hyperlocomotion than those previously treated with vehicle (VEH). In contrast, those previously treated with CLZ showed a weaker inhibition than the VEH controls. When assessed in adulthood, the enhanced sensitivity to OLZ and the decreased sensitivity to CLZ were detected on ~P 76, even on ~P 91 in the case of OLZ. These findings suggest that adolescent OLZ or CLZ exposure can induce long-term alterations in antipsychotic response that persist into adulthood.

Clozapine impairs insulin action by up‐regulating AKT phosphorylation and Ped/Pea‐15 protein abundance

Clinical and experimental evidence indicates that atypical antipsychotics impair glucose metabolism. We investigated whether clozapine may directly affect insulin action by analyzing insulin signaling in vitro and in vivo. Clozapine reduced insulin-stimulated glucose uptake in PC12 and in L6 cells, representative models of neuron and skeletal muscle, respectively. Consistently, clozapine reduced insulin effect on insulin receptor (IR) by 40% and on IR substrate-1 (IRS1) tyrosine phosphorylation by 60%. Insulin-stimulated Akt phosphorylation was also reduced by about 40%. Moreover, insulin-dependent phosphorylation of protein kinase C-ζ (PKC-ζ) was completely blunted in clozapine-treated cells. Interestingly, clozapine treatment was accompanied by an insulin-independent increase of Akt phosphorylation, with no change of IR, IRS1, and PKC-ζ basal phosphorylation. The cellular abundance of Ped/Pea-15, an Akt substrate and inducer of insulin resistance, was also increased following clozapine exposure, both in the absence and in the presence of cyclohexymide, a protein synthesis inhibitor. Similar as in cellular models, in the caudate–putamen and in the tibialis muscle of clozapine-treated C57/BL/KsJ mice, Akt phosphorylation and Ped/Pea-15 protein levels were increased and PKC-ζ phosphorylation was decreased. Thus, in these experimental models, clozapine deranged Akt function and up-regulated Ped/Pea-15, thereby inhibiting insulin stimulation of PKC-ζ and of glucose uptake. J. Cell. Physiol. 227: 1485–1492, 2012. © 2011 Wiley Periodicals, Inc.

Antipsychotics During Pregnancy

Knowledge about the effects of exposure to the newer antipsychotics during pregnancy is limited. To investigate the effects of maternal use of antipsychotics during pregnancy on gestational diabetes and fetal growth. Population-based cohort study comparing women exposed and not exposed to antipsychotics during pregnancy. Exposure was defined as prescriptions filled. Swedish national health registers. All women giving birth in Sweden from July 1, 2005, through December 31, 2009, grouped by filled prescriptions for (1) olanzapine and/or clozapine, the most obesogenic and diabetogenic antipsychotics (n = 169), (2) other antipsychotics (n = 338), or (3) no antipsychotics (n = 357,696). Odds ratios (ORs) with 95% CIs for gestational diabetes and being small for gestational age (SGA) and large for gestational age for birth weight, birth length, and head circumference. Exposure to other antipsychotics was associated with an increased risk of gestational diabetes (adjusted OR, 1.77 [95% CI, 1.04-3.03]). The risk increase with olanzapine and/or clozapine was of similar magnitude but not statistical significance (adjusted OR, 1.94 [95% CI, 0.97-3.91]). Infants exposed to either group of antipsychotics had increased risks of being SGA on birth weight, whereas only exposure to other antipsychotics yielded increased risks of being SGA for birth length and head circumference. None of the risks for SGA measurements remained significant after adjusting for maternal factors. There were no increased risks of being large for gestational age for birth weight or birth length after exposure to olanzapine and/or clozapine, but the risk increased for head circumference (OR, 3.02 [95% CI, 1.60-5.71]). Women who used antipsychotics during pregnancy had increased risks of gestational diabetes. The increased risks of giving birth to an SGA infant seemed to be an effect of confounders, such as smoking. Except for macrocephaly, olanzapine and/or clozapine exposure was not associated with anabolic fetal growth.

