Br J Clin Pharmacol 1998; 46: 453–459

Pharmacokinetics of clozapine and its metabolites in psychiatric patients:

plasma protein binding and renal clearance
G. Schaber,1 I. Stevens,2 H. J. Gaertner,2 K. Dietz3 & U. Breyer-Pfaff1
1
Department of Toxicology, 2Department of Psychiatry, and 3Department of Medical Biometry, University of Tübingen, Tübingen, Germany

Aims N-Desmethylclozapine and clozapine N-oxide are major metabolites of the

atypical neuroleptic clozapine in humans and undergo renal excretion. The aim of
this study was to investigate to what extent the elimination of these metabolites in
urine contributes to the total fate of clozapine in patients and how they are handled
by the kidney.
Methods From 15 psychiatric patients on continuous clozapine monotherapy, blood
and urine samples were obtained during four 2 h intervals, and clozapine and its
metabolites were assayed in serum and urine by solid-phase extraction and h.p.l.c.
Unbound fractions of the compounds were measured by equilibrium dialysis.
Results The following unbound fractions in serum were found (geometric means):
clozapine 5.5%, N-desmethylclozapine 9.7%, and clozapine N-oxide 24.6%. Renal
clearance values calculated from unbound concentrations in serum and quantities
excreted in urine were for clozapine on average 11% of the creatinine clearance,
whereas those of N-desmethylclozapine and clozapine N-oxide amounted to 300
and 640%, respectively. The clearances of unbound clozapine and N-desmethylclozap-
ine increased with increasing urine volume and decreasing pH. All renal clearance
values exhibited large interindividual variations. The sum of clozapine and its
metabolites in urine represented on average 14% of the dose.
Conclusions Clozapine, N-desmethylclozapine and clozapine N-oxide are highly
protein-bound in serum. Clozapine is, after glomerular filtration, largely reabsorbed
in the tubule, whereas the metabolites undergo net tubular secretion. Metabolic
pathways alternative or subsequent to N-demethylation and N-oxidation must make
major contributions to the total fate of clozapine in patients.
Keywords: clozapine N-oxide, clozapine, N-desmethylclozapine, plasma protein binding,
renal clearance, tubular secretion

17±8 h) [2, 6]. The two so-called ‘major’ metabolites,

Introduction
which can be measured in plasma [7–10] are N-desmethyl-
After the reintroduction of clozapine into the market and clozapine and clozapine N-oxide (Figure 1). Their enzymatic
the opening of new markets for clozapine in the late 1980s, formation in human liver microsomes or by expressed
clinical and scientific interest in this atypical neuroleptic was cytochrome P450 (CYP) species has been the subject of
reawakened. Its high therapeutic efficacy and the virtual some recent investigations. N-Oxidation of clozapine was
absence of adverse effects on extrapyramidal motor func- mainly catalysed by CYP3A4 [11, 12] and a flavin
tion—a phenomenon originally detected in Central Europe monooxygenase [13]. As indicated in Figure 1, the reaction
around 1970 [1]—has been confirmed in numerous recent is reversible in vivo [14]. Major contributions to demethyl-
studies; moreover, clozapine has proven efficacious in some ation by CYP3A4 and CYP1A2 were found in one study
patients refractory to other neuroleptics [see 2–4 for reviews]. [11], whereas kinetic data of expressed enzymes indicated
Clozapine’s clinical importance has stimulated investi- an additional important part of CYP2C19 and CYP2D6
gation of its kinetics in patients [2], although knowledge is [12]. Single-dose kinetics of clozapine in volunteers were,
still incomplete, particularly with regard to the metabolic however, not affected by deficiencies of CYP2C19 or
fate. When administered orally, clozapine was absorbed CYP2D6 [15]. Clozapine and the two metabolites have
nearly completely [5], but mean values reported for its been quantified in urine collected from five patients and a
systemic availability were 27% and 47% only [6], apparently ratio of clozapine: N-desmethylclozapine: clozapine N-
due to a highly variable first-pass metabolism. Elimination oxide of 15152 has been reported [16]. Recent measurements
half-lives in patients were shorter after single doses (8 h) in three patients resulted in a mean ratio of 15458.7 and
than after achievement of steady-state (14 h, 16 h or recoveries corresponding to 1.7–13% of the dose within
12 h [17]. Apart from these data, no information is available
Correspondence: Dr U. Breyer-Pfaff, Department of Toxicology, University of on the quantitative importance of the two metabolites
Tübingen, D-72074 Tübingen, Germany. for the overall metabolic fate of clozapine in patients.

