Clozapine OpTimal Timing for Optimal moNitoring and Side Effects
Conditions: Cardiovascular Side Effects of Clozapine, Neutropenia Due to Clozapine, General Side Effects of Clozapine, Constipation Due to Clozapine, Symptoms of Schizophrenia, Optimal Blood Sampling Time for Clozapine in Patients Who Receive Clozapine Once and Twice Daily, Clozapine and Norclozapine Plasma Level Concentrations
Sponsor: GGZ Noord-Holland-Noord

 
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Clozapine OpTimal Timing for Optimal moNitoring 
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and Side effects (COTTONS) 
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PROTOCOL TITLE ‘Clozapine OpTimal Timing for Optimal moNitoring’ 
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Protocol ID 
COTTONS 
Short title 
Clozapine OpTimal Timing for Optimal moNitoring 
and Side effects 
EudraCT number  
n.a.  
Version 
2.0 
Date 
22 November 24 
Coordinating investigator 
A. Abdul Roda, MSc 
E-mail: a.abdulroda@amsterdamumc.nl 
Phone: +31(0)617789903 
Co-investigators 
Participating sites: 
GGZ NHN, Alkmaar 
- L. Kaya, MD 
- M. Musters, MD 
 
Noordwest ziekenhuisgroep, Alkmaar 
- F. Ph. Mulder, PharmD 
- E. ten Boekel, PhD 
- Dr. S. Simsek, MD, PhD 
- R. Fijn, PharmD, PhD 
 
Albert Schweitzer ziekenhuis, Dordrecht 
M.M. Beex-Oosterhuis, PharmD, PhD 
 
University Medical Center Groningen, Groningen 
prof. D.J. Touw, PharmD, PhD 
 
Principal investigator(s) (in 
Dutch: hoofdonderzoeker/ 
uitvoerder) 
 
Noordwest ziekenhuisgroep, Alkmaar 
P.C.D. Bank, PharmD, PhD 
Email: pcd.bank@nwz.nl 
Phone: 088 085 5110 
 
GGZ Noord-Holland-Noord 
S.R.T. Veerman, MD, PhD 
Email: s.veerman@ggz-nhn.nl 
 
Amsterdam UMC, locatie AMC 
Prof. R.A.A. Mathôt, PharmD, PhD 

 
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Email: r.mathot@amsterdamumc.nl 
Phone: 020 566 3327 
 
 
Sponsor (in Dutch: 
verrichter/opdrachtgever) 
GGZ NHN 
 
 
Subsidising party 
N.A. 
Independent expert (s) 
N.A. 
Laboratory sites  
NWZ Alkmaar 
 
Juliana van Stolberglaan 13, 1814 HB Alkmaar 
072 548 4444 
Pharmacy  
N/A 
 
 

 
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PROTOCOL SIGNATURE SHEET 
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Name 
Signature 
Date 
Sponsor: 
 
GGZ NHN 
 
S.R.T. Veerman, MD, PhD 
psychiatrist GGZ NHN 
board member Dutch Clozapine 
Collaboration Group 
 
 
 
 
 
 
18-11-2024 
Principal Investigator: 
 
P.C.D. Bank, PharmD, PhD  
hospital pharmacist &  
clinical pharmacologist,  
co-chair clinical pharmaceutical  
laboratory, North West Clinics  
Member Dutch Clozapine 
Collaboration Group 
 
Prof. R.A.A. Mathôt, PharmD, PhD 
hospital pharmacist &  
clinical pharmacologist,  
Amsterdam UMC, location AMC 
 
 
18-11-2024 
 
 
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TABLE OF CONTENTS 
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INTRODUCTION AND RATIONALE 
8OBJECTIVES
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13STUDY DESIGN
25 
 
14STUDY POPULATION
26 
 
15Population (base)
27 
 
15Inclusion criteria
28 
 
15Exclusion criteria
29 
 
15Sample size calculation
30 
 
15TREATMENT OF SUBJECTS
31 
 
16INVESTIGATIONAL PRODUCT
32 
 
16NON-INVESTIGATIONAL PRODUCT
33 
 
16METHODS
34 
 
17Study parameters/endpoints
35 
 
17Main study parameter/endpoint
36 
 
17Secondary study parameters/endpoints
37 
 
17Other study parameters
38 
 
18Randomisation, blinding and treatment allocation
39 
 
18Study procedures
40 
 
18Withdrawal of individual subjects
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18Specific criteria for withdrawal (if applicable)
42 
 
18Replacement of individual subjects after withdrawal
43 
 
18Follow-up of subjects withdrawn from treatment
44 
 
18Premature termination of the study
45 
 
18SAFETY REPORTING
46 
 
19STATISTICAL ANALYSIS
47 
 
20Primary study parameter(s)
48 
 
20Secondary study parameter(s)
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22Other study parameters
50 
 
23Interim analysis (if applicable)
51 
 
23ETHICAL CONSIDERATIONS
52 
 
24Regulation statement
53 
 
24Recruitment and consent
54 
 
24Objection by minors or incapacitated subjects (if applicable)
55 
 
24Benefits and risks assessment, group relatedness
56 
 
24Compensation for injury
57 
 
24Incentives (if applicable)
58 
 
24ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION
59 
 
25Handling and storage of data and documents
60 
 
25Monitoring and Quality Assurance
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26Amendments
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26Annual progress report
63 
 
26Temporary halt and (prematurely) end of study report
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26Public disclosure and publication policy
65 
 
26STRUCTURED RISK ANALYSIS
66 
 
26TIME SCHEDULE
67 
 
26REFERENCES
68 
 
27 
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LIST OF ABBREVIATIONS AND RELEVANT DEFINITIONS  
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ABR 
General Assessment and Registration form (ABR form), the 
application form that is required for submission to the 
accredited Ethics Committee; in Dutch: Algemeen 
Beoordelings- en Registratieformulier (ABR-formulier) 
AUC 
Area Under the Curve 
Cl 
Clearance 
Cmax 
Maximum Concentration 
CV 
Coefficient of Variation  
EHR 
Electronic Health Record 
GCP 
Good Clinical Practice 
GDPR 
General Data Protection Regulation; in Dutch: Algemene 
Verordening Gegevensbescherming (AVG) 
IC 
Informed Consent 
ICC 
Class Correlation Coefficient  
IIV 
Interindividual Variability (synonyms are interpatient of 
between-patient variability) 
IOV 
Interoccasion Variability (synonyms are intrapatient of 
within-patient variability) 
Ke 
The elimination rate constant, is the fraction of a drug that 
is eliminated from the body over a set unit of time 
MIPD 
Model-Informed Precision Dosing 
NONMEM 
Nonlinear Mixed-Effects Modelling 
PANSS 
Positive and Negative Syndrome Scale 
PD 
Pharmacodynamics 
PE 
Prediction Error 
PK 
Pharmacokinetics 
PopPK 
Population pharmacokinetics 
RMSE 
Root Mean Square Error  
SPC 
Summary of Product Characteristics; in Dutch: officiële 
productinformatie IB1-tekst 
Sponsor 
The sponsor is the party that commissions the 
organisation or performance of the research, for example a 
pharmaceutical company, academic hospital, scientific 
organisation or investigator. A party that provides funding 
for a study but does not commission it is not regarded as 
the sponsor, but referred to as a subsidising party. 
Steady state 
Equilibrium where drug intake equals elimination, 
maintaining constant average concentration (= approx. 4-5x 
the half-life). 
T1/2 
Half-life, time it takes for the concentration to be halved 
TDM 
Therapeutic Drug Monitoring 
Therapeutic window 
Range of plasma drug concentrations spanning the 
minimum concentration for clinical efficacy and toxicity 
Tmax 
Time to Maximum concentration 
Trough 
Lowest concentration of a drug in the bloodstream, usually 
just before intake of the next dose 
UAVG 
Dutch Act on Implementation of the General Data 
Protection Regulation; in Dutch: Uitvoeringswet AVG 
Vd 
Distribution of volume 
WMO 
Medical Research Involving Human Subjects Act; in Dutch: 
Wet Medisch-wetenschappelijk Onderzoek met Mensen 