Electroencephalographic Abnormalities in Clozapine-Treated Patients: A Cross-Sectional Study

The objective of our study was to examine the electroencephalogram (EEG) abnormalities associated with clozapine treatment. It was a cross-sectional study on 87 psychiatric patients on clozapine treatment. 32 channel digital EEG was recorded and analysed visually for abnormalities. EEG abnormalities were observed in 63.2% of patients. Both slowing and epileptiform activities were noted in 41.4% of patients. The EEG abnormalities were not associated with dose or duration of clozapine exposure.

An Archival, Follow-Forward Exploration of the Metabolic Syndrome in Randomly Selected, Clozapine-Treated Patients

Objectives: Cross-sectional studies indicate that clozapine is associated with unusually high rates of the metabolic syndrome (MetS) in schizophrenia. These studies cannot address the extent to which schizophrenia or other factors are major risks for the MetS, independent of clozapine exposure. The objectives of this study were to longitudinally examine metabolic risk factors before and after clozapine initiation: 1) to determine MetS prevalence rates during firstgeneration antipsychotic (FGA) and clozapine treatment; 2) to identify metabolic changes contributing to the MetS; and, 3) to evaluate the extent to which prior treatment and subject variables contributed to increased MetS prevalence rates. Methods: Using an archival, follow-forward design, metabolic risk factors were sampled on a quarterly basis from medical records of twenty-five randomly selected inpatients. The sampling period was six years (three years of FGA and three years of clozapine). All subjects had been treated only with FGAs prior to clozapine exposure. Results: During clozapine treatment 16 of 25 (64%) subjects met MetS criteria; however, half (8 of 16) of the subjects already met MetS criteria during FGA treatment. Increased MetS prevalence with clozapine resulted from increases in fasting glucose and triglyceride levels and increased systolic BP. BMI was stable over time. Gender, age of clozapine initiation, and clozapine dose and duration may have contributed to long-term MetS risk. Conclusions: Clozapine-treated patients are at increased risk for the MetS. When observed longitudinally, however, it is clear that a significant proportion of the metabolic risk involves factors other than clozapine exposure alone.

Clozapine Exposure and the Impact of Smoking and Gender: A Population Pharmacokinetic Study

The primary objective of this study was to evaluate the magnitude and variability of concentration exposure to clozapine and norclozapine in a real-world clinical setting, with a focus on smoking status, using population pharmacokinetic methodologies. A retrospective review of plasma clozapine and norclozapine concentrations taken from inpatients at the Centre for Addiction and Mental Health, Toronto, from 2001 to 2007 was conducted. A nonlinear mixed-effects model was developed using NONMEM, including age, gender, weight, smoking status, and dosage formulation as covariates. Pharmacokinetic parameters and interindividual and residual variabilities were estimated with 1- and 2-compartment models. A total of 519 plasma clozapine concentrations from 197 patients (138 males; mean +/- SD age, 38 +/- 13 years; schizophrenia spectrum disorder 98.2%) were included for the analysis. A 1-compartment model with first-order absorption and elimination best described the data. Apparent volume of distribution was fixed to a previously reported value in the literature of 7 L/kg.The population-predicted oral clearance of clozapine and norclozapine was 18.0 and 39.0 L/h, respectively; both the predicted clearance values vary nearly 6-fold (range, 9.18-59.06 and 16.29-97.84 L/h, respectively). For clozapine, smokers and males showed increased oral clearance by 6.0 and 4.5 L/h, respectively. For norclozapine, smokers and male gender were associated with an increased oral clearance of 11.3 and 7.6 L/h, respectively. The formulation of clozapine administered had an impact on the absorption rate with a Ka of 0.14/h for tablet and 10.3/h for the suspension form.The data suggest that smoking and male gender are associated with lower exposure to clozapine and norclozapine due to the higher oral clearance. These findings may account for some of the variability in clozapine exposure and have important implications for individualized drug dosing and therapeutic drug monitoring.

