Clozapine Exposure Research Articles

Longitudinal changes in DNA methylation associated with clozapine use in treatment-resistant schizophrenia from two international cohorts

The second-generation antipsychotic clozapine is used as a medication for treatment-resistant schizophrenia. It has previously been associated with epigenetic changes in pre-clinical rodent models and cross-sectional studies of treatment-resistant schizophrenia. Cross-sectional studies are susceptible to confounding, however, and cannot disentangle the effects of diagnosis and medication. We therefore profiled DNA methylation in sequential blood samples (n = 126) from two independent cohorts of patients (n = 38) with treatment-resistant schizophrenia spectrum disorders who commenced clozapine after study enrolment and were followed up for up to six months. We identified significant non-linear changes in cell-type proportion estimates derived from DNA methylation data - specifically B-cells - associated with time on clozapine. Mixed effects regression models were used to identify changes in DNA methylation at specific sites associated with time on clozapine, identifying 37 differentially methylated positions (DMPs) (p < 5 × 10-5) in a linear model and 90 DMPs in a non-linear quadratic model. We compared these results to data from our previous epigenome-wide association study (EWAS) meta-analysis of psychosis, finding evidence that many previously identified DMPs associated with schizophrenia and treatment-resistant schizophrenia might reflect exposure to clozapine. In conclusion, our results indicate that clozapine exposure is associated with changes in DNA methylation and cellular composition. Our study shows that medication effects might confound many case-control studies of neuropsychiatric disorders performed in blood.

Impacts of exposure to and subsequent discontinuation of clozapine on tripartite synaptic transmission.

Clozapine is an effective antipsychotic for treatment-resistant schizophrenia, but its discontinuation leads to discontinuation syndrome/catatonia complicated by benzodiazepine-resistance and rhabdomyolysis. This study determined time-dependent effects of exposure and subsequent discontinuation of clozapine on expression of connexin43, 5-HT receptors, intracellular L-β-aminoisobutyrate (L-BAIBA) and 2nd-messengers and signalling of AMPK, PP2A and Akt in cultured astrocytes and rat frontal cortex. Intracellular L-BAIBA levels increased during clozapine exposure but immediately recovered after discontinuation. Both exposure to clozapine and L-BAIBA increased connexin43 and signalling of AMPK/Akt time-dependently, but reduced PP2A signalling, 5-HT receptor expression and IP3 level. These changes recovered within 2 weeks after discontinuation, while 5-HT receptors and IP3 transiently increased during the recovery process. L-BAIBA activated AMPK signalling, leading to attenuated PP2A signalling. Astroglial D-serine release was increased by clozapine exposure but continued to increase within 1 week after discontinuation via activation of IP3 receptor function. Clozapine discontinuation restored PP2A signalling due to decreased L-BAIBA, increased 5-HT receptor expression via probably enhanced 5-HT receptor recycling, but increased astroglial D-serine release persisted by transiently activated IP3 receptors via transiently increased IP3 level. Decreased L-BAIBA caused by clozapine discontinuation is, at least partially, involved in the transiently increased 5-HT receptor and astroglial D-serine release.

Association Between Clozapine Exposure and Risk of Hematologic Malignancies in Veterans With Schizophrenia.

Association Between Clozapine Exposure and Risk of Hematologic Malignancies in Veterans With Schizophrenia.