 
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SUMMARY 
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Rationale:  
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Therapeutic drug monitoring (TDM) is essential for clozapine and can enhance therapeutic 
76 
outcomes and minimize side effects. As of yet, research on (nor)clozapine concentrations 
77 
and their association to metabolic side effects is limited and inconclusive. Unfortunately, not 
78 
enough is known about individual risk factors for developing metabolic side effects to 
79 
personalize clozapine treatment. It would be desirable to have another way to predict which 
80 
clozapine users are at increased risk of developing severe side effects. Current guidelines 
81 
are based on limited evidence, potentially resulting in inconsistent or suboptimal monitoring 
82 
and management. 
83 
 
84 
Objective:  
85 
The primary objective is to evaluate the correlation between (nor)clozapine kinetics and 
86 
serum level HbA1c. Secondary objectives include validating an existing population 
87 
pharmacokinetic model, assessing the validity of the current 12-hour post-intake sampling 
88 
practice, determining the optimal sampling window for once-daily clozapine TDM and 
89 
assessing the correlation of other metabolic and multiple laboratory parameters and 
90 
influence of demographic and clinical parameters on the pharmacokinetics and -dynamics of 
91 
clozapine and norclozapine.  
92 
 
93 
Study design:  
94 
This is a non-interventional single-center cohort study involving prospective data collection 
95 
for the primary (pharmacodynamic) analysis. This will be supplemented with retrospective 
96 
population pharmacokinetic analyses. Using pharmacokinetic/pharmacodynamic modelling 
97 
and statistical analysis will be used to evaluate the relation to side effects as well as optimize 
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the sampling time for clozapine. Demographic, clinical and laboratory data are collected 
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routinely based on current standard practice.  
100 
 
101 
Study population:  
102 
The study includes patients (18-70 years) diagnosed with treatment-resistant schizophrenia 
103 
or schizoaffective disorder who are on a registered, stable dose of clozapine. Participants 
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must have a known smoking status and have their times of intake and sampling registered. 
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To ensure comparability of (nor)clozapine concentrations, pregnant women will be excluded 
106 
as well as plasma levels affected by acute inflammation/infection/intoxication and/or 
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cessation or dose change of interacting co-medication or tobacco containing products seven 
108 
days prior to sampling. Clinical parameters such as baseline severity of illness, duration of 
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illness and duration of clozapine treatment will be assessed to describe the study population. 
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Main study parameter/endpoint:  
112 
The main study parameter is to evaluate the correlation between (nor)clozapine kinetics and 
113 
serum level HbA1c. The correlation between (nor)clozapine concentrations and ratio and 
114 
HbA1c will be assessed using a PK-PD turn over model. 
115 
 
116 
Nature and extent of the burden and risks associated with participation, benefit and 
117 
group relatedness. 
118 
Not applicable for this retrospective non-interventional study.    
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INTRODUCTION AND RATIONALE 
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Background clozapine 
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Clozapine has proven to be superior to other antipsychotics in treatment of refractory 
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schizophrenia. Therapeutic drug monitoring (TDM) is strongly recommended for clozapine, a 
125 
relatively well-studied compound among antipsychotics. As clozapine levels show large inter- 
126 
and intraindividual variation, TDM helps to increase efficacy and reduce prevalence of side 
127 
effects and toxicity (1-4). A systematic review and meta-analysis has shown a higher risk of 
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non-response in the treatment of refractory schizophrenia or schizoaffective disorder when 
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plasma levels are below the threshold of 350 to 400 µg/l. Other publications have shown that 
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increased plasma levels (> 750 µg/L) are associated with anticholinergic side effects and the 
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occurrence of seizures (4). Clozapine is known to cause more serious metabolic side effects 
132 
than other antipsychotics (5). Up to 80% of the patients are overweight and 45 to 58% have 
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metabolic syndrome, while hyperglycemia and hypertension are often undertreated. 
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Cardiometabolic disorders are therefore the most common cause of premature death in 
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schizophrenia. Clozapine-induced agranulocytosis (CIA) is much rarer, occurring in 0.4% of 
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clozapine users and the risk of death is 10% (6). Nevertheless, fear of CIA is often a reason 
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not to choose clozapine. Norclozapine, clozapine’s main metabolite, may contribute to some 
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extent to the therapeutic effect and side effects of clozapine. Though, its precise role is yet 
139 
inconclusive (7-10). Due to its unknown clinical relevance, many laboratories do not measure 
140 
norclozapine. It is likely that norclozapine, due to its stronger effect as an inverse agonist on 
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the serotonin 5-HT2C receptor, causes weight gain and metabolic abnormalities, which are 
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associated with a lower quality of life and somatic comorbidities such as diabetes mellitus 
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type II, cardiovascular diseases and higher mortality (7). This is further supported by other 
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small studies where norclozapine caused insulin resistance in animals(11), an association 
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between norclozapine levels and elevated triglycerides, elevated cholesterol, and weight gain 
146 
(12) was found or with a higher waist circumference and higher HbA1c (8). Studies with 
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either CYP1A2 induction through smoking (8) or inhibition through fluvoxamine (13) show 
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norclozapine concentrations to be associated with waist circumference, HbA1c, and body 
149 
mass index (BMI) (8) or a lower weight and metabolic parameters (insulin, glucose and 
150 
triglycerides) (8, 13, 14). Finally, two studies contradict this association as a 
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clozapine/norclozapine ratio > 2 appears to be most beneficial regarding cardiometabolic 
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outcomes according to Polcwiartek et al. (15), whereas Lu et al. could not confirm this at a 
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ratio of 3.7 (14). Regarding the level of granulocytes, only one study has shown a higher 
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norclozapine concentration to be more beneficial than a higher clozapine concentration after 
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long-term use of clozapine (16). As of yet, research on (nor)clozapine concentrations and 
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their association to metabolic side effects is limited and inconclusive. For both patients and 
157 
healthcare providers, blood tests and fear of side effects are the main reasons to postpone or 
158 
even abandon clozapine treatment (17). Unfortunately, not enough is known about individual 
159 
risk factors for developing metabolic side effects to truly personalize clozapine treatment. It 
160 
would be desirable to have another way to predict which clozapine users are at increased 
161 
risk of developing severe side effects. Because of this, current guidelines differ in their advice 
162 
regarding metabolic screening. 
163 
 
