Kari Raaska- Pharmacokinetic Interactions of Clozapine in Hospitalized Patients
Transcript of Kari Raaska- Pharmacokinetic Interactions of Clozapine in Hospitalized Patients
Department of Clinical Pharmacology
University of Helsinki
Finland
PHARMACOKINETIC INTERACTIONS OF CLOZAPINE
IN HOSPITALIZED PATIENTS
by
KARI RAASKA
ACADEMIC DISSERTATION
To be presented, with the permission of the Medical Faculty of the University of Helsinki, for
public examination in the Auditorium of Lapinlahti Hospital, on 31 October, 2003, at 12 noon.
HELSINKI 2003
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Supervisor: Professor Pertti Neuvonen, MD
Department of Clinical Pharmacology
University of Helsinki
Helsinki, Finland
Reviewers: Docent Kimmo Kuoppasalmi, MD
Department of Mental Health and Alcohol Research
National Public Health Institute
Helsinki, Finland
Docent Arja Rautio, MD
Department of Pharmacology and Toxicology
University of Oulu
Oulu, Finland
Official opponent: Professor Esa Leinonen
Medical School
University of Tampere
Tampere, Finland
ISBN 952-91-6491-2 (paperback)
ISBN 952-10-1437-7 (PDF)
Helsinki 2003
Yliopistopaino
This dissertation is available online at http://ethesis.helsinki.fi
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To Hanna, Eveliina, Antti, and Iida
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CONTENTSABBREVIATIONS ……………………………………………………..……6
LIST OF ORIGINAL PUBLICATIONS ………………………………… 8
ABSTRACT …………………………………………………………………… 9
INTRODUCTION ……………………………………………………………. 11
REVIEW OF THE LITERATURE ……………………………………….. 13
1. Pharmacokinetics ……………………………………………………………. 13
1.1. Drug elimination ………………………………………………………… 13
1.1.1. Metabolism ………………………………………………………..13
1.1.2. Excretion …………………………………………………………. 14
1.1.3. Cytochrome P450 (CYP) ………………………………………… 15
1.1.3.1. CYP1A subfamily ………………………………………... 16
1.1.3.2. CYP2C subfamily ………………………………………... 17
1.1.3.3. CYP2D subfamily ………………………………………... 18
1.1.3.4. CYP3A subfamily ………………………………………... 18
1.1.4. P-glycoprotein ……………………………………………………. 19
2. Metabolic drug interactions in psychiatry …………………………………….22
3. Schizophrenia, other psychotic disorders, and their drug treatment …………. 23
4. Clozapine …………………………………………………………………….. 25
4.1. Pharmacokinetics ………………………………………………………... 26
4.1.1. Pharmacokinetic interactions …………………………………….. 31
4.1.1.1. SSRIs and clozapine ………………………………………31
4.1.1.2. Other inhibitors of CYP enzymes and clozapine ………… 32
4.1.1.3. Inducers of CYP enzymes and clozapine …………………34
4.2. Pharmacodynamics ……………………………………………………… 35
4.3. Adverse effects ………………………………………………………….. 38
4.4. Therapeutic drug monitoring (TDM) …………………………………….39
5. Drugs and other factors as inhibitors of clozapine metabolism ……………... 40
5.1. Itraconazole ………………………………………………………………40
5.2. Ciprofloxacin ……………………………………………………………. 42
5.3. Risperidone ……………………………………………………………… 44
5.4. Influenza vaccination ……………………………………………………. 46
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5.5. Caffeine and coffee-drinking …………………………………………… 47
AIMS OF THE STUDY ………………………………………………........... 51
MATERIALS AND METHODS ………………………………………….. 53
1. Subjects ………………………………………………………………………. 53
2. Design of the studies …………………………………………………………. 55
3. Blood sampling ………………………………………………………………. 57
4. Determination of serum drug, caffeine, and CRP concentrations …………… 58
4.1. Clozapine and its metabolites …………………………………………… 58
4.2. Itraconazole and hydroxyitraconazole …………………………………... 59
4.3. Ciprofloxacin ……………………………………………………………. 59
4.4. Caffeine and paraxanthine ………………………………………………. 59
4.5. C-reactive protein (CRP) ………………………………………………... 60
5. Statistical analysis ……………………………………………………………. 60
6. Ethical considerations ………………………………………………………... 60
RESULTS ………………………………………..…………………………….. 63
1. Itraconazole (Study I) ………………………………………………………... 63
2. Ciprofloxacin (Study II) ……………………………………………………… 63
3. Influenza vaccination (Study III) …………………………………………….. 64
4. Risperidone (Study IV) ………………………………………………………. 64
5. Coffee-drinking (Study V) …………………………………………………… 65
DISCUSSION ………………………………………………………………… 70
1. Methodological considerations ………………………………………………. 70
2. Effect of itraconazole on serum clozapine concentration ……………………. 71
3. Effect of ciprofloxacin on serum clozapine concentration ……………………72
4. Effect of risperidone on serum clozapine concentration ……………………...73
5. Effect of influenza vaccination on serum clozapine concentration …………...75
6. Effect of coffee-drinking on serum clozapine concentration ………………… 76
7. General discussion …………………………………………………………… 78
CONCLUSIONS ……………………………………………………………… 81
ACKNOWLEDGEMENTS ………………………………………………….82
REFERENCES …………………………………………………………………85
ORIGINAL PUBLICATIONS ……………………………………………... 111
A B B R E V I A T I O N S
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ABBREVIATIONS
AhR aryl hydrocarbon receptor
Arnt AhR nuclear translocator protein
AUC area under drug concentration-time curve
AUC(0- ) area under drug concentration-time curve (time 0 to infinity)
b.i.d. twice daily
BPRS Brief Psychiatric Rating Scale
C/D ratio ratio of serum drug concentration / daily dose of the drug
CL clearance
Cmax peak concentration
CNS central nervous system
CRP c-reactive protein
CYP cytochrome P450
CV coefficient of variation
D dopamine
DSM-IV Diagnostic and Statistical Manual of Mental Disorders, 4th ed.
EM extensive metabolizer
EPS extrapyramidal symptoms
F bioavailability
FMO flavine-containing monooxygenase
HPLC high-performance liquid chromatography
5-HT serotonin (5-hydroxytryptamine)
IM intermediate metabolizer
Ki inhibition constant
Km Michaelis-Menten constant
NADPH reduced form of nicotinamide adenine dinucleotide phosphate
ND not determined or unknown
NS statistically non-significant
PAH polycyclic aromatic hydrocarbon
P-gp P-glycoprotein
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PM poor metabolizer
PXR pregnane X receptor
RXR retinoid X receptor
SD standard deviation
SSRI selective serotonin reuptake inhibitor
T½ elimination half-life
TDM therapeutic drug monitoring
Tmax time to peak concentration
UKU Udvalg for Kliniske Undersøgelser side-effect rating scale
v volume
VCFS velo-cardio-facial syndrome
Vd apparent volume of distribution
L I S T O F O R I G I N A L P U B L I C A T I O N S
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LIST OF ORIGINAL PUBLICATIONS
This thesis is based on the following publications, which will be referred to in the text by
Roman numerals I to V.
I Raaska K, Neuvonen PJ. Serum concentration of clozapine and N-
desmethylclozapine are unaffected by the potent CYP3A4 inhibitor
itraconazole. Eur J Clin Pharmacol 1998;54:167-170
II Raaska K, Neuvonen PJ. Ciprofloxacin increases serum clozapine and N-
desmethylclozapine: a study in patients with schizophrenia. Eur J Clin
Pharmacol 2000;56:585-589
III Raaska K, Raitasuo V, Neuvonen PJ. Effect of influenza vaccination on serum
clozapine and its main metabolite concentrations in patients with
schizophrenia. Eur J Clin Pharmacol 2001;57:705-708
IV Raaska K, Raitasuo V, Neuvonen PJ. Therapeutic drug monitoring data:
Risperidone does not increase serum clozapine concentration. Eur J Clin
Pharmacol 2002;58:587-591
V Raaska K, Raitasuo V, Laitila J, Neuvonen PJ. Effect of caffeine-containing
versus decaffeinated coffee on serum clozapine concentrations in hospitalised
patients. Pharmacol Toxicol. In press.
The original published articles are reprinted with permission of the copyright holder
(Springer-Verlag GmbH & Co. KG).
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ABSTRACT
ackground: Clozapine is an atypical antipsychotic drug that was first introduced into
clinical use in the 1970’s. After a few years it was withdrawn from the market
worldwide because of fatal cases of agranulocytosis cases first reported in Finland. Because
of its superior efficacy over typical antipsychotics in treatment-refractory schizophrenia,
however, clozapine was reintroduced under a strict blood-cell-monitoring requirement.
Today, clozapine is the gold standard in treatment-refractory schizophrenia. It is eliminated
mainly by cytochrome P450 (CYP) enzyme-mediated metabolism in the liver. The CYP
enzymes involved in the metabolism include CYP1A2, CYP2C19, CYP2D6, and CYP3A4.
Despite its relatively long clinical history, understanding of its pharmacokinetic interactions
has until recently been based largely on case-reports and in vitro studies. The case-reports
suggest that erythromycin (CYP3A4 inhibitor), ciprofloxacin (CYP1A2 inhibitor),
risperidone (CYP2D6 substrate) and caffeine (CYP1A2 substrate) are able to increase serum
clozapine concentration.
ethods: Effects of itraconazole (I), ciprofloxacin (II), risperidone (IV), influenza
vaccination (III), and coffee-drinking (V) on serum concentrations of clozapine or
its main metabolites were investigated in five studies (I-V) in hospitalized clozapine-treated
patients with schizophrenia or other psychotic disorders. Three of the Studies (I, II, and V)
were randomized clinical crossover trials, Study III was an open prospective trial, and Study
IV an analysis of retrospective therapeutic drug monitoring (TDM) data.
esults and discussion: Potent CYP3A4 inhibition by itraconazole showed no effect
on serum clozapine or its metabolite N-desmethylclozapine concentration. This was
in contradiction to reported cases of apparent interactions between clozapine and another
CYP3A4-inhibitor, erythromycin. Based on these reports, CYP3A4-inhibition was widely
assumed to increase clozapine concentration. A low dose of the moderate CYP1A2-inhibitor
ciprofloxacin elevated serum clozapine (P < 0.01) and N-desmethylclozapine (P < 0.05)
concentrations by about 30%, confirming assumptions based on case-reports. Contrary to
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current belief based on case-reports, another atypical antipsychotic, risperidone, had no
effect on serum clozapine concentrations in the present retrospective analysis of TDM data.
Influenza vaccination had no effect on serum clozapine, or on its two main metabolites, N-
desmethylclozapine or clozapine-N-oxide. In Study III, a minor infection occurred during
the study period in two patients. In both of them, during the infection, serum clozapine
concentrations increased. Coffee-drinking caused a minor enhancement of effect on serum
clozapine concentrations, suggesting that “normal” amounts of coffee have a low propensity
to cause any clinically significant increase in serum clozapine concentration.
onclusions: Potent inhibition of CYP3A4 by itraconazole does not affect serum
concentrations of clozapine or N-desmethylclozapine to a clinically significant
degree. Coffee-drinking has a low interaction potential with clozapine. Even a low dose of
the CYP1A2 inhibitor ciprofloxacin, however, raises serum clozapine concentrations to a
degree which may have clinical relevance for some of the patients. Clozapine serum
concentrations seem to be left unaltered by risperidone and influenza vaccination. Although
clozapine is metabolized by both CYP1A2 and CYP3A4, it seems that, in vivo, only
CYP1A2 inhibition can significantly elevate serum clozapine concentrations.
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INTRODUCTION
lozapine and many other antipsychotic drugs are extensively metabolized by
cytochrome P450 (CYP) enzymes, whose activity can be suppressed (inhibition) or
enhanced (induction) by some drugs and environmental factors. Inhibition and induction of
CYP enzymes are the most important mechanisms for drug interactions that may raise or
reduce serum concentrations of drugs, and lead to undesirable outcomes. Genes for drug-
metabolizing CYP enzymes exist in many allelic variants, which can cause profound
differences in specific CYP activities between individuals (Coutts & Urichuk 1999). Some
of the drug-metabolizing CYPs are polymorphically expressed, meaning that individuals are
either extensive (EM) or poor (PM) metabolizers in respect to that enzyme. Both EMs and
PMs are common in all populations (Coutts & Urichuk 1999). In the case of antipsychotic
drugs, induction of the CYPs mainly responsible for their metabolism may lead to relapse or
worsening of psychotic symptoms, and inhibition of the same enzymes may lead to the
intensification of adverse effects and of toxicity. The safety of antipsychotics in
combination with other drugs is important because patients treated with these drugs often
need them for years. During that time, other psychotropic drugs may be necessary to control
their psychiatric symptoms, and patients may also need different types of drugs, for
example, to treat infections. Among other factors that could possibly affect clozapine
pharmacokinetics are age, sex, tobacco-smoking, coffee-drinking, and vaccination. For
example, tobacco-smoking induces CYP1A2 and reduces serum clozapine concentrations
(Seppälä et al. 1999).
Several case-reports suggest that clozapine concentrations may be affected by some drugs
and environmental factors. These reports, together with what is known about clozapine
metabolism, imply that the most likely mechanisms in these apparent interactions involve
CYP enzymes. This is suggested by the fact that the potent CYP1A2 inhibitor fluvoxamine
seems to elevate clozapine concentrations by up to 10-fold (Hiemke et al. 1994, Koponen et
al. 1996, Dequardo & Roberts 1996, DuMortier et al. 1996, Bender & Eap 1998, Wetzel et
al. 1998). Serious adverse effects and increase in plasma clozapine concentrations have
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occurred in co-administration with the CYP1A2 inhibitor ciprofloxacin (Markowitz et al.
1997). Another antibacterial drug, the potent CYP3A4 inhibitor erythromycin, is reported to
double clozapine concentrations (Funderburg et al. 1994, Cohen et al. 1996). A similar
degree of inhibition of clozapine metabolism is suggested by an antipsychotic drug–
risperidone–which is a substrate for and a weak inhibitor of CYP2D6 (Koreen et al. 1995,
Tyson et al. 1995). Paroxetine, a strong CYP2D6 inhibitor, apparently also elevates
clozapine concentrations (Centorrino et al. 1996, Joos et al. 1997, Spina et al. 2000). All of
these CYP enzymes are to some extent involved in the metabolism of clozapine, although
CYP2D6 seems to be far less important than CYP1A2 and CYP3A4. In vitro, CYP1A2 is
the most important enzyme in the formation of N-desmethylclozapine (Pirmohamed et al.
1995). The formation of another main metabolite, clozapine-N-oxide, is catalyzed
principally by CYP3A4 (Eierman et al. 1997).
Assumptions as to the pharmacokinetic interactions of clozapine were until recently mostly
based on sporadic case-reports, and on conclusions drawn from clozapine metabolism
studies. Administration of antimicrobial drugs like itraconazole or ciprofloxacin to treat
infections, or of risperidone to augment the antipsychotic efficacy of clozapine could
theoretically put patients at risk for adverse effects due to increasing clozapine
concentrations. The possibility that influenza vaccination may affect CYP1A2-mediated
drug metabolism (Renton et al. 1980, Meredith et al. 1985) raises the question of possible
effects of influenza vaccination on serum clozapine concentrations. Although coffee-
drinking seems to have some effect on serum clozapine concentrations, its mean effect in
patients in clinical setting remains elusive.
The purpose of the present studies was to investigate in hospitalized patients the effects of
itraconazole (CYP3A4 inhibitor), ciprofloxacin (CYP1A2 inhibitor), risperidone, influenza
vaccination, and coffee-drinking on serum concentrations of clozapine and its main
metabolites.
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REVIEW OF THE LITERATURE
1. Pharmacokinetics
oth pharmacokinetics and pharmacodymamics study drug-body interactions. Whereas
pharmacodynamics deals with the effects of drugs on the body, pharmacokinetics
deals with the effects of the body on drugs. Pharmacokinetic parameters describe how drugs
are absorbed from different sites of administration, how they are distributed, and how they
are eliminated from the body. Bioavailability (F) is the percentage (0-100%) of an
administered drug that reaches the systemic circulation; it is affected by the extent of
absorption and pre-systemic elimination. The apparent volume of distribution (Vd, the unit
is L/kg) of a drug describes how the drug is distributed between plasma and tissue.
Clearance (CL), which describes the elimination of a drug, is the plasma volume from
which the drug is totally removed in one minute.
1.1. Drug elimination
rugs and other foreign compounds (xenobiotics) are eliminated either by metabolism
(yielding metabolites), or by excretion from the body as unchanged parent
compounds. Metabolites are either excreted or further metabolized. Each metabolite has its
own pharmacokinetic characteristics that determine its distribution and its routes and the
rate of elimination.
1.1.1. Metabolism
ost orally administered drugs are lipid-soluble (lipophilic) compounds. This
facilitates their passage through biological membranes. Metabolism transforms
them to more water-soluble (hydrophilic) molecules that can be effectively excreted from
the body. Although metabolites are generally less toxic than their parent compounds,
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sometimes metabolism yields highly toxic compounds. Some drugs are administered as
biologically inactive prodrugs that require metabolism to convert them into active drugs.
Drugs are metabolized most importantly in the liver. In addition, the small intestine has
considerable metabolic activity towards a number of drugs. Other tissues contribute less to
overall drug metabolism, although they may have some local importance in protection from
organ-specific toxicity. Drug metabolism is often divided into phase I and phase II
enzymatic reactions. Together phase I and II reactions make up a form of protective
detoxification system that transforms lipophilic molecules into less toxic hydrophilic
compounds. Phase I (functionalization) reactions consist of oxidation, reduction, and
hydrolysis, which usually lead to metabolites that are more polar than is the parent
compound (Gibson & Skett 2001). Of phase II, glucuronidation, sulphation, acetylation, and
conjugation to glutathione and amino acids are the major conjugation reactions (Gibson &
Skett 2001). All drugs do not require phase I metabolism before they can take part in
conjugation reactions. Furthermore, as an exception to the rule, some conjugates such as
morphine-6-glucuronide possess greater biological activity than the parent drug, and some
are chemically highly reactive (Tephly & Green 2000).
1.1.2. Excretion
enal excretion of drugs and metabolites into the urine is the most important excretion
route. This may be passive, or be an active energy-consuming process. Generally,
compounds have to be hydrophilic in order to be effectively excreted. Along with some
other conjugates, most glucuronide conjugates are excreted in the bile (Roberts et al. 2002).
After they enter, with the bile, the small intestine, they can either leave the body with the
feces, or the conjugate can be cleaved by bacterial -glucuronidase enzymes back to the
parent drug (Roberts et al. 2002). The drug can be reabsorbed from the gut into the systemic
circulation. This absorption-biliary excretion-reabsorption cycle is called the enterohepatic
circulation.