Associations of the Gut Microbiome With Treatment Resistance in Schizophrenia

There is growing interest in the role of gut microbiome composition in schizophrenia. However, lifestyle factors are often neglected, and few studies have investigated microbiome composition in treatment-resistant schizophrenia. To explore associations between the gut microbiome and schizophrenia diagnosis, treatment resistance, clozapine response, and treatment-related adverse effects while adjusting for demographic and lifestyle factors. In this case-control study of adults aged 20 to 63 years, stool samples and data on demographic characteristics, lifestyle, and medication use were collected and gut microbiome measures obtained using shotgun metagenomics. Participants with a schizophrenia diagnosis were referred through psychiatric inpatient units and outpatient clinics. Data were collected for 4 distinct groups: control individuals without a psychiatric diagnosis (past or present), individuals with treatment-responsive schizophrenia taking nonclozapine antipsychotic medications, clozapine-responsive individuals with treatment-resistant schizophrenia, and clozapine-nonresponsive individuals with treatment-resistant schizophrenia. Participants were recruited between November 2020 and November 2021. Control individuals were recruited in parallel through posters and online advertisements and matched for age, sex, and body mass index (BMI) to the individuals with schizophrenia. Participants were excluded if taking antibiotics in the past 2 months, if unable to communicate in English or otherwise follow study instructions, were pregnant or planning to become pregnant, or had any concomitant disease or condition making them unsuited to the study per investigator assessment. Data were analyzed from January 2022 to March 2023. Omics relationship matrices, α and β diversity, and relative abundance of microbiome features. Data were collected for 97 individuals (71 [74%] male; mean [SD] age, 40.4 [10.3] years; mean [SD] BMI, 32.8 [7.4], calculated as weight in kilograms divided by height in meters squared). Significant microbiome associations with schizophrenia were observed at multiple taxonomic and functional levels (eg, common species: b2, 30%; SE, 13%; adjusted P = .002) and treatment resistance (eg, common species: b2, 27%; SE, 16%; adjusted P = .03). In contrast, limited evidence was found for microbiome associations with clozapine response, constipation, or metabolic syndrome. Significantly decreased microbial richness was found in individuals with schizophrenia compared to control individuals (t95 = 4.25; P < .001; mean [SD] for control individuals, 151.8 [32.31]; mean [SD] for individuals with schizophrenia, 117.00 [36.2]; 95% CI, 18.6-51.0), which remained significant after a covariate and multiple comparison correction. However, limited evidence was found for differences in β diversity (weighted UniFrac) for schizophrenia diagnosis (permutational multivariate analysis of variance [PERMANOVA]: R2, 0.03; P = .02), treatment resistance (R2, 0.02; P = .18), or clozapine response (R2, 0.04; P = .08). Multiple differentially abundant bacterial species (19) and metabolic pathways (162) were found in individuals with schizophrenia, which were primarily associated with treatment resistance and clozapine exposure. The findings in this study are consistent with the idea that clozapine induces alterations to gut microbiome composition, although the possibility that preexisting microbiome differences contribute to treatment resistance cannot be ruled out. These findings suggest that prior reports of microbiome alterations in individuals with chronic schizophrenia may be due to medication or lifestyle factors and that future studies should incorporate these variables in their design and interpretation.

Evaluating the epidemiology of clozapine-associated neutropenia among people on clozapine across Australia and Aotearoa New Zealand: a retrospective cohort study

Clozapine is associated with the risk of serious neutropenia. However, this risk might decrease over time, meaning that indefinite absolute neutrophil count (ANC) monitoring could be unnecessary. We aimed to determine the epidemiology and timing of clozapine-associated neutropenia outcomes, to investigate variables that might contribute to the odds of neutropenia, and to determine risk of competing neutropenic events during clozapine treatment. We performed a retrospective analysis of the Australian and New Zealand Viatris Pharmacovigilance system (one of two monitoring databases for these two countries) between June 6, 1990, and Oct 25, 2022. Patients were excluded from analysis if they commenced clozapine before 1990, did not have a haematology test within 2 weeks of commencement date, or had no follow-up. We measured minor neutropenia (ANC 1·0-1·5 × 109 per L) and serious neutropenia (ANC <1·0 × 109 per L) leading to cessation of clozapine within 6 weeks of the neutropenic event. We determined the rates of minor and serious neutropenia and calculated odds ratios (ORs) for the likelihood of neutropenia leading to cessation. For serious neutropenia leading to cessation, we used time-to-event to calculate rolling weekly averages and to perform competing risk analysis of outcomes using Cox proportional hazards models and a Fine-Gray subdistribution hazards regression model. For the subset of data where information on previous clozapine use was available, we did an analysis for participants who did and did not have previous clozapine exposure. We included 26 630 people, with 2·6 million ANC values. Within the total cohort, 17 585 people (66%) were male, 9025 (33·9%) female, and 20 (0·1%) other gender, and the mean age was 36·1 years (SD 13·7). We did not have data on race or ethnicity. Of the 26 630 people taking clozapine, 1146 (4·3%) had minor neutropenia, 313 (1·2%) had serious neutropenia leading to cessation, and 223 (0·8%) had serious neutropenia unrelated to clozapine without cessation. In people with no previous exposure to clozapine (n=15 973), the cumulative incidence of serious neutropenia leading to cessation was 0·9% at 18 weeks and 1·4% at 2 years; the weekly incidence rate for serious neutropenia leading to cessation peaked at 9 weeks (0·128%) and fell to a rolling average weekly incidence of 0·001% by 2 years. For minor neutropenia, the cumulative incidence was 1·7% at 18 weeks and 3·5% at 2 years; the weekly incidence rate peaked at 9 weeks (0·218%) and fell to a stable rolling average of 0·01%. The median time to a serious neutropenic event leading to cessation was 17 weeks (IQR 9·96-102). Previous clozapine exposure reduced the risk of serious neutropenia leading to cessation (OR 0·19, 95% CI 0·12-0·31; p <0·0001). Most serious neutropenia leading to clozapine cessation occurs within 18 weeks of treatment and becomes negligible after 2 years. Weekly haematological monitoring after the first 18 weeks could be safely reduced to once every 4 weeks and ceased after 2 years unless clinically indicated. Clozapine retrial after interruption with 2 cumulative years of unremarkable testing might not require further haematological monitoring. A serious neutropenia ANC threshold of ≤1·0 × 109 per L could be used in more jurisdictions. None.