164 
Therapeutic drug monitoring of clozapine 
165 
Based on the information mentioned above, the recommended window for therapeutic drug 
166 
monitoring for clozapine is 350-700 μg/L for both once- and twice-daily dosing. Some 
167 
patients respond well on lower or higher levels; the latter is acceptable as long as higher 
168 
levels are tolerable. The therapeutic window is based on trough concentrations (i.e., 
169 
approximately 12 hours after the evening dose) from studies using a divided dosing 
170 
regimen(18, 19). Similar to twice-daily dosing, current practice for once-daily dosing of 
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clozapine advises a sample to be collected at least 12 hours after intake or within a window 
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of 10-14 hours after intake. It is however unknown if the concentration-effect relations of 
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once- and twice-daily dosing are similar. Additionally, clinical laboratories do not have a 
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uniform general practice regarding sampling times, resulting in variability in preferred 
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sampling times and windows (1, 3, 4, 20).  
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As of yet, while often used in daily practice, there is no conclusive evidence as to the 
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adequacy and suitability of this sampling time for once-daily dosing. Clozapine’s 
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pharmacokinetic properties further question this suitability. It has a biphasic elimination with a 
179 
mean elimination half-life of 12-16 hours (3). The range is 6-26 hours, which after a single 
180 
dosage of 75 mg was reported to be 7.9 hours that increased to 14.2 hours after reaching 
181 
steady state (after one week) (21). Theoretically, a mean half-life of 12 hours and sampling 
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time of 12 hours could lead to inadequate and inconsistent samples. This might be suitable 
183 
for twice-daily dosing, where a 12-hour sample would be a trough, but this might not be the 
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case for once-daily dosing. With once-daily dosing concentrations at 12 hours after ingestion 
185 
may be adequate (within the therapeutic window) but subtherapeutic after 24 hours. 
186 
Furthermore, smoking influences the metabolism such that a shorter half-life is reported (21).  
187 
 
188 
Pharmacokinetics of clozapine in relation to dosing 
189 
One way to understand the pharmacokinetic variability of such compounds and provide 
190 
individualised treatment strategies is by using population pharmacokinetics (popPK) models. 
191 
These serve as useful tools within the framework of the model-informed precision dosing 
192 
(MIPD). A previous study determined the comparability of once- and twice-daily dosing 
193 
based on popPK analyses as well as the effect of once-daily dosing on clozapine and 
194 
norclozapine concentrations (22). Their simulations show a 30% higher 12 hr-post dose 
195 
concentration with once-daily (400 mg) dosing versus twice-daily (2x 200mg) dosing and a 
196 
30% lower trough (24 hr-post dose) when dosed once-daily compared to twice-daily. A 
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higher once-daily dosage, 700 mg, shows 12 hr-post dose concentrations to lie above the 
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upper limit and a trough (24 hr-post dose concentration) to be within the therapeutic window 
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but almost half the 12 hr-post dose concentration (788 μg/L vs 425 μg/L). This suggests the 
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current sampling time of 12 hr-post dose to be inadequate for once-daily dosing. This is 
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further supported by Procyshyn et al. as well as Zwaag et al. who noted simulated 12 hr-post 
202 
dose concentrations to be 23% higher after a single evening dose versus twice-daily dosing 
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(23, 24). Neither of these two studies included norclozapine in their assessment. This in 
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contrast to the study by Kitagawa et al. where no significant differences were found in 
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estimated peak and trough samples of clozapine and norclozapine dosed once- versus 
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twice-daily (400 mg/day) (25). The Dutch Clozapine Working group recommends increasing 
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the lower limit of clozapine’s therapeutic range by 23% for 12-hr post evening dose samples 
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in patients with once-daily dosing based on the study by Zwaag et al (4, 23).  
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For a valid interpretation of the clozapine/norclozapine ratio, the clozapine dosage regimen, 
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and blood sampling interval are important. Sources of large variability in pharmacokinetics of 
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clozapine that affect CYP1A2 metabolism are smoking behaviour (polycyclic aromatic 
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hydrocarbons in cigarette smoke induce CYP1A2), caffeine consumption (CYP1A2 
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substrate), and estrogen and progesterone levels (inhibit CYP1A2). Excessive smoking is 
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correlated with reduced plasma levels of both CLO and NCLO. Coffee consumption is 
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correlated with increased CLO and NCLO plasma levels and negatively correlated with the 
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CLO/NCLO plasma level ratio. In premenopausal women CLO levels are higher compared to 
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postmenopausal women (26). Pharmacokinetic drug-drug interactions are caused by the 
218 
inhibition or induction of CYPs. Fluvoxamine increases the CLO/NCLO ratio through inhibition 
219 
of the CYP1A2 enzyme. The demethylation of CLO to NCLO is reduced, resulting in an 
220 
increase of the CLO plasma level and decrease of the NCLO plasma level (26). 
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Pro-inflammatory cytokines interleukin (IL)-6 and IL-1 inhibit CYP1A2, which can be detected 
222 
indirectly using the biomarker C-Reactive Protein (CRP).  Furthermore, CLO is highly bound 
223 
to the acute phase protein alpha-1-acid glycoprotein (AGP) and less to serum albumin (95% 
224 
of CLO binds to AGP). During inflammation, concentrations of AGP increase, resulting in a 
225 
decreased CLO unbound fraction. Pharmacogenetics of the  also affects pharmacokinetics of 
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clozapine. For example, asian ancestry is associated with lower CYP1A2 activity compared 
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to other ethnic groups. Additionaly, genetics may also account for variability in response tot 
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clozapine. It is reported that transcriptional activity of the M1 receptor gene is correlated with 
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anticholinergic burden (26). 
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Finally, CLO clearance is affected by age, sex, ethnicity, and smoking status. Higher age 
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results in a reduction in CLO clearance for both male and female smokers and non-smokers 
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of all ethnicities. Females have decreased CLO clearance compared to males. Patients of 
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Afro-Caribbean ethnicity have increased CLO clearance, whereas patients of Asian ethnicity 
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have decreased CLO clearance compared to their White counterparts. Additionally, smokers 
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have increased CLO clearance compared to non-smokers (26). 
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In addition to the CLO/NCLO ratio additional biomarkers to predict the CYP1A2 activity could 
237 
be useful in clinical practice. Pu et al have reported that in rats the ratio of the concentrations 
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of the amino acids phenylalanine and tyrosine in serum is highly correlated with the 
239 
expression of the CYP1A2 gene and the activity of the CYP1A2 enzyme. This biomarker 
240 
could be useful in prediction of the clearance of clozapine by this enzyme (27). 
241 
 