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1.1.3. Cytochrome P450 (CYP)
reduced pigment of the NADPH/O2-dependent oxidation system has an absorption
band with a max at 450 nm after binding to carbon monoxide (Klingenberg 1957,
Garfinkle 1958). This pigment was first characterized as a P450 hemoprotein (Omura &
Sato 1961). Over the next 20 years, it became evident that this microsomal mixed function
monooxygenase system consists of multiple forms of P450 enzymes (CYPs). Currently 18
CYP families and 43 subfamilies have been identified in human being
(www.drnelson.utmem.edu/famcount.html, and www.imm.ki.se/CYPalleles/, Nelson 2003)
(Table 1). CYP enzymes metabolize both endogenic (fatty acids and steroids) and exogenic
(xenobiotics) compounds (Gonzalez 1988, Nebert & Russell 2002) (Table 1). Drugs are
among the exogenic compounds, as are carcinogens such as polycyclic aromatic
hydrocarbons (PAH) and arylamines (Gonzalez 1988, Nebert & Russell 2002). Only the
families CYP1, CYP2, and CYP3 seem to be important in human drug metabolism (Tables
1 and 2).
Individual CYP forms show affinity towards structurally unrelated compounds. Many
substrates can be significantly metabolized by more than one CYP form (Gonzalez 1988).
The principal function of CYP enzymes is the mono-oxygenation of various substrates
(Gonzalez 1988). This requires molecular oxygen and a supply of reducing equivalents from
the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) (Gibson &
Skett 2001).
The nomenclature of CYP enzymes is based on their identity in amino acid sequences
(Nelson et al. 1996). The CYP superfamily is divided into families and subfamilies. The
abbreviation “CYP” for the cytochrome P450 superfamily is followed by the number of that
family, e.g., CYP1. The protein sequences of the enzymes within each family are at least
40% identical (e.g., CYP2C8 and CYP2D6), and within each subfamily they are > 55%
identical (e.g., CYP2C8 and CYP2C9). An individual enzyme is identified by a number
following the letter for a subfamily (e.g., CYP1A2).
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Subfamilies CYP1A, CYP2A, CYP2C, CYP2D, CYP2E, and CYP3A include important
drug-metabolizing enzymes (Nebert & Russell 2003, Tables 1 and 2). It seems that all of
them except CYP2D6 are inducible (Table 2). For one or more isoenzymes belonging to
these subfamilies, several drugs act as inhibitors (Table 2). Inhibition of the metabolizing
enzymes may be either reversible (competitive, non-competitive or uncompetitive) or
irreversible (stable complex formation or suicide inhibition) (Thummel et al. 2000).
1.1.3.1. CYP1A subfamily
YP1A1 is expressed mainly in extrahepatic tissue, and CYP1A2 is liver-specific
(Raunio et al. 1995). CYP1A2 accounts for about 15% of the total hepatic CYP
content (Pelkonen et al. 1998). Although the CYP1A2-mediated caffeine N-3-demethylation
rate in 22 human livers was shown to vary 54-fold (Butler et al. 1989), CYP1A2 is not
polymorphically expressed (Smith et al. 1998, Miners & McKinnon 2000, Table 2). CYP1A
enzymes transform some procarcinogens to carcinogens such as PAH compounds and
aflatoxin B1 (Miners & McKinnon 2000). Dioxin is the prototypic inducer for CYP1A
(Pelkonen et al. 1998, Miners & McKinnon 2000). The large interindividual variation in
CYP1A1 inducibility is probably linked to genetic polymorphism in the aryl hydrocarbon
receptor (AhR) (Miners & McKinnon 2000), and the inducers of CYP1A2 are ligands of
this transcription factor (Schrenk 1998). Studies suggest an increased risk for malignancies
in human beings who belong to the high-affinity AhR phenotype (Miners & McKinnon
2000). It seems that CYP1A induction may be either AhR-mediated (ligand-dependent or
ligand-independent) or AhR-independent (Miners & McKinnon 2000). After AhR-ligand
binding in the cytoplasm, the complex binds to the AhR nuclear translocator protein (Arnt)
in the nucleus to form a dimer that acts as a nuclear transcription factor which enhances the
transcription of the CYP1A2 gene by binding to Ah-responsive element sequences (Schrenk
1998, Miners & McKinnon 2000).
CYP1A2 is an important drug-metabolizing enzyme (Table 2), for which caffeine is often
used as a probe substance (Butler et al. 1989, Miners & McKinnon 2000). The caffeine N-3-
demethylation that yields paraxanthine is a suitable index of CYP1A2 activity in vivo
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(Butler et al. 1989, Fuhr & Rost 1994, Miners & McKinnon 2000). Some of the commonly
used drugs inhibit or induce CYP1A2 activity (Table 2); fluvoxamine, in particular, is a
potent CYP1A2 inhibitor (Brøsen et al. 1993, Rasmussen et al. 1995).
1.1.3.2. CYP2C subfamily
YP2C, the most complex of the drug-metabolizing CYP subfamilies (Rettie et al.
2000), includes CYP2C8, CYP2C9, CYP2C18, and CYP2C19 (Rettie et al. 2000).
Due to pharmacogenetic polymorphism, 2% to 5% of Caucasians, but 13% to 23% of
Asians, are “poor metabolizers” (PM) of CYP2C19, and the rest are “extensive
metabolizers” (EM) (Nakamura et al. 1985, Rettie et al. 2000). In PMs, diazepam
(CYP2C19 substrate) produces prolonged sedation (Goldstein 2001). Of the other members
of this subfamily, at least CYP2C9 and CYP2C18 are polymorphically expressed (Rettie et
al. 2000, Goldstein 2001) (Table 2). The CYP2C subfamily makes up about 20% of total
hepatic CYP content (Pelkonen et al. 1998), the most abundant member is CYP2C9 (Rettie
et al. 2000).
Paclitaxel can serve as a prototypic substrate and trimetoprim as a selective inhibitor of
CYP2C8 (Dai et al. 2001, Wen et al. 2002). Sulfaphenazole is a prototypic competitive
inhibitor of CYP2C9 (Rettie et al. 2000). The clinical importance of the CYP2C18 isoform
seems to be minor, but it is–for example, with its low Michaelis-Menten constant (Km)–
capable of catalyzing diazepam N-demethylation in vitro (Rettie et al. 2000). Hydroxylation
of S-mephenytoin can serve as a prototypic reaction for CYP2C19 (Goldstein et al. 1994).
At high concentrations, S-mephenytoin is suitable as a selective CYP2C19 inhibitor (Rettie
et al. 2000).
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1.1.3.3. CYP2D subfamily
YP2D6 is polymorphically expressed, 7% of Caucasians being PMs, and thus carrying
two non-functional alleles (Coutts & Urichuk 1999). The level of CYP2D6 protein
expression in the liver varies among individuals from undetectable (PMs), low levels
(intermediate metabolizers, IMs), and “normal” (EMs), to levels of expression more than
100-fold higher than in the majority of EMs; on average, CYP2D6 expression accounts for
about 2% to 5% of total CYP content (Zanger & Eichelbaum 2000), but CYP2D6
metabolizes about 30% of all drugs (Anzenbacher & Anzenbacherová 2001). Potent
inhibitors of CYP2D6 include quinidine, ritonavir, and paroxetine (Zanger & Eichelbaum
2000, Shapiro & Shear 2002). Since CYP2D6 seems not to be inducible, gene duplications
may be one way to adapt to environmental chemical pressures (Ingelman-Sundberg et al.
1999).
1.1.3.4. CYP3A subfamily
YP3A takes part in the metabolism of more than half of all drugs (Wrighton &
Thummel 2000). The isoenzymes CYP3A4, CYP3A5, and CYP3A7 together account
for about 30% of total hepatic CYP content, making CYP3A the most prominent of the CYP
enzymes in the liver (Pelkonen et al. 1998, Wrighton & Thummel 2000). By far the most
important of the CYP3A enzymes is CYP3A4, whose expression varies 20-fold in the
human liver, but is not polymorphically expressed (Table 2). Wrighton & Thummel (2000)
discuss the following characteristics: CYP3A4 is the most prominent CYP also in the small
intestine, CYP3A5 is absent from or poorly expressed in about 70% of human livers with a
level of expression usually much lower than CYP3A4 expression, but still it is the major
CYP3A form in about 5% of the livers. CYP3A4, CYP3A5, and CYP3A7 seem to show
similar substrate specificity. CYP3A7 is poorly expressed in the adult human liver, but it
represents about 50% of the total CYP content in the human fetal liver (Wrighton &
Thummel 2000).
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Midazolam 1-hydroxylation and testosterone 6-hydroxylation reactions, among others, can
serve as probe reactions for CYP3A (Wrighton & Thummel 2000, Pelkonen et al. 1998).
Induction of CYP3A4 is mediated by nuclear receptors: pregnane X receptor (PXR),
constitutive androstane receptor (CAR), and glucocorticoid receptor (Gibson & Skett 2001).
In order to activate the CYP3A4 gene, PXR and CAR have first to bind to an inducer, and
thereafter form heterodimers with the retinoid X receptor (RXR) which binds to specific
gene regulation elements (Gibson & Skett 2001).
1.1.4. P-glycoprotein
-glycoprotein (P-gp) is a transmembrane transporter which is an ATP-dependent efflux
pump that transports a wide variety of chemically unrelated substances. A high
concentration of P-gp is located in the epithelial cells lining the luminal surface of
enterocytes in the small intestine and the kidney, in the biliary canalicular membranes of
hepatocytes, and in the luminal surface of the endothelial cells making up the blood-brain
barrier (Shapiro & Shear 2002). Although P-gp and CYP3A4 have overlapping substrate
specificity (Lin & Yamazaki 2003), important exceptions to this “rule” exist: Digoxin,
which is eliminated almost entirely by excretion (Mooradian 1988), is a prototypical P-
glycoprotein substrate susceptible to clinically relevant drug interactions with P-gp
inhibitors (Partanen et al. 1996, Fromm et al. 1999, Silverman 2000). Midazolam, not a P-
gp substrate (Kim et al. 1999), is a prototypical CYP3A4 substrate.
It seems that intact P-gp activity is of special importance in preventing central nervous
system (CNS) -adverse effects of drugs (Lin & Yamazaki 2003). Generally, P-gp inhibition
leads to a much greater increase in drug concentrations in the brain than in plasma (Lin &
Yamazaki 2003). Healthy volunteers tolerated the P-gp substrate loperamide well when it
was ingested with placebo, but when administered with the P-gp inhibitor quinidine, it
caused respiratory depression (Sadeque et al. 2000).
P
R E V I E W O F T H E L I T E R A T U R E
20
Table 1. Human CYP families, subfamilies, locations of functional genes, and main
functions of corresponding CYP enzymes.
CYPfamily1
Subfamilies1 Location offunctional
genes(chromosome)1
Main functions 2-6
1A 15 Xenobiotic metabolismCYP11B 2 Estradiol 4-hydroxylation
2A, 2B, 2F, 2G, 2S,2T
19 Xenobiotic metabolism (2A, 2B)
2C, 2E 10 Xenobiotic metabolism2D 22 Xenobiotic metabolism2J 1 Arachidonic acid and xenobiotic metabolism2R 11 Unknown2U 4 Unknown
CYP2
2W 7 UnknownCYP3 3A 7 Xenobiotic metabolism
4A, 4B 1 Fatty acid, arachidonic acid and leukotriene metabolism4F 19 Fatty acid and arachidonic acid metabolism4V 4 Unknown4X 1 Unknown
CYP4
4Z 1 UnknownCYP5 5A 7 Thromboxane A2 synthesisCYP7 7A, 7B 8 Bile acid synthesis
8A 20 Prostacyclin synthesisCYP88B 3 Bile acid synthesis
11A 15 Cholesterol side-chain cleavageCYP1111B 8 Steroid 11 -hydroxylation, aldosterone synthesis
CYP17 17A 10 Steroid 17 -hydroxylationCYP19 19 15 Steroid aromatization (estrogen synthesis)CYP20 20 2 UnknownCYP21 21A 6 Steroid 21-hydroxylationCYP24 24 20 Vitamin D metabolism
26A, 26C 10 Vitamin A metabolismCYP2626B 2 Vitamin A metabolism
27A, 27C 2 Vitamin D synthesis, bile acid synthesisCYP2727B 12 Vitamin D synthesis
CYP39 39A 6 Bile acid synthesisCYP46 46 14 Cholesterol 24-hydroxylationCYP51 51 7 Sterol 14-demethylation
1 Nelson:http://drnelson.utmem.edu/human.genecount.html, 2 Nebert & Russell 2002, 3 Lund et al. 1999,4Yoshida et al. 2000, 5Matsumoto et al. 2002, 6Bylund et al. 2002.
RE
VIE
W O
F T
HE
LIT
ER
AT
UR
E
21
Tab
le 2
. Gen
etic
pol
ymor
phis
m, s
elec
ted
subs
trate
s, in
hibi
tors
, and
indu
cers
of t
he m
ain
drug
-met
abol
izin
g C
YP
enzy
mes
.
GEN
ETIC
PO
LYM
OR
PHIS
M
CY
P1A
2C
YP2
C8
CY
P2C
9C
YP2
C19
CY
P2D
6C
YP3
A4
No1,
2Su
gges
ted3
Yes
3Y
es3
Yes
4N
o5, 6
SUB
STR
AT
ES
CY
P1A
2C
YP2
C8
CY
P2C
9C
YP2
C19
CY
P2D
6C
YP3
A4
Caf
fein
e2, 7
Clo
zapi
ne8,
9
Imip
ram
ine7
Lido
cain
e10
Map
rotil
ine7
Ola
nzap
ine9
Prop
rano
lol7
Rop
ivac
aine
11
R-w
arfa
rin2
Tacr
ine2
Theo
phyl
line2
Car
bam
azep
ine3
Cer
ivas
tatin
12, 1
3
Ibup
rofe
n3
Pacl
itaxe
l3
Rep
aglin
ide14
Ros
iglit
azon
e15
Tolb
utam
ide3
Dic
lofe
nac3
Fluv
asta
tin7
Ibup
rofe
n3
Losa
rtan3
Nap
roxe
n3
Phen
ytoi
n3
Piro
xica
m3
S-w
arfa
rin3
Tolb
utam
ide3
Clo
mip
ram
ine7
Dia
zepa
m3
Imip
ram
ine7
Om
epra
zole
3
Phen
ytoi
n3
Prop
rano
lol7
Prog
uani
l3
Am
itrip
tylin
e4
Clo
mip
ram
ine4
Cod
eine
4
Fluo
xetin
e7
Fluv
oxam
ine4
Hal
oper
idol
4
Met
opro
lol4
Nor
tript
ylin
e4
Paro
xetin
e4
Ris
perid
one4,
9, 1
6
Thio
ridaz
ine4
Am
ioda
rone
7
Am
itrip
tylin
e7
Alp
razo
lam
7
Car
bam
azep
ine7
Cic
losp
orin
7
Eryt
hrom
ycin
7, 1
7
Felo
dipi
ne7
Lova
stat
in7
Mid
azol
am7,
17
Nef
azod
one7
Qui
nidi
ne7
INH
IBIT
OR
S
CY
P1A
2C
YP2
C8
CY
P2C
9C
YP2
C19
CY
P2D
6C
YP3
A4
Cim
etid
ine7
Cip
roflo
xaci
n7, 1
8
Fluv
oxam
ine2,
19
Gre
paflo
xaci
n7
Gem
fibro
zil12
, 13
Trim
etho
prim
20A
mio
daro
ne3
Fluc
onaz
ole3,
7
Mic
onaz
ole3
Sulp
haph
enaz
ole3
Fluo
xetin
e3
Fluv
oxam
ine3
Om
epra
zole
3
Flec
aini
de4
Fluo
xetin
e4
Paro
xetin
e4
Qui
nidi
ne4
Eryt
hrom
ycin
17
Itrac
onaz
ole17
Nef
azod
one17
Rito
navi
r17
IND
UC
ERS
CY
P1A
2C
YP2
C8
CY
P2C
9C
YP2
C19
CY
P2D
6C
YP3
A4
Car
bam
azep
ine21
Dio
xin2
Phen
obar
bita
l7, 2
2
Rifa
mpi
cin2,
23
Toba
cco
smok
e3, 7
, 22
Phen
obar
bita
l24
Rifa
mpi
cin23
, 24
Phen
obar
bita
l7, 2
4
Rifa
mpi
cin7,
23,
24
Phen
obar
bita
l24
Rifa
mpi
cin7,
23,
24
Unk
now
n4C
arba
maz
epin
e17
Dex
amet
haso
ne17
Phen
obar
bita
l17
Phen
ytoi
n17
Rifa
mpi
cin7,
23
1 Smith
et a
l. 19
98, 2 M
iner
s & M
cKin
non
2000
, 3 Ret
tieet
al.
2000
, 4 Zang
er &
Eic
helb
aum
200
0, 5 La
mba
et a
l. 20
02, 6 G
arci
a-M
artin
et a
l. 20
02, 7
Stoc
kley
200
2, 8Ta
ylor
199
7, 9 de
Van
e &
Mar
kow
itz 2
000,
10W
ang
et a
l. 20
00, 11
Arla
nder
et a
l. 19
98, 12
Wan
g et
al.
2002
b, 13
Bac
kman
et a
l. 20
02, 14
Nie
mi e
t al.
2003
b, 15
Nie
mi e
t al.
2003
a, 1
6 Yas
ui-F
uruk
ori e
t al.
2001
, 17W
right
on &
Thu
mm
el 2
000,
18 Fu
hr e
t al.
1992
,19
Brø
sen
et a
l. 19
93, 20
Wen
et a
l. 20
02, 21
Park
eret
al.
1998
, 22 Pe
lkon
enet
al.
1998
, 23N
iem
i et a
l. 20
03c,
24G
erba
l-Cha
loin
et a
l. 20
01.
R E V I E W O F T H E L I T E R A T U R E
22
2. Metabolic drug interactions in psychiatry
sychiatric patients are at increased risk for metabolic drug-drug interactions, because
only a minority who are treated for such conditions as depression or schizophrenia are
on monotherapy (Rittmannsberger et al. 1999). In Austrian psychiatric clinics, patients with
schizophrenia were using on average 2.5 to 3.5 psychotropic drugs, and a mean total of 3 to
4 drugs. About half these patients with schizophrenia each received two antipsychotic drugs.
Depending on the clinic, 6% to 35% of the patients with schizophrenia were receiving
antidepressants, 29% to 74% receiving anxiolytics, and 6% to 9% receiving anticonvulsants
(Rittmannsberger et al. 1999).
Many of the newer antidepressants are potent inhibitors, while some of the anticonvulsants
are potent inducers of the drug-metabolizing CYP enzymes. Furthermore, some of the
psychotropic drugs, especially lithium and tricyclic antidepressants, have narrow therapeutic
indices. Tricyclics are, in addition, subject to considerable pharmacogenetic variability.
Nearly half of all patients with schizophrenia have a comorbic medical condition that
requires drug-treatment but is often either misdiagnosed or undiagnosed (Goldman 1999).