Add-On Filgrastim During Clozapine Rechallenge in Patients With a History of Clozapine-Related Granulocytopenia/Agranulocytosis

Back to table of contents Previous article Next article Letters to the EditorFull AccessAdd-On Filgrastim During Clozapine Rechallenge in Patients With a History of Clozapine-Related Granulocytopenia/AgranulocytosisGRIGORI JOFFE M.D., Ph.D.,SAANA ESKELINEN M.D.EILA SAILAS M.D.,GRIGORI JOFFE M.D., Ph.D.Search for more papers by this author,SAANA ESKELINEN M.D.Search for more papers by this authorEILA SAILAS M.D.Search for more papers by this author,Published Online:1 Feb 2009https://doi.org/10.1176/appi.ajp.2008.08081295AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InEmail To the Editor: Clozapine treatment is often discontinued as a result of clozapine-related granulocytopenia/agranulocytosis, even among patients who are resistant to other antipsychotics. Moreover, clozapine re-exposure is sometimes undertaken among patients with a history of clozapine-related granulocytopenia/agranulocytosis whose previous good response to the drug cannot be maintained with other treatments. Prior to a recent report of the failure to prevent clozapine-related granulocytopenia/agranulocytosis with concomitant granulocyte colony stimulating factor filgrastim (1) , we used a clozapine-filgrastim combination to treat five patients with a history of clozapine-related granulocytopenia/agranulocytosis. Our attempts were encouraged by several earlier successful case reports (2) . We examined five schizophrenia patients who were resistant to numerous antipsychotic drugs (demographic and clinical characteristics of these patients are presented in a supplemental table that accompanies the online version of this letter). During previous clozapine exposure, these patients achieved a clinical response rating (physician’s global impression based on patient records) of at least “fair.” In all cases, treatment with clozapine had to be discontinued as a result of clozapine-related granulocytopenia/agranulocytosis. One patient (patient 2) also developed sepsis, which was successfully treated.Despite usual treatment, severe and prolonged psychotic states re-emerged in the patients. Consequently, a clinical decision for the use of clozapine rechallenge with concomitant filgrastim was made following a thorough risk-benefit consideration. After complete description of the possible risks was given, oral informed consent was obtained from each patient. In four patients, clozapine (step-wise increase) and filgrastim (dosage was according to clinical judgment, with no standard schedule applied) were started simultaneously, and in one patient, filgrastim was added to clozapine 2 weeks after reonset of clozapine-related granulocytopenia/agranulocytosis.For each patient, a clinical response of at least “fair” was reachieved. However, four patients redeveloped clozapine-related granulocytopenia/agranulocytosis. Among these, clozapine treatment was discontinued for two patients whose granulocyte count dropped below the critical <1xE/l level. At the time of the present report, clozapine treatment was continued for the three remaining patients, despite the reoccurrence of noncritical clozapine-related granulocytopenia/agranulocytosis in two of these patients.The patient (who had sepsis at first clozapine exposure) whose clozapine treatment was discontinued developed full-blown agranulocytosis, with peritonsillitis, epiglottitis, and sepsis. The condition was resolved later, with no sequelae.As a result of our small data size, we were unable to identify a clear-cut relationship between filgrastim dosing and a clozapine-related granulocytopenia/ agranulocytosis protecting effect. However, both patients who received a filgrastim dose of >0.3 mg/week were able to continue clozapine treatment, despite re-emergence of clozapine-related granulocytopenia/agranulocytosis, and one patient who received a dose of 0.3 mg/week developed agranulocytosis and sepsis.It has been previously suggested that severe clozapine-induced agranulocytosis is caused by an immunoallergic idiosyncratic mechanism that is unrelated to a granulocyte colony stimulating factor, while mild granulocytopenia results from prolonged clozapine exposure and decreased granulocyte production by hypoplastic bone marrow.We feel that, despite an add-on filgrastim, clozapine rechallenge is potentially dangerous, at least for patients who have a history of severe clozapine-related agranulocytosis and sepsis. In the future, if clinicians decide to use the clozapine-filgrastim combination, it is important that they seek to publish their cases, since a greater number of cases in the literature may enable the detection of plausible risk factors.Helsinki, FinlandKellokoski, FinlandThe authors report no competing interests.This letter (doi: 10.1176/appi.ajp.2008.08081295) was accepted for publication in November 2008.