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Pharmacokinetic modelling 
243 
Population PK models may be used to determine the sampling time or window that yields the 
244 
most precise estimation of pharmacokinetic parameters through simulations and optimization 
245 
techniques. Since several models have been developed for clozapine, a careful selection 
246 
based on suitability is applied. In this case, a valid popPK model should be developed 
247 
including Dutch patients, once-daily dosing regimen, trough samples of both clozapine and 
248 
norclozapine (up until 24 hours after ingestion) and include sufficient information during the 
249 
elimination phase. Geers et al. describes a popPK model for clozapine based on Dutch 
250 
patients. However, this model only included samples of patients who are dosed twice-daily, 
251 
fewer samples were taken between 8-24 hours after intake (and thus during the elimination 
252 
phase) and their model was based on two-time point sampling strategies for AUC (28). 
253 
Perera et al. studied optimal sampling points through modeling, but did so for twice-daily 
254 
dosing regimen based on AUC based monitoring as well and concluded three-time point 
255 
strategies. They also concluded that trough concentrations show an excellent correlation to 
256 
exposure (29). Geers et al. concluded the same for twice-daily dosing regimen (AUC0-12) 
257 
(28). Shang et al. used population PK/PD modeling to study the relationship between 
258 
exposure of clozapine (AUC) and the Positive and Negative Syndrome Scale (PANSS) (30). 
259 
They found the AUC to be a better predictor as PANSS score showed little relationship with 
260 
trough concentrations of clozapine. However they mention that this is probably due to the 
261 
slow onset of antischizophrenic effect after reaching steady state. Furthermore they only 
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included Chinese patients with a twice-daily dosing regimen. In addition none of these 
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models included norclozapine. The recent model by Beex et al. is based on Dutch patients, 
264 
trough samples of both clozapine and norclozapine as well as once- and twice-daily dosing 
265 
regimen (22). They mention model misspecification in the absorption phase. This might be 
266 
due to the occurrence of a first-pass effect and could have been described if more samples 
267 
had been taken during the absorption phase. However, this should not be relevant to this 
268 
study which focuses on sampling times during the elimination phase. Therefore this model is 
269 
most suitable to be used to study and find the optimal sampling time or window. Its suitability 
270 
will be further examined by performance of an external validation. 
271 
 
272 
Pharmacokinetics/pharmacodynamics and metabolic side effects 
273 
Metabolic side effects of clozapine comprise weight gain, dyslipidemia and hyperglycemia, 
274 
which can result in a metabolic syndrome, associated with an increased risk for type 2 
275 
diabetes mellitus and cardiovascular disease (5). NCZ is hypothesised due to its stronger 
276 
action as an inverse agonist on the serotonin 5-HT2C receptor to cause weight gain and 
277 
metabolic changes, which are associated with a lower quality of life, somatic comorbidity, 
278 
and a higher mortality (7).    
279 
In a small low-quality meta-analysis (n=120) a positive correlation was found between 
280 
clozapine concentrations and elevated triglycerides, heart rate, and overall combined 
281 
adverse drug reaction (12). Furthermore, a positive correlation was found between 
282 
norclozapine plasma levels and triglycerides, cholesterol, and weight gain. In short, at 
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present there is too little evidence to identify the role of the clozapine/norclozapine plasma 
284 
level ratio in the minimisation of clozapine side effects. In addition, the correlation of 
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CLO(&CNLO) concentration with metabolic markers might be affected by cortisol and 
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genetics of the glucocorticoid receptor (31). Finally, other genetic markers known to 
287 
modulate the basic risk of metabolic syndrome might play a role in this correlation between 
288 
the correlation of the CLO(&CNLO) concentration and the increase in metabolic markers 
289 
(32). 
290 
 
291 
Personalised clozapine treatment 
292 
Since clozapine is mostly given to patients with treatment-resistant schizophrenia, a clear 
293 
defined sample time is required in order to interpret their plasma concentrations and optimize 
294 
their treatment through TDM. Inadequate interpretation may lead to suboptimal treatment 
295 
efficacy or increased risk of adverse effects. Furthermore the relation of adverse effects, 
296 
especially metabolic and granulocyte, to (nor)clozapine concentration has yet to be 
297 
researched well in order to improve screening and optimize treatment. Based on earlier 
298 
studies it is suggested that current sample times may fit a twice-daily dosing schedule and 
299 
not a once-daily dosing schedule. Thus in this study the difference between the 
300 
(nor)clozapine concentrations at 12-hours post intake and the concentration at other time 
301 
points within the 10-14 hours sampling window will be evaluated for once-daily dosed 
302 
clozapine. To this end, the model by Beex et al. will be validated with independent data. 
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Through predicted concentrations, we aim to contribute novel insights into the optimal timing 
304 
of sample collection as well as insight into the development of metabolic and granulocyte 
305 
side effects, addressing the existing knowledge gap and enhancing the precision of 
306 
therapeutic drug monitoring in this population. 
307 
This research will not only provide more knowledge about risk factors for developing general 
308 
side effects of clozapine, metabolic side effects, constipation, and chronic neutropenia, but 
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will also improve metabolic screening in clozapine users through better implementation of 
310 
metabolic screening.  
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OBJECTIVES 
315 
 
316 
Primary Objective:  
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● To evaluate the correlation of (nor)clozapine kinetics and serum level HbA1c 
318 
 
319 
Secondary Objectives: 
320 
● To validate an existing population model with independent data. 
321 
● To study the validity of current practice (12-hr post intake) sampling times within a 
322 
window of 4 hours (10-14 hours post intake) for TDM of once daily dosed clozapine 
323 
● To identify the optimal sampling time or window for TDM of clozapine based on 
324 
predicted concentration-time profiles up until 24-hours post intake. 
325 
 
326 
● To investigate the influence of the following covariates on the pharmacokinetics of 
327 
clozapine, the metabolite norclozapine (and CZ/NCZ ratio): 
328 
o age 
329 
o estradiol (indirectly through demographic data [premenopausal or 
330 
postmenopausal female / male),  
331 
o comedication with fluvoxamine (dosage) and other medication 
332 
o ethnicity (Asian ancestry is associated with lower CYP1A2 activity compared 
333 
to other ethnic groups). 
334 
o pharmacogenetics of the metabolic enzymes CYP2C19 and CYP3A4  
335 
o smoking behaviour (yes/no and amount) 
336 
o inflammation markers (C-reactive protein (CRP) and Erythrocyte 
337 
Sedimentation (BSE)) 
338 
o phenylalanine and tyrosine (a higher ratio of phenylanlanine/tyrosine is 
339 
associated with a higher CYP1A2 activity) (27). 
340 
  