© 1998 Blackwell Science Ltd 453

G. Schaber et al.

Patients and study design

Twelve inpatients and three outpatients with an ICD diagnosis
of schizophrenia (Table 1) gave their written consent for
participation in the study after a half hour discussion with a
psychiatrist. None of them suffered from cardiac, renal or
hepatic disease. Clinical laboratory surveillance revealed
elevated liver enzyme values in some patients, but in no case
did the values exceed 150% of the upper limit of the reference
range. Patient TP was treated with prednisolone for idiopathic
thrombocytopenic purpura. In all of the patients, clozapine
was the only psychotropic drug taken; the dose and dosing
schedule were held constant for at least 7 days and were not
changed during the investigation. Inpatients took their
clozapine doses with supervision at 08.00 h, 11.45 h, 17.45 h,
and 21.30 h provided that four daily doses were given. Dosing
schedules were individualized according to clinical need and
tolerability. Seven patients received a morning dose that
corresponded to 20–33% of the daily dose (designated as the
group with even distribution of doses), in three patients the
morning dose was 15–18% of the daily dose and five patients
took clozapine at bedtime only or with an additional dose in
the afternoon. At 08.00 h, 10.5 h after the last dose and
Figure 1 Structural formulas of clozapine and its metabolites and
before ingestion of the morning dose, the patients were asked
biotransformation pathways interconnecting them. to empty the bladder and 18 ml blood was taken for the
preparation of serum that was frozen at −20° C. After 2, 4,
6, and 8 h the patients collected urine and additional blood
samples were drawn. The volume and pH of the urine were
Furthermore, nothing has apparently been published on the measured and aliquots were stored at −20° C. One of the
renal mechanisms that govern the excretion of clozapine authors stayed with the patients during the investigational
and its metabolites. period in order to ensure compliance with the time schedule.
When a single clozapine dose was given to volunteers,
they excreted in urine further major metabolites that had
been formed by aromatic hydroxylation with or without
Analytical methods
removal of the Cl substituent [5]. Some of these had The procedure for solid-phase extraction of clozapine and
previously been identified in patient urine [18]. Nothing is its metabolites from serum was modified from that of
known about the enzymes that catalyse their formation or Weigmann & Hiemke [8]. Cartridges with C18-silica gel
on their quantitative importance in patients. were washed with 1 ml 0.5 m HCl, 1 ml acetonitrile, 2 ml
In the present investigation, clozapine, N-desmethylcloza- methanol, and 3 ml water. Serum (1 ml) was mixed with
pine and clozapine N-oxide were analysed in timed serum 1 ml 1% (w/v) Na2CO3 and applied with suction. After
and urine samples of 15 patients during steady-state treatment washing with 2×0.5 ml water and 0.5 ml 50% (v/v)
in order to elucidate the quantitative role of their renal methanol, substances were eluted with 2×1 ml 0.1 m acetic
excretion for the total fate of clozapine and their handling acid in methanol. The eluate was evaporated under a stream
by the human kidney. of nitrogen at 35–40° and the residue dissolved in 0.2–1 ml
of h.p.l.c. eluent, 0.1 ml of which was injected.
Separation was achieved on a 4.6×200 mm h.p.l.c.
Methods
column with C18-silica gel as the stationary phase and
10 mm perchloric acid adjusted with NaOH to
Materials
pH 2.5/acetonitrile (65535, v/v) as the mobile phase, flow
−1
Clozapine, N-desmethylclozapine and clozapine N-oxide 1 ml min . Peaks were detected by their absorption at
were kindly donated by Wander (Bern, Switzerland). 290 nm and quantified by their heights relative to those of
Cartridges with 100 mg C18-silica gel (Bond Elut C18 ) external standards containing 0.025–0.2 nmol (about
produced by Analytichem (Harbor City, CA, USA) were 8–70 ng ) in 0.1 ml. Retention times were 7 min for
purchased from ict Handelsgesellschaft (Frankfurt am Main, clozapine, 5.8 min for N-desmethylclozapine and 8.6 min
Germany). Thin-layer chromatography (t.l.c.) glass plates for clozapine N-oxide. From 10 samples of blank
coated with nonfluorescent silica gel (Kieselgel 60) and serum spiked with concentrations of 0.05–1 mm (about
−1
organic solvents were obtained from Merck (Darmstadt, 20–350 ng ml ), the three compounds were recovered by
Germany), C18-silica gel for h.p.l.c. (Nucleosil 5 C18 ) from 95±2%, 82±3% and 97±4% (means±s.d.), respectively.
Macherey-Nagel (Düren, Germany). Blank plasma or serum Standard curves constructed from the results of these
and blank urine were obtained from five healthy drug-free recovery experiments were linear with coefficients of
donors recruited from laboratory staff. correlation of 0.994–0.998 and went through the origin.