Liver and renal diseases may directly slow drug elimination, and like the
pharmacogenetically determined poor metabolism genotype, they put patients at risk for
high drug concentrations. It is also common for patients on antipsychotic medication not to
take their drugs as prescribed. About 40% of patients stop taking antipsychotics within one
year, and about 75% within 2 years (Perkins 1999). In addition, patients may use over-the-
counter drugs or herbal products that interact with the prescribed drugs. Psychiatric
disorders are present in almost all completed suicides, with schizophrenia and depression
demonstrating an especially a high suicide risk (Isometsä 2001, Joukamaa et al. 2001). Drug
overdose with multiple drugs, often at least one psychotropic drug, is a common method of
suicide (Baca-García et al. 2002), in which case consequences of overdose are worse due to
interaction between drugs.
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3. Schizophrenia, other psychotic disorders, and their drug treatment
bout 1% of the world population is affected by schizophrenia, a severe psychiatric
disorder (Norquist & Narrow 2000). The diagnostic and statistical manual of mental
disorders, 4th edition (DSM-IV) describes the characteristic symptoms as delusions,
hallucinations, disorganized speech, grossly disorganized (or catatonic) behavior, and
negative symptoms (i.e., affective flattening, alogia, or avolition) (American Psychiatric
Association 1994). The illness begins typically in early adulthood (Schultz & Andreasen
1999). Generally, the symptoms lead to marked social dysfunction, with the majority of the
affected individuals unable to continue to work or study (Schultz & Andreasen 1999,
Cancro & Lehman 2000). Susceptibility to schizophrenia is determined by both genetic and
environmental factors (Norquist & Narrow 2000), with the heritability is estimated at about
80% (Harrison & Owen 2003). Several genes are implicated as susceptibility loci for
schizophrenia, but none, so far, have been adequately confirmed (Harrison & Owen 2003).
The genetic syndrome velo-cardio-facial syndrome (VCFS, or DiGeorge syndrome) is
associated with a high rate of schizophrenia (Murphy 2002). The characteristic
microdeletion on chromosome 22 is present in about 85% of VCFS patients, and in about
1/4000 live births (Murphy 2002). The implicated early-life environmental factors
associated with later occurrence of schizophrenia include exposure to some viruses,
nutritional deficiencies, and obstetric complications (Schultz & Andreasen 1999, Norquist
& Narrow 2000). In addition, to clarify the ethiopathogenesis of schizophrenia, which is still
largely unknown, mechanisms of action of antipsychotic drugs, as well as the occurrence of
neuroanatomical abnormalities, are undergoing study (Egan & Hyde 2000, Sawa & Snyder
2002). At the level of neurotransmitters, involvement of at least dopamine, serotonin, and
glutamate is strongly implicated in schizophrenia (Olney & Farber 1995, Goff & Coyle
2001, Sawa & Snyder 2002, Meltzer 2002).
All antipsychotics are effective against positive symptoms of schizophrenia, reducing
disturbed thoughts and hallucinations (Schultz & Andreasen 1999). Older, typical
antipsychotics, or neuroleptics, although they are effective for positive symptoms, may even
worsen anhedonia, withdrawal, and other negative symptoms (Karow & Naber 2002).
A
R E V I E W O F T H E L I T E R A T U R E
24
Common adverse effects of typical antipsychotics include extrapyramidal and
anticholinergic symptoms. The newer atypical antipsychotics, which are also called
serotonin-dopamine antagonists, are as effective in the treatment of positive symptoms as
the typical antipsychotics, but they reduce negative symptoms of schizophrenia more
effectively (Kane 1996, Schultz & Andreasen 1999, Marder 2000). They have considerably
fewer motor adverse effects, but some of them cause weight gain, insulin resistance, and
lipid abnormalities (Henderson 2001, Lindenmeyer et al. 2003). Compared to the effect of
typical antipsychotics, atypical antipsychotics seem to lead to better subjective well-being
and quality of life (Karow & Naber 2002). Antipsychotic drugs can be administered in
maintenance treatment either as daily oral doses or as depot injections. Other medications
often used in combination with antipsychotic drugs include lithium, benzodiazepines,
anticonvulsants, and antidepressants (Kane 1996). Although antipsychotics are critical in the
acute and maintenance treatment of schizophrenia, so are various psychosocial interventions
(Bustillo et al. 2000).
At least 20% of all patients with schizophrenia are resistant to drug treatment (Conley &
Kelly 2001). The most well-accepted criteria define treatment-resistant schizophrenia as at
least moderately severe and persistent illness with no stable periods of good social or
occupational functioning within the previous 5 years, plus persistent positive psychotic
symptoms, all despite at least three adequate treatment trials with typical antipsychotics
from at least two chemical classes (Kane et al. 1988). The only drug with demonstrated
efficacy in treatment resistance is clozapine; other atypical antipsychotics than clozapine
may also be more effective than typical antipsychotics in treatment-resistant patients
(Conley & Kelly 2001).
Patients with schizoaffective disorder suffer from both schizophrenia-like symptoms and
prominent mood symptoms (major depressive, manic, or mixed episodes) (DSM-IV). Most
of them are treated with a combination of antipsychotic, mood stabilizing, or antidepressant
drugs, or a combination of these (Levinson et al. 1999). Antipsychotic drugs are also used to
treat patients with other diagnostically specified or unspecified psychoses.
R E V I E W O F T H E L I T E R A T U R E
25
4. Clozapine
lozapine, an orally administered antipsychotic drug, was the first of the antipsychotics
to be called “atypical.” It was synthesized in Switzerland in 1958 and subsequently
identified in 1959 (Meyer & Simpson 1997, Hippius 1999). It was introduced into clinical
practice in Finland in February 1975, but in the same summer, eight Finnish patients who
were taking clozapine died from agranulocytosis (Idänpään-Heikkilä et al. 1975), a
granulocyte count less than 500 cells/mm3. This led the manufacturer to voluntarily
withdraw clozapine from the market. After two comparative trials demonstrated the superior
efficacy of clozapine in treatment-resistant patients (Kane et al. 1988, Claghorn et al. 1987),
however, clozapine was approved by the Food and Drug Administration (FDA) in 1990 for
clinical practice in the United States. Since then, a large number of studies have been
conducted on various aspects of clozapine. Until recently, however, controlled studies on
the pharmacokinetic interactions involving clozapine have been scarce.
Today clozapine has a unique place among antipsychotics due to its superior efficacy in
reducing both positive and negative symptoms of schizophrenia, and due to an adverse-
effect profile often more favorable than those of typical antipsychotics.
Clozapine may, however, cause agranulocytosis. For this reason, its use is restricted to
treatment-resistant schizophrenia, and to patients who are intolerant of typical
antipsychotics. Clozapine’s efficacy is superior to that of chlorpromazine, and at 6 weeks it
is effective in 30% of treatment-resistant patients (Kane et al. 1988). It reduces both positive
and negative symptoms, and improves the impaired cognition (Kuoppasalmi et al. 1993,
Wagstaff & Bryson 1995, McGurk 1999). Emerging reports indicate that clozapine reduces
suicidality (Meltzer & Okayli 1995, Meltzer et al. 2003), violence, and persistent aggression
and substance abuse (Volavka 1999) among patients with schizophrenia. In 2003 clozapine
was approved by the FDA in USA for reducing the risk of suicidal behavior in patients with
schizophrenia or schizoaffective disorder (FDA Consumer 2003).
C
R E V I E W O F T H E L I T E R A T U R E
26
4.1. Pharmacokinetics
lozapine has a linear, or first-order, kinetics in therapeutically relevant concentrations
(Perry et al. 1998, Guitton et al. 1998, Haring et al. 1989, 1990). This means that its
rates of absorption and elimination are proportional to drug concentrations (Sjöqvist et al.
1997). The absolute bioavailability of clozapine is widely variable. After oral
administration, 27 to 47% of the clozapine dose reaches the systemic circulation unchanged
(Cheng et al. 1988, Choc et al. 1990). Peak concentration (Cmax) is reached in 1 to 3 hours,
mean elimination half-life (T½) is 10 to 17 hours (range 5-60 hours), mean volume of
distribution (Vd) is 2 to 5 L/kg (range 1-10 L/kg) and mean plasma clearance (CL) is 13 to
57 L/h (range 11-435 L/h) (Byerly & De Vane 1996) (Table 3).
Table 3. Pharmacokinetic parameters of clozapine (references in text).
Parameter Value
Oral bioavailability (F) 27-47%Time to peak concentration (Tmax) 1-3 hoursElimination half-life (T½) 10-17 hoursVolume of distribution (Vd) 2-5 L/kgPlasma clearance (CL) 13-57 L/hUnbound fraction 5.5%
Clozapine is principally biotransformed by N-demethylation and N-oxidation of the
piperazine ring, yielding N-desmethylclozapine and clozapine N-oxide metabolites (Jann et
al. 1993, Pirmohamed et al. 1995, Fang et al. 1998; Figure 1). In addition, hydroxylated and
chemically reactive metabolites are formed (Jann et al. 1993, Pirmohamed et al. 1995).
Clozapine-N-oxide may be reduced back to clozapine in the presence of NADPH, but the
reaction is inhibited in vitro by ascorbic acid (Pirmohamed et al. 1995). The enzyme
responsible for this conversion remains unknown. The interconversion of clozapine and
clozapine-N-oxide was demonstrated also in vivo in a randomized crossover study in which
formation of clozapine-N-oxide from clozapine was slower than the formation of clozapine
from clozapine-N-oxide (Chang et al. 1998).
C
R E V I E W O F T H E L I T E R A T U R E
27
Unbound fractions in serum for clozapine, N-desmethylclozapine, and clozapine-N-oxide
are approximately 5.5%, 9.7%, and 24.6% (Schaber et al. 1998). The mean ratio
clozapine:N-desmethylclozapine:clozapine-N-oxide varies greatly, but in plasma it is about
3:2.5:1, and in urine about 1:43:77 (Wagstaff & Bryson 1995, Schaber et al. 1998). At least
80% of a clozapine dose is eliminated in metabolites in the urine and feces (Baldessarini &
Frankenburg et al. 1991). Enterohepatic recycling of clozapine is suggested by the biphasic
manner of its elimination, with a second increase in concentration taking place within a few
hours after Tmax (Lin et al. 1994, Dahl et al. 1994). If a very high dose of clozapine is
ingested (intentional poisoning), the N-demethylation pathway is saturated (Reith et al.
1998). Clozapine is a poor substrate for P-gp, as suggested by the minimal effects of some
P-gp inhibitors on its plasma concentrations (Lane et al. 2001a, 2001b, Taylor et al. 1999)
and by the in vitro Km value of 58 M for P-gp ATPase activity towards clozapine (Boulton
et al. 2002).
Figure 1. Clozapine metabolism
Several in vitro studies suggest that in clozapine metabolism CYP1A2 is the main enzyme
catalyzing the formation of N-desmethylclozapine, and CYP3A4 is mostly responsible for
the formation of clozapine-N-oxide (Tugnait et al. 1999, Eiermann et al. 1997, Pirmohamed
et al. 1995, Tugnait et al. 1997, Linnet & Olesen 1997, Olesen & Linnet 2001) (Table 4).
One study found CYP3A4 to be the principal enzyme in the formation of both N-
desmethylclozapine and clozapine N-oxide (Fang et al. 1998). In addition, a few other CYP
N
N
N
NCH3
Cl
H
O
N
N
N
NCH3
Cl
H
N
N
N
N
Cl
H
H
Other metabolites
Clozapine-N-oxide Clozapine N-desmethylclozapine
CYP1A2CYP3A4CYP3A3CYP2C19
CYP1A2FMO
R E V I E W O F T H E L I T E R A T U R E
28
enzymes, and flavine-containing monooxygenase type 3 (FMO3), have been shown to
metabolize clozapine to N-desmethylclozapine or clozapine-N-oxide (Tugnait et al. 1997)
(Table 4). An earlier study showed that in human liver microsomal preparations and in
recombinant RT2D6 cells, clozapine was metabolized by CYP2D6 to unknown metabolites
other than N-desmethylclozapine or clozapine-N-oxide (Fischer et al. 1992). In the overall
metabolism of clozapine, CYP2D6 seems to be of only minor importance. The formation of
N-desmethylclozapine, clozapine-N-oxide, 7-hydroxyclozapine, and two unidentified
metabolites was similar in those human liver microsomes prepared from the livers of poor
or of extensive metabolizers of CYP2D6 (Pirmohamed et al. 1995).
Table 4. Studies on in vitro oxidation of clozapine to its main metabolites N-desmethylclozapine and clozapine N-oxide.
Enzymes responsible for metabolism of
clozapine to
Source Method Clozapineconcentration
N-desmethylclozapine Clozapine-N-oxide
Pirmohamed et al. 1995 Human livermicrosomes
2 M CYP1A2 CYP3A4, FMO
Eiermann et al. 1997 Human livermicrosomes
50 M CYP1A2, 3A4 CYP3A4
Linnet & Olesen 1997 cDNA-expressedhuman CYPs
0.5-50 M CYP3A4, 1A2, 2C,2C19, 2D6
CYP3A4
Fang et al. 1998 Human livermicrosomes
20 M300 M
CYP1A2, 3A4CYP3A4, 1A2
CYP3A4, 1A2CYP3A4
Human livermicrosomes
1-1000 M CYP1A2, 3A4 CYP3A4, 1A2Tugnait et al. 1999
cDNA-expressedhuman CYPs
350 M CYP2D6, 1A2, 3A4 CYP3A4, 1A2
Olesen & Linnet 2001 Human livermicrosomes
5 M50 M
CYP1A2, 2C19CYP3A4, 1A2, 2C19
Not studied
Clozapine clearance and CYP1A2 activity are similarly distributed in a Swedish population
(Jerling et al. 1997). About 70% of variance in the oral clearance of clozapine is explained
by CYP1A2 activity, determined by the caffeine N3-demethylation index (Bertilsson et al.
R E V I E W O F T H E L I T E R A T U R E
29
1994). Very high (ultrarapid) CYP1A2 activity, caused by homozygous alleles with C A
polymorphism in intron 1 of the CYP1A2 gene (CYP1A2*1F), is linked to exceptionally
low serum clozapine concentration and non-response to clozapine (Bender & Eap 1998,
Sachse et al. 1999, Özdemir et al. 2001). A study by Carrillo et al. (1998) showed that the
plasma concentration ratio of N-desmethylclozapine to clozapine is a fairly good estimate of
individual CYP1A2 activity. Using that as an estimate of CYP1A2 activity, Dailly et al.
(2002) found in their population pharmacokinetic analysis that CYP1A2 activity has a major
and a linear effect on clozapine clearance that could explain inter- and intraindividual
variation in clozapine plasma concentrations.
Serum clozapine concentrations were similar in healthy volunteers who each took a single
oral dose of 10 mg clozapine, irrespective of their polymorphic hydroxylation phenotype for
either debrisoquine or S-mephenytoin, indicating minimal involvement of CYP2D6 and of
CYP2C19, respectively (Dahl et al. 1994). Furthermore, serum clozapine concentrations
were similar in patients on clozapine monotherapy, and those treated with both CYP2D6
inhibitors and clozapine (Jerling et al. 1994).
The pharmacokinetics of clozapine is largely variable both inter- and intraindividually
(Byerly & De Vane 1996, Kurz et al. 1998, Schaber et al. 1998, Wagstaff & Bryson 1995,
Cheng et al. 1988, Choc et al. 1990). In a 26-week prospective study, the mean
intraindividual coefficient of variation (CV) for dose- and weight-corrected plasma
clozapine concentration was 54.9% (+ SD 23.9), even though patients (n = 41) did not use
other medications known to interact significantly with clozapine (Kurz et al. 1995). In
another study of shorter duration, the intrapatient variation was 27%, and the interpatient
weight-corrected variation 50.9%, for 44 outpatients in weekly monitoring (Centorrino et al.
1994a). In a study of 14 previously psychotropic drug-naive Chinese patients, interpatient
variability for the AUC of clozapine was over 6-fold (Lin et al. 1994). The range for Tmax
was 1.0 to 5.0 hours, for oral clearance 19 to 123 L/h, for the apparent volume of
distribution 7 to 22 L/kg, and for T½ 5.5 to 35 hours (Lin et al. 1994). The Tmax for N-
desmethylclozapine was between 1.5 and 40 hours, and the metabolite was eliminated in
parallel with clozapine, suggesting formation-rate-limited elimination of the N-
R E V I E W O F T H E L I T E R A T U R E
30
desmethylclozapine metabolite (Lin et al. 1994). In that case, the T½ of N-
desmethylclozapine has to be shorter than that of clozapine (Rowland & Tozer 1995).
Men seem to have higher clearance of clozapine than do women, whose dose- and weight-
corrected plasma concentrations of clozapine are 40% higher than in men (Haring et al.
1989, Centorrino et al. 1994a). In children and adolescents (aged 9-16 years, n = 6) on
clozapine monotherapy and a caffeine-free diet, N-desmethylclozapine serum
concentrations were, unlike in adults, higher than those of clozapine, but dose- and weight-
corrected serum clozapine concentrations were similar to those in adults (Frazier et al.
2003). Hepatic diseases may significantly reduce the metabolism of many drugs
metabolized by CYP enzymes (e.g., theophylline) and increase their plasma concentrations
(Gibson & Skett 2001, Kraan et al. 1988). Metastases in the liver were associated in one
report with a 10-fold increase in serum clozapine concentration (Uges et al. 2000). Renal
disease is not expected to reduce significantly the clearance of clozapine, and no
adjustments in clozapine dose are necessary (Bennett 1997). Plasma concentration of
clozapine in one newborn infant was reported to be 2-fold higher than in the maternal
plasma or amniotic fluid. In the same mother’s breast milk, during the first week after
delivery, clozapine concentration was about 3- to 4-fold higher than in her plasma (Barnas
et al. 1994).
Animal studies. In line with their differing lipophilicity, clozapine and its metabolites in
rats were not equally distributed between the brain and peripheral tissues: after repeated oral
ingestion of clozapine, concentrations of N-desmethylclozapine were 1.5- to 2.2-fold higher
in serum than those of clozapine. In these rat brains, however, clozapine concentrations
were 3- to 4-fold higher than those of N-desmethylclozapine (Weigmann et al. 1999).
Recently, fatty-acid derivatives of clozapine have been developed and tested preclinically in
the rat, and they show a more selective distribution to the brain, lower concentrations in
peripheral tissues, higher potency, and a longer duration of action than does clozapine
(Baldessarini et al. 2001).
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4.1.1. Pharmacokinetic interactions
4.1.1.1. SSRIs and clozapine
ntil recently, knowledge of the pharmacokinetic interactions of clozapine has largely
relied on sporadic case-reports (Taylor 1997). Most impressive among the reports of
clozapine interactions are those reporting highly increased clozapine concentrations during
concomitant treatment with normal doses of the selective serotonin reuptake inhibitor
(SSRI) fluvoxamine. Such increases in serum clozapine concentrations have been high,
sometimes even more than 10-fold (Hiemke et al. 1994, Jerling et al. 1994, Dequardo &
Roberts 1996, DuMortier et al. 1996, Koponen et al. 1996, Wetzel et al. 1998, Szegedi et al.