341 
● To evaluate the correlation of (nor)clozapine concentrations and/or ratio on: 
342 
o Other metabolic parameters such as: fasting glucose, insulin, HOMA-IR, 
343 
triglycerides, HDL, LDL, total cholesterol, BMI, abdominal circumference, 
344 
blood pressure and heart rate.   
345 
o Absolute neutrophil count (ANC) and total white cell count (TWC). 
346 
o General side effects of clozapine using the Glasgow Antipsychotic Side-effect 
347 
Scale for Clozapine (GASS-C) (33). 
348 
o Defecation frequency per week and consistency using the Bristol Stool Form 
349 
Scale (BSFS). 
350 
o Positive and Negative Syndrome Scale (PANSS-6), regarding severity of 
351 
schizophrenia 
352 
 
353 
● To evaluate the effect of cortisol concentration and genetics of known relevant loci 
354 
related on the correlation of (nor)clozapine concentrations and/or ratio on the 
355 
metabolic markers (31,32) 
356 
 
357 
 
358 
 
 
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STUDY DESIGN 
360 
This is a (non-interventional) single-center cohort study. (Out)patient data will be collected 
361 
from the mental healthcare organization GGZ-NHN and the laboratory of NorthWest clinics. 
362 
For all endpoints a  baseline value is required. The inclusion period will start 01-12-2024 and 
363 
will last until 01-12-2025. For each patient two years of follow-up will be collected (until a 
364 
maximum of 01-12-2027). For the secondary endpoint pertaining to the sample times 
365 
retrospective data will be collected from 01-02-2024 up to 01-02-2025.  Remnant material will 
366 
be collected in the period during the follow-up period after obtaining informed consent 
367 
 
368 
 
 
369 

 
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STUDY POPULATION 
370 
Population (base)  
371 
The population includes adult patients (age between 18 and 70) with treatment resistant 
372 
schizophrenia of schizoaffective disorder, treated with oral, once or twice-daily clozapine. 
373 
Data will be collected from mental healthcare organization GGZ-NHN. All the patients have 
374 
been treated with clozapine as part of standard care, as well as sampled routinely as part of 
375 
standard care.  
376 
 
377 
Inclusion criteria 
378 
In order to be eligible to participate in this study, a subject must meet all of the following 
379 
criteria and provide informed consent. These criteria are partially based on covariates used 
380 
the model by Beex et al., as it will be used in this study (22):  
381 
● Standard treatment of a stable, oral dose of clozapine for at least one week (at steady 
382 
state).  
383 
● Age between 18-70 years. 
384 
● Registered time of intake as well as sampling time and dosage. 
385 
● Registered smoking status (yes/no). 
386 
● At least two samples in the elimination phase of clozapine with both clozapine and 
387 
norclozapine measured. 
388 
● Measurement of the white cell count at least every three months or more often. 
389 
● Routine metabolic screening performed at moment of inclusion 
390 
● Subjects should be able to understand the study information and procedures and give 
391 
informed consent or when incapacitated subjects are not reliably able to give 
392 
informed consent their legal representatives should give informed consent under the 
393 
condition that these subjects are willing to participate.  
394 
 
395 
 
396 
Exclusion criteria 
397 
A potential subject or specific samples that meet any of the following criteria will be excluded 
398 
from this study: 
399 
● Pregnancy. 
400 
● Malignancy or treatment with immunosuppressive medication. 
401 
● Samples where cessation, start or dose change of interacting co-medication (such as 
402 
valproic acid, gemfibrozil, fluvoxamine, omeprazole and cyclic oral contraceptives [21 
403 
on, 7 days off]) or changes in use of tobacco containing products occurred within 
404 
seven days prior to blood sampling  (21, 28, 34, 35). 
405 
● Acute inflammation, infection or samples shortly after intoxication. In case this 
406 
information is unknown, it may be derived by large unexpected change in levels 
407 
compared to previous or target levels.  
408 
● Not sampled at Starlet (blood collection site) or sampled by dried blood spot 
409 
● Unknown status of smoking (including vaping). 
410 
● Unknown time of intake of clozapine. 
411 
● Unknown time of blood sampling. 
412 
● If informed consent is not obtained by the patient or by a legal representative in a 
413 
mentally incapacitated patient, i.e. a legally incompetent adult. 
414 
 
415 
 
416 
Sample size calculation 
417 
No calculation was performed since the effect of clozapine on the increase of metabolic 
418 
markers is unknown. A rule of thumb with pharmacokinetic studies is an amount of 30 
419 
patients, however with the combination of pharmacokinetics and pharmacodynamics the 
420 
variability in the endpoint will increase. Based on a previous PK/PD study which studied the 
421 
relationship of the drug adalimumab with the PASI-score, a population of approximately 60 
422 
patients was sufficient for establishing a correlation with the pharmacokinetics of adalimumab 
423 

 
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and the clinical response measured by the PASI (36). For the secondary endpoint 
424 
investigating the effect a minimum of 30 patients with 90-120 samples is likely sufficient.  
425 
 
426 
 
427 
TREATMENT OF SUBJECTS 
428 
This chapter is not applicable as this is a non-interventional study.  
429 
 
430 
INVESTIGATIONAL PRODUCT  
431 
This chapter is not applicable as this is a non-interventional, retrospective study that involves 
432 
standard care.  
433 
 
434 
NON-INVESTIGATIONAL PRODUCT 
435 
This chapter is not applicable as this is a non-interventional, retrospective study that involves 
436 
standard care.  
437 
 
 
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METHODS 
439 
Study parameters/endpoints 
440 
Main study parameter/endpoint 
441 
Correlation between serum level HbA1c and (nor)clozapine concentrations and ratio 
442 
will be assessed using a PK-PD turn over model. 
443 
 
444 
Secondary study parameters/endpoints  
445 
● The predictive performance of the model by Beex et al. (22) for our population will be 
446 
assessed through calculations of the prediction error for each observation as well as 
447 
the root mean square error to measure the overall prediction accuracy.  
448 
 
449 
In order to study the main study point, the model by Beex et al. will have to be validated for 
450 
our population first. However, since this is not the primary objective, it is noted as a 
451 
secondary objective that will be performed before researching the primary endpoint. The 
452 
cutoff value for the mean and median values of prediction errors is also based on clinical 
453 
practice as well as the study by Veerman et al. (37) and thus set at 20%.  
454 
 
455 
● Percentage difference in clozapine and norclozapine median concentrations 
456 
predicted 10, 11, 13 and 14-hour post intake (based on clinical samples) compared to 
457 
median concentrations at current reference sampling time of 12-hour post intake.    
458 
 