454 © 1998 Blackwell Science Ltd Br J Clin Pharmacol, 46, 453–459

 Renal clearance of clozapine and metabolites

Table 1 Demographic and clinical data

of patients. Clozapine Abnormal
Age Weight ICD-10 dose Additional laboratory
Name Sex (years) (kg ) Smoker diagnosis (mg day−1) medication data

TE F 27 57 No F 23.9 200 Dihydroer- —

gotamine
MR F 29 79 No F 20.0 250 Oxybutynin, —
desmopressin
DG F 30 59 No F 20.2 400 — —
EL F 33 60 No F 20.3 350 — —
SH F 36 62 No F 20.0 75 OC —
PB F 37 74 Yes F 20.0 175 OC —
TW M 19 59 No F 20.0 600 — —
WM M 20 79 Yes F 20.0 350 Antacid Alk.
phosphatase
GR M 26 73 Yes F 20.0 650 — GLDH, alk.
phosphatase
EK M 27 77 Yes F 20.0 300 — —
MG M 27 91 No F 20.4 350 — —
TP M 35 80 No F 20.3 350 Prednisolone Thrombocytes
VM M 39 82 Yes F 20.1 200 — ALAT
DW M 56 71 Yes F 20.5 400 — —
TR M 57 108 No F 20.0 125 — —

OC, oral contraceptive; GLDH, glutamate dehydrogenase; ALAT, alanine aminotransferase.