Clozapine and neutrophil response in patients of African descent: A six-month, multinational, prospective, open-label clinical trial

IntroductionClozapine is the most effective antipsychotic for treatment-resistant schizophrenia, but it is markedly underutilized, particularly in the US Black population, partly because of concern over clozapine-associated low absolute neutrophil count (ANC). People of African descent have a lower normative ANC range than the White population, which is associated with a specific “ACKR1-null” (“Duffy null”) CC genotype (SNP rs2814778) on the ACKR1 gene, termed benign ethnic neutropenia (BEN). The range of ANC variability and safety of clozapine have not been established in people with BEN or examined prospectively in people of African descent. MethodsWe completed a multisite, 6-month, prospective, open-label clinical trial of clozapine treatment in people of African descent with schizophrenia spectrum disorders for whom clozapine was clinically indicated, with or without the ACKR1-null genotype. We examined clozapine safety and weekly ANC during clozapine treatment and evaluated ANC variability by ACKR1-null genotype, sex, study site, and clozapine dosing using repeated measures analysis of covariance. Genotype was assayed using TaqMan® technology. ResultsWe enrolled 274 participants, of whom 227 (82.8 %) completed 6 months of clozapine treatment. There was one case of severe neutropenia (<500 cells/mm3) (0.36 %) over 1467.6 person-months of clozapine exposure. This participant recovered without sequelae after discontinuation of clozapine. Of the 249 participants with known genotypes, 199 (79.9 %) had the ACKR1-null genotype. Neutropenia (<1500 cells/mm3) occurred significantly more often in the ACKR1-null group (33 % [65/199]) than in those with the T allele (6 % (3/50); p < 0.001). Fourteen (5 %) patients discontinued due to adverse events. Rates of infection and fever were low and sialorrhea was the commonest side effect (N = 187, 68 %). ConclusionTo our knowledge, this is the largest prospective clozapine trial in people of African descent. Severe neutropenia was rare, despite the high prevalence (80 %) of the ACKR1-null genotype. Our findings suggest that clozapine can be used safely in Black patients including those with BEN.

Vitamin D impact in affecting clozapine plasma exposure: A potential contribution of seasonality

Schizophrenia affects approximately 24 million people worldwide and clozapine is the most effective antipsychotic drug. Nevertheless, its use in therapy is limited due to adverse effects.Therapeutic drug monitoring is a clinical tool useful to reduce the clozapine toxicity. In the literature, papers showed how psychiatric disorders could be associated with low vitamin D levels, but a few studies focusing on its role in affecting clozapine exposure are available. A TDM repository was analyzed: clozapine and vitamin D levels measured with liquid chromatography were considered. 1261 samples obtained from 228 individuals were evaluated: 624 patients (49.5%) showed clozapine plasma levels in therapeutic range (350–600 ng/mL). Clozapine toxic plasma levels (>1000 ng/mL) were more present in winter (p = 0.025), compared to other seasons. Concerning vitamin D, a sub-analysis of 859 samples was performed: 326 (37.81%) were deficient ( ng/mL), 490 (57.12%) had insufficient concentrations (10–30 ng/mL), while 43 (5.02%) had sufficient (>30 ng/mL) levels. A correlation between vitamin D and clozapine plasma levels (p = 0.007, Pearson coefficient=0.093) was observed. The role of seasonal variation in clozapine plasma exposure in psychiatric patients treated with clozapine was suggested. Further studies in larger cohorts are needed in order to clarify these aspects.

