Clozapine is the most preferred antipsychotic drug for the management of treatment-resistant schizophrenia (TRS) because of its superior clinical efficacy (Kane et al., 1988), its ability to reduce negative symptoms and its low propensity for movement disorders (Tandon and Fleischhacker, 2005). However, it may lead to a suboptimal response in 40–70% patients (Remington et al., 2005) and serious adverse events such as seizures, agranulocytosis and dyslipidaemia (Henderson et al., 2000). Fixed oral doses of clozapine can produce up to 45-fold interindividual variability in serum levels (Potkin et al., 1994). Many patients with suboptimal therapeutic responses to clozapine have inadequate serum clozapine levels and their responses can be enhanced by ensuring serum clozapine levels above a minimum threshold value (Miller et al., 1994; Potkin et al., 1994; Kronig et al., 1995). Moreover, serum clozapine levels above a maximum threshold value increase the risk for its toxicity (Ulrich et al., 2003) and seizures (Cooper, 1996). Such a therapeutic window and the predisposition for high interindividual variability mandate the estimation of serum clozapine levels, especially in patients who show poor response to treatment with clozapine, who are intolerant to usual therapeutic doses and who require clozapine dose titration above 600 mg/day (Cooper, 1996; Bell et al., 1998).
The lower limit of this therapeutic window for serum clozapine levels is considered between 250 and 420 ng/ml (Miller et al., 1994; Potkin et al., 1994; Kronig et al., 1995; Cooper, 1996; Bell et al., 1998; Perry et al., 1998; Ulrich et al., 2003). The minimum threshold value for serum clozapine level is suggested to be 350 ng/ml (Miller et al., 1994; Kronig et al., 1995; Perry et al., 1998) for acute treatment with clozapine, which can be lower for maintenance therapy (Gaertner et al., 2001). Above this cut-off value, the relationship between serum clozapine level and clinical response to clozapine remains uncertain (Kronig et al., 1995; Cooper, 1996; Wong et al., 2006). Besides, routine therapeutic drug monitoring of clozapine is not feasible in many clinical settings, especially in low-income and middle-income countries (Greenwood-Smith et al., 2003). Hence, identification of clinical predictors, which act as proxy measures of serum clozapine levels, is desirable during everyday clinical practice in such settings (Perry et al., 1998).
Studies examining the clinical factors associated with serum clozapine levels have focused on age, sex or on smoking so far, and their results have not been consistent (Haring et al., 1989; Centorrino et al., 1994; Lane et al., 1999; Palego et al., 2002; Rostami-Hodjegan et al., 2004; Tang et al., 2007). Pharmacogenetic studies evaluating the association between serum clozapine levels and single nucleotide polymorphisms of the specific candidate genes, responsible for the metabolism of clozapine, have also been inconclusive (Van der Weide et al., 2003; Kootstra-Ros et al., 2005; Jaquenoud Sirot et al., 2009). Most of these studies did not exclusively recruit patients with TRS and evaluated only a very limited number of variables. They also did not carry out structured assessments of cognition, disability and adverse effects (Haring et al., 1989; Centorrino et al., 1994; Lane et al., 1999; Palego et al., 2002; Van der Weide et al., 2003; Rostami-Hodjegan et al., 2004; Kootstra-Ros et al., 2005; Tang et al., 2007; Jaquenoud Sirot et al., 2009). Despite the presence of ethnic differences in the serum clozapine levels (Ng et al., 2005), there is a dearth of relevant data from Asian countries, where there is a clinical need for proxy measures of serum clozapine levels (Tang et al., 2007). Hence, we aimed to investigate the associations between various clinical variables and serum clozapine levels in patients with TRS in India.
We used a cross-sectional study to evaluate the clinical factors associated with serum clozapine levels. We used a case–control design framework to identify the clinical factors associated with inadequate serum clozapine levels below 350 ng/ml.
We carried out this study at the Department of Psychiatry, Christian Medical College, Vellore, India, a tertiary referral centre for the management of psychiatric disorders. The hospital has short-term inpatient services, daily outpatient and regular follow-up clinics. Patients with schizophrenia are initially treated with either dopamine antagonists or serotonin dopamine antagonists. Clozapine is never used as the first-line antipsychotic drug and is reserved for patients with TRS. Detailed medical records of treatment are maintained for all patients. Leucocyte counts and metabolic parameters of the patients receiving clozapine are monitored periodically. Most of our outpatients with schizophrenia live in the community with their families. Their medications are directly provided by their first-degree relatives or spouses, who report any degree of nonadherence to the treating psychiatrists during periodically scheduled follow-up visits.