With each series of analyses, two control samples of blank tube and the residue concentrated in the tip. For t.l.c., it
serum were run, to one of which 1 mm clozapine and was dissolved in 50 ml hexane/1-propanol of which 40 ml
desmethylclozapine and 0.5 mm clozapine N-oxide had been was applied as a spot on a 20×20 cm plate. Samples and
added, while the other one was spiked with half these standards containing 250–2000 ng clozapine were applied
concentrations. In 12 series, the recoveries from these in alternating fashion and the plate was developed in
samples were 98±1.5% for clozapine, 83±2.5% for 2-propanol/tert-butyl methyl ether/25% ammonia/water
desmethylclozapine, and 97±1.5% for clozapine N-oxide. (125650.7550.75, v/v/v/v) to a height of 12 cm above
Values measured in patient serum were corrected by the baseline. The dried plate was scanned at 290 nm by
recoveries of the control samples run in parallel. All analyses reflectance photometry [7] and the clozapine peaks (Rf
were performed in duplicate with coefficients of variation 0.75) were quantified by the trapezoidal rule. Standard
found to be 4% for clozapine, 5% for desmethylclozapine curves were linear up to 750 ng clozapine. In 11 series,
and 10% for clozapine N-oxide (n=51). Concentrations as mean recoveries from two control samples each were
−1
low as 16 ng ml could be quantified without a loss 100±3%. Duplicate analyses of urine samples gave a
of accuracy. The detection limit with a signal/noise ratio coefficient of variation of 14% (n=56). A control consisting
−1
of 3–4 was 2 ng ml for all three substances, and no of blank urine with clozapine N-oxide added in a quantity
interferences were observed in blank serum. corresponding to that found in the patient sample was run
N-Desmethylclozapine and clozapine N-oxide were ana- with each urine sample. A mean of 0.2% of the N-oxide
lysed in urine as described above, but samples were diluted was found as clozapine, and this amount (corresponding on
25 fold with water before solid-phase extraction. Standard average to 16% of the clozapine originally present) was
curves constructed from six samples with final concentrations subtracted from the clozapine quantity measured.
of 0.25–2 mm desmethylclozapine and clozapine N-oxide Protein binding of clozapine and its metabolites was
were linear and went through the origin; recoveries from determined by equilibrium dialysis in 2×1 ml Teflon cells
these samples amounted to 92±4.5% and 91±4%, respect- with serum adjusted to pH 7.4 by CO2 [19]. Equilibrium
ively. In 11 series of analyses, mean recoveries from two with the buffer compartment containing 100 mm sodium
control samples each of which were spiked with final phosphate pH 7.4 and 30 mm NaCl was achieved within
concentrations of 1 or 0.5 mm desmethylclozapine and 2 or 4 h. The pH was checked regularly after dialysis and found
1 mm clozapine N-oxide, recoveries were 94±1% and to deviate from 7.4 by not more than 0.05 units. Weighed
93±1%, respectively. Coefficients of variation in duplicate aliquots of serum and buffer were stored frozen and
analyses amounted to 6 and 7%, respectively (n=56). analysed by the procedure described above. Unbound
A different extraction procedure was required for fractions (fu ) were calculated by dividing the concentrations
measuring clozapine in urine, because artifactual reduction in the buffer by those in the serum after dialysis. When
of clozapine N-oxide had to be kept to a minimum. Urine five blank plasma or serum samples were spiked with
(3 ml) was adjusted to pH 7.5 with 1% Na2CO3 and 0.25–1.5 mm clozapine and desmethylclozapine and
immediately extracted with 2 ml hexane/1-propanol (9753, 0.125–0.75 mm clozapine N-oxide, the recoveries from the
v/v) by shaking for 0.5 min and centrifuging. A measured two compartments amounted to 104±6%, 83±6% and
aliquot of the organic layer was evaporated in a conical 104±4%, respectively.