Modeling Approach with Therapeutic Drug Monitoring Data to Describe Time Course of Clozapine Exposure and Positive and Negative Syndrome Scale Scores

The Positive and Negative Syndrome Scale (PANSS) is commonly used to assess the severity of the clinical symptoms of schizophrenia (SCZ). This study aimed to develop a pharmacokinetic (PK)/pharmacodynamic (PD) model based on therapeutic drug monitoring (TDM) data to characterize the relationship between clozapine exposure and the PANSS scores in patients with SCZ. TDM data for clozapine and PANSS scores from 45 patients with SCZ were included in this modeling analysis using NONMEM. Based on published data, intensive PK sampling data collected up to 12 hours postdose from 23 patients was incorporated into the PK data set to improve the fitting of absorption and disposition. For PD model development, the PANSS score was assessed at baseline, followed by 8 and 18 weeks after the initiation of clozapine dosing. Visual predictive check plots, the precision of parameter estimates, and decreases in the minimum objective function values were used for the model evaluation. A 2-compartment model with an absorption lag and a combined error model adequately described the PK of clozapine. The implementation of disease progression with placebo and drug effects improved the model's ability to describe the time course of the PANSS scores. In the final PK/PD model, Weibull and maximum effect (E max ) models were selected as disease progression models for the placebo and drug effect models, respectively. The model evaluation results supported the adequacy of the final model. A clozapine PK/PD model based on clinical settings adequately described the PANSS time course in patients with SCZ. These findings may aid the development of treatment strategies for patients with SCZ.

Correlation between assessment of cytochrome P450 1A2 activity and enzyme activity scores, and their relation to clozapine exposure.

Cytochrome P450 1A2 (CYP1A2) is involved in the metabolism of antipsychotic drugs such as clozapine and olanzapine. Personalization of these treatments requires an accurate estimation of CYP1A2 activity. In this study, we aimed (1) to evaluate the correlation between activity score (AS), covariate-corrected activity score (CCS) and the phenotype of CYP1A2 using a caffeine test probe and (2) to investigate their relationship with dose-adjusted clozapine concentrations in a subgroup of the cohort. A multicentric, retrospective and observational study was carried out in the French university hospitals of Marseille and Tours. CYP1A2 activity was calculated by the paraxanthine/caffeine (17X/137X) ratio determined 4h after an oral intake of 100 mg caffeine. AS was calculated according to the CYP1A2*1F alleles. CCS was calculated according to the CYP1A2*1F alleles, smoking status and the presence of concomitant inhibitors. As expected, among the 89 patients included, the 17X/137X ratio was significantly higher in patients who smoked. We found a significant but modest correlation between the 17X/137X ratio and CCS (R2 = 0.3, P = 1.74 × 10-8 ) but none between the 17X/137X ratio and AS (R2 = -0.007, P = 0.52). AS was not correlated with dose-adjusted clozapine levels, contrary to CCS (R2 = 0.19, P = 0.016) and especially the 17X/137X ratio (R2 = 0.42, P = 1.7 × 10-5 ). Correlation with clozapine concentrations showed the advantage of the 17X/137X ratio over the CCS in clozapine dose optimization. CYP1A2 activity, especially when determined by the caffeine probe, may be used to personalize clozapine dosing for patients experiencing treatment failure.

Relationship between clozapine exposure and the onset of appendicitis in schizophrenia patients: a retrospective cohort study

ObjectiveClozapine may cause serious side effects despite benefits in patients with schizophrenia. Thus, an accurate understanding of the side-effect profile of clozapine is extremely important in the management of its administration to patients with schizophrenia. Our aim was to validate the relationship between clozapine exposure and appendicitis onset in patients with schizophrenia.MethodsIn this study, we retrospectively compared the incidence and cumulative incidence of appendicitis in patients with schizophrenia with and without a history of clozapine exposure. Among the patients with schizophrenia who visited our hospital between June 2009 and August 2021, we extracted those with a history of clozapine treatment. Patients with a history of taking clozapine were defined as the clozapine exposure group, while the others were defined as the clozapine non-exposure group. Patients with a history of appendectomy before their initial visit to our hospital or with a history of clozapine use at other hospitals were excluded.ResultsThere were 65 patients in the clozapine exposure group and 400 patients in the clozapine non-exposure group who met the inclusion criteria. The exposure group exhibited a remarkably higher incidence of appendicitis during the observation period than the non-exposure group (863 cases vs. 124 cases per 100,000 person-years). In particular, if limited to the period of clozapine exposure, the incidence of appendicitis is extremely high, at 2,086 cases per 100,000 person-years. Moreover, multivariable analysis showed that clozapine exposure was an independent factor contributing to the onset of appendicitis.ConclusionsClozapine exposure is associated with appendicitis onset in patients with schizophrenia.