Recruitment of participants
We invited all consecutive patients who fulfilled the following eligibility criteria: (i) Diagnostic and Statistical Manual of Mental Disorders, 4th ed., Text Revision diagnosis of schizophrenia, (ii) treatment resistance established after failure to respond to at least two adequate antipsychotic trials, as documented by treating psychiatrists. An adequate antipsychotic trial was defined by 600 mg chlorpromazine equivalents for a duration of at least 6 weeks, with good drug compliance. The two adequate antipsychotic trials included at least one adequate trial with an serotonin dopamine antagonist. (iii) On stable dose regimen of clozapine for at least 12 weeks, with good drug compliance during that period. Participants with severe neurological illnesses, intellectual disability and sensory impairment, before the assessment, were excluded.
Serum clozapine assay
Peripheral venous blood samples were collected from all participants by venipuncture 12 h after their last clozapine dose. Serum clozapine levels were measured by deproteinization with diethyl ether and subsequent high-performance liquid chromatography with ultraviolet detection (Wongsinsup et al., 2010). The serum clozapine levels were expressed as ng/ml.
We used a structured questionnaire to collect sociodemographic, clinical and treatment data. We recorded data on comorbidity, comedication, caffeine as well as grape juice consumption, smoking and anthropometric measures. We collected self-reported data on clozapine-related adverse effects using a check list of adverse effects. We also used the following instruments: (i) the Brief Psychiatric Rating Scale (BPRS) to assess psychopathology (Overall and Gorham, 1962), (ii) the Abnormal Involuntary Movements Scale (AIMS) to measure neuroleptic induced dyskinesia, if present (Guy, 1976), (iii) Addenbrooke’s Cognitive Examination-Revised (ACE-R), a brief cognitive test battery to evaluate cognitive functioning (Mioshi et al., 2006), and (iv) WHO Disability Assessment Scale-II (WHODAS-II), to quantify the disability (WHO, 2001).
The protocol of the study was approved by the Institutional Review Board of Christian Medical College, Vellore, India. We provided a fact sheet about the details of this study to all participants. We discussed these details and obtained written informed consent from the participants and from their first-degree relatives or spouses. We assessed every participant for psychopathology using the BPRS. Another independent investigator, who was blinded to the serum clozapine levels, assessed other clinical variables through detailed personal interviews with the participants and their primary caregivers. She accessed the medical records of all participants with their consent. We followed standard quality control procedures to ensure the accuracy of our data collection, data entry and of the serum clozapine assay.
We initially analysed the study variables using descriptive statistics. We checked whether all continuous variables were normally distributed using one-sample Kolmogorov–Smirnov tests. As the distribution of serum clozapine levels was nonparametric, we used Spearman’s correlation analyses to assess the bivariate correlation between serum clozapine levels and continuous clinical variables. We studied the associations between serum clozapine levels and various hypothesized clinical explanatory variables with robust regression models, using the STATA rreg command. Robust regression models are valid, despite the presence of influential outliers and the non-normality of residuals. Ordinary least squares regression was initially performed to compute absolute residuals, which were scaled by the median residual value. After estimating Huber weights and Tukey biweights, iteratively reweighted least squares regression was performed to estimate the regression coefficients. We also determined multiple robust regression statistics to adjust for the effects of age, sex and oral clozapine dose. We developed a multivariate model with all predictors that were significant during bivariate analyses.
Then, we dichotomously categorized the serum clozapine levels into adequate levels and inadequate levels on the basis of the minimum threshold value of 350 ng/ml (Miller et al., 1994; Kronig et al., 1995; Perry et al., 1998). The outcome variable was inadequate serum clozapine level and the predicting variables were the hypothesized clinical variables. We calculated the odds ratios (ORs) with 95% confidence intervals (CIs). We used multiple logistic regression models to calculate adjusted ORs accounting for the effects of age, sex and oral clozapine dose. We used Nagelkerke pseudo R2 statistics to determine the proportion of variability explained by the model and the Hosmer–Lemeshow test to assess the goodness of fit of the model. We analysed our data using the statistical software packages, SPSS 16.0 (IBM, New York, New York, USA) and STATA 12.1 (StataCorp, College station,Texas, USA).
Sample size estimation
An earlier study has documented a correlation coefficient, between serum clozapine level and oral clozapine dose, as high as 0.7 (Centorrino et al., 1994). We estimated the required sample size with an anticipated correlation coefficient of 0.3, for the clinical variables, 80% power and 5% α error as 79 for a two-sided test.