© 1998 Blackwell Science Ltd Br J Clin Pharmacol, 46, 453–459 455

G. Schaber et al.

−1 −1
In two serum samples from each patient and in all urine (mg kg ) ). Geometric means were significantly lower
samples, creatinine concentrations were measured using an in the eight evaluable male than in the six female patients
−1 −1 −1
enzymatic test kit (Creatinin PAP, Boehringer, Mannheim, (94 vs 145 ng ml (mg kg ) , F1,37=70.2, P<0.0001).
Germany). Lower mean values were also observed in the six smokers
−1
compared with eight nonsmokers (80 vs 144 ng ml
−1 −1
(mg kg ) , F1,37=187, P<0.0001). In addition, there
Pharmacokinetic evaluation
was a highly significant difference in the N-desmethylclozap-
Renal clearances of creatinine (CLCR ) and of clozapine and ine/clozapine ratios in serum between smokers and nonsmo-
its metabolites (CLR ) were calculated by dividing their kers (0.78 vs 0.48, F1,37=143, P<0.0001). Relative serum
excretion rates measured as the quantities in urine per 2 h clozapine levels did not differ between patients with an
interval by the mean serum concentration of the respective even distribution of doses over the day and those receiving
compound (Cpl ) during this interval. Division of the no or a small morning dose. Clozapine N-oxide was
excreted quantities by the unbound concentrations in serum measurable in all serum samples, though at a mean
(Cpl×fu ) resulted in the renal clearances of unbound concentration of 19% only of the clozapine level. The
clozapine and metabolites (CLRu ). The ratio of CLRu and logarithms of the mean concentrations of N-desmethylcloza-
the creatinine clearance CLCR (CLRu/CLCR ) served to pine and clozapine N-oxide in the individual patients
estimate the extent of net renal secretion or reabsorption. showed close linear correlations with the values for clozapine
Drug and metabolite quantities in urine as a percentage of (r=0.92 and 0.88, respectively, P<0.001).
daily dose were obtained by extrapolation from the 8 h The free fractions of clozapine, N-desmethylclozapine
sampling period to 24 h. In five patients, complete 24 h and clozapine N-oxide in serum or plasma were independent
urine samples were available and the desmethylclozapine and of the concentrations in the range investigated. Values
clozapine N-oxide quantities measured in them proved to measured in plasma from four volunteers were in the same
be on average 108% and 85%, respectively, of the values ranges as those in serum from patients (Table 2). Inter-
obtained by extrapolation. As a measure of the individual individual differences were generally small, though they
elimination capacity for clozapine, the drug concentration exceeded intraindividual differences in repeated measure-
−1
in the serum was divided by the daily dose in mg kg . ments (Table 2). In one patient’s serum, the free fraction of
−1
This dose-related serum concentration (in ng ml clozapine N-oxide was as low as 9.5% and in a sample taken
−1 −1
(mg kg ) ) is proportional to the reciprocal of the four weeks later, it was 10.2%, whereas in all other patients
apparent oral clearance. and volunteers the values varied between 21 and 34%.
Patient WG had to be excluded from evaluations relating Measurements of compounds excreted in urine within four
to the daily dose because of suspected noncompliance with 2 h intervals revealed minor quantities of clozapine that
the prescribed drug regimen. His initial serum clozapine and (when extrapolated to 24 h) represented 0.5% of the dose or
desmethylclozapine levels were more than 50% lower than less (mean 0.12%), whereas the quantities of N-desmethylcloz-
some values measured on routine drug monitoring during apine and the N-oxide corresponded on average to about 5
the preceding weeks. Also then compliance seemed to be and 8% (Table 2); the sum of all three compounds was always
unreliable as reflected by large serum level fluctuations. Of below 25%. Seven patients with an even distribution of doses
the 60 urine samples, five from three patients could not be excreted a higher quantity of clozapine N-oxide than the
included in calculations of absolute clearance values, because seven evaluable patients who received the major doses in the
sampling was considered incorrect. The criterion was a evening (11.4 vs 8.1% of the dose, P<0.05), possibly due to
creatinine clearance that deviated by more than 50% from preferential N-oxidation during first-pass metabolism; no
the mean of the other sampling intervals. The ratios other difference was detected. The mean clozapine: N-
CLRu/CLCR were, however, included in the evaluation. desmethylclozapine: clozapine N-oxide ratios in urine were
1543577 in the total group and 1544585 in the patients with
an even distribution of doses.
Statistical evaluation
The mean renal clearances (CLR ) of N-desmethylclozap-
One-way analyses of variance served for calculations of ine and clozapine N-oxide were about 80- and 400-fold,
interindividual differences and multiple regression analyses for respectively, that of clozapine (Table 2). When the clearances
the establishment of correlations. Original data were log were calculated for the unbound compounds, mean CLRu
transformed in order to obtain homogeneous variances. values attained by the metabolites were found to exceed the
Geometric means are given in the Results section and in creatinine clearance by a factor of 3 in the case of N-
Table 2 coefficients of variation calculated from untransformed desmethylclozapine and 6.4 in the case of clozapine N-
data, whereas in the description of methods data are arithmetic oxide, whereas that of clozapine was one-ninth only of
means with standard deviations. Statistical analysis was CLCR (Table 2). Concentrations in urine exceeded the
performed using JMP (SAS Institute Inc., Cary, NC, USA). unbound concentrations in serum by a mean factor of 7.4
for clozapine, 336 for N-desmethylclozapine and 737 for
clozapine N-oxide.
Results
All renal clearance data exhibited large interindividual
The steady-state concentrations of clozapine and its metab- variations that by analysis of variance significantly exceeded
olites in serum varied greatly among patients (Table 2). intraindividual variation (Table 2). There was no significant
Lowest and highest levels of clozapine relative to the contribution of sex or age to total variation (data not
−1
daily dose differed by a factor of 6 (41–237 ng ml shown). No significant correlation existed between dose-