459 
Based on the study of Veerman et al. an increase of 100 μg/L is regarded as clinically 
460 
relevant (37). However, measurement errors may also impact the differences between 
461 
concentrations and are expressed in a percentage. Therefore, clinically relevant differences 
462 
will be expressed as a percentage as well. The increase of 100 μg/L is set off against the 
463 
therapeutic window of 350-700 μg/L up to the alert level of 1000 μg/L. As the midpoint of this 
464 
range would be 525 μg/L, 100 μg/L would correspond with a difference of 20%. This 
465 
difference is defined as clinically relevant for this study, according to which concentrations 
466 
would no longer be deemed similar and thus corresponding to the same time window or an 
467 
adequate sampling time or window. 
468 
 
469 
● Percentage difference in clozapine and norclozapine concentrations based on 
470 
different sampling times across entire concentration-time profiles to identify optimal 
471 
sampling time or window within current therapeutic window of 350-700 μg/L.  
472 
 
473 
To identify the optimal sampling time or window, expected concentration-time profiles will be 
474 
constructed on basis of individual PK parameters obtained by Bayesian analysis. In these 
475 
profiles sampling times during which the concentration will be within the therapeutic window 
476 
of 350-700 μg/L will be identified. These graphical ‘reference ranges’ could point to an 
477 
optimal sampling time or window. In order for a sampling time window to be found, 
478 
concentrations within that window would have to be deemed similar. As a percentage 
479 
difference of 20% between concentrations will be regarded as clinically relevant, 
480 
concentrations within the same window are allowed a difference up to 20%. As such, a new 
481 
sampling time or window might be found instead of the current window of 10 to 14 hours post 
482 
intake. 
483 
 
484 
● Effect of covariates on the pharmacokinetics of clozapine, the metabolite 
485 
norclozapine (and CZ/NCZ ratio). Covariates to be studied include: age, estradiol, 
486 
comedication (e.g. fluvoxamine), ethnicity, pharmacogenetics, smoking behaviour, 
487 
inflammation markers, phenylalanine and tyrosine. 
488 
 
489 
● Correlation between other metabolic parameters (such as fasting glucose, insulin, 
490 
HOMA-IR, triglycerides, HDL, LDL, total cholesterol, BMI, abdominal circumference, 
491 
blood pressure and heart rate), ANC and TWC, GASS-C, BSFS and PANSS-6 
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regarding severity of schizophrenia and the (nor)clozapine concentrations and ratio 
493 
will be assessed using a PK-PD turn over model.  
494 
 
495 
● The effect of cortisol concentration and genetics on the correlation of (nor)clozapine 
496 
concentrations and/or ratio on the metabolic markers 
497 
  
498 
 
499 
Other study parameters 
500 
Demographics 
501 
- Sex (male/female). 
502 
- Age (year). 
503 
- Estradiol indirectly (premenopausal/postmenopausal female / male). 
504 
- Ethnicity (Caucasian/African-Caribbean/Asian/other). 
505 
 
506 
Clinical parameters 
507 
- Indication for clozapine use (diagnosis treatment resistant schizophrenia or schizoaffective 
508 
disorder). 
509 
- Duration of illness. 
510 
- Duration of clozapine treatment. 
511 
- Dosage scheme. 
512 
- Baseline PANSS-6, regarding severity of schizophrenia  
513 
 
514 
Other parameters 
515 
- Tobacco smoking behaviour (yes/no, including amount (prospectively)) and vaping 
516 
(yes/no). 
517 
- Interacting co-medication such as strong CYP3A4-inductors (gemfibrozil, apalutamide, 
518 
carbamazepine, efavirenz, enzalutamide, phenobarbital, phenytoin, St. John’s wort, 
519 
lumacaftor, mitotane, nevirapine, primidone, rifabutin, rifampicin) or level increasing drugs 
520 
such as fluvoxamine, fluoxetine or sertraline, CYP1A2 inhibitors such as ciprofloxacin, or 
521 
inductors such as omeprazole, oral contraceptives and valproic acid). 
522 
- Gemfibrozil and valproic acid use, as both inhibit renal clearance of norclozapine (38). 
523 
- Patient specific target level if this falls outside of the therapeutic window of 350-700 μg/L, in 
524 
order to be able to identify these levels as expected normal target levels instead of toxic or 
525 
caused by (in)adherence, interactions or infection.  
526 
- Reason for blood collection if available (routine or otherwise: possible toxicity, infection, 
527 
CRP and BSE if recently measured and known, change in dosage or co-medication). 
528 
 
529 
  
530 
Randomisation, blinding and treatment allocation 
531 
Not applicable 
532 
Study procedures 
533 
Not applicable, patients will not undergo any (additional) procedures for research 
534 
purposes. All samples will be taken as part of standard care for treatment purposes.  
535 
Withdrawal of individual subjects 
536 
Not applicable 
537 
Specific criteria for withdrawal (if applicable) 
538 
Not applicable 
539 
Replacement of individual subjects after withdrawal 
540 
Not applicable 
541 
Follow-up of subjects withdrawn from treatment 
542 
Not applicable 
543 
Premature termination of the study 
544 
Not applicable 
545 
 
 
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SAFETY REPORTING 
547 
This chapter is not applicable as this is a non-interventional, retrospective study that involves 
548 
standard care.  
549 
 
 
550 

 
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STATISTICAL ANALYSIS 
551 
 
552 
Primary study parameter(s) 
553 
 
554 
PK analyses will be performed using nonlinear-mixed-effects modelling (NONMEM) software 
555 
(version 7.4.2, ICON Development Solution, Gaithersburg, MD, USA). Software like R 
556 
(www.rproject.org), Pirana and Xpose (version 4.3.2, Niclas Jonsson and Mats Karlsson, 
557 
Uppsala, Sweden) will be used for graphical display and evaluation of the models (including 
558 
goodness of fit plots).  
559 
 
560 
Previously Beex et al., while studying the comparability of pharmacokinetics of clozapine and 
561 
norclozapine with once- and twice-daily dosing, developed a population PK model using 
562 
NONMEM (13). Furthermore, their popPK model has incorporated patient-specific covariate 
563 
smoking to further refine pharmacokinetic parameter estimates and improve the accuracy of 
564 
dosing. Their base model describes clozapine by a two-compartment model with a transit 
565 
compartment describing the delay in absorption and linear elimination consisting of combined 
566 
excretion and formation of the metabolite norclozapine. This model was extended with a one-
567 
compartment model for norclozapine with first-order elimination (Figure 2, excerpt from their 
568 
study).  
569 
 
570 
 
571 
 
572 
Their final model tested for several covariates: total body weight, BMI, age, sex, AGP 
573 
concentrations, albumin concentrations, CRP, and smoking status. Only smoking had a 
574 
significant effect on clozapine clearance (not on norclozapine clearance). Based on this 
575 
model, clearance of clozapine is 55% higher in smokers than in non-smokers. Table 2 is an 
576 
excerpt of the overview of parameter estimates of both base and final model. 
577 
 