1999, Fabrazzo et al. 2000, Özdemir et al. 2001) (Table 5). Fluvoxamine raised the serum
elimination T½ of clozapine from 17.3 to 50.6 hours (Wetzel et al. 1998). Furthermore,
adding fluvoxamine to clozapine therapy has produced extrapyramidal symptoms (Kuo et
al. 1998). Fluvoxamine is a potent inhibitor of CYP1A2 (Ki 0.12 to 0.24) and a moderate
inhibitor of CYP2C19 (Brøsen et al. 1993, Jeppesen et al. 1996a). The 2000 study by
Olesen & Linnet suggested that in vivo fluvoxamine significantly inhibits N-demethylation
of clozapine but not the N-oxidation pathway. In a clozapine-treated patient with ultrarapid
CYP1A2 activity, fluvoxamine 50 mg/day reduced his CYP1A2 activity by approximately
50% and raised serum concentration of clozapine about 4-fold (Özdemir et al. 2001).
Fluoxetine and its main metabolite norfluoxetine are both potent inhibitors of CYP2D6
(Brøsen & Skjelbo 1991). Fluoxetine seems to raise serum clozapine concentrations by 30%
to 76% (Centorrino et al. 1994b, Centorrino et al. 1996, Spina et al. 1998), but a case
showing no change in serum clozapine concentration during fluoxetine has also been
reported (Eggert et al. 1994). A fatal pharmacokinetic interaction between clozapine and
fluoxetine was suggested in the case of a patient with a high postmortem plasma clozapine
concentration who died from pulmonary edema, visceral vascular congestion, paralytic
ileus, gastroenteritis, and eosinophilia (Ferslew et al. 1998).
Studies on the effect of paroxetine and sertraline on serum clozapine concentrations are
controversial (Table 5). The potent CYP2D6 inhibitor paroxetine is reported to show a
U
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32
moderate enhancement effect on clozapine concentrations of 30% to 57%, and in one case-
report an enhancement of up to 2-fold (Centorrino et al. 1996, Joos et al. 1997, Wetzel et al.
1998, Spina 2000). One study found no effect by paroxetine on serum clozapine
concentrations (Wetzel et al. 1998). One study found 30% higher serum clozapine
concentrations in patients treated with sertraline than in controls (Centorrino et al. 1996). In
another study in which patients served as their own controls, sertraline showed no effect on
clozapine concentrations (Spina et al. 2000).
A weak inhibition of clozapine metabolism by the SSRI citalopram, which is not a strong
inhibitor of any of the major drug-metabolizing enzymes, is suggested in one case-report
(Borba & Henderson 2000), but was not observable in two studies (Taylor et al. 1998).
4.1.1.2. Other inhibitors of CYP enzymes and clozapine
indings on effects of valproate on serum clozapine concentration have been
controversial (Spina & Perucca 2002). While some found a decrease of up to 51% in
serum clozapine concentration, others have found a mean increase of 39% or even no effect
at all (Finley & Warner 1994, Centorrino et al. 1994b, Longo & Salzman 1995, Facciolà et
al. 1999) (Table 5).
The potent CYP3A4 inhibitor erythromycin has been linked to increased serum clozapine
concentrations and adverse effects in two case-reports of patients with acute infections
(Funderburg et al. 1994, Cohen et al. 1996). In one randomized crossover study,
erythromycin did not affect clozapine concentrations (Hägg et al. 1999b) (Table 5).
The antidepressants nefazodone (CYP3A4 inhibitor) and reboxetine seem to have no effect
on serum clozapine concentrations, although elevated clozapine concentrations were
reported during 300 mg, but not during 200 mg of nefazodone (Taylor et al. 1999, Khan &
Preskorn 2001, Spina et al. 2001) (Table 5).
F
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Koreen et al. (1995) reported on a female patient whose serum clozapine concentration
more than doubled, and whose N-desmethylclozapine to clozapine ratio decreased when
risperidone 2 mg daily was added to her clozapine treatment. In another patient, plasma
clozapine concentration increased by 74% after addition of a 2-mg daily dose of risperidone
(Tyson et al. 1995) (Table 5).
Cimetidine, a H2-receptor antagonist that inhibiting several CYP enzymes, has raised
clozapine serum concentrations and its adverse effects (Szymanski et al. 1991). Elevated
clozapine concentrations appeared also with lamotrigine and lisinopril (Abraham et al.
2001, Kossen et al. 2001). In a study by Palego et al. (2002), those clozapine patients co-
treated with typical antipsychotics exhibited no increase in dose- and weight-corrected
plasma concentrations of clozapine and N-desmethylclozapine. In a study with only two
patients on clozapine, the anti-obesity drug orlistat had no effect on plasma concentrations
of clozapine (Hilger et al. 2002). In a retrospective analysis of clozapine patients who used
concomitant omeprazole, and whose omeprazole was later switched to pantoprazole, serum
concentrations during both drugs were similar (Mookhoek & Loonen 2002) (Table 5).
In an open study, abstinence from dietary caffeine resulted in a 47% decrease in patients’
mean concentration of serum clozapine (Carrillo et al. 1998). In healthy volunteers, oral
caffeine raised the mean clozapine AUC (0, ) by 19%, and reduced mean oral clearance of
clozapine by 14% (Hägg et al. 2000).
Grapefruit juice (CYP3A4 inhibitor) seems to have no effect on serum clozapine or its main
metabolite concentrations (Vandel et al. 2000, Özdemir et al. 2001, Lane et al. 2001a,
2001b). Neither grapefruit juice nor ketoconazole 400 mg, potent inhibitors of CYP3A4,
seem to have any significant effect on plasma concentrations of clozapine or of its two main
metabolites (Lane et al. 2001a).
Clozapine as an inhibitor. Clozapine is a weak inhibitor (Ki 30-95 M) of CYP1A2-
mediated theophylline metabolism in vitro (Rasmussen et al. 1995). Fischer et al. (1992)
found in their study in human liver microsomal preparations and in recombinant RT2D6
cells that clozapine inhibits CYP2D6 (Ki 4-19 M). The serum nortripyline (CYP2D6
R E V I E W O F T H E L I T E R A T U R E
34
substrate) concentration was reported to increase by 2-fold after the initiation of 150 mg of
clozapine (Smith & Riskin 1994). Clozapine seems to raise serum cocaine concentration in
a dose-dependent manner, but it diminishes subjective cocaine effects (the “high” or “rush”)
(Farren et al. 2000). Cocaine is metabolized by hepatic and plasma esterases rather than by
CYP enzymes (in Farren et al. 2000).
4.1.1.3. Inducers of CYP enzymes and clozapine
everal inducers of CYP enzymes seem to enhance clozapine clearance. In a group
comparison of TDM data, patients who used both clozapine and carbamazepine had
68% lower plasma clozapine concentrations, and 43% lower clozapine concentration/dose
(C/D) ratios than did patients taking only clozapine; the C/D ratio was inversely correlated
with the daily dose of carbamazepine (Jerling et al. 1994). In the same study, all eight
patients suitable for intraindividual comparison had a lower C/D ratio when they were on
than off carbamazepine (Jerling et al. 1993). In two patients, 2 weeks after discontinuation
of carbamazepine, clozapine plasma concentrations increased by 71% and 100% (Raitasuo
et al. 1993). In a report of two cases, after phenytoin was initiated, clozapine concentrations
decreased by 65 to 85% (Miller 1991). After phenobarbital was discontinued, plasma
clozapine concentration was reported to increase by about 75% (Lane et al. 1998). Facciolà
et al. (1998) found that patients treated with clozapine and phenobarbital had lower plasma
concentrations of clozapine and higher N-desmethylclozapine to clozapine ratios than did
patients treated with clozapine alone. In one forensic patient, plasma concentration of
clozapine decreased to one-sixth 2 to 3 weeks after the initiation of rifampicin 600 mg/day,
and when ciprofloxacin was substituted for rifampicin were 60% higher than before
rifampicin (Joos et al. 1998). Another case-report also suggests that the CYP1A2 inhibitor
ciprofloxacin may raise the plasma concentration of clozapine and cause serious adverse
effects (Markowitz et al. 1997) (Table 5).
Several reports suggest that clozapine metabolism is induced by cigarette-smoking (Seppälä
et al. 1999, Hasegawa et al. 1993, Meyer 2001) (Table 5). Aspiration pneumonia occurred
in one patient who had a high clozapine concentration after smoking cessation (Meyer
S
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35
2001). One clozapine patient was admitted to hospital unconscious 2 weeks after cessation
of heavy smoking: He salivated excessively and before recovery was pulseless with a
systolic blood pressure of about 40 mmHg. The authors suggested that these symptoms were
due to clozapine intoxication caused by normalization of induced clozapine metabolism
after smoking cessation (Skogh et al. 1999). In another patient, plasma clozapine
concentration increased after cessation of both tobacco- and cannabis-smoking (Zullino et
al. 2002).
4.2. Pharmacodynamics
lozapine is an antagonist, with Ki < 10 nM, for serotonin 5-HT2A, 5-HT2C, 5-HT6, and
5-HT, for dopamine D4, muscarine M1, and adrenergic 1 receptors, but unlike the
typical antipsychotics, clozapine is only a weak antagonist (Ki 172 nM) for D2 receptors
(Meltzer 1994). One of the most popular hypotheses on the mechanism of action of
clozapine is the dopamine-serotonin hypothesis, which suggests that the combination of the
weaker D2 with the more potent 5-HT2A is vital for its superior efficacy (Meltzer 1989,
Meltzer 2002). Genetic polymorphisms in dopamine and serotonin receptors are suggested
as factors influencing patients’ responsiveness to clozapine (Mancama et al. 2002).
One of the two main metabolites of clozapine, N-desmethylclozapine, is biologically active
(Kuoppamäki et al. 1993, Young et al. 1998). Its contribution to overall antipsychotic
effects of the drug seems, however, to be minor. The other main metabolite, clozapine-N-
oxide, is not biologically active (Kuoppamäki et al. 1993).
C
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36
Table 5. Pharmacokinetic interactions of clozapine with drugs (a) and other factors (b)
a.
Interacting drug
Daily dose
Subjects (nb),Study design
Effect on serum CLZconcentration
Source
SSRIs
Citalopram 40 mg Patients (8), sequential No change Avenoso et al. 199820 mg Patients (5), sequential No change Taylor et al. 1998
Fluoxetine Mean 36 mg Patients (6, 17 controls),parallel
Increase, 76% Centorrino et al. 1994b
80 mg Patient, case-report No change Eggert et al. 1994Mean 39 mg Patients (14, 40 controls),
parallelIncrease, 30% Centorrino et al. 1996
20 mg Patients (10), sequential Increase, 58% Spina et al. 1998Fluvoxamine 100 mg Patient, case-report Increase, 8-fold Hiemke et al. 1994
100 to 150 mg Patients (2), case-report Increase, 5- to 10 -fold Jerling et al. 199425 mg Patient, case-report Increase, 4- to 9 -fold Dequardo & Roberts 1996100 to 200 mg Patients (3), case-report Increase, 3- to 9 -fold DuMortier et al. 1996150 mg Patients (2), case-report Increase, 5- to 10 -fold Koponen et al. 199650 mg Patients (16), sequential Increase, 3-fold Wetzel et al. 199850 mg Patients (16), sequential Increase, 2- to 5-fold Szegedi et al. 1999100 mg Patients (16), sequential Increase, 5-fold Fabrazzo et al. 200050 mg Patient, case study Increase, 3-fold Özdemir et al. 2001
Paroxetine Mean 31 mg Patients (16, 40 controls),parallel
Increase, 57% Centorrino et al. 1996
20 mg Patient, case-report Increase, 30% to 2-fold Joos et al. 199720 mg Patients (14), sequential No change Wetzel et al. 199820 to 40 mg Patients (9), sequential Increase, 31% Spina et al. 2000
Sertraline Mean 92 mg Patients (10, 40 controls),parallel group
Increase, 30% Centorrino et al. 1996
300 mg Patient, case-report Increase, 2-fold Pinninti & de Leon 199750 to 100 mg Patients (8), sequential No change Spina et al. 2000
OtherinhibitorsCimetidine 800 mg Patient, case-report Increase, 60% Szymanski et al. 1991Ciprofloxacin 1000 mg Patient, case-report Increase, 2-fold Markowitz et al. 1997
1000 mg Patient, case-report Increase, 60% Joos et al. 1998Erythromycin 1000 mg Patient, case-report Increase, 2-fold Funderburg et al. 1994
1000 mg Patient, case-report Increase, 3-fold Cohen et al. 19961500 mg Healthy (12), randomized
crossoverNo change Hägg et al. 1999b
Ketoconazole 400 mg Patients (5), sequential No change Lane et al. 2001aLamotrigine 100 mg Patient, case-report Increase, 3-fold Kossen et al. 2001Lisinopril 5 to 10 mg Patient, case-report Increase, 2- to 3 -fold Abraham et al. 2001Nefazodone 400 mg Patients (6), sequential No change Taylor et al. 1999
200 mg Patient, case-report No change Khan & Preskorn 2001300 mg Patient, case-report Increase, 75% Khan & Preskorn 2001
Orlistat 360 mg Patients (2), sequential No change Hilger et al. 2002Ranitidine 300 mg Patient, case-report No change Szymanski et al. 1991Reboxetine 8 mg Patients (7), sequential No change Spina et al. 2001Risperidone 2 mg Patient, case-report Increase, 2-fold Koreen et al. 1995
2 mg Patient, case-report Increase, 74% Tyson et al. 1995
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37
Interacting drug
Daily dose
Subjects (nb),Study design
Effect on serum CLZconcentration
Source
Valproate 750 to 1000 mg Patients (4), sequential Decrease, 23 to 51% Finley & Warner 1994Mean 1060 mg Patients (11, 17 controls),
parallelIncrease, 39% Centorrino et al. 1994b
Mean serumconcentration62 mg/L
Patients (7), sequential Decrease, 15% Longo & Salzman 1995
900 to 1200 mg Patients (6), sequential No change Facciolà et al. 1999600 to 1500 mg Patients (15, 22 controls),
parallelNo change Facciolà et al. 1999
Inducers
Carbamazepine 600 to 800 mg Patients (2), case-report Decrease, 42 to 50% Raitasuo et al. 1993Not stated Patients (17, 124 controls),
retrospective, parallelDecrease, 68% Jerling et al. 1994
500 to 1500 mg Patients (12), sequential Decrease, 47% Tiihonen et al. 1995Phenobarbital 60 mg Patient, case-report Decrease, 43% Lane et al. 1998
Concentration12 to 31 g/mL
Patients (7, 15 controls),parallel
Decrease, 35% Facciolà et al. 1998
Phenytoin 300 mg Patients (2), case-report Decrease, 65 to 85% Miller 1991Rifampicin 600 mg Patient, case-report Decrease, 83% Joos et al. 1998
b.Interacting factor
Daily dose
Subjects (nb),Study design
Effect on serum CLZconcentration
Source
Caffeine + coffee Patient, case-report Increase, 2-fold Odom-White & de Leon 1996Coffee 150 to 1100 mg
of caffeinePatients (7), sequential Increase, 2-fold Carrillo et al. 1998
Caffeine 400 to 1000 mg Healthy (12), open randomizedcrossover
AUC increased 19% Hägg et al. 2000
Cigarette-smoking Patients (38, 18 controls ),parallel
Decrease, 20% Hasegawa et al. 1993
Patients (34, 10 controls),parallel
Decrease, 38% Seppälä et al. 1999
Patients (11), sequential Decrease, 41% Mayer 2001Grapefruit juice 500 mL Patients (9), sequential No change Vandel et al. 2000
250 mL Patients (21), sequential No change Lane et al. 2001a500 mL Patients (15), sequential No change Lane et al. 2001b1125 mL Patient, case study No change Özdemir et al. 2001
Infection or inflammation Patients (5), case-report Increase, 2- to 10-fold van der Molen-Eijgenraamet al. 2001
Patient, case-report Increase, 3-fold Raaska et al. 2002b
Patient, case-report Increase, 2-fold de Leon & Diaz 2003
Patients (4), case-report Increase, 2-fold Haack et al. 2003
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4.3. Adverse effects
lozapine has a low propensity to cause extrapyramidal symptoms, and it does not raise
serum prolactin or corticotrophin levels (Wagstaff & Bryson 1995). On the other
hand, common adverse effects of clozapine are sedation, hypersalivation, bodyweight gain,
constipation, tachycardia, and seizures, and most of these are dose-dependent (Wagstaff &
Bryson 1995). In one study, patients found clozapine-related adverse effect more acceptable
than neurologic symptoms they had encountered during treatments with typical
antipsychotics (Centorrino et al. 1994a).
About 1% of clozapine-treated patients develop agranulocytosis, a granulocyte count less
than 500 cells/mm3 (Lieberman & Alvir 1992), which is the reason for mandatory weekly,
and after the first 18 weeks, monthly white blood-cell count measurements as long as
patients are using the drug (Baldessarini & Frankenburg et al. 1991). As a rare but severe
acute reaction to clozapine, some patients develop myocarditis with eosinophilic infiltrates
and myocytolysis (Killian et al. 1999). A mild elevation of serum transaminases occurs in
up to 50% of patients, but toxic hepatitis is not commonly encountered (Kellner et al. 1993).
As a rare allergic manifestation, clozapine may cause acute interstitial nephritis (Elias et al.
1999). EEG abnormalities are more common with clozapine than with other antipsychotics
(Pisani et al. 2002, Centorrino et al. 2002). About 30% of clozapine recipients gain weight
(Meltzer et al. 2003). Clozapine is linked to increased risk for type 2 diabetes, and for lipid
abnormalities (Henderson 2001). Some patients suffer from nocturnal enuresis that may be
related to urinary retention caused by the anticholinergic effects of clozapine (Centorrino et
al. 1994a, Cohen et al. 1994).
Clozapine overdoses seem to be relatively rare (Reith et al. 1998, Wagstaff & Bryson 1995).
Coma, lethargy, tachycardia, agitation, and confusion are common symptoms in
intoxications (Wagstaff & Bryson 1995), with seizures and metabolic acidosis also reported
(Hägg et al. 1999a). Some patients with a lethal clozapine overdose suffer cardiac failure,
aspiration pneumonia or renal failure (Wagstaff & Bryson 1995). In one case of clozapine
intoxication, the ingestion of 12.5 g of clozapine resulted in a measured concentration of
C
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39
9100 g/L with a calculated peak value between 35 000 g/L and 24 000 g/L for an
assumed absorption T½ of 0.5 and 4 hours, respectively (Sartorius et al. 2002). For both
absorption half-lives, the elimination T½ was calculated to be about 24 h (Sartorius et al.
2002). Surprisingly, sometimes even with a lethal concentration of serum clozapine, typical
adverse effects of clozapine toxicity are absent or go unnoticed (Uges et al. 2000).
Clozapine should not be given to any healthy subject. In one bioequivalence study, when 17
healthy volunteers each received a single 25-mg dose of clozapine, several had serious
adverse effects uncommon in patients at similar doses. Orthostatic hypotension, 60/29
mmHg at the lowest, occurred in 10 subjects, bradycardia below a pulse rate of 40 beats per
minute in eight subjects, and in two subjects, cardiac arrest lasting 10 and 60 seconds
(Pokorny et al. 1994).