456 © 1998 Blackwell Science Ltd Br J Clin Pharmacol, 46, 453–459

 Renal clearance of clozapine and metabolites

Table 2 Renal creatinine clearance and

kinetic data of clozapine and metabolites Interindividual
measured during four 2 h intervals in Geometric CV
15 psychotic patients. The degrees of Parameter Substance mean (%) F P
freedom for variation between patients
were 14, while those used for the CLCR Creatinine 1.7 34 6.9 <0.0001
estimation of intraindividual variation (ml min−1 kg−1 )
were 39 (16–19 in the case of unbound Cpl Clozapine 387 88 86 <0.0001
fractions). (ng ml−1 ) N-Desmethylclozapine 227 78 150 <0.0001
Clozapine N-oxide 73 73 48 <0.0001
fu Clozapine 5.5 19 7.6 <0.0001
(%) N-Desmethylclozapine 9.7 25 2.5 0.03
Clozapine N-oxide 24.6 21 12 <0.0001
CLR Clozapine 0.0065 100 5.8 <0.0001
(ml min−1 kg−1 ) N-Desmethylclozapine 0.51 41 3.5 0.0011
Clozapine N-oxide 2.7 47 6.7 <0.0001
CLRu Clozapine 0.109 111 6.5 <0.0001
(ml min−1 kg−1 ) N-Desmethylclozapine 5.0 50 3.9 0.0004
Clozapine N-oxide 10.6 46 5.4 <0.0001
CLRu /CLCR Clozapine 0.109 120 6.5 <0.0001
N-Desmethylclozapine 3.0 98 15 <0.0001
Clozapine N-oxide 6.4 49 17 <0.0001
Quantity in Clozapine 0.12 83
urine N-Desmethylclozapine 5.3 46
(% of the dose) Clozapine N-oxide 8.3 45
sum 14.1 33

CV, coefficient of variation; CLCR, renal creatinine clearance; Cpl, serum concentration; fu, free
fraction in serum; CLR, renal clearance; CLRu, renal clearance of unbound compound.

related serum clozapine levels and renal clearance values. [23] could be confirmed as well as the finding of higher N-
The clearances of the unbound compounds divided by that desmethylclozapine/clozapine ratios in smokers [25]. Since
of creatinine (CLRu/CLCR ) did not correlate with the tobacco smoke induces cytochrome P450 1A2 (CYP1A2),
serum concentrations of the respective compounds. When the present data are also compatible with the close correlation
they were investigated for a relationship to urine volume between clozapine oral clearance and CYP1A2 activity
and pH, significant negative regression coefficients with determined by a caffeine test in volunteers [26].
respect to pH and positive coefficients with respect to The extent of protein binding of clozapine and its
volume were revealed for clozapine and to a lesser extent metabolites in serum is similar to that of structurally related
for N-desmethylclozapine, while no significant regression drugs; for instance, the mean unbound fraction of the
coefficients were found for the N-oxide (Table 3). phenothiazine neuroleptic perazine was 4.6% [19], that of
amitriptyline 7.8% [19] or 5.7% [27], and that of imipramine
11.5% [19], 9.4% [27] or 10.9% [28]. The somewhat larger
Discussion
free fraction of the secondary amine metabolite as compared
The dose-related serum clozapine levels were in agreement with that of the tertiary amine parent compound was also
with those found previously [20, 21], and this also applied analogous to the behaviour of other tricyclic psychoactive
to the relations between metabolite and parent drug drugs [19, 27]. Regarding their N-oxide metabolites, binding
concentrations [8, 9, 22]. The observation that dose-related to our knowledge has been measured only for amitriptylinox-
serum clozapine levels were lower in men compared with ide, which is 71–84% bound in plasma [29]. This is in
women [23, 24] and in smokers compared with nonsmokers accordance with 66–79% binding of clozapine N-oxide in