578 

 
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579 
 
580 
Subsequently, their developed final population PK model will be used to predict 
581 
concentration-time profiles for clozapine and norclozapine under various dosages and 
582 
sampling schedules on basis of individual PK parameters obtained by Bayesian analysis. 
583 
Collected data will be inserted into R as well as relevant patient-specific demographic, 
584 
clinical and laboratory data. A new database suitable for PK analyses of clozapine will be 
585 
constructed in NONMEM. In order to do this the clozapine dose, measured concentration 
586 
data along with time of intake and sampling time will be taken into account. Subsequently, 
587 
PK profiles will be predicted. The reference window to be targeted will be 350-700 μg/L for 
588 
clozapine. Deviation from this window will be allowed for patients who, according to their 
589 
psychiatrist or nurse specialist or other practitioner, have a higher or lower target level than 
590 
the defined therapeutic window with good therapeutic effect. This will only be allowed based 
591 
on the psychiatrist/nurse specialist/practitioner’s information and help identify these levels as 
592 
expected normal target levels instead of toxic or caused by inadherence, interactions or 
593 
infection.  
594 
 
595 
The first step before researching the primary endpoint, is the performance of an external 
596 
validation (see secondary study parameter(s) below). Afterwards, the current study 
597 
population will be studied in order to research the primary endpoint. To analyse this, the 
598 
concentration-time data will be used to develop PK/PD turn-over models to estimate the 
599 
parameters (Kin, Kout, Emax and EC50) linking the pharmacokinetics of clozapine and 
600 
norclozapine to HbA1c serum levels. Once the structural PK/PD model is developed, the 
601 
between subject variability and residual unexplained variability of the estimated 
602 
pharmacokinetic parameters will be assessed.  
603 

 
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604 
A univariate covariate analysis will be performed to check for associations between the 
605 
patient-related factors on parameters of the PD model using a χ2 test-statistic. Covariates 
606 
with a significant association in the univariate analysis will be included in the multivariate 
607 
analysis. Covariates that remain significant in the multivariate analysis will be included in the 
608 
final pharmacodynamic model. This final model will be validated using visual predictive check 
609 
(VPC’s) and using a bootstrap procedure the robustness of the model will be tested. The 
610 
latter two procedures will be performed using Piraña and Pearl speaks NONMEM (PsN) 
611 
software. 
612 
 
613 
Secondary study parameter(s)  
614 
 
615 
For the external validation of the model, predicted profiles will be compared to the observed 
616 
data of our population (which is regarded as an external data set). Prediction error (PE) for 
617 
each predicted concentration (PEi) was defined through the following equation: 
618 
 
619 
 
620 
 
621 
where Observedi and Predictedi  represent the ith observed and predicted concentrations, 
622 
respectively. The root mean square error (RMSE) prediction error was also calculated using 
623 
the following equation: 
624 
 
625 
where n represents the total number of concentrations. A population PK model was regarded 
626 
as valid for our clinical settings when both the mean and median values of PE were less than 
627 
20%. As stated earlier, the external validation of the model by Beex et al. will be performed 
628 
before researching our primary goal. 
629 
 
630 
Based on the predicted concentration-time profile for all participants, the percentage 
631 
difference in predicted clozapine and norclozapine concentrations between different 
632 
sampling times will be calculated. The 12-h time point will be set as reference and compared 
633 
to concentrations predicted (or measured if available) at 10, 11, 13 and 14 hours-post intake 
634 
concentrations. Plasma concentrations taken at steady state should be comparable to others 
635 
within an acceptable time window. As stated earlier, concentrations are regarded as 
636 
comparable if the total percentage difference does not exceed 20%. This includes expected 
637 
bias and imprecision in measurement and interoccassion and interindividual variability. This 
638 
difference is defined as clinically relevant for this study, according to which concentrations 
639 
would no longer be deemed similar and thus corresponding to the same time window or an 
640 
adequate sampling time or window.  
641 
Statistical significance will be tested through generalized linear mixed models for proportions, 
642 
in which the proportion of clinically relevant differences per time point versus t=12 will be 
643 
compared. For descriptive statistics, continuous outcomes are presented as medians and 
644 
ranges between minimum and maximum values with corresponding confidence intervals, 
645 
when applicable. Categorical outcomes are reported as numbers and proportions (n (%)). 
646 
Two tailed p-values of < 0.05 based on χ2 with one degree of freedom were considered 
647 
statistically significant. 
648 
 
649 
Based on the clinical patient data, predicted concentration-time profiles, averaged for each 
650 
subject, will be portrayed for different oral dosages. The (new) optimal sampling window will 
651 
be based on the time window during which the difference in measured clozapine and 
652 

 
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norclozapine concentrations in the predicted profile is smaller than 20% as well as within the 
653 
defined therapeutic window of 350-700 μg/L for clozapine. If needed, deviation from this 
654 
window will be allowed for patients with different target levels as described before. This will 
655 
be only be allowed based on the psychiatrist/nurse specialist/practitioner’s information. If a 
656 
window cannot be found, then sampling times will be noted during which clozapine 
657 
concentrations fall within the defined therapeutic window. The student’s t-test for paired data 
658 
was used to analyse differences in median serum concentrations across sampling times.  
659 
 
660 
Aside from the metabolic parameters mentioned in the primary endpoint, correlation with 
661 
other metabolic parameters (such as fasting glucose, insulin, HOMA-IR, triglycerides, HDL, 
662 
LDL, total cholesterol, BMI, abdominal circumference, blood pressure and heart rate), ANC, 
663 
TWC, GASS-C and BSFS with the concentrations will be studied. The concentration-time 
664 
data will be used to develop PK/PD turn-over models to estimate the parameters (Kin, Kout, 
665 
Emax and EC50) linking the pharmacokinetics of clozapine and norclozapine to these 
666 
parameters. Once the structural PK/PD model is developed, the between subject variability 
667 
and residual unexplained variability of the estimated pharmacokinetic parameters will be 
668 
assessed. A univariate covariate analysis will be performed to check for associations 
669 
between the patient-related factors on parameters of the PD model using a χ2 test-statistic. 
670 
Covariates with a significant association in the univariate analysis will be included in the 
671 
multivariate analysis. Covariates that remain significant in the multivariate analysis will be 
672 
included in the final pharmacodynamic model. This final model will be validated using visual 
673 
predictive check (VPC’s) and using a bootstrap procedure the robustness of the model will 
674 
be tested. The latter two procedures will be performed using Piraña and Pearl speaks 
675 
NONMEM (PsN) software. 
676 
 
677 
Using an univariate analysis in NONMEM the association of patient-related factors on the 
678 
pharmacokinetic parameters of clozapine will be tested using a χ2 test-statistic. The 
679 
covariates that will be included in the univariate analysis include age, gender, 
680 
premenopauzal/postmenopausal female/male, ethnicity, weight, height, CRP, BSE, smoking 
681 
behaviour, comedication with fluvoxamine (dosage) and genotypes. Covariates with a 
682 
significant association in the univariate analysis will be included in the multivariate analysis. 
683 
Covariates that remain significant in the multivariate analysis will be included in the final 
684 
pharmacokinetic model. This final model will be validated using visual predictive check 
685 
(VPC’s) and using a bootstrap procedure the robustness of the model will be tested. The 
686 
latter two procedures will be performed using Piraña and Pearl speaks NONMEM (PsN) 
687 
software. The final pharmacokinetic model will then be used to create concentration-time 
688 
curves for each individual participating in the PK/PD study. 
689 
 