4.4. Therapeutic drug monitoring (TDM)
onitoring of serum clozapine concentrations is recommended in optimizing of
therapeutic dosage (Freeman 1997, Olesen 1998, Buur-Rasmussen & Brøsen 1999).
Based on several studies (Perry et al. 1991, Miller et al. 1994, Potkin et al. 1994, Kronig et
al. 1995, Hasegawa et al. 1993), a therapeutic threshold value of about 400 g/L has been
suggested (Freeman 1997). However, in one randomized study, serum clozapine
concentrations in the range of 350 to 450 g/L offered no advantage over concentrations in
the range of 200 to 300 g/L (VanderZwaag et al. 1996). In one prospective study with
repeated TDM of serum clozapine and its two main metabolite concentrations, dose-
corrected clozapine concentrations were significantly lower in responders (n = 21) than in
non-responders (n = 13), although the concentration data collected could not differentiate
responders from non-responders (Dettling et al. 2000). Interestingly, clozapine seems to be
effective in L-DOPA-induced psychosis in Parkinson’s disease at much lower
concentrations (< 20 g/L ) than in schizophrenia (Meltzer et al. 1995).
During clozapine treatment, TDM is also useful in avoiding unnecessarily high clozapine
concentrations. Serum clozapine concentrations above 1000 g/L (3060 nmol/L) increase
M
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40
risk for confusion, delirium, and generalized seizures (Freeman 1997, Buur-Rasmussen &
Brøsen 1999). To aid in dose optimization, a dosing nomogram has been developed for
prediction of steady-state clozapine plasma concentration (Perry et al. 1998). This
nomogram is based on the effect of sex, smoking status, and dose on steady-state plasma
clozapine concentration (Perry et al. 1998).
5. Drugs and other factors as inhibitors of clozapine metabolism
5.1. Itraconazole
Figure 2. Chemical structure of itraconazole
n plasma, itraconazole is mainly bound to albumin, leaving only 0.2% of the drug
unbound (De Beule & Van Gestel 2001). It has an apparent volume of distribution of 11
L/kg (De Beule & Van Gestel 2001). Itraconazole accumulates in the kidney, liver, bone,
stomach, spleen, the female genital tract, and in muscle, and keratinous tissues (De Beule &
Van Gestel 2001). It is eliminated mostly by metabolism (De Beule & Van Gestel 2001),
with CYP3A4 the principal enzyme metabolizing it (Backman et al. 2000). Itraconazole
metabolism is induced by rifampicin, carbamazepine, and phenytoin (Backman et al. 2000).
Itraconazole has saturable elimination with a terminal T½ of 20 to 24 hours after the usual
I
N N NNNCH2O
OCHCH2CH3
CH3
H
O ON
N N
Cl
Cl
R E V I E W O F T H E L I T E R A T U R E
41
single dose, but in the steady state T½ raises to about 30 hours or even more (De Beule &
Van Gestel 2001). Itraconazole is a highly lipophilic and poorly soluble weak base (Gupta et
al. 1994). In order to be absorbed from the gastrointestinal tract, it has to be transformed by
acid in the stomach into a soluble hydrochloride salt. Its absorption is reduced with reduced
gastric acidity (Gupta et al. 1994). Oral bioavailability improves if the drug is taken with
food.
It is a triazole antifungal agent with a broad spectrum of activity against fungal pathogens,
including dermatophytes, Candida spp., and Aspergillus spp. (Grant & Clissold 1989, Meis
& Verweij 2001). It is primarily fungistatic (Gupta et al. 1994). Its mechanism of action is
inhibition of the fungal cytochrome P450 (CYP) enzyme 14-demethylase that is responsible
for 14 -demethylation of lanosterol in the ergosterol synthesis pathway (Meis & Verweij
2001, De Beule & Van Gestel 2001). Ergosterol is a vital component of the fungal cell
membrane (De Beule & Van Gestel 2001).
Oral itraconazole 200 to 400 mg/day for one week is used in the treatment of extensive
superficial fungal infections, or if a topical treatment has failed and is also used in treatment
of fungal infections of the nails (onychomycosis), because it accumulates in the nails and
other keratinous tissues. In the nails, therapeutic concentrations of itraconazole persist for
up to 9 months after the end of therapy (Meis & Verweij 2001). Itraconazole is also
recommended as first-choice treatment for many systemic fungal infections and duration of
itraconazole treatment may be very long: For example, in treatment of meningeal
coccidioidomycosis in immunocompromised patients, the dose is itraconazole 400 mg/day
for the rest of one’s life (Meis & Verweij 2001).
Itraconazole is a potent inhibitor of CYP3A4, with Ki values < 1 M for many CYP3A4-
catalyzed reactions (Backman et al. 2000), and strongly inhibits the metabolism of many
orally administered CYP3A4 substrates such as midazolam (Olkkola et al. 1994), triazolam
(Varhe et al. 1994), and buspirone (Kivistö et al. 1997). For example, itraconazole 200 mg
daily will raise the AUC of oral midazolam by 10- to 15-fold, and its T½ by 3- to 4-fold
(Olkkola et al. 1994). Among other CYP3A4 substrates susceptible to a considerable
increase in oral bioavailability when used in combination with itraconazole are: lovastatin
R E V I E W O F T H E L I T E R A T U R E
42
(> 20-fold AUC) and lovastatin acid (AUC 20-fold) (Neuvonen & Jalava 1996), felodipine
(6-fold) (Jalava et al. 1997a), simvastatin and simvastatin acid (> 10-fold) (Neuvonen et al.
1998). Oral itraconazole can also raise the AUC of the inhaled CYP3A4 substrate
budesonide by about 4-fold (Raaska et al. 2002a).
Itraconazole is also a potent inhibitor of P-glycoprotein (Backman et al. 2000, Wang et al.
2002a), and it raises serum concentration of digoxin and reduces its renal clearance
(Partanen et al. 1996, Jalava et al. 1997b).
5.2. Ciprofloxacin
Figure 3. Chemical structure of ciprofloxacin
he oral bioavailability of ciprofloxacin ranges from 55 to 85%. Tmax values range
from 47 to 90 min. The volume of distribution is between 1.7 and 2.5 L/kg. Although
tissue penetration of ciprofloxacin in most tissues is good, concentration in cerebrospinal
fluid (CSF) is less than 50% of serum concentration. Only about 20 to 40% of plasma
concentration is bound to plasma proteins. Ciprofloxacin is excreted mainly in its
unchanged form via the kidneys, in bile, and through the intestinal mucosa. T½ is between 3
and 5 hours (Aminimanizani et al. 2001).
In the gastrointestinal tract, ciprofloxacin forms insoluble cation chelates with magnesium-,
aluminium- and calcium-containing antacids and with sucralfate, which may reduce its
bioavailability to 15%; the same mechanism applies to oral iron and zinc preparations
(Aminimanizani et al. 2001). Ferrous sulphate reduced the AUC and peak plasma
T
N
OF
NNH
COOH
R E V I E W O F T H E L I T E R A T U R E
43
concentration of ciprofloxacin in one controlled study by more than 50% (Lehto et al.
1994). Concomitant ingestion of milk or yogurt has reduced the bioavailability of
ciprofloxacin by a respective 30% and 36% (Neuvonen et al. 1991).
Ciprofloxacin was introduced in the 1980s as the first fluoroquinolone antibacterial agent
(Aminimanizani et al. 2001). It has bactericidal activity against both Gram-negative and
Gram-positive bacteria, and its mechanism of action is in its interactions with DNA gyrase
and topoisomerase IV enzymes that lead to cleavage of bacterial DNA (Hooper 1999).
The most common adverse effects are diverse gastrointestinal symptoms, especially nausea,
with an incidence of 10%, and diarrhea (Bertino 2000). Skin reactions are common, as well
(Bertino 2000). CNS symptoms such as dizziness, headache, tremor, and restlessness occur
in about 3% of ciprofloxacin-treated patients (Bertino 2000). In spontaneously reported
adverse reactions, fluoroquinolones are linked, at a much higher percentage than are other
systemic antimicrobials, to reactions involving the musculoskeletal system (15% vs. 0.3%)
and CNS (12% vs. 4%), and involving psychiatric symptoms (9% vs. 2%) (Leone et al.
2003). Fluorokinolones as a group are associated with Achilles tendon disorders, which
seem to affect especially those who are over 60 years of age and use corticosteroids (van der
Linden et al. 2002).
Ciprofloxacin is a CYP1A2 inhibitor, although metabolism is not a major route in its
elimination (Fuhr et al. 1992). It inhibits the metabolism of CYP1A2 substrates such as
theophylline, caffeine, and ropivacaine (Batty et al. 1995, Nicolau et al. 1995, Jokinen et al.
2003).
R E V I E W O F T H E L I T E R A T U R E
44
5.3. Risperidone
Figure 4. Chemical structure of risperidone
isperidone is almost completely absorbed in the gastrointestinal tract. After oral
administration, time to Cmax is about 0.8 to 1 hour (Byerly & De Vane 1996). Both
in oral and intravenous administration, the T½ of risperidone is 3 to 24 hours, depending on
CYP2D6 activity (Heykants et al. 1994, Byerly & De Vane 1996). The Vd of risperidone is
1.1 L/kg (Byerly & De Vane 1996). Risperidone undergoes first-pass metabolism resulting
in the formation of the active metabolite 9-OH-risperidone, another main metabolite, 7-OH-
risperidone, and a number of less important metabolites (Byerly & De Vane 1996, Heykants
et al. 1994, DeVane & Markowitz 2000). The sum of risperidone and 9-OH-risperidone is
referred to as the antipsychotic fraction, or the active moiety (Heykants et al. 1994). The T½
and AUC (0- ) for the active moiety between intravenous, intramuscular, and oral routes of
administration do not vary (Huang et al. 1993). About 10% of risperidone is excreted
unchanged in the urine (Heykants et al. 1994).
The metabolism of risperidone is stereoselective, with CYP2D6 being mainly responsible
for (+)-9-hydroxylation, and CYP3A4 for (-)-9-hydroxylation (Yasui-Furukori et al. 2001).
The importance of these two enzymes for risperidone metabolism is supported by the
concentration of plasma risperidone and 9-OH-risperidone in patients genotyped for
CYP2D6, and the effect of drugs on the activity of these enzymes (Bork et al. 1999,
DeVane & Nemeroff 2001). In both extensive and poor metabolizers of CYP2D6, the T½ of
the active moiety is 20 hours (Heykants et al. 1994). In ultrarapid metabolizers, however,
risperidone concentration is very low, and the 9-OH-risperidone concentration may also be
R
N
N CH3
OCH2
CH2
N ON
F
R E V I E W O F T H E L I T E R A T U R E
45
slightly lower than expected, which in some patients may explain non-response to
risperidone (Güzey et al. 2000). The 9-OH-risperidone is eliminated mainly by renal
excretion (Huang et al. 1993). In the steady state, the concentration of 9-OH-risperidone
exceeds the concentration of the parent risperidone (Huang et al. 1993), which makes it
pharmacologically more important than risperidone in extensive metabolizers of CYP2D6.
That P-gp ATPase activity towards risperidone has an in vitro Km value of 12.4 M
(Boulton et al. 2002) suggests that P-gp may contribute to the pharmacokinetics of
risperidone.
Risperidone is the first of the new atypical antipsychotics specifically developed to have a
clozapine-like strong affinity for postsynaptic 5-HT2 and a weaker affinity for D2 receptors.
Risperidone and 9-OH-risperidone have high affinities for 5-HT2A receptors (cloned human
5-HT2A, Ki 0.4) and lower affinities for D2 receptors (Ki 3-7) (Leysen et al. 1994). In the rat
brain, the active fraction has an about 4-fold higher half-life in the frontal cortex than in
plasma, supposedly due to its binding to 5-HT2 and D2 receptors (Heykants et al. 1994).
In schizophrenia, risperidone shows efficacy for both positive and negative symptoms
(Marder et al. 1997, Marder & Meibach 1994), and is better than haloperidol in the
prevention of relapses (Csernansky et al. 2002). In schizophrenia, the optimal benefit is
usually obtained at doses of 4 to 8 mg per day (Citrome & Volavka 2002).
Risperidone is generally well tolerated. It causes fewer extrapyramidal symptoms (EPS)
than does haloperidol, but more than clozapine does (Owens 1994, Tuunainen et al. 2002,
Csernansky et al. 2002). Other adverse effects with an incidence of 5% or more include
somnolence, agitation, and hyperkinesias (Csernansky et al. 2002). Risperidone is
associated with less weight gain than are clozapine or olanzapine, but more than are
haloperidol, quetiapine, or ziprasidone (Nasrallah 2003, Csernansky et al. 2002).
Risperidone elevates plasma prolactin concentration more than do haloperidol, clozapine or
olanzapine (Kleinberg et al. 1999, Turrone et al. 2002). Its prolactin response correlates
with plasma concentration of the active moiety (Huang et al. 1993).
R E V I E W O F T H E L I T E R A T U R E
46
Risperidone does not seem to increase plasma concentrations of amitriptyline, lithium,
valproate or digoxin; citalopram, mirtazapine or reboxetine do not affect its plasma
concentration (DeVane & Nemeroff 2001, Spina et al. 2001). The overall interaction
potential is relatively low with risperidone, but some possibly significant interactions have
been described (DeVane & Nemeroff 2001). Fluoxetine and paroxetine, inhibitors of
CYP2D6, have a significant effect in increasing plasma concentration of risperidone in
extensive metabolizers (DeVane & Nemeroff 2001, Bork et al. 1999). Carbamazepine, an
inducer of CYP3A4 and of some other CYP enzymes, may decrease plasma concentration
of 9-OH-risperidone to one-half (de Leon & Bork 1997). Based on two case-reports,
risperidone may significantly raise plasma concentrations of clozapine (Koreen et al. 1995,
Tyson et al. 1995), but the mechanism for this apparent interaction remains unknown.
Risperidone is a weak inhibitor of CYP2D6 but does not significantly inhibit CYP1A2 or
CYP3A4 (Eap et al. 2001). A mild form of neuroleptic malignant syndrome has occurred in
a man with first-episode schizophrenia, after clozapine was combined with risperidone
(Kontaxakis et al. 2002).
5.4. Influenza vaccination
nfluenza vaccines are given to millions of people worldwide each year. As a preventive
measure, their risk-benefit ratio should be extremely favorable, and they should offer
minimal risk, and be of clear benefit to individuals and to the population in general. For
instance, they should not have clinically important effects on the pharmacokinetics of drugs.
Vaccination is a targeted immunostimulation that leads to immunization, but the
immunological response to vaccination is complex. The immunological response to a
number of factors such as infection and influenza vaccination can modulate the activity of
CYP enzymes, and the general assumption is that this effect is mediated by cytokines
(Morgan 2001). Both in vitro and in vivo studies show that administration of
proinflammatory cytokines (e.g., interleukins 1 and 6, interferon , transforming growth
factor- 1) causes a downregulation of CYP mRNA and a decrease in the corresponding
CYP protein (Haas 2001, Morgan 2001). These findings suggest that regulation of gene
I
R E V I E W O F T H E L I T E R A T U R E
47
transcription is a major mechanism of decreased CYP activity following administration of
cytokines (Haas 2001, Morgan 2001). Post-transcriptional mechanisms, for example, one
that requires the presence of the nuclear receptor PPAR , are also involved (Morgan 2001).
Plasma concentrations of the CYP1A2 substrate theophylline may increase due to an
influenza infection (Kraemer et al. 1982). Similarly, during an acute infection, serum
clozapine concentration may increase by several-fold (van der Molen-Eijgenraam et al.
2001, Raaska et al. 2002b, de Leon & Diaz 2003, Haack et al. 2003). In one case, a patient
who died of septicemia resulting from pneumonia with high fever (39.9 C) had at the same
time a very high serum clozapine concentration (4034 g/L); the same patient was found in
autopsy also to have a cancer with metastases to the liver (Uges et al. 2000). Several case-
reports have suggested that influenza vaccination inhibits theophylline metabolism (Renton
et al. 1980, Meredith et al. 1985, Tjia et al. 1996), but in some studies influenza vaccination
showed no effect on theophylline pharmacokinetics (Jonkman et al. 1988, Stults &
Hashisaki 1983).
5.5. Caffeine and coffee-drinking
Figure 5. Chemical structure of caffeine
affeine is completely absorbed after oral administration (Carrillo & Benítez 2000).
Time to Cmax is about 1 hour, volume of distribution is about 0.7 L/kg (Carrillo &
Benítez 2000). It is mainly eliminated through the metabolism by CYP1A2 to paraxanthine
(Butler et al. 1989, Carrillo & Benítez 1994, Jeppesen et al. 1996a). Other demethylated
metabolites, theobromine and theophylline, are produced via CYP1A2 and CYP2E1
C
N
NN
N
O
O
CH3
CH3
CH3
R E V I E W O F T H E L I T E R A T U R E
48
pathways. These main metabolites undergo further demethylation and oxidation (Carrillo &
Benítez 1994, Lelo et al. 1986a).
Caffeine has a T½ of about 4 hours and paraxanthine about 3 hours (Lelo et al. 1986b,
Kaplan et al. 1997). After a single 500-mg oral dose of caffeine, the T½ of caffeine was
longer (4.74 + 0.6 h vs. 3.94 + 0.5 h), and its clearance was reduced (1.64 + 0.3 mL/min/kg
vs. 2.07 + 0.4 mL/min/kg) compared to a 250-mg oral caffeine dose (Kaplan et al. 1997).
Caffeine is a central nervous system stimulant which increases alertness and improves
performance on some tasks if consumption is not excessive (Smith 2002). These desirable
effects can be explained by its inhibition of A1- and A2a-type adenosine receptors in the
brain. A1 inhibits the release of excitatory neurotransmitters, and A2a enhances the activity
of dopamine D2 receptors (Fredholm 1995). Excess consumption of caffeine, especially by
patients with anxiety disorders, may cause caffeine intoxication with such signs as
restlessness, psychomotor agitation, insomnia, increased diuresis, tachycardia, and cardiac
arrythmia (DSM-IV, Smith 2002).
The main source of caffeine in adults is coffee (Mandel 2002), and consumption varies
greatly among world regions and countries. For example, the mean consumption of raw
coffee in 1989 was 2.0 kg/person in Britain, and 12.0 kg/person in Scandinavia
(http://www.coffeeresearch.org/market/consumption.htm). It seems that patients with
schizophrenia have a higher caffeine intake than does the general population (Hughes et al.
1998b), and high caffeine consumption is associated also with institutionalization (Hughes
& Boland 1992). About 25% of psychiatric inpatients are reported to drink daily more than
5 cups of coffee (Winstead 1976). Although it is unclear whether caffeine is a drug of abuse
for some individuals, many seem to exhibit dependence-like behaviors (Hughes et al.
1998a): Abrupt cessation of caffeine intake produces withdrawal symptoms in 11% to 100%
of individuals (Dews et al. 2002). Symptoms of caffeine withdrawal may include headache,
irritability, sleepiness or insomnia, anxiety, muscle pain, and delirium (Dews et al. 2002).