Acute clozapine exposure in vivo induces lipid accumulation and marked sequential changes in the expression of SREBP, PPAR, and LXR target genes in rat liver

Several antipsychotic drugs (APDs) have high propensity to induce weight gain and dyslipidemia in patients, with clozapine and olanzapine as the most potent drugs. These lipid-related effects have been attributed to drug-mediated blockade or antagonism of histamine H1 and serotonin 5-HT2 receptors as well as activation of hypothalamic AMP-activated protein kinase. We recently showed that APDs activate lipid biosynthesis in cultured liver cells through stimulation of the sterol regulatory element-binding protein (SREBP) transcription factors. The objective of the study was to search for clozapine-related lipogenic effects in peripheral tissues in vivo using rat liver as target organ. Adult female Sprague-Dawley rats were administered single intraperitoneal injections of clozapine (25 and 50 mg/kg). Hepatic lipid levels were measured during a 48-h time course. Real-time quantitative PCR was used to analyze expression of genes involved in lipid biosynthesis, oxidation, efflux, and lipolysis. We identified an initial up-regulation of central lipogenic SREBP target genes, followed by a marked and sustained down-regulation. We also observed a sequential transcriptional response for fatty acid beta-oxidation and cholesterol efflux genes, normally controlled by the peroxisome proliferator activated receptor alpha and liver X receptor alpha transcription factors, and also down-regulation of genes encoding major lipases. The transcriptional responses were associated with a significant accumulation of triacylglycerol, phospholipids, and cholesterol in the liver. These results demonstrate that acute clozapine exposure affects SREBP-regulated lipid biosynthesis as well as other lipid homeostasis pathways. We suggest that such drug-induced effects on lipid metabolism in peripheral tissues are relevant for the metabolic adverse effects associated with clozapine and possibly other APDs.

Case report: clozapine given in the context of chemotherapy for lung cancer

Despite the frequent use of the antipsychotic medication, clozapine, in chronic treatments of psychiatric patients, there is limited clinical evidence available to guide clinicians in the problematic situation of a chemotherapy-induced blood dyscrasia. To perform a literature review and add a case report to the available clinical evidence. We gathered evidence through literature searches on Medline and with the assistance of a medical information specialist from Novartis who searched their internal database. We also report the case of a patient maintained on clozapine treatment despite full-dose chemotherapy (cisplatin and etoposide) for an extensive lung cancer. The searches returned seven clinically relevant references. These references do not establish a synergistic effect of clozapine and chemotherapy on blood counts. However, it has been shown that clozapine exposure activates common apoptotic pathways shared with anticancer drugs. Although the meagre clinical evidence precludes drawing any general conclusion as to the safety of maintaining clozapine administration during chemotherapy, it does not point to an obvious worsening of the haematological outcomes.

Clozapine and Dilated Cardiomyopathy

Clozapine is effective in treatment-resistant schizophrenia, but carries a number of risks, including the rare but potentially fatal complication of cardiomyopathy. We report the case of a long-term inpatient with schizophrenia who improved significantly with clozapine therapy, but eighteen months after initiating treatment expired from sudden cardiac death. Autopsy revealed four chamber dilated cardiomyopathy, with no evidence of inflammation and normal coronary arteries. By history and pathological examination the finding of cardiomyopathy was thought to be associated with clozapine exposure, and the small body of literature on clozapine-associated dilated cardiomyopathy strongly suggests a direct effect of clozapine on the myocardium by an as yet unidentified mechanism. Based upon the published studies of cardiac adverse effects from clozapine exposure, possible hypotheses for this effect include selenium deficiency, prior myocarditis, or persistent supraventricular tachycardia. Importantly, several ca...