Clozapine placental passage at delivery: an update

IntroductionClozapine is an effective second-generation antipsychotic that is approved for treatment-resistant schizophrenia and risk reduction of recurrent suicidal behavior in schizophrenia or schizoaffective disorder. Its available pregnancy pharmacikinetics data remain limited, which presents a challenge for clinicians managing women taking clozapine during perinatal period .ObjectivesThe aim of this study was to provide new data of clozapine and norclozapine placental passage and neonatal outcomes.MethodsWe retrospectively studied a consecutive case series of six pregnancies where there was clozapine exposure (5 in politherapy and 1 in monotherapy). Clozapine and norclozapine serum concentrations were determined in the mother-infant pairs on the day of delivery (intrapartum maternal blood and umbilical cord blood respectively) and measured using a validated high-performance liquid chromatography method. The within- and between-day precision expressed as the coefficient of variation (CV)% were both &lt;10%. The limit of quantification (LoQ) was 5 ng/mL. Neonatal outcomes were reviewed from pediatric records.ResultsThe mean infant-mother clozapine and norclozapine ratio at delivery were 0.44 (SD=0.13) and 0.28 (SD=0.05) respectively. There was a weak positive correlation between maternal and umbilical cord clozapine and norclozapine serum concentratios (Pearson correlation coefficient 0.183, p=0.769 and 0.827, p=0.084 respectively). The rate of neonatal complications was 16%. One neonate (16%) , whose mother had drug abuse history during pregnancy, presented with a generalized neurodevelopment delay and the consequent need for continuous intensive care.ConclusionsIn our study, placental passage of clozapine and norclozapine was partial during delivery. Statistical power was limited for examining te association between neonatal clozapine levels and neonatal outcomes.DisclosureNo significant relationships.

Effects of a Novel UGT2B Haplotype and UGT1A4*3 Allele Variants on Glucuronidation of Clozapine In vivo.

Glucuronidation is an important metabolic pathway of clozapine (CLZ), but the impact of various uridine 5'diphospho-glucuronosyltransferases (UGT) polymorphisms on the exposure and metabolism of CLZ in vivo is unclear. The objective of this study was to investigate the impact of UGT2B haplotype and UGT1A4*3 allele variants on the formation of CLZ glucuronide metabolites (5N- and N+-glucuronide) and CLZ exposure in patients' serum after adjusting for sex, age, and smoking habits. The study was based on serum samples from CLZ-treated patients (n=79) subjected to routine therapeutic drug monitoring (TDM) at Diakonhjemmet Hospital, Oslo, Norway. From the same patients, the following UGT variants were genotyped using Real-Time PCR: UGT2B:GA haplotype (defined as UGT2B:GA; rs1513559A>G and rs416593T>A) and UGT1A4*3 (rs2011425T>G). Serum concentrations of CLZ 5N- and N+-glucuronide were measured by UPLC high-resolution mass spectrometry. None of the genotypes had significant impact on CLZ exposure (p>0.05). However, compared to UGT2B:AT/AT and UGT1A4*1/*1, the 5N-glucuronide exposure was reduced in UGT2B:GA/GA carriers (-75 %, p=0.03) while the exposure was non-significantly increased in UGT1A4*3 carriers (+100 %, p=0.14), respectively. The N+-glucuronide exposure was unchanged in UGT1A4*3 vs. noncarriers (p=0.28), but significantly reduced in heterozygous (-50 %, p=0.016) and homozygous carriers (-70 %, p=0.021) of UGT2B:GA compared to UGT2B:AT/AT carriers, respectively. The UGT2B:GA and UGT1A4*3 variants had no impact on CLZ exposure but were associated with differences and preferences in CLZ glucuronidation. The latter might be of potential relevance for CLZ tolerability since levels of the N+-glucuronide metabolite may reflect the generation and trapping of reactive metabolites involved in CLZ-induced toxicity.

Clozapine- and non-clozapine-associated neutropenia in patients with schizophrenia: a retrospective cohort study.

Introduction:The antipsychotic drug clozapine remains underutilized partly because of the risk of life-threatening adverse effects, such as neutropenia. Therefore, an extensive hematological monitoring program was set up to detect neutropenia.Methods:In this retrospective cohort study, we used registry-based data from the Capital Region of Denmark to investigate incidence rates of neutropenia among patients with a diagnosis of schizophrenia or other psychotic disorders and treated with clozapine for the first time. In a within-subject design, we compared rates of neutropenia in time periods where patients were exposed to clozapine versus time periods, where they were not exposed to clozapine. We also investigated whether the lengths of clozapine-associated neutropenia (CAN) were related to discontinuation of clozapine treatment.Results:Data from 520 clozapine users were included. The incidence rate of CAN was 3.2 cases per 100 person-years (95% confidence interval [CI]: 2.1–4.8) throughout the entire study. There was no significant difference in incidence rates of neutropenia during clozapine exposure and non-clozapine exposure, with an incidence rate ratio of 0.7 (95% CI: 0.4–1.3). One episode of severe neutropenia was detected. Episodes of CAN with only one sub-threshold neutrophil count were not associated with higher clozapine discontinuation (26%) than CAN episodes of more than one sub-threshold neutrophil count (28%).Conclusion:In the present study, we could not confirm that clozapine treatment was associated with neutropenia.

Population pharmacokinetic model and limited sampling strategy for clozapine using plasma and dried blood spot samples.

Background:To improve efficacy, therapeutic drug monitoring is often used in clozapine therapy. Trough level monitoring is regular, but trough levels provide limited information about the pharmacokinetics of clozapine and exposure in time. The area under the concentration time curve (AUC) is generally valued as better marker of drug exposure in time but calculating AUC needs multiple sampling. An alternative approach is a limited sampling scheme in combination with a population pharmacokinetic model meant for Bayesian forecasting. Furthermore, multiple venepunctions can be a burden for the patient, whereas collecting samples by means of dried blood spot (DBS) sampling can facilitate AUC-monitoring, making it more patient friendly.Objective:Development of a population pharmacokinetic model and limited sampling strategy for estimating AUC0-12h (a twice-daily dosage regimen) and AUC0-24h (a once-daily dosage regimen) of clozapine, using a combination of results from venepunctions and DBS sampling.Method:From 15 schizophrenia patients, plasma and DBS samples were obtained before administration and 2, 4, 6, and 8 h after clozapine intake. MwPharm® pharmacokinetic software was used to parameterize a population pharmacokinetic model and calculate limited sampling schemes.Results:A three-point sampling strategy with samples at 2, 6, and 8 h after clozapine intake gave the best estimation of the clozapine AUC0-12h and at 4, 10, and 11 h for the AUC0-24h. For clinical practice, however, a two-point sampling strategy with sampling points at 2 and 6 h was sufficient to estimate AUC0-12h and at 4 and 11 h for AUC0-24h.Conclusion:A pharmacokinetic model with a two–time point limited sampling strategy meant for Bayesian forecasting using DBS sampling gives a better prediction of the clozapine exposure in time, expressed as AUC, compared to trough level monitoring. This limited sampling strategy might therefore provide a more accurate prediction of effectiveness and occurrence of side effects compared to trough level monitoring. The use of DBS samples also makes the collection of clozapine samples easier and wider applicable.

Combining Therapeutic Drug Monitoring and Pharmacokinetic Modelling Deconvolutes Physiological and Environmental Sources of Variability in Clozapine Exposure.

Background: Clozapine is a key antipsychotic drug for treatment-resistant schizophrenia but exhibits highly variable pharmacokinetics and a propensity for serious adverse effects. Currently, these challenges are addressed using therapeutic drug monitoring (TDM). This study primarily sought to (i) verify the importance of covariates identified in a prior clozapine population pharmacokinetic (popPK) model in the absence of environmental covariates using physiologically based pharmacokinetic (PBPK) modelling, and then to (ii) evaluate the performance of the popPK model as an adjunct or alternative to TDM-guided dosing in an active TDM population. Methods: A popPK model incorporating age, metabolic activity, sex, smoking status and weight was applied to predict clozapine trough concentrations (Cmin) in a PBPK-simulated population and an active TDM population comprising 142 patients dosed to steady state at Flinders Medical Centre in Adelaide, South Australia. Post hoc analyses were performed to deconvolute the impact of physiological and environmental covariates in the TDM population. Results: Analysis of PBPK simulations confirmed age, cytochrome P450 1A2 activity, sex and weight as physiological covariates associated with variability in clozapine Cmin (R2 = 0.7698; p = 0.0002). Prediction of clozapine Cmin using a popPK model based on these covariates accounted for <5% of inter-individual variability in the TDM population. Post hoc analyses confirmed that environmental covariates accounted for a greater proportion of the variability in clozapine Cmin in the TDM population. Conclusions: Variability in clozapine exposure was primarily driven by environmental covariates in an active TDM population. Pharmacokinetic modelling can be used as an adjunct to TDM to deconvolute sources of variability in clozapine exposure.

Clozapine Treatment During Pregnancy and the Postpartum Period: A Systematic Literature Review.