Table 3 Results of multiple regression

analysis of the relationships between Compound Parameter Estimate F P
urinary pH and volume and renal
clearances of unbound clozapine and Clozapine m 9.3
metabolites divided by the creatinine 2
r =0.882 b −8.4 43.6 <0.0001
clearance. c 0.84 38.1 <0.0001
Estimates of parameters and significances N-Desmethylclozapine m 3.0
were calculated by the formula ln 2
r =0.884 b −1.5 8.0 0.007
(CLRu/CLCR )=m+ai+bΩln(urine c 0.18 10.1 0.003
pH)+cΩln(urine volume) with ai as the Clozapine N-oxide m 3.2
patient-dependent intercept. 2
r =0.861 b −0.7 2.1 0.16 (n.s.)
r2 is the fraction of total variance c 0.01 0.02 0.88 (n.s.)
explained by the model when individual
a values are taken into account. For abbreviations see Table 2.

© 1998 Blackwell Science Ltd Br J Clin Pharmacol, 46, 453–459 457

G. Schaber et al.

14 out of 15 patients. The reason for the much higher desmethylclozapine in urine must have been eliminated by
percentage bound in repeated samples of one female patient this route. The excess of renal clearance over the creatinine
is unclear. This patient was being treated with a third- clearance was even more marked with unbound clozapine
generation oral contraceptive (composed of desogestrel N-oxide, possibly due to lack of reabsorption. Tubular
0.15 mg and ethinylestradiol 0.03 mg) and her dose-related secretion was estimated to contribute a mean 86% of renal
−1 −1 −1
clozapine serum level (228 ng ml (mg kg ) ) was at N-oxide excretion. Net tubular secretion could also be
the upper end of the group investigated. demonstrated for amitriptylinoxide [34].
Drug concentrations in cerebrospinal fluid (CSF) that are As an alternative explanation for the high renal clearances
assumed to roughly equal unbound concentrations in plasma of desmethylclozapine and clozapine N-oxide, their formation
are available for clozapine from two studies. Of nine patients from clozapine in renal tissue can be considered. Such a
−1
with serum clozapine levels between 43 and 165 ng ml , mechanism is, however, rather unlikely, because human
−1
seven had concentrations in CSF between 20 and 39 ng ml kidneys have very low or nondetectable contents of
corresponding to 26±11% of the levels in serum [30]. In cytochrome P450 [35] that would be required for metabolite
19 patients, a positive correlation was detected between formation. Even if it is assumed that the metabolites were
clozapine levels in plasma and CSF [31]; mean values were partially formed in renal tubular cells, a difference remains
−1 −1
290 ng ml in plasma and 5.6 ng ml in CSF (1.9% of between their handling and that of clozapine. Tubular
the plasma concentration). None of these discrepant results secretion of organic bases involves an uphill transport by a
is in accordance with the 2.6–9.3% (mean 5.5%) free cation/proton antiporter located at the luminal membrane
fraction of clozapine found in this study. [36]. Since the metabolites were concentrated in urine relative
The low renal clearance of clozapine resulted in only to unbound concentrations in serum more than 300-fold
0.12% of the dose on the average being excreted unchanged (desmethylclozapine) and more than 700-fold (clozapine N-
in urine. This agrees with a value of 0.5% found by previous oxide), it must be concluded that they are better substrates of
investigators [5, 32]. In contrast, Gauch & Michaelis [16] the antiporter than the parent drug. Whether the latter is
reported a clozapine: N-desmethylclozapine: clozapine N- transported uphill at the luminal membrane at all, cannot be
oxide ratio of 15152 in a pooled urine sample from five derived from the present data. The 7-fold higher concentration
patients. During their work-up procedure considerable in urine than in plasma water can result from incomplete
quantities of clozapine N-oxide were probably reduced to tubular reabsorption of filtered clozapine at a high urine flow
parent drug. A certain extent of reduction may also have and low urine pH. According to the relationship depicted in
taken place on automated analysis of clozapine and metab- Table 3, a 3-fold higher clozapine concentration can be
olites including solid-phase extraction of patient urine explained by the lower pH in urine (mean 6.5) than in
samples; the resulting ratio of 15458.7 [8] was clearly plasma. There was no indication of saturation of tubular
different from the ratio 1543577 determined now. transport in the range of clozapine metabolite concentrations
Artifactual reduction of clozapine N-oxide in alkalinized occurring in the patient group.
plasma has been studied in detail by Lin et al. [10], and in N-Desmethylclozapine and clozapine N-oxide usually
the present study it could be shown to take place in a small termed the ‘major’ metabolites of clozapine were excreted in
percentage during solid-phase extraction of urine. Because urine in quantities corresponding on average to 5.3 and 8.3%,
of the enormous excess of clozapine N-oxide over clozapine respectively, of the dose during continuous therapy. In single-
in urine, values of the latter can be grossly distorted unless dose experiments, volunteers excreted 1.1 and 5.1% only in
reduction is avoided or controlled for. Using solvent the form of these metabolites, while some aromatically
extraction at near-neutral pH, clozapine values could be hydroxylated derivatives represented larger percentages of the
corrected for the small percentage resulting from N-oxide dose [5]. Nothing as yet is known on their quantitative
reduction. With these precautions, a renal clearance of importance in patients nor on that of the quaternary
unbound clozapine was obtained that averaged 11% of the ammonium glucuronide identified in patient urine [37].
creatinine clearance. Since the latter is by 87% due to In conclusion, clozapine, N-desmethylclozapine and cloz-
glomerular filtration [33], the 11% of the total renal apine N-oxide are on the average protein-bound in patient
creatinine clearance corresponds to 13% of the clearance by serum by 95, 90 and 75%, respectively. When the free
filtration, and thus a mean of 87% of the filtered clozapine fractions are assumed to undergo glomerular filtration in the
underwent net tubular reabsorption. This fraction was kidney, nearly 90% of the filtered clozapine must have been
decreased by a high urine flow and low pH, as can be reabsorbed in the tubule. In contrast, the quantities excreted
expected for a lipophilic base. in urine exceeded the filtered quantities more than 3-fold
In contrast to the parent drug, N-desmethylclozapine and for N-desmethylclozapine and about 7-fold for clozapine
clozapine N-oxide had mean renal clearances of their N-oxide, apparently due to secretion by a tubular cation/
unbound fractions exceeding that of creatinine 3- and proton antiporter. Though these two metabolites are the
6.4-fold, respectively. Thus, their renal disposition was only ones that have been detected in patient plasma, their
governed by net tubular secretion. Desmethylclozapine combined quantities in urine corresponded to 14% only of
probably also underwent reabsorption, as can be surmised the dose, such that other biotransformation products must
from the enhancing effects of higher urine volumes and play a major role in the overall fate of clozapine.
lower pH on the clearance of the unbound compound
relative to creatinine clearance (Table 3). However, the
balance was in favour of secretion, and, on average, at The authors thank Mr K. Nill for expert advice concerning
least 71% (calculated as [(3–0.87)/3]×100%) of the N- h.p.l.c. technique.

458 © 1998 Blackwell Science Ltd Br J Clin Pharmacol, 46, 453–459

 Renal clearance of clozapine and metabolites

Volpicelli SA, Flood JG. Clozapine and metabolites:

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© 1998 Blackwell Science Ltd Br J Clin Pharmacol, 46, 453–459 459