Are 1 or 2 dangerous? Clozapine and olanzapine exposure in toddlers

Clozapine (Clozaril ®) and olanzapine (Zyprexa ®) are two relatively new atypical antipsychotics that are structurally and pharmacologically related. There are currently no therapeutic indications for these pharmaceuticals in infants and toddlers.Presumably, as the usage of these medications in adults increases, the frequency of unintentional pediatric ingestions will increase. In 2001 the annual report of the American Association of Poison Control Centers Toxic Exposure Surveillance System included a separate subcategory for atypical antipsychotics under the heading of sedatives/hypnotics/antipsychotics. The toxidrome resulting from these drugs is predominately central nervous system depression and anticholinergic effects. Although the desirable lack of extrapyramidal symptoms in adults results in their greatest clinical utility, several reports of toxic ingestions in small children are noteworthy for having extrapyramidal manifestations. We review here the available reported clinical experience with toxic doses of these medications that in small children may amount to as little as a single tablet. Although such doses may be lethal, supportive care and gastrointestinal decontamination in this population will generally lead to a good outcome.

The Dynamin-dependent, Arrestin-independent Internalization of 5-Hydroxytryptamine 2A (5-HT2A) Serotonin Receptors Reveals Differential Sorting of Arrestins and 5-HT2A Receptors during Endocytosis

5-Hydroxytryptamine 2A (5-HT2A) receptors, a major site of action of clozapine and other atypical antipsychotic medications, are, paradoxically, internalized in vitro and in vivo by antagonists and agonists. The mechanisms responsible for this paradoxical regulation of 5-HT2A receptors are unknown. In this study, the arrestin and dynamin dependences of agonist- and antagonist-mediated internalization were investigated in live cells using green fluorescent protein (GFP)-tagged 5-HT2A receptors (SR2-GFP). Preliminary experiments indicated that GFP tagging of 5-HT2A receptors had no effect on either the binding affinities of several ligands or agonist efficacy. Likewise, both the native receptor and SR2-GFP were internalized via endosomes in vitro. Experiments with a dynamin dominant-negative mutant (dynamin K44A) demonstrated that both agonist- and antagonist-induced internalization were dynamin-dependent. By contrast, both the agonist- and antagonist-induced internalization of SR2-GFP were insensitive to three different arrestin (Arr) dominant-negative mutants (Arr-2 V53D, Arr-2-(319-418), and Arr-3-(284-409)). Interestingly, 5-HT2A receptor activation by agonists, but not antagonists, induced greater Arr-3 than Arr-2 translocation to the plasma membrane. Importantly, the agonist-induced internalization of 5-HT2A receptors was accompanied by differential sorting of Arr-2, Arr-3, and 5-HT2A receptors into distinct plasma membrane and intracellular compartments. The agonist-induced redistribution of Arr-2 and Arr-3 into intracellular vesicles and plasma membrane compartments distinct from those involved in 5-HT2A receptor internalization implies novel roles for Arr-2 and Arr-3 independent of 5-HT2A receptor internalization and desensitization.

Clozapine inhibits limbic system kindling: Implications for antipsychotic action

Clozapine and haloperidol were tested for their ability to influence the acquisition of kindled seizures following electrical stimulation of the amygdala and ventral hippocampus. While haloperidol pretreatment did not alter kindling genesis from either limbic region, preexposure to clozapine delayed the rate at which kindling evolved. Anatysis of the number of seizure behaviors expressed during epileptogenesis revealed that clozapine produced a relative antagonism of seizure development arresting kindling at the stage-3 level. The kindling inhibition was dependent upon the daily administration of clozapine during the kindling process and was not evident after withdrawal from a chronic schedule of clozapine exposure. A subconvulsive dose of pilocarpine (80.0 mg/kg) produced an overall enhancement of kindling rate, a finding consistent with the excitatory role of acetylcholine (ACh) in kindling. Lower doses of pilocarpine (20.0 and 40.0 mg/kg) that did not alter seizure advancement partially antagonized the clozapine-elicited inhibition of amygdaloid kindling. Pilocarpine, however, did not affect the clozapine-induced increase in the number of stage-3 behaviors exhibited during amygdaloid kindling, suggesting that other neurochemical effects of clozapine, not related to its anticholinergic properties, modulate the kindling suppression. Clozapine's unique actions on limbic system sensitization were discussed in relation to its effectiveness as an antipsychotic agent.