690 
Other study parameters 
691 
 
692 
Baseline characteristics will be depicted by means of summary statistics: mean ± SD for 
693 
normally distributed continuous variables and median ± interquartile range for non-normally 
694 
distributed continuous variables. Proportions will be calculated for categorical variables. 
695 
 
696 
Interim analysis (if applicable) 
697 
Not applicable. 
 
698 

 
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ETHICAL CONSIDERATIONS 
699 
This is an observational cohort study in which the data will be collected retrospectively. The 
700 
subjects included in this study will not undergo any additional procedures. After gaining 
701 
permission by the Advisory Committee on Local Feasibility of the GGZ-NHN, all subjects will 
702 
be asked informed consent before participation. Informed consent will be asked in 
703 
accordance with the General Data Protection Regulation (GDPR). Their Electronic Health 
704 
Record (EHR) will be viewed by the treating psychiatrist, physician, nurse specialist, or other 
705 
doctor/practitioner. Data collected by the practitioner will be used for the dataset. 
706 
Additionally, information such as plasma levels is collected through Labosys rapports for 
707 
which informed consent is given according to standard procedure for blood collection. 
708 
Remnant material from laboratory procedures performed as part of routine clinical care will 
709 
be collected and stored.  
710 
 
711 
Regulation statement 
712 
This research will be conducted in accordance with Good clinical practice (GCP) and GDPR. 
713 
 
714 
Recruitment and consent 
715 
This study will be submitted to the local review committee of the GGZ-NHN. Letter and 
716 
informed consent are attached as separate documents.  
717 
 
718 
Objection by minors or incapacitated subjects (if applicable) 
719 
Not applicable. 
720 
Benefits and risks assessment, group relatedness 
721 
Not applicable. 
722 
Compensation for injury 
723 
Not applicable. 
724 
Incentives (if applicable) 
725 
Not applicable. 
726 
 
727 
 
728 
 
 
729 

 
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ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION 
730 
Handling and storage of data and documents 
731 
 
732 
Data regarding sex, age, plasma levels of both clozapine and norclozapine, time of intake 
733 
and sampling time will be collected through reports from Labosys (Alkmaar). This data is 
734 
sourced from the blood collection site, where requesting psychiatrists, physicians, nurse 
735 
specialists, or other doctors/practitioners are required to fill in this information. All specified 
736 
laboratory parameters (see above) from both GGZ-NHN and other sources of requests for 
737 
laboratory diagnostic procedures (i.e. general practitioner, specialist internal medicine) within 
738 
the period of follow-up will be used for analysis. Time of intake will be collected from the 
739 
patients before the sampling time at the collection site (Starlet) according to standard 
740 
practice. Due to new ISO regulations, there are upcoming changes in standard practice for 
741 
blood collection at the main site. These changes will require requesting doctors to fill in more 
742 
information at every blood collection request such as: smoking (yes/no), dosage, reason for 
743 
blood collection (routine or otherwise: possible toxicity, infection, change in dosage). Once 
744 
these changes have followed through, this data can also be collected through Labosys. Co-
745 
medication will be retrieved through the patient’s public pharmacy.  
746 
 
747 
Before implementation of these changes, information on smoking, dosage and reason for 
748 
blood collection will be collected through the treating psychiatrist/nurse specialist/practitioner 
749 
who has access to the patient’s electronic health record if needed. Other information to be 
750 
gathered by the treating psychiatrist/nurse specialist/practitioner includes: weight and BMI , 
751 
ethnicity, diagnosis (of schizophrenia or other psychiatric disorder), pregnancy, interacting 
752 
co-medication (including newly started, stopped medication and changes in dosages within a 
753 
week of sampling time). Data regarding metabolic parameters such as fasting glucose, 
754 
serum level HbA1c, triglycerides, HDL, LDL, total cholesterol, abdominal circumference, BMI 
755 
and blood pressure and heart rate will be collected through reports through Nexus and 
756 
mConsole (Alkmaar), as well as the absolute neutrophil count (ANC) and the outcomes of 
757 
the Bristol Stool Form Scale (BSS) assessment, the Glasgow Antipsychotic Side-effect Scale 
758 
for Clozapine (GASS-C) questionnaire and the Positive and Negative Syndrome Scale 
759 
(PANSS-6) questionnaire will be collected through Nexus and mConsole (Alkmaar). Clinical 
760 
data needed from the dedicated includers (co-investigators, with an affiliation to the GGZ-
761 
NHN) will be collected through a simple case report form. This information will be collected at 
762 
the moment of inclusion and at the end of the study (retrospectively) in case any changes 
763 
occur during the study period. Biologic material collected as part of laboratory investigation 
764 
as part of routine clinical care after obtaining informed consent will be stored at the 
765 
laboratories of the North West Clinics until all laboratory procedures pertaining the 
766 
investigation are completed (metabolic enzymes, the genes CYP2C19 and CYP3A4 and 
767 
genes related to metabolic markers). 
768 
 
769 
Missing data (unless described in the inclusion or exclusion criteria) will be allowed. Native 
770 
speakers, if available, will be asked to deduct a patient’s ethnicity based on their name in 
771 
case the ethnicity is unknown before regarding this as missing data. If data, such as BMI or 
772 
CRP, are not reported in the patient’s medical file the event will be recorded as ‘missing data’ 
773 
or as not occurred.   
774 
 
775 
During the study period, data collected from the psychiatrist/nurse 
776 
specialist/practitioner/dedicated includer will be linked to data collected from Labosys 
777 
according to date of birth and name. Patient data will be encoded. The key will be 
778 
safeguarded by the coordinating investigator and principal investigators. Furthermore 
779 
collected data will be stored for 15 years. 
780 
 
781 
The results, discussion and conclusion of the study will be reported and shared in an 
782 
abstract, manuscript and registration presentation in accordance to the principles of the 
783 

 
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ICMJE guidelines. Information concerning this study’s processes and data is confidential and 
784 
its rights belong to GGZ-NHN, NWZ Alkmaar and Amsterdam UMC.  
785 
 
786 
 
787 
Monitoring and Quality Assurance  
788 
Not applicable 
789 
Amendments  
790 
Not applicable 
791 
Annual progress report 
792 
Not applicable 
793 
Temporary halt and (prematurely) end of study report 
794 
Not applicable 
795 
Public disclosure and publication policy 
796 
Not applicable 
797 
 
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STRUCTURED RISK ANALYSIS  
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Not applicable  
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