Several drugs have been reported to reduce the clearance of caffeine, and for the majority of
these interactions, the apparent mechanism seems to be inhibition of CYP1A2 activity
R E V I E W O F T H E L I T E R A T U R E
49
(Carrillo & Benítez 2000). For example, fluvoxamine reduces the clearance of caffeine to
one-fifth (Jeppesen et al. 1996b). Although caffeine has a relatively high Km (200 M) for
the CYP1A2-mediated caffeine-3-demethylation reaction, caffeine serum concentrations in
vivo are high enough (50 M) to suggest that caffeine can have some interaction potential as
a CYP1A2 inhibitor (Pelkonen et al. 1998). In fact, in healthy subjects, caffeine-containing
instant coffee has reduced clearance of the CYP1A2 substrate theophylline by 23% (Sato et
al. 1993). Abstinence from dietary caffeine resulted in a 47% decrease in mean
concentration of serum clozapine, and caffeine reduced mean oral clearance of clozapine by
14% (Carrillo et al. 1998, Hägg et al. 2000) (Tables 5 and 6).
A pharmacodynamic interaction between clozapine and caffeine is suggested by Vainer &
Chouinard (1994) in the case of a patient who had acute psychotic exacerbations each time
he took his clozapine with two cups of coffee. The suggested mechanism was a receptor-
receptor interaction between adenosine 2 and dopamine receptors 1 and 2 (Vainer &
Chouinard 1994).
RE
VIE
W O
F T
HE
LIT
ER
AT
UR
E
50
Tab
le 6
. Exa
mpl
es o
f pha
rmac
okin
etic
dru
g in
tera
ctio
ns w
ith c
affe
ine,
cip
roflo
xaci
n, it
raco
nazo
le, a
nd ri
sper
idon
e as
inhi
bito
rs.
Inhi
bito
r
D
aily
dos
eSu
bjec
ts (n
), st
udy
desi
gnSu
bstr
ate
Eff
ect o
nph
arm
acok
inet
ics o
fsu
bstr
ate
Sour
cePr
obab
le m
echa
nism
App
roxi
mat
e m
ean
300
mg
in c
offe
ePa
tient
s (7)
, seq
uent
ial
Clo
zapi
neIn
crea
se in
seru
mco
ncen
tratio
n of
2-f
old
Car
rillo
et a
l. 19
98In
hibi
tion
of C
YP1
A2
400
to 1
000
mg
mea
n 55
0 m
g pe
r day
Hea
lthy
(12)
, ope
n ra
ndom
ized
cros
sove
rC
loza
pine
AU
C in
crea
sed
by 1
9%H
ägg
et a
l. 20
00In
hibi
tion
of C
YP1
A2
4 to
8 c
ups o
f cof
fee
Patie
nts (
11),
sequ
entia
lLi
thiu
mM
este
r et a
l. 19
95In
hibi
tion
of p
roxi
mal
tubu
lar
reab
sorp
tion
of li
thiu
m
Caf
fein
e
2 to
7 c
ups o
f ins
tant
coff
eeH
ealth
y (6
), se
quen
tial
Theo
phyl
line
Red
uced
seru
m c
once
ntra
tion
by 2
4%
Red
uced
mea
n or
al c
lear
ance
by 2
3%, T
½ in
crea
sed
32%
Sato
et a
l. 19
93In
hibi
tion
of C
YP1
A2
1000
mg
Hea
lthy
(24)
, sin
gle-
blin
d ra
ndom
ized
cros
sove
rC
affe
ine
Red
uced
AU
C 2
-fol
dN
icol
au e
t al.
1995
Inhi
bitio
n of
CY
P1A
2
1000
mg
Hea
lthy
(9),
doub
le-b
lind
rand
omiz
edcr
osso
ver
Rop
ivac
aine
Red
uced
mea
n cl
eara
nce
by31
%Jo
kine
n et
al.
2003
Inhi
bitio
n of
CY
P1A
2
Cip
roflo
xaci
n
1000
mg
Hea
lthy
(9),
sequ
entia
lTh
eoph
yllin
eR
educ
ed m
ean
oral
cle
aran
ceby
19%
Bat
ty e
t al.
1995
Inhi
bitio
n of
CY
P1A
2
200
mg
Hea
lthy
(8),
doub
le-b
lind
rand
omiz
edcr
osso
ver
Bus
piro
neR
aise
d A
UC
19-
fold
Kiv
istö
et a
l. 19
97In
hibi
tion
of C
YP3
A4
200
mg
Hea
lthy
(10)
, dou
ble-
blin
d ra
ndom
ized
cros
sove
rD
igox
inR
aise
d m
ean
seru
mco
ncen
tratio
n 80
%Pa
rtane
n et
al.
1996
Inhi
bitio
n of
P-g
p
200
mg
Hea
lthy
(9),
doub
le-b
lind
rand
omiz
edcr
osso
ver
Felo
dipi
neR
aise
d A
UC
6-f
old
Jala
va e
t al.
1997
aIn
hibi
tion
of C
YP3
A4
200
mg
Hea
lthy
(12)
, dou
ble-
blin
d ra
ndom
ized
cros
sove
rLo
vast
atin
Rai
sed
Cm
ax >
20-f
old
Neu
vone
n &
Jala
va19
96In
hibi
tion
of C
YP3
A4
Lova
stat
in a
cid
Rai
sed
Cm
ax 1
3-fo
ld a
nd A
UC
23-f
old
200
mg
Hea
lthy
(9),
doub
le-b
lind
rand
omiz
edcr
osso
ver
Mid
azol
amR
aise
d A
UC
10-
fold
Olk
kola
et a
l. 19
94In
hibi
tion
of C
YP3
A4
Itrac
onaz
ole
200
mg
Hea
lthy
(9),
doub
le-b
lind
rand
omiz
edcr
osso
ver
Tria
zola
mR
aise
d A
UC
27-
fold
Var
he e
t al.
1994
Inhi
bitio
n of
CY
P3A
4
2 m
gPa
tient
, cas
e-re
port
Clo
zapi
neR
aise
d se
rum
con
cent
ratio
n by
2-fo
ldK
oree
n et
al.
1995
Unk
now
nR
ispe
ridon
e
2 m
gPa
tient
, cas
e-re
port
Clo
zapi
neR
aise
d se
rum
con
cent
ratio
n by
74%
Tyso
n et
al.
1995
Unk
now
n
A I M S O F T H E S T U D Y
51
AIMS OF THE STUDY
lozapine is eliminated mainly through CYP enzyme-mediated metabolism, and in
vitro studies indicate that it is metabolized mainly by CYP1A2 and CYP3A4.
Knowledge of the metabolic drug interactions of clozapine has been based almost entirely
on case-reports which suggest that strong inhibition of CYP3A4 by erythromycin could
considerably elevate serum clozapine concentrations and lead to significant adverse effects.
Case-reports show strong inhibition of CYP1A2 by fluvoxamine to increase serum
clozapine concentrations by several-fold. Two open studies and some case-reports suggest
that another CYP1A2 substrate, caffeine, may competitively inhibit clozapine metabolism
and lead to elevated serum clozapine concentrations, and caffeine withdrawal may, in some
patients, considerably reduce serum clozapine concentrations.
Similarly, modest inhibition of CYP1A2 by ciprofloxacin is suggested to raise serum
clozapine concentrations and intensify its adverse effects. CYP1A2-mediated metabolism of
theophylline may be lower during influenza epidemics and lead to elevated theophylline
concentrations and adverse effects. Whether influenza or other infections, or influenza
vaccination has any effect on serum clozapine concentrations has been unknown. Case-
reports suggest that even low-dose risperidone may double the serum concentration of
clozapine.
C
A I M S O F T H E S T U D Y
52
The specific aims of the studies were to study whether, in clozapine-treated patients:
1. Strong inhibition of CYP3A4 by itraconazole (CYP3A4 inhibitor, antimicromic drug)
has any clinically significant effect on serum concentrations of clozapine and its
metabolite N-desmethylclozapine.
2. A low dose of ciprofloxacin (CYP1A2 inhibitor, antimicrobic drug), commonly used in
urinary tract infections, has any clinically significant effect on serum concentrations of
clozapine and its metabolite N-desmethylclozapine.
3. An influenza vaccination with the commonly used trivalent subunit vaccine has any
clinically significant effect on serum concentrations of clozapine and its metabolites N-
desmethylclozapine and clozapine-N-oxide.
4. Concomitantly used risperidone is linked to increased serum clozapine concentrations.
5. Coffee-drinking (caffeine, CYP1A2 substrate) has any clinically significant effect on
serum concentrations of clozapine and its metabolites N-desmethylclozapine and
clozapine-N-oxide.
M A T E R I A L S A N D M E T H O D S
53
MATERIALS AND METHODS
1. Subjects
ll the subjects participating in Studies I, II, III, and V were clozapine-using patients (age
range 18-50 years; weight 64-138 kg) currently hospitalized in Kellokoski Hospital. A total
of 38 (12 females, 26 males) participated in Studies I to III and V. Four of these patients
participated in two of the studies. One of the 18 patients included in Study IV also participated in
two of the other four studies. The characteristics of the patients in each Study are shown in Table 7.
The characteristics data for patients in the register-based Study IV were obtained through records in
Kellokoski Hospital
Inclusion and exclusion: Only patients that were currently being treated with clozapine could be
included. Before entering the studies all patients were required to have had stable medication for a
minimum of 2 weeks. Those who took drugs that significantly inhibit CYP enzymes were excluded.
In Studies I to III and V, patients with acute physical or mental illnesses or with unstable chronic
diseases were excluded. Their evaluation for inclusion included a psychiatric interview and
examination, blood chemistry, and an electrocardiogram. Before entering the study all patients
understood a description of the study and gave their written informed consent.
Table 7. Characteristics of the patients, all with schizophrenia unless otherwise (n = 3) noted.StudyNo.
Female/male
(F/M)
Age
(years)
Weight*
(kg)
Smoker
(yes/no)
Coffee drinker(yes/no)
Clozapinedaily dose*
(mg)
Clozapinemonotherapy
(yes/no)1 I M 25 74 No ND 400 No2 I M 20 66 Yes ND 400 No3 I M 36 76 Yes ND 350 No4 I M 22 70 No ND 200 No5 I F 29 71 Yes ND 200 Yesasc
6 I M 27 64 Yes ND 550 Yesasc
7 I M 36 80 Yes ND 250 Yes8 II M 28 94 Yes Yes 150 No9 F 38 85 Yes Yes 350 No
F 39 ND Yes Yes 400 No{ IIIVV F 42 85 Yes Yes 600 No
10 II M 36 100 Yes Yes 275 Yesasc
11 II M 27 92 Yes Yes 450 No12 II F 35 65 Yes Yes 200 YesIUD
13 II M 32 102 No Yes 400 No14 II F 46 70 Yes Yes 300 No
A
M A T E R I A L S A N D M E T H O D S
54
Table 7. continued.
StudyNo.
Female/male
(F/M)
Age
(years)
Weight*
(kg)
Smoker
(yes/no)
Coffee drinker(yes/no)
Clozapinedaily dose*
(mg)
Clozapinemotherapy(yes/no)
15 III M 48 81 No Yes 275 No16 M 47 75 No Yes 650 Yes{ III
V M 48 71 No Yes 650 Yes17 III F 50 64 Yes Yes 400 No18 III F 45 88 No Yes 400 No19 III M 48 84 Yes Yes 500 No20 M 35 85 No Yes 300 Yes{ III
V M 36 89 No Yes 300 Yes21 III M 26 64 No Yes 750 No22 III F 46 114 No Yes 400 No23 III F 43 80 Yes Yes 450 No24 III F 35 75 No No 450 No25 III F 34 100 Yes Yes 450 No26 M 34 135 Yes Yes 375 No{ III
V M 35 138 Yes Yes 375 No27 III M 21 83 No Yes 375 Yes28 III M 22 80 Yes Yes 550 No29 III M 45 83 No Yes 300 Yes30 III F 34 100 No Yes 800 No31 IV M 24 112/110 Yes Yes 450/425 No32 IV M 53 98/74 Yes Yes 700 No33 IV M 49 ND/96 Yes Yes 600 No34 IV M 49 ND Yes Yes 800/700 No35 IV F 34 85 Yes Yes 425 No361 IV M 53 72/ND Yes ND 450/900 No37 IV M 30 89/90 Yes ND 300/200 No38 IV M 43 69/ND Yes Yes 450/500 Yes39 IV M 51 ND/88 Yes Yes 500 No40 IV F 45 ND/80 Yes Yes 350 No41 IV M 37 ND/84 Yes ND 600 No42 IV M 35 ND/113 Yes Yes 400/600 No43 IV F 30 ND Yes ND 300/225 No442 IV F 32 100/98 No No 200/250 No45 IV F 39 ND/70 Yes Yes 400 No46 IV M 48 118 Yes Yes 450/300 No47 IV M 18 96/90 No ND 100 No48 V M 39 92 Yes Yes 450 No49 V M 26 71 Yes Yes 350 No50 V M 41 85 Yes Yes 250 No51 V M 27 100 Yes Yes 750 No523 V F 22 75 Yes Yes 300 No53 V M 48 79 Yes Yes 500 No54 V M 18 92 Yes Yes 400 No55 V M 38 68 Yes Yes 450 No
* if the variable differs between study phases, the value is given for both phases (control phase/inhibitor phase)asc patient on clozapine monotherapy, but taking ascorbic acid dailyIUDpatient on clozapine monotherapy and wearing an intrauterine device (hormone)1Psychotic disorder NOS2Delusional disorder3Schizoaffective disorderND – not determined or unknown
M A T E R I A L S A N D M E T H O D S
55
2. Design of the studies
tudies I, II, and V were randomized, placebo-controlled crossover studies with two periods.
Study III was a prospective open-label study, and Study IV a retrospective registered-based
study (Table 8).
Study I. Seven patients (1 female, 6 males) received either itraconazole 100 mg or placebo orally at
8 a.m. and 8 p.m. daily for 7 days. For the next 7 days itraconazole was changed to placebo and vice
versa. Venous blood samples (10 mL) for determination of serum concentrations of
clozapine, N-desmethylclozapine, itraconazole, and hydroxyitraconazole were drawn 12 h after the
previous evening’s dose of clozapine and itraconazole/placebo on study days 0, 3, 7, 10, and 14.
Serum was separated within one hour and stored at -20°C until analyzed (Table 8).
Study II. Seven patients (3 females, 4 males) received either ciprofloxacin 250 mg or placebo
orally at 8:00 h and 20:00 h daily for 7 days, followed by a 7 days’ wash-out period with no
ciprofloxacin or placebo administered. For the next 7 days the previously administered
ciprofloxacin was changed to placebo and vice versa. During both phases venous blood samples (10
mL) for determination of serum concentrations of clozapine, N-desmethylclozapine, and
ciprofloxacin were drawn 12 h after the previous evening’s dose of clozapine on Day 1, before the
first dose of ciprofloxacin or placebo and on study days 3 and 8 (i.e., the morning after the last dose
of ciprofloxacin or placebo). Serum was separated within 1 hour and stored at -20°C until analyzed
(Table 8).
Study III. Sixteen patients (7 females, 9 males) on study Day 0 received intramuscularly (into the
deltoid muscle) the recommended dose of 0.5 mL of the 2000-2001 formula of a conventional
trivalent (influenza A subtypes H1N1 and H3N2, and influenza B) influenza subunit vaccine
(Influvac ®, Solvay Pharma). Venous blood samples (10 mL) for determination of serum
concentrations of clozapine, N-desmethylclozapine, clozapine-N-oxide, and c-reactive protein
(CRP) were collected 12 h after the previous evening’s dose of clozapine at 0800 hours on
vaccination day and 2, 4, 7, and 14 days after vaccination. Serum was separated within 1 h and
stored at -20 ºC until analyzed (Table 8).
S
M A T E R I A L S A N D M E T H O D S
56
Study IV. The electronic data base in Kellokoski Hospital served for collection of therapeutic drug
monitoring (TDM) data on all serum clozapine concentration measurements in that hospital
between January 1995 and August 2001 (a total of 2490 clozapine measurements for 414 patients).
The concomitant medications at the sampling time of each clozapine concentration measurement
were recorded. For 37 patients who had used both clozapine and risperidone at the times of
sampling, patient-related data were collected concerning each 327 serum clozapine concentration
sampling date. The following variables were registered: sex, age, time of the sampling, clozapine
concentration, daily dose of clozapine and risperidone, ratio of concentration to daily dose (C/D) of
clozapine, duration of treatment with the current dose of clozapine and risperidone, and
concomitant drugs and their daily doses. Entries in follow-up charts provided data on patients’
cigarette-smoking and coffee-drinking habits around the time of the relevant clozapine
concentrations, as well as to exclude concentrations taken during a period of non-compliance with
medication. In addition, ward staff was interviewed regarding the smoking- and coffee-drinking
habits of these patients (Table 8).
Inclusion and exclusion criteria: Only morning clozapine trough concentrations were included. A
minimum of 7 days’ treatment at the current dose of clozapine and risperidone was required.
Samples were excluded if patient was concomitantly using drugs known significantly to inhibit or
induce CYP1A2 or CYP3A4 enzymes in vivo in humans. Patients whose tobacco-smoking or
coffee-drinking habits were known to differ between the phases were excluded. If more than one
serum clozapine concentration with concomitant risperidone was available for one of the patients,
the following order was used to select one sample for comparison: If the patient’s daily dose of
risperidone was higher at one date of sampling than at others, then the concentration of that
measurement was selected. If this criterion (1), the highest daily dose of risperidone, could be
applied to more than one concentration, the selection was continued between them with the second
criterion (2), the highest daily dose of clozapine, and if necessary with the third criterion (3), the
most recent date of sampling. The matching control sample without concomitant risperidone was
selected by the following criteria: 1) The closest matching daily dose of clozapine, 2) The most
similar concomitant medication, 3) The closest matching date of sampling.
Study V. The 12 patients (2 females, 10 males) who were habitual coffee-drinkers drank either
regular caffeine-containing instant coffee or decaffeinated instant coffee (Nescafe Gold Blend® or
Nescafe Gold Blend Decaffeinated®), for 7 days (Table 8). For the next 7 days caffeine-containing
coffee (caffeine period) was changed to decaffeinated coffee (control period) and vice versa. No
M A T E R I A L S A N D M E T H O D S
57
other sources of caffeine, such as any other coffee, caffeine tablets, tea, chocolate, or cola drinks
were allowed. Patients were advised to prepare their coffee servings similarly during both study
periods. Venous blood samples (10 mL) for the determination of serum concentrations of clozapine,
N-desmethylclozapine, clozapine-N-oxide, and CRP were collected 12 h after the previous
evening’s dose of clozapine before the first cup of coffee on study days 1, 4 and 8 (i.e., the morning
after the last day of each study week) of both study phases. The serum was separated within 1 h and
stored at -20 ºC until analyzed. Patients were not allowed to drink study coffee for 12 hours before
each blood sample. Other than that, the patients were free to drink study coffee at all times in
amounts of their own choosing. (Randomized coffee was always delivered to the patients in
identical packages.)