Objective: The objective of this systematic review was to provide a critical appraisal of the evidence related to the safety of clozapine for schizophrenia during pregnancy and lactation. Data Sources: PubMed/MEDLINE, Embase, and the Cochrane Library were searched from inception through December 2020. Reference lists of included studies were hand-searched. The International Clinical Trials Registry Platform and ClinicalTrials.gov were searched for unpublished trials, and PROSPERO was searched for unpublished reviews. The current marketing authorization holder of the originator brands Clozaril and Leponex was also contacted for pharmacovigilance data. Study Selection: Original reports published in English, German, French, or Dutch containing clinical and preclinical data were included if they provided data on maternal, fetal, and neonatal outcomes after clozapine exposure during pregnancy or lactation. Data Extraction: Two reviewers independently extracted relevant data. Results: A total of 860 records were identified, and the full texts of 117 articles were reviewed. Forty-two studies met the inclusion criteria. Data on perinatal clozapine exposure are of limited quality and quantity. Although clozapine demonstrates partial placental passage, data thus far do not support that clozapine is teratogenic; that it increases the risk of stillbirth, abortion, or fetal disorders; or that it increases the risk of delivery complications or premature birth. Information about clozapine exposure through breast milk is scarce, but based on its chemical properties, it is likely that clozapine enters the breast milk of nursing mothers taking clozapine. Conclusions: When weighing the risks and benefits of clozapine continuation during pregnancy and lactation versus switching to another antipsychotic, one should include severity of illness and treatment history but also be aware of the limitations of the available safety data regarding perinatal clozapine use, including the fact that there are few studies.

Environmentally relevant concentrations of clozapine induced lipotoxicity and gut microbiota dysbiosis in Chinese rare minnow (Gobiocypris rarus)

Clozapine (CLZ) is a neuroactive pharmaceutical that is frequently detected in aquatic environments. Although the cardiotoxicity, developmental toxicity, and neurotoxicity of CLZ in aquatic non-target organisms have been reported, its lipotoxicity and underlying mechanism are unknown. Therefore, in this study, 2-month-old Chinese rare minnows were exposed to 0, 0.1, 1, and 10 μg/L CLZ for 90 days. Overt dyslipidemia was observed after CLZ exposure, whereas the body weights of females significantly increased after CLZ exposure (p < 0.05). In addition, obvious hepatocyte vacuolization and hepatic lipid droplet accumulation were observed at all treatment groups (p < 0.05). The activities of sterol regulatory element binding proteins 1 (SREBP1) and fatty acid synthase (FAS) were significantly upregulated at the 1 and 10 μg/L CLZ treatment groups (p < 0.05). Moreover, evident cell boundary disintegration of the intestinal villi and increasing mucus secretion were observed at all treatment groups (p < 0.05). Furthermore, the diversity of the gut microbiota increased, whereas the relative abundances of Proteobacteria, Firmicutes and Bacteroidetes significantly increased after CLZ exposure (p < 0.05). Furthermore, significantly increased bacterial secondary bile acid biosynthesis activity in Chinese rare minnows was observed after 1 μg/L CLZ exposure (p < 0.05). Therefore, our findings confirmed that CLZ induced lipotoxicity by stimulating SREBP1 and affecting the bacterial secondary bile acid biosynthesis activity in Chinese rare minnows.

Neonatal and infant outcomes of clozapine exposure in pregnancy: A consecutive case series

IntroductionClozapine is a second-generation antipsychotic agent approved for treatment-resistant schizophrenia and risk reduction of recurrent suicidal behavior in schizophrenia and schizoaffective disorder. Given the known negative consequences of relapse of severe mental disorders for both mother and infant, the maintenance of clozapine during pregnancy is recommended.1 Studies of pregnancy regarding to clozapine have demonstrated a heterogenous range of neonatal and infant complications.2ObjectivesTo evaluate neonatal and infants outcomes of clozapine exposure in pregnancy.MethodsWe report three cases of infants exposed to clozapine politherapy throughout pregnancy. The dose range for all women on clozapine was 200-600 mg/day. Infants were evaluated between 4-6 months of chronological age with the Bayley-III infant development scale (BSID-III)3 and with the Alarme Détresse Bébé Scale (ADBB)4 for the detection of early-signs of withdrawal.ResultsWomen remained stable during pregnancy but presented obesity and gestational diabetes. Clozapine Newborn were born to term by caesarean section due to breech presentation (N=2) or instrumental delivery due to loss of fetal well-being (N=1). They presented normal weight (3500-3800 gr). Two presented Apgarmin1-5 9/10 and one Apgarmin1-5 6/8 which showed lethargy and low alertness during the first weeks of life. All showed normal capacity for sociability, reciprocity and development of language and communication. However, one baby had scores in the low normal zone for cognition and another for motor skills.ConclusionsThe infant’s risks of clozapine exposure during pregnancy should be discussed with women and weighed against those associated with other treatments and/or with untreated severe mental illness.

Clozapine Induced Developmental and Cardiac Toxicity on Zebrafish Embryos by Elevating Oxidative Stress.

Clozapine is one of the antipsychotic drugs for treating schizophrenia, but its cardiotoxicity was the primary obstacle for its clinical use, due to the unknown mechanism of clozapine-induced cardiotoxicity. In this study, we studied the cardiotoxicity of clozapine by employing zebrafish embryos. Acute clozapine exposure showed dose-dependent mortality with the LC50 at 59.36μmol L-1 and 49.60μmol L-1 when determined at 48 and 72h post exposure, respectively. Morphological abnormalities like pericardial edema, incompletely heart looping, and bradycardia were detected after clozapine exposure in a time- and dose-dependent manner. Clozapine treatment also resulted in a slower heart rate and disturbed rhythm in zebrafish embryos. Also, oxidative stress was observed after clozapine exposure by measurement of ROS (reactive oxygen species), MDA (a lipid peroxidation marker), antioxidant enzyme activities, and oxidative stress-related gene expression. The elevation of inflammation coincided with oxidative stress by the assay of inflammation-related genes expression accompanied by clozapine incubation. Collectively, the data indicate that clozapine might achieve cardiotoxic effect in zebrafish larva through increasing oxidative stress, attenuation in antioxidant defense, and up-regulation of inflammatory cytokines. The data could provide experimental explanations for myocarditis and pericarditis induced by clozapine in clinics, and help find an effective solution to reduce its cardiotoxicity.

S202. PROXIMAL AND DISTAL FACTORS ASSOCIATED WITH CLOZAPINE EXPOSURE IN A SAMPLE OF TREATMENT RESISTANT AND TREATMENT RESPONSIVE SCHIZOPHRENIA PATIENTS

BackgroundClozapine is the gold standard for Treatment Resistant Schizophrenia (TRS) and its use is strongly recommended after failure of two/three antipsychotics. Nonetheless, prescribing trends do not appear to follow the global guidelines and switching to clozapine is often delayed or improper. Reasons for clozapine underuse are quite understood, whereas little is known on clozapine misuse. In fact, more stringent operative criteria to define TRS should be used in clinical practice, taking advantage of standardized assessment tools in order to avoid false TRS positive.In these perspectives, we studied the patterns of clozapine prescription in a sample of TRS and non-TRS patients, in order to identify psychopathological variables associated with lifetime exposure to clozapine.Methods198 consecutive patients have been screened for eligibility criteria from 2016 until 2019 at Treatment Resistant Psychosis Centre of the University of Naples “Federico II”. Only stabilized schizophrenic patients aged between 18 and 65 were enrolled. All relevant clinical-demographic data were recorded. TRS diagnosis was based on TRRIP working group operational criteria and clear pseudo-resistance factors were excluded. Patients were subdivided into two groups: those having received or currently under clozapine treatment (eCLZ) and those never treated by clozapine (nCLZ). Patients were assessed for psychotic symptoms (PANSS), cognitive performances (BACS), neurological soft signs (NES), functional capacity (UPSA), social functioning (PSP and SLOF scales), and severity of illness (CGI-S).ResultsAmong the patients included (92), eCLZ were 50 (54.3%), of which 20% being non-TRS. Compared to nCLZ, eCLZ had a significantly earlier age at onset, more hospitalizations, higher daily antipsychotic doses; eCLZ patients had significantly higher scores on the CGI-S, PANSS total, PANSS Negative, General Psychopathology and PAUSS subscale and, according to the Five Factor PANSS Model, higher score on Negative and Disorganization Factors; eCLZ had also more impaired Working Memory performances, higher NES Total score, lower PSP and all SLOF areas scores, except Area4.Then we use univariate and multivariate analysis to delineate predictors of clozapine exposure and to evaluate whether these factors may organize in proximal (independent) and distal (mediated) associations. We found five independently associated variables that mediate all the other ones: high antipsychotic doses, higher disease severity, suicide attempts, impaired working memory, more severe Stereotyped Thinking.DiscussionWhile it is expected that some TRS patients might not receive clozapine, the observation that non-TRS patients had been exposed to clozapine may surprise. A possible explanation may be the lack of systematized procedure to diagnose TRS, which is often merely coincident with a nonspecific lack of response to antipsychotics.Due to mediation analysis, we further gained insights into the hierarchy of these variables’ impact on clozapine prescription and we finally compose a complex network of proximal and distal factors predicting clozapine exposure in schizophrenic patients.The above-mentioned independent variables significantly associated with clozapine exposure let intend that eCLZ patients may belong to a subset of more severely ill ones, beyond reaching the response threshold.Earlier onset patients may suffer from a more severe disease, be more prone to suicide attempts, and less responsive to antipsychotics.This result needs to be replicated in wider samples, and this relevant therapeutic issue should be further debated in order to gather robust evidence to support or discourage clozapine off-label use and misuse.