We assessed 113 consecutive patients who fulfilled the eligibility criteria. We excluded six patients, who were not completely compliant with clozapine, within the past 12 weeks. One patient with severe Parkinson’s disease and another with moderate mental retardation were also excluded. Among the 105 patients found to be eligible, 101 consented to participate, yielding a participation rate of 96.2%. Common reasons for refusing consent were lack of interest in study objectives and reluctance to provide blood samples. Participants (n=101) and those who were excluded (n=12) did not differ significantly in terms of sex (χ2=0.04; d.f.=1; P=0.84), age (t=−1.41; d.f.=111; P=0.16) and duration of illness (t=−1.27; d.f.=111; P=0.21). Table 1 presents the sociodemographic and clinical profiles of all participants. The majority of the participants were single or separated men, unemployed and were living in urban areas (n=58; 57.4%). Most of the participants had a chronic continuous course of paranoid schizophrenia. The oral doses of clozapine ranged from 100 to 650 mg/day, with a median value of 350 mg/day. The duration of clozapine treatment ranged from 4 to 174 months, with a median value of 28 months. More than half of the participants (53.5%) had received clozapine for more than 2 years.
Clinical variables associated with serum clozapine levels
Serum clozapine levels of the participants ranged from 104 to 2547 ng/ml, with a median value of 428 ng/ml. The mean serum clozapine level was 550.5 (SD 378.5) ng/ml. Twenty-nine participants (28.7%) had serum clozapine less than 350 ng/ml. Twenty-four participants (23.8%) had serum clozapine levels above 750 ng/ml. The mean serum clozapine level/oral dose ratio was 1.71 (SD 1.12). There was a 30-fold interindividual variability among the participants, with their clozapine level/dose ratio ranging between 0.28 and 8.29. We present the association between oral doses of clozapine and serum clozapine levels in Fig. 1. Table 2 presents the bivariate as well as multivariate analyses for the associations between various clinical variables and serum clozapine levels. Oral doses of clozapine and Valproate comedication were associated positively with serum clozapine levels. Those who consumed three or more cups of coffee or tea everyday had significantly lower serum clozapine levels. Age, sex, body weight and smoking were not associated with serum clozapine levels in our sample. Our participants were on the following comedications: Risperidone (n=10; 9.8%), Haloperidol (n=6; 5.9%), Quetiapine (n=2; 2.0%), Aripiprazole (n=4; 3.9%), Trihexyphenidyl (n=12; 11.8%) and Clonazepam (n=7; 6.9%). None of these comedications was significantly associated with serum clozapine levels in our sample. Table 3 shows a multiple robust regression model predicting serum clozapine levels (F=9.78; P<0.001). We derived the following equation from this model: serum clozapine level (ng/ml)=194.51+0.91 (oral clozapine dose in mg/day)−130.45 (if high caffeine intake is present)+247.93 (if Valproate comedication is present).
Multiple logistic regression models examining the clinical variables associated with serum clozapine levels below 350 ng/ml showed that age, sex, body weight, smoking, caffeine intake, duration of illness, age of onset of illness, duration of treatment with clozapine, tardive dyskinesia, fluoxetine or sertraline and Valproate comedication were not significantly associated with serum clozapine levels below 350 ng/ml. Oral doses of clozapine less than 250 mg/day increased the risk of inadequate serum levels by four times (OR 4.27; 95% CI 1.47−12.41; P=0.008), after adjusting for the effects of age and sex. All participants who were on Valproate comedication had serum clozapine levels above 350 ng/ml. A multiple logistic regression model including an oral clozapine dose below 250 mg/day, Valproate comedication and high caffeine intake could predict the inadequate serum clozapine levels correctly in 73.3% of participants. The Hosmer–Lemeshow test confirmed the goodness of fit of this model (χ2=0.08; d.f.=2; P=0.96; Nagelkerke pseudo R2=0.196).