During the run-in period and study periods all patients recorded on a form the times and the number
of cups of coffee drunk and cigarettes smoked each day. After each of the 3 weeks, all patients were
rated with the Brief Psychiatric Rating Scale (BPRS) and the Udvalg for Kliniske Undersøgelser
(UKU) side-effect rating scale–the self-rating version in Finnish (UKU-SERS-Pat-Finnish)–by one
of the authors (KR). The BPRS (18 items) was scaled from 1 to 7 (1 = not present, 7 = extremely
severe) and UKU (46 items) from 0 to 3 (0 = not present, 3 = severe) for each item. Most studies
use a > 20% change in BPRS score as an indicator of treatment response (Lachar et al. 1999).
Compliance was assessed by measuring serum caffeine and paraxanthine concentrations.
Measurement of CRP concentration was done in order to ensure that patients had no infections that
could possibly inhibit clozapine metabolism.
3. Blood sampling
enous blood samples (10 mL) for determination of serum concentrations of clozapine, N-
desmethylclozapine, and clozapine-N-oxide, and concentrations of all the inhibitors studied
and of CRP were drawn 12 h after the previous evening’s dose of clozapine, before the ingestion of
clozapine or of the inhibitors. Serum was separated within one hour and stored at -20°C until the
samples were analyzed.
V
M A T E R I A L S A N D M E T H O D S
58
4. Determination of serum drug, caffeine, and CRP concentrations
erum concentrations of clozapine and N-desmethylclozapine in Studies I, II, and IV were
determined in the Laboratory of Helsinki University Central Hospital. Clozapine and its
metabolite concentrations, other drug concentrations, caffeine and paraxanthine concentrations, and
CRP concentrations were determined in the Analytical Laboratory of the Department of Clinical
Pharmacology, University of Helsinki.
4.1. Clozapine and its metabolites
erum concentrations of clozapine, N-desmethylclozapine, and clozapine-N-oxide were
determined by automated solid-phase extraction and subsequent high-performance liquid
chromatography with an electrochemical detector (Humpel et al. 1989, Lovdahl et al. 1991,
Weigmann & Hiemke 1992, Schulz et al. 1995). Analysis was performed on a Merck LiChrospher
60 RP-select B column (125 mm x 4 mm) and the electrochemical detector ESA Model 5100 A
Coulochem. The mobile phase consisted of acetonitrile-methanol-0.02 M potassiumphosphate
buffer (150:450:400, volume (v)/v/v), which was adjusted to pH 6.6. Imipramine served as an
internal standard.
In Study I, the quantitation limit was 100 nmol/L for clozapine and N-desmethylclozapine. The day-
to-day CV for clozapine at 390 nmol/L was 10.7%.
In Studies II and IV, The quantitation limit was 50 nmol/L for both compounds and the within-day
and day-to-day CVs at relevant concentrations were less than 5%.
In Study III, The quantitation limit for clozapine, N-desmethylclozapine and clozapine-N-oxide was
30 nmol/L. The day-to-day CV was for clozapine 12% and 3.3% at mean concentrations of 220
nmol/L and 1200 nmol/L, respectively; for N-desmethylclozapine 12% and 3.5% at 200 nmol/L and
1050 nmol/L; and for clozapine-N-oxide 13% and 9% at 160 nmol/L and 880 nmol/L.
S
S
M A T E R I A L S A N D M E T H O D S
59
In Study V, The quantitation limit for clozapine, N-desmethylclozapine and clozapine-N-oxide was
30 nmol/L. The day-to-day CV was for clozapine 13% and 7% at mean concentrations of 180
nmol/L and 970 nmol/L, respectively; for N-desmethylclozapine 4% and 2% at 170 nmol/L and
1040 nmol/L; and for clozapine-N-oxide 7% and 1.5% at 190 nmol/L and 1170 nmol/L.
4.2. Itraconazole and hydroxyitraconazole
oncentrations of itraconazole and hydroxyitraconazole were determined by high-performance
liquid chromatography (HPLC) with a fluorescence detector (Remmel et al. 1988, Allenmark
et al. 1990). A 15 cm x 3.9 mm I.D. reverse-phase column (Waters Nova-Pak C18) was used. The
mobile phase was water-acetonitrile (50:50), containing 0.28% of triethylamine. The mobile phase
was adjusted to pH 2.3. The quantitation limit was 10 ng/mL. The CV for itraconazole was 1.6% (at
194 ng/mL) and for hydroxyitraconazole 1.2% (at 194 ng/mL).
4.3. Ciprofloxacin
oncentrations of ciprofloxacin were determined by liquid chromatography (PERKIN ELMER
Series 200) tandem mass spectrometry using electrospray ionization (PE-SCIEX API 3000)
(Volmer et al. 1997). The chromatographic analysis was performed on a Waters Symmetry C-8
column (50 mm x 2.1 mm). The mobile phase was acetonitrile-formic acid (5:95, v/v). The
quantitation limit was 1 ng/mL. The day-to-day CV for ciprofloxacin was 3.7% at 800 ng/mL and
7.6% at 30 ng/mL.
4.4. Caffeine and paraxanthine
erum concentrations of caffeine and paraxanthine were determined by high-performance liquid
chromatography (HPLC) (Pickard et al. 1986, Holland et al. 1998). The analysis was
performed with an automated HP 1100 liquid chromatograph on a Waters Symmetry C8 column
(150 mm x 4.6 mm) and ultaviolet detector. The mobile phase consisted of methanol-15 mM
potassiumphosphate buffer (17.5:82.5, v/v), which was adjusted to pH 5.0. Beta-
hydroxyethyltheophylline served as the internal standard. The quantitation limit for caffeine and
C
C
S
M A T E R I A L S A N D M E T H O D S
60
paraxanthine was 20 g/L. The day-to-day CV was for caffeine 9.8%, 13.25%, and 2.7% at mean
concentrations of 135 g/L , 1260 g/L, and 15 400 g/L; for paraxanthine 153 g/L, 1460 g/L,
and 15 030 g/L.
4.5. C-reactive protein
erum concentration of CRP was determined with a validated high-sensitivity
immunoturbidimetric assay (Markkola et al. 2000) in the laboratory of Helsinki University
Central Hospital.
5. Statistical analysis
tudy results are given as mean values + SD, or mean + range. Statistical analysis of continuous
data was performed with Student’s two-tailed t-test for paired data. Differences were
considered statistically significant when P values were less than 0.05. The Wilcoxon Signed Ranks
Test was used for non-parametric data. Correlations between continuous data are given as the
Pearson correlation coefficient. Consumer’s risk was evaluated by taking a equivalence approach
(Steinijans et al. 1991). In testing equivalence, no interaction was assumed if the ratio test/ reference
and the corresponding 90% confidence intervals fell into the equivalence range 0.8 to 1.25. The data
are given as mean ratio test/ reference ( test = mean value during tested treatment, reference = mean
value during control treatment) and 90% confidence intervals.
6. Ethical considerations
or ethical reasons, none of these studies involved healthy volunteers, because serious adverse
effects have occurred in healthy subjects even after single low doses of clozapine (Pokorny et
al. 1994). All studies were thus carried out with the patients who were already using clozapine. All
the study protocols of these studies comply with the current laws and ethical standards of Finland.
The protocols of Studies I and II were approved by the Ethics Committee of the Hyvinkää Health
Care District and by the National Agency for Medicines (Finland). The study protocols of Studies
S
S
F
M A T E R I A L S A N D M E T H O D S
61
III and V were approved by the Ethics Committee of pediatrics and psychiatry in the hospital
district of Helsinki and Uusimaa. The study protocol of Study IV, which was a register-based study
in which the patients were not contacted, was approved by the chief physician of Kellokoski
Hospital. All the patients in Studies I to III and V received oral and written information, and after
the minimum 4 days’ reconsideration period, they gave their written informed consent before
entering the study. The ability of any patient to give an informed consent was evaluated through a
psychiatric interview by the author and through consulting the psychiatrist in charge of the
treatment of each patient.
MA
TE
RIA
LS
AN
D M
ET
HO
DS
62
Tab
le 8
. Stu
dy d
esig
n. T
he a
im w
as to
inve
stig
ate
any
poss
ible
eff
ect o
f fiv
e fa
ctor
s (in
hibi
tors
or c
o-dr
ugs)
on
seru
m c
once
ntra
tion
of c
loza
pine
and
its m
etab
olite
s in
hosp
italiz
ed p
atie
nts.
Stud
yno
.Pa
tient
sfe
mal
es/m
ales
Stru
ctur
eIn
hibi
tor /
co-
drug
Con
trol
Stud
y du
ratio
n
In
= 7
1 / 6
rand
omiz
ed, p
lace
bo-c
ontro
lled
cros
sove
r stu
dy w
ith tw
oph
ases
itrac
onaz
ole
100
mg
b.i.d
(CY
P3A
4 in
hibi
tor)
plac
ebo
7 da
ys p
er p
hase
IIn
= 7
3 / 4
rand
omiz
ed, p
lace
bo-c
ontro
lled
cros
sove
r stu
dy w
ith tw
oph
ases
cipr
oflo
xaci
n 25
0 m
g b.
i.d(C
YP1
A2
inhi
bito
r)pl
aceb
o7
days
per
pha
se +
7da
ys w
asho
ut p
erio
dbe
twee
n th
e ph
ases
III
n =
167
/ 9op
en, p
rosp
ectiv
e; p
atie
nts a
sth
eir o
wn
cont
rols
influ
enza
vac
cina
tion
(stim
ulat
es c
ytok
ine
prod
uctio
n,cy
toki
nes i
nhib
it C
YPs
)
none
(bef
ore
the
influ
enza
vacc
inat
ion)
14 d
ays
IVn
= 18
6 / 1
2re
trosp
ectiv
e; p
atie
nts a
s the
irow
n co
ntro
lsris
perid
one
2-4
mg
daily
(sub
stra
te o
f CY
P2D
6 an
d 3A
4)no
ne(w
ithou
t ris
perid
one)
paire
d sa
mpl
es0.
3-48
mon
ths a
part
Vn
= 12
2 / 1
0ra
ndom
ized
, pla
cebo
-con
trolle
dcr
osso
ver s
tudy
with
two
phas
es
caff
eine
-con
tain
ing
coff
eead
libi
tum
(caf
fein
e is
a C
YP1
A2
subs
trate
)
deca
ffei
nate
d co
ffee
7 da
ys ru
n-in
per
iod
+ 7
days
per
pha
se
R E S U L T S
63
RESULTS
Effects of itraconazole, ciprofloxacin, risperidone,influenza vaccination, and coffee-drinking on clozapineconcentrations
1. Itraconazole (Study I)
7-day treatment with itraconazole 100 mg twice daily had no statistically significant
effect on serum concentrations of clozapine or N-desmethylclozapine. During the
itraconazole phase, the mean clozapine concentration was 3% lower (non-significant, NS, P
= 0.79) (Figure 6) and N-desmethylclozapine 7% higher (NS, P = 0.43) than during the
placebo phase. The N-desmethylclozapine to clozapine ratio was 8% higher (P < 0.05)
during the itraconazole phase (Table 9).
Compliance with itraconazole ingestion was good in each patient as shown by serum
itraconazole concentrations. No change was reported in patients’ symptoms or in adverse
effects during the study.
2. Ciprofloxacin (Study II)
7-day treatment with ciprofloxacin 250 mg twice daily raised mean serum clozapine
concentration by 29% (P < 0.01; range 6-57%) (Figure 6) and that of N-
desmethylclozapine by 31% (P < 0.05; range 0-63%). The mean N-desmethylclozapine to
clozapine concentration ratio remained unchanged (NS, P = 0.83). The correlations between
serum ciprofloxacin concentrations and the increase (%) in serum clozapine or N-
desmethylclozapine concentrations were non-significant (r = 0.73; P < 0.10 and r = 0.74; P
< 0.10, respectively). The correlation between the increase (%) in the sum of clozapine + N-
desmethylclozapine serum concentrations and serum ciprofloxacin concentration was
A
A
R E S U L T S
64
significant (r = 0.90; P < 0.01). The correlation was significant between the increase (%) in
serum clozapine concentrations during the ciprofloxacin phase and the ratios of N-
desmethylclozapine to clozapine concentrations determined on the 8th day of the placebo
phase (r = 0.89; P < 0.01) (Table 9).
Compliance with ciprofloxacin ingestion was good in each patient, as shown by serum
ciprofloxacin concentrations.
3. Influenza vaccination (Study III)
erum concentrations of clozapine, N-desmethylclozapine or clozapine-N-oxide did not
significantly differ from baseline concentrations at any time-points during the study.
Mean serum concentration of clozapine tended to increase by 5% (NS, P = 0.22), 2% (NS, P
= 0.65), and 1% (NS, P = 0.87), 2, 4, and 7 days after vaccination (Figure 6). No significant
increase in CRP appeared after the vaccination (Table 9).
Two patients (a male and a female) were excluded from statistical analysis because both had
suffered an acute infection during the study period after the vaccination. Both had elevated
serum clozapine concentrations, and a decreased serum concentration ratio N-
desmethylclozapine to clozapine during the period of increased CRP (Figure 7).
The clinical efficacy of clozapine remained unchanged throughout the study for all 16
patients.
4. Risperidone (Study IV)
nalysis was based on the results from 18 patients who met the inclusion criteria, of
the total 37 patients who took both clozapine and risperidone in their daily
medication. Characteristics of the patients are shown in Table 7.
Neither the clozapine concentration to dose ratio nor clozapine concentration significantly
differed during risperidone from the control value. During risperidone, the mean serum
S
A
R E S U L T S
65
clozapine concentration was 3% lower (NS, P = 0.75) (Figure 6), and the clozapine
concentration to dose ratio was 8% lower (NS, P = 0.46) than during control. The ratio
test reference for clozapine concentration to dose ratios indicated equivalence with 90% CI
0.82-1.15 within limits of statistical significance (90% CI 0.80-1.25) (Table 9).
The time-range between the individual paired samples was 0.3 to 48 months (median 15.2
months). During risperidone, at the time of clozapine TDM sampling, the mean durations of
unchanged clozapine and risperidone treatments were 320 + 743 days (range 7 to 3120) and
52 + 59 days (range 7 to 193), respectively. The mean dose of risperidone was 2.9 + 0.9
mg/day (range 2 to 4 mg/day). During the control phase (without risperidone), at the time of
clozapine sampling, the mean duration of unchanged clozapine treatment was 209 + 564
days (range 7 to 2409).
5. Coffee-drinking (Study V)
erum paraxanthine concentrations indicated that of the 12 patients, six were compliant
in caffeine use throughout this study, and four up to the fourth study day (partially
compliant patients). Thus, 10 of 12 patients were included in the statistical analysis. Day 4
data were analyzed for 10 patients, and Day 8 data for six patients. In addition, the Day 4
data from 4 partially compliant patients (who were non-compliant on Day 8) and the Day 8
data from 6 fully compliant patients were pooled to increase statistical power in analysis of
the combined data.
Analysis of Day 4 data. Serum concentrations of clozapine (Figure 6), N-
desmethylclozapine, or clozapine-N-oxide were not significantly changed between the
caffeine and control phases. During the control phase, the mean serum concentration of
caffeine was 12%, and that of paraxanthine 13% of the corresponding concentrations during
the caffeine phase.
Analysis of Day 8 data. During the caffeine phase the mean serum concentration of
clozapine was 26% (NS, P = 0.07) (Figure 6) , N-desmethylclozapine, 6% (P = 0.03), and
S
R E S U L T S
66
clozapine-N-oxide, 7% (NS, P = 0.22) higher than during the control phase. The N-
desmethylclozapine to clozapine ratio was 13% (NS, P = 0.06) and the clozapine-N-oxide to
clozapine, 7% (NS, P = 0.19) lower than during the caffeine phase. Mean serum caffeine
and paraxanthine concentrations during the control phase were 5% and 8% of the
corresponding concentrations during the caffeine phase (Table 9).
Analysis of the combined data. The mean serum concentration of clozapine was 20% (P =
0.03) (Figure 6), and N-desmethylclozapine, 7% (P = 0.02) higher during the caffeine phase
than during the control phase, but the mean serum clozapine-N-oxide concentration was not
significantly increased (P = 0.11). During the caffeine phase, the ratio of N-
desmethylclozapine to clozapine was 9% (NS, P = 0.06) lower than during the control
phase.
Patients’ consumption of cigarettes did not significantly differ between study phases. Mean
coffee consumption, BPRS, and UKU ratings were non-significantly higher during the
control than caffeine phase (P > 0.05).
R E S U L T S
67
Figure 6. Serum clozapine concentration (% of control) during itraconazole (ITRA) and
ciprofloxacin (CIPRO), 2, 4, and 7 days after influenza vaccination (VAC), and after
caffeine-containing coffee for 3 and 7 days, and serum clozapine concentration to clozapine
daily dose ratio during risperidone (RISP). Medians ( ) are shown, and 95% confidence
intervals are indicated by short horizontal lines.
0
20
40
60
80
100
120
140
160
C
loza
pine
con
cent
ratio
n
(%
of c
ontr
ol)
I II III IV V1261201049210110210512997
Study
ITR
A
CIP
RO
2 D
AYS
AFTE
R V
AC
RIS
P
CO
F 3
DAY
S
CO
F C
OM
B
CO
F 7
DAY
S
4 D
AYS
AFTE
R V
AC
7 D
AYS
AFTE
R V
AC
RE
SU
LT
S
68
Tab
le 9
. Mai
n re
sults
of t
he st
udie
s.
Stud
yno
.In
hibi
tor
/ co-
drug
Mea
n ef
fect
s of i
nhib
itors
or
co-d
rugs
on
seru
m c
once
ntra
tions
of
C
loza
pine
N
-des
met
hylc
loza
pine
Clo
zapi
ne-N
-oxi
deI
Itra
cona
zole
100
mg
b.i.d
(CY
P3A
4 in
hibi
tor)
Unc
hang
edU
ncha
nged
Not
stud
ied
IIC
ipro
floxa
cin
250
mg
b.i.d
(CY
P1A
2 in
hibi
tor)
Incr
ease
29%
(P <
0.0
1)In
crea
se 3
1%(P
< 0
.05)
Not
stud
ied
III
Influ
enza
vac
cina
tion
(stim
ulat
es c
ytok
ine
prod
uctio
n:cy
toki
nes i
nhib
it C
YPs
)
Unc
hang
edU
ncha
nged
Unc
hang
ed
IVR
ispe
rido
ne 2
-4 m
g da
ily(s
ubst
rate
of C
YP2
D6
and
3A4)
Unc
hang
edN
ot st
udie
dN
ot st
udie
d
VC
affe
ine-
cont
aini
ng c
offe
ead
libitu
m(c
affe
ine
is a
CY
P1A
2su
bstra
te)
Non
-sig
nific
ant i
ncre
ase
26%
(P =
0.0
7)In
crea
se 6
%(P
< 0
.05)
Unc
hang
ed
R E S U L T S
69
Figure 7. Serum concentrations of clozapine ( ), CRP ( ), and N-desmethylclozapine to
clozapine ratios ( ) of the two excluded patients in Study III on Day 0 before vaccination
and 2, 4, 7, and 14 days after vaccination.
