Development of an optimal sampling strategy for clozapine to predict area under the concentration-time curve ratios and the clinical validation of the dried blood spot analysis for clozapine

Clozapine is one of the most effective antipsychotic drugs currently available
that is particularly used in patient who are refractory or intolerant to other
antipsychotics. The plasma concentration of clozapine is very important for the
treatment efficacy of the medicine. Unfortunately, no clear linear correlation
between the dose and plasma concentration of clozapine is observed. This is
caused by various genetic, individual and environmental factors and also
co-morbidity may influence the plasma concentration.
Since many factors affect the clozapine plasma concentration in patients,
therapeutic drug monitoring (TDM) is highly recommended and prevailed in
clozapine treatment. Concentration measurements have become common practice in
the treatment of a patient with clozapine. The clozapine plasma concentration
is generally determined at the start of the therapy, when there is a poor
response on the drug, in case of expected interactions, the suspicion of an
intoxication, infections, the stop or start of smoking or control therapy in
case of higher doses. Plasma levels above 350 µg/L seem to give a better
therapy-response, although response may also occur below 350 µg/L.
By TDM, both the clozapine plasma levels and the levels of its major metabolite
desmethylclozapine are determined using the trough level concentration. The
relationship between the clozapine and desmethylclozapine concentrations gives
an impression of the pharmacokinetic profile of clozapine However, trough-level
monitoring for clozapine/ desmethylclozapine is probably not optimal in all
cases. Patients with the same trough concentration may not have the same drug
exposure as judged by the pharmacokinetic profile. The area under the plasma
concentration time curve (AUC) might be a better indicator of clozapine
exposure. However, clozapine AUC-0-12h measurement requires multiple samples
from patients over 12 hours, what makes it intensive, time-consuming and
uneconomical. A limited-sampling model that can estimate the AUC of clozapine
from a reduced number of blood samples may help to overcome these problems. In
this research, we are going to determine which sample point or combination of
sample point(s) gives the best estimate of the AUC of clozapine using a
population PK model of clozapine.
In order to make TDM even more easier and patient friendly, Dried Blood Spot
(DBS) sampling will be used. DBS samples are dry, more stable and easier to
obtain as compared to conventional samples. We assume that the DBS method is
suitable for determining clozapine plasma levels in patients with schizophrenic
disorders especially in the ambulatory setting. However, the DBS method needs
to be validated in a clinical setting to ensure its reliability. Therefore in
this research protocol, analysis of venous blood samples is compared with
analysis of dried blood spots.
In future research the correlation between the clozapine plasma concentration,
the given dosage and the clinical effect will be examined. A model to predict
the exposure as determined by AUC based on a population PK model with limited
sampling, will be very useful in this research. Furthermore, DBS sampling will
be a great advantage in collecting and transporting blood samples in different
settings, DBS samples are dry and stable and easy to transport with usual
transport systems (postal mail). In addition, they can be stored at ambient
conditions and a refrigerator is not requirement as is with liquid samples.

- To establish a model for TDM and calculate the AUC in patients treated with
clozapine.
- To clinically validate the DBS analysis method for clozapine.
- To determine which sample points or combination of sample point(s) gives the
best estimate of the AUC using the developed population PK model.
- To perform an external validation of the model using
concentration-time data of clozapine from Russian patients.

a) Recruitment of 15 schizophrenic patients (male or female; Caucasian
ethnicity; aged 18 till 55 years) under stable mono-therapy with 200 - 600 mg
clozapine in a single morning dose for at least two weeks and excluding the
usage off all predefined concomitant medication. All used co-medication should
be recorded including dosage used and generic names.

b) Collecting venous blood samples of about 5 ml before intake of clozapine and
at 2, 4, 6, 8, and 12 hrs after oral administration of clozapine (when 12 hrs
is feasible).

c) Finger prick DBS (2-3 blood drops) before intake of clozapine and at 2, 4,
6, 8 and 12 hrs after oral administration of clozapine.

d) Venous DBS before intake of clozapine and at 2, 4, 6, 8 and 12 hrs after
oral administration of clozapine prepared by pipetting 3 * 50 µl of the venous
blood sample onto a dried blood spot paper.

e) Storage of all obtained venous plasma samples at -80C.

f) Clozapine serum concentration-time data for external validation will be made
available by
The Mental Health Research Institute Tomsk, as part of a collaborative
partnership.

Patients may experience minor discomfort from blood sampling and the finger
prick. The finger prick is however widely applied for self-test glucose
monitoring in diabetic patients and self-test INR control in patients using
anticoagulants and discomfort is estimated to be mild. There are no advantages
for the participant in this study.