Clinical utility of serum clozapine levels
The relationship between serum clozapine levels and psychopathology of the participants, as measured by the BPRS total scores, was not significant, after adjusting for the oral dose of clozapine (β=0.12; SE=2.21; t=0.06; P=0.95). Higher serum clozapine levels were associated with worse cognitive functioning, as measured by ACE-R total scores, after adjusting for the effects of age and oral clozapine dose (β=−0.011; SE=0.005; t=−2.07; P=0.04). However, this relationship was not significant when psychopathology of the participants was also included in this model (β=−0.006; SE=0.005; t=−1.17; P=0.25). Similarly, serum clozapine levels were not associated with disability, as measured by WHODAS-II total scores, after adjusting for the effects of age, oral clozapine dose and psychopathology (β=0.006; SE=0.004; t=1.68; P=0.10). Among those who had serum clozapine levels below 350 ng/ml (n=29), BPRS total scores were significantly associated with worse cognitive outcomes (β=−0.65; SE=0.20; t=−3.33; P=0.003). Serum clozapine levels were not associated with AIMS total scores after adjusting for the effects of age and oral clozapine dose (β=−0.003; SE=0.002; t=−1.46; P=0.15). Serum clozapine levels above 750 ng/ml increased the risk of seizures by five times after adjusting for the effects of age and oral clozapine dose (OR 5.15; 95% CI 1.11–23.88; P=0.03). Other adverse effects such as hypersomnolence, sialorrhoea, neutropenia, sexual dysfunction, constipation, nausea, nocturnal enuresis and obesity were not associated with serum clozapine levels.
This study examined the clinical predictors of serum clozapine levels among patients with TRS. The strengths of this study include exclusive recruitment of treatment-resistant patients, adequate sample size, minimal refusal rate, structured assessment of clinical variables and the use of nonparametric robust regression models for analyses. Consecutive sampling strategy minimized the possibility of selection bias. An independent assessment of serum clozapine levels and clinical variables by two different investigators reduced the possibility of observer bias. Interview of primary caregivers of the participants and verification of their follow-up medical records reduced the possibility of recall bias on the reported clinical variables. The potential limitations of this study are its cross-sectional design and lack of assessment of serum levels of the clozapine metabolite, norclozapine. The optimum study design for evaluating the lower limit of the therapeutic window for serum clozapine levels is a prospective double-blind trial, where participants are randomly assigned to different groups and are treated with varying oral doses of clozapine, which would yield predefined ranges of serum clozapine levels (VanderZwaag et al., 1996; Hiemke et al., 2011).
Clinical predictors of serum clozapine levels
Our results show that higher oral doses of clozapine as well as Valproate comedication are associated with higher serum clozapine levels and that high caffeine intake reduces serum clozapine levels. Previous studies have reported that the oral clozapine dose is the only clinical variable consistently associated with serum clozapine levels, but is insufficient to explain the high interindividual variability of serum levels (Haring et al., 1989; Centorrino et al., 1994; Perry et al., 1998; Lane et al., 1999; Palego et al., 2002; Rostami-Hodjegan et al., 2004; Tang et al., 2007). Female sex has been associated with higher clozapine levels (Haring et al., 1989; Centorrino et al., 1994; Lane et al., 1999; Ulrich et al., 2003; Tang et al., 2007), but this association has been questioned (Palego et al., 2002, Diaz et al., 2008). Increasing age has been associated with higher clozapine levels (Haring et al., 1989; Ulrich et al., 2003), but evidence to the contrary also exists (Centorrino et al., 1994; Palego et al., 2002; Tang et al., 2007; Diaz et al., 2008). Our study did not find any association between serum clozapine levels and age or sex in our sample. There are studies supporting (Haring et al., 1989; Perry et al., 1998) and challenging (Tang et al., 2007) the association between smoking and lower serum clozapine levels. As the prevalence of smoking was low in our sample (18.8%) and we did not have any female smokers, because of cultural reasons, our study was probably underpowered to detect any association between smoking and serum clozapine levels.
We confirmed the association between Valproate comedication and higher serum clozapine levels (Facciola et al., 1999; Wong et al., 2006; Diaz et al., 2008). The literature available on the association between caffeine consumption and serum clozapine levels is contradictory. Previous smaller studies have suggested that caffeine either reduces (Dratcu et al., 2007) or increases (Hagg et al., 2000; Raaska et al., 2004) serum clozapine levels, because of the competitive inhibition of the CYP1A2 hepatic enzyme. Our results have clarified that caffeine consumption is associated with lower serum clozapine levels. Underlying pharmacokinetic pathways warrant further investigation. As smoking and drinking caffeinated beverages are positively correlated (Rihs et al., 1996), their combined influence over CYP1A2 hepatic enzyme activity needs to be investigated in more detail. Constituents in the caffeinated beverages, other than caffeine, may also induce CYP1A2 enzyme activity. Selective serotonin reuptake inhibitors (SSRI) such as Fluvoxamine (Diaz et al., 2008) and Paroxetine (Spina et al., 2000) have been reported to be associated with higher serum clozapine levels. Eight of our participants, who were on SSRI comedication, were receiving either Fluoxetine or Sertraline. We did not find any association between these SSRI comedications and serum clozapine levels.
Serum clozapine levels and treatment-resistant schizophrenia
Higher serum clozapine levels have been reported to be associated with greater cognitive impairment in patients with chronic schizophrenia (Adler et al., 2002) after adjusting for the effects of age, sex and oral dose (Rajji et al., 2010). We replicated this finding in our sample, but further clarified that this observed association was confounded by the psychopathology of the participants. We documented that the bivariate positive correlation, between psychopathology and serum clozapine levels, was spurious, because of the confounding effect of oral clozapine doses. Patients who have severe psychopathology develop more cognitive deficits, and they require more oral doses of clozapine, producing higher serum clozapine levels. Hence, we argue against any direct causal relationship between serum clozapine levels and cognitive impairment in patients with chronic schizophrenia. Similarly, our findings suggest that the observed relationship between serum clozapine levels and disability in patients with chronic schizophrenia was also confounded by the psychopathology of the participants. A previous study has hypothesized that patients with tardive dyskinesia would differ in their metabolism of clozapine and would have higher serum clozapine levels (Pollack et al., 1993). Our results disprove this hypothesis with appropriate multivariate analyses.
The literature and our results suggest the following:
Serum clozapine levels, above the minimum threshold value of its therapeutic window, maximize the clinical response to clozapine (Miller et al., 1994; Potkin et al., 1994; Kronig et al., 1995), reduce the lag time to response (Perry et al., 1998) and reduce the relapse rates (Ulrich et al., 2003). We presented an equation to predict serum clozapine levels using three clinical variables. In clinical settings where routine therapeutic drug monitoring of clozapine is not feasible, this equation can aid the treating psychiatrists during dose adjustments of clozapine.
We highlight the clinical need for appropriate clozapine dose reductions while adding Valproate comedication to reduce level-dependent adverse effects (Nielsen et al., 2011).
Patients worldwide consume caffeinated beverages everyday and their importance is often neglected during clinical dose adjustments of clozapine (Dratcu et al., 2007). Patients should be advised to inform their psychiatrists when they make any abrupt changes in their caffeine habits (Carrillo et al., 1998). Our dosing equation may help the psychiatrists to consider appropriate clozapine dose adjustments in such situations.
Oral doses of clozapine above 600 mg/day are known to increase the risk of seizures (Cooper, 1996). We identified a threshold value of serum clozapine levels (750 ng/ml) above which the risk of seizures increases by five-fold. If patients who are on maintenance treatment with clozapine have serum clozapine levels above 750 ng/ml, appropriate dose reductions or adjustment of comedications should be considered.
We found that oral clozapine doses below 250 mg/day were four times more likely to produce inadequate serum clozapine levels (<350 ng/ml). Hence, we suggest the minimum clinical dose for clozapine should be 250 mg/day unless the patients develop intolerable adverse effects on lower doses.
Because of high interindividual variability, serum clozapine levels do not usually follow Gaussian distributions among clinical samples. Hence, dosing nomograms on the basis of linear regression models (Haring et al., 1989; Perry et al., 1998) are often misleading (Lane et al., 1999). We recommend that clinical psychiatrists and future researchers to pursue dosing equations developed from appropriate nonparametric regression models.
The pharmacokinetics of clozapine is complex. Hence, statistical models with clinical predictors often explain only a fraction of the variability observed in the serum clozapine levels (Perry et al., 1998; Rostami-Hodjegan et al., 2004). We should consider the differing findings of the studies on this topic as not contradictory but as contributing to each other. Routine therapeutic drug monitoring of clozapine is desired in all clinical settings (Ulrich et al., 2003). Until it becomes a reality in resource-poor settings, we should combine the available clinical predictors for serum clozapine levels to aid appropriate clozapine dose adjustments. When we progress towards genome-wide pharmacogenetic studies to explain the interindividual variability of serum clozapine levels, we should not overlook the valuable contribution by the clinical predictors. We recommend that future longitudinal studies investigate both clinical and pharmacogenetic factors together to develop better models for predicting serum clozapine levels.
A.P.R., B.P. and K.S.J. conceived this study and wrote the study protocol. A.P.R., C. Chitra and Dr S. Bhuvaneshwari carried out the data collection. A.P.R. and K.S.J. analysed the data and wrote the manuscript. All authors were involved in revising the manuscript. The authors are grateful to Dr P. Thangadurai, S.D. Manoranjitham, Dr Binu Susan Mathew, K. Saravanakumar and S. Velvizhi, Christian Medical College, Vellore, for help and support. They thank all participants and their families.
This study was funded by a fluid research grant (22X356) by the Christian Medical College, Vellore, India.
Conflicts of interest
There are no conflicts of interest.
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