DM
CLZ
/CLZ
-rat
io
0 2 4 7 140
1000
2000
3000
4000C
loza
pine
and
met
abol
ites
(nm
ol/l)
20
40
60
CR
P (m
g/l)
Days after vaccination
0.3
0.6
0.9
CRP
DMCLZ/CLZ-ratio
Clozapine
Pharyngitis
FEMALE, age 34
DM
CLZ
/CLZ
-rat
io
0 2 4 7 140
200
400
600
800
1000
1200
1400
Clo
zapi
ne c
once
ntra
tion
(nm
ol/l)
0
20
40
60
80
CR
P (m
g/l)
0.1
0.2
0.3
0.4
0.5
CRP
DMCLZ/CLZ-ratio
Abdominal pain
CLOZAPINE
Days after vaccination
Sedation
MALE, age 21
D I S C U S S I O N
70
DISCUSSION
1. Methodological considerations
hese studies were conducted only with patients, because healthy volunteers should
never take clozapine (Pokorny et al. 1994). Between these patients concomitant
medication and the daily dose of clozapine varied. Patients who had medications known (or
suspected) to affect the clozapine disposition were excluded. In statistical analysis, a similar
percentage change in patients’ clozapine concentrations thus had more variable effects on
the mean concentration results than if each patient within each study had taken an identical
dosage. Within each subject, medication remained unchanged during the study phases,
making comparisons of effect for each patient relevant.
All study subjects were currently residing in one hospital under regular close surveillance.
The majority took their medication at fixed times of day under the surveillance of staff
members, and the minority resided on wards where taking medication was mostly the
patients’ own responsibility. Ward staff was informed about the study protocols, and, if
needed, they helped patients follow the protocols as planned. This ensured prompt reporting
of any significant change in subjects’ well-being.
In all of the studies, subjects served as their own controls, which reduces variability between
paired samples and reduces the number of subjects needed to attain sufficient statistical
power. The number of patients in Studies I, II, and V was relatively small (7,7, and 12). This
increases the probability of error (i.e., reduces the power to detect a true difference), but
this number of patients is sufficient to reveal an about 35% to < 30% change with 80%
power in serum concentrations between study phases ( = 0.05 and = 0.20 and within-
patient SD < 20%). The number of patients in Studies III and IV (16 and 18) was sufficient
to reveal a 20% change with 80% power between the study phases.
Much effort was put into the informed consent process, not only to ensure the ethics, but to
include only patients who were well motivated and educated about the study protocol in
T
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order to maximize compliance. Compliance with the supposed inhibitor of clozapine
metabolism was assessed by measurement of its serum concentration in Studies I
(itraconazole and metabolite), II (ciprofloxacin), and V (caffeine and metabolite).
Compliance was generally good, perhaps because of careful patient selection. Only in Study
V were some of the patients found to be non-compliant. That study required one week’s
abstinence from caffeine, which often produces withdrawal symptoms and the desire to
resume coffee-drinking.
Only a few study subjects had a diagnosis other than schizophrenia. There is, however, no
reason to suspect any profound differences in basic pharmacokinetic interaction
mechanisms, such as CYP-mediated ones, between different diagnostic categories of mental
disorders. Results of these studies can therefore be applied not only to patients with
schizophrenia, but also to clozapine-treated patients in general.
2. Effect of itraconazole on serum clozapine concentration
n this study the potent CYP3A4 inhibitor itraconazole 200 mg a day had no significant
effects on serum clozapine or N-desmethylclozapine concentrations, although
itraconazole 200 mg a day has greatly increased the AUC of many substrates of CYP3A4
(Neuvonen & Jalava 1996, Varhe et al. 1994, Olkkola et al. 1994, Kivistö et al. 1997).
Compliance in the use of itraconazole was good, confirmed by determination of its serum
concentrations. It thus seems that inhibition of CYP3A4 (or P-gp) in general does not
significantly elevate serum concentrations of clozapine or N-desmethylclozapine.
Two case-reports suggest that concomitant use of erythromycin with clozapine can elevate
serum concentrations of clozapine, leading to signs of clozapine toxicity (Funderburg et al.
1994, Cohen et al. 1996). Erythromycin is a relatively specific CYP3A4 inhibitor. Authors
of both reports suggest a pharmacokinetic interaction caused by erythromycin as the most
likely explanation for high clozapine concentrations. On the other hand, itraconazole clearly
I
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72
causes a more pronounced increase in plasma concentrations of various CYP3A4 substrates
than does erythromycin, e.g., of midazolam (Olkkola et al. 1994, Olkkola et al. 1993),
triazolam (Varhe et al. 1994), and buspirone (Kivistö et al. 1997, Kivistö et al. 1999).
The complete lack of effect of itraconazole on serum concentrations of clozapine and N-
desmethylclozapine in this prospective study does not support the hypothesis of a CYP3A4-
inhibition-mediated interaction between clozapine and erythromycin. Influenza (Kraemer et
al. 1982) and influenza vaccines (Renton et al. 1980) may inhibit CYP1A2-mediated drug
metabolism. Possibly infection could explain elevated clozapine concentrations in the two
cases of Funderburg et al. (1994) and Cohen et al. (1996).
It appears that CYP3A4 inhibition normally has little effect on serum clozapine
concentrations. If the CYP1A2 pathway is inhibited, however, CYP3A4 may become a
major pathway in clozapine metabolism. In that case, additional CYP3A4 inhibition could
lead to highly elevated clozapine concentrations.
3. Effect of ciprofloxacin on serum clozapine concentration
he low dose of ciprofloxacin (250 mg b.i.d.) elevated mean concentrations of both
clozapine and N-desmethylclozapine by about 30%. An increase of that magnitude in
serum clozapine concentration is generally well tolerated, but an increase of about 50% is
enough to cause significant adverse effects in some sensitive patients. The smoking and
coffee-drinking habits of the study patients remained unchanged during this study, so those
factors could not have influenced results.
The extent of the interaction was related to the serum ciprofloxacin concentration, which
varied considerably between patients. It is likely that higher doses of ciprofloxacin (500 mg
or 750 mg b.i.d.) than used in this study would cause a greater increase in clozapine and N-
desmethylclozapine concentrations. Probably due to the small sample size, the percentage
increase in clozapine or N-desmethylclozapine concentrations did not quite correlate with
ciprofloxacin concentrations, but the correlation nearly reached significance. The pooled
T
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73
concentration of clozapine and N-desmethylclozapine correlated well, however, with
ciprofloxacin concentrations, despite the small number of patients. Although an increase in
serum clozapine is clinically more important, an increase in N-desmethylclozapine
concentration may also be significant for some of the patients. It, too, has biological
activity, demonstrated by its clozapine-like effect in elevating fos-protein in rat brain
(Young et al. 1998).
The ratio of serum N-desmethylclozapine to clozapine correlates with CYP1A2 activity
measured by the urinary caffeine test (Carrillo et al. 1998). In this study, the individual
ratios of N-desmethylclozapine to clozapine at Day 8 of the placebo phase correlated well
with the percentage increase in clozapine concentrations at Day 8 of the ciprofloxacin phase
(r = 0.90; P < 0.01). That is, this interaction was strongest in patients whose CYP1A2
activity was highest. This suggests that inhibition of CYP1A2 is the most likely mechanism
behind this interaction. This is in line with the fact that ciprofloxacin is a CYP1A2 inhibitor
(Fuhr et al. 1992).
The suggested mechanism in this interaction is inhibition of the CYP1A2 enzyme by
ciprofloxacin. Those patients who have both a high ratio of serum N-desmethylclozapine to
clozapine concentration and a high serum concentration of ciprofloxacin seem to comprise a
risk group for a clinically relevant ciprofloxacin-clozapine interaction.
4. Effect of risperidone on serum clozapine concentration
isperidone (2-4 mg/day) had no effect on the mean serum clozapine concentration, or
on the ratio of serum clozapine concentration to clozapine dose. In fact, the mean
serum clozapine concentration was slightly lower (NS, P = 0.75) during risperidone than
during the control phase, despite the fact that the mean clozapine dose was higher (451 +
207 mg vs. 438 + 168 mg; P = 0.66), and the patients were, on average, 0.4 years older
during the risperidone phase. Because clozapine concentrations tend to increase with age
(Haring et al. 1989), this insignificant age-difference does not impair the conclusions.
R
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74
The results do not support the assumption that risperidone added to clozapine treatment
would increase serum clozapine concentrations. That assumption of a significant
risperidone-clozapine interaction is based on two case-reports (Koreen et al. 1995, Tyson et
al. 1995), which suggest that even low 2-mg daily doses of risperidone may double serum
clozapine concentrations. In the light of the present study, it seems unlikely that in those
cases the increase in clozapine concentrations was caused by risperidone or the other co-
medications. On the other hand, the results of this study are supported by a study
demonstrating that risperidone is neither a CYP1A2 nor CYP2C19 inhibitor, and that it is
only a weak CYP2D6 inhibitor (Eap et al. 2001).
Of the 18 patients in the present study, 16 were smokers, and at least 13 drank coffee daily
during both phases. No entries in the follow-up charts of any of the 18 patients suggested
any significant changes in their smoking or coffee-drinking habits during that time period.
Thus, it is unlikely that changes in these habits would have impaired the results.
Furthermore, to ensure that the results were not compromised by concomitant medication,
all patients who used clinically significant inhibitors or inducers of CYP1A2 or CYP3A4
were excluded.
Although the difference between mean clozapine concentrations was insignificant, in four of
the patients clozapine concentration to dose ratios showed an over 50% change from the
control to the risperidone phase. Differences between their co-medication, age, smoking, or
coffee-drinking seem not to explain this variability. Possibly, the explanation involves
minor changes in the co-medication, compliance or dietary factors. Although entries in the
follow-up charts did not suggest it, infections could have changed their serum clozapine
concentration to dose ratios (Raaska et al. 2002b).
Two open trials of 13 and 12 patients suggest that some patients may benefit from a
combination of clozapine and risperidone (Taylor et al. 2001, Henderson & Goff 1996). In
one of these studies, serum clozapine concentrations (measured in 7 patients) increased only
insignificantly, by 2.2%, during 4 weeks of risperidone treatment (Henderson & Goff 1996).
Together with the fact that risperidone does not inhibit the main clozapine-metabolizing
D I S C U S S I O N
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enzymes (Eap et al. 2001), it is unlikely that risperidone would cause any significant
increase in serum clozapine concentrations.
5. Effect of influenza vaccination on serum clozapine concentration
n the present study, influenza vaccination had no effect on the serum clozapine, N-
desmethylclozapine or clozapine-N-oxide concentrations. The vaccination did not
elevate CRP. However, two of the patients were excluded from the statistical analysis
because of infections during the study period after the vaccination. Interestingly, during
their high CRP levels both had higher clozapine concentrations than at baseline. It is
probable that the concentrations increased at least in part due to the reduced volume of
distribution of clozapine, because elevated acute-phase 1 acid glycoprotein during
infections is expected to bind more clozapine in plasma. But these elevated serum clozapine
concentrations may, in part, have been caused by infections inhibiting clozapine-
metabolizing enzymes.
Because several of the factors that stimulate a cytokine response may inhibit CYP enzymes
(Morgan 2001), it is important to know whether influenza vaccination with the trivalent
vaccine currently used can cause drug interactions. Influenza virus vaccination stimulates
cytokine production (Meredith et al. 1985, Saurwein-Teissl et al. 1998, Bernstein et al.
1998), but the cytokine response is, however, dependent on the type of influenza vaccine
(Saurwein-Teissl et al. 1998). Whole-virus vaccines stimulate a more pronounced response
than the type of subunit vaccines used in this trial (Saurwein-Teissl et al. 1998). It appears
that these trivalent subunit influenza vaccines cause no significant alterations in the
pharmacokinetics of clozapine.
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76
6. Effect of coffee-drinking on serum clozapine concentration
n this study, caffeine-containing coffee elevated mean serum clozapine concentration by
20% to 26%. Although non-significantly, the N-desmethylclozapine to clozapine ratio
fell by 9% to 13%, which suggests that caffeine or other ingredients in the coffee reduced
CYP1A2 activity.
Clozapine has higher affinity for CYP1A2 than does caffeine (Km values 60 mol/L vs.
200-500 mol/L) (Pelkonen et al. 1998, Carrillo & Benítez 2000), suggesting that in order
to inhibit CYP1A2-mediated clozapine metabolism, caffeine has to be at much higher molar
concentrations than clozapine in hepatocytes where the metabolism takes place. It is
important to keep in mind, however, that their relative concentrations in serum may differ
from those in hepatocytes. In the present study, patient 26 in Table 7 had the largest
percentage increase (+41%) in serum clozapine concentration, and his caffeine and
paraxanthine concentrations at baseline were the highest among the study patients. His
caffeine concentrations during the caffeine phase (about 13 000 nmol/L) and at baseline
(over 30 000 nmol/L) exceeded his clozapine trough concentrations by 10- to 30-fold,
although caffeine concentrations were measured 14 hours after the previous cup of coffee.
The volumes of coffee servings and their caffeine contents probably varied considerably
between the patients, making it difficult to approximate caffeine intake. Serum paraxanthine
concentration, rather than caffeine concentration, served as an indicator of caffeine intake,
because it fluctuates less during the day (Kaplan et al. 1997). Serum paraxanthine
concentrations suggest that mean caffeine intake during the caffeine phase was about 60%
of that at baseline. The mean serum concentrations of clozapine at baseline (1287 + 749
nmol/L) and during the caffeine phase (1310 + 724 nmol/L) were, however, very similar.
Mean caffeine and paraxanthine concentrations during the control phase were 3.5% to 8%
of the corresponding concentrations at baseline or during the caffeine phase, as is expected
if caffeine-containing coffee is switched to identically prepared decaffeinated coffee.
The pharmacokinetics of caffeine is non-linear with increasing doses (Kaplan et 1997).
Probably non-linearity is reached with lower caffeine concentrations than normally, if it has
I
D I S C U S S I O N
77
to compete with clozapine for metabolism, or if a patient’s CYP1A2 activity is low. Those
patients with higher clozapine concentration to dose ratios at baseline, suggesting lower
clozapine clearance, seemed to be more sensitive to the caffeine-clozapine interaction. On
the other hand, cigarette-smoking induces CYP1A2, which elevates the clearances of
clozapine (Seppälä et al. 1999) and caffeine (Carrillo & Benítez 1996). In theory, if
CYP1A2 is induced, higher caffeine concentrations are needed to saturate the clozapine
metabolism by this pathway. But if caffeine concentrations are high enough to inhibit the
induced CYP1A2 significantly, the highest possible increase in serum clozapine
concentration should be higher than in a non-induced state of clozapine metabolism. A
likely example of this is the patient in a caffeine withdrawal study (Carrillo et al. 1998),
who had the highest daily consumption of both coffee (1100 mg per day) and cigarettes (40
per day), and whose clozapine concentrations fell to one-fifth after the caffeine withdrawal,
being the largest decrease seen in that study.
Two open trials have studied the effect of either coffee-drinking or caffeine intake on serum
clozapine concentrations (Carrillo et al. 1998, Hägg et al. 2000). In the study by Carrillo et
al. (1998) in 7 hospitalized patients on clozapine monotherapy, caffeine withdrawal reduced
the mean serum concentration of clozapine in 5 days by 47%. Clozapine concentration
decreased in each one of the patients (range -29 to -80%). In the study by Hägg et al. (2000)
with 12 non-smoking healthy male volunteers, caffeine increased the mean clozapine AUC
(0, ) by 19% (range -14% to +97%, P = 0.05), and reduced the mean oral clearance of
clozapine by 14% (range -49% to +7%, P = 0.05). These healthy subjects ingested a single
12.5-mg dose of clozapine with caffeine (mean 550 mg per day). The effect of caffeine on
serum clozapine concentrations in the present study was more modest than in the Carrillo et
al. (1998) study, but quite similar to the effect in Hägg et al. (2000).
This interaction is best explained by the inhibitory effect of caffeine on the CYP1A2
enzyme. Although coffee-drinking has the minor effect of raising serum clozapine
concentrations in most of the patients, genetic factors, for example, may make some
individuals more sensitive to this interaction.
D I S C U S S I O N
78
7. General discussion
roblems due to drug interactions occur in about 10% of all patients, and in almost 40%
of elderly patients (Stockley 2002). Clinically the most important mechanisms of
pharmacokinetic drug interactions are those involving CYP enzymes that eliminate the
majority of drugs (Bertz & Granneman 1997). In this respect, the most important of them is
CYP3A4, followed by CYP2D6, the CYP2C family, and CYP1A2 (Bertz & Granneman
1997). All of these are involved in many clinically significant drug interactions. Problems
may arise, especially if a drug meant, for example, to treat psychosis, is metabolized mainly
by only one of these enzymes. The usual dose of a drug may lead to toxic drug
concentrations if the patient who takes the drug is either a poor metabolizer of that enzyme
(CYP2D6, CYP2C family), or the normally functional enzyme is inhibited by another drug
(or factor). Often important interactions are recognized first through case-reports when the
drug is already on the market.
These studies demonstrate that some of the generally accepted conclusions (Taylor 1997) on
the pharmacokinetic interactions of clozapine that were drawn from case-reports
(Funderburg et al. 1994, Koreen et al. 1995, Tyson et al. 1995, Cohen et al. 1996), were
incorrect. Although CYP3A4 is involved in clozapine metabolism, its contribution to overall
clozapine elimination seems to be minimal in vivo. When studied under controlled
conditions, several potent CYP3A4 inhibitors have no clinically significant effects on serum
clozapine concentrations (Raaska & Neuvonen 1998, Hägg et al. 1999b, Taylor et al. 1999,
Vandel et al. 2000, Lane et al. 2001a, Lane et al. 2001b, Özdemir et al. 2001).
Recent reports support the hypothesis presented in Studies I and III that infections may
elevate serum clozapine concentrations (van der Molen-Eijgenraam et al. 2001, Raaska et
al. 2002b, de Leon & Diaz 2003, Haack et al. 2003). In one female patient a more than 3-
fold increase in serum clozapine concentration occurred during a probable bacterial
pneumonia (Raaska et al. 2002b). During this increase, the serum concentration ratio of N-
desmethylclozapine to clozapine had decreased, implying CYP1A2 inhibition by the
infection (Raaska et al. 2002b). The Netherlands Pharmacovigilance Foundation has
received five reports of elevated serum clozapine concentrations (3- to 10-fold) during
P
D I S C U S S I O N
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inflammation, and followed by normalization of the elevated clozapine concentrations after
recovery (van der Molen-Eijgenraam et al. 2001). In addition, five other patients with
inflammation had elevated serum clozapine concentrations (de Leon & Diaz 2003, Haack et
al. 2003).
These reports suggest that infections may raise total serum clozapine concentrations as
much as does fluvoxamine, but more reports and studies are needed to either confirm or
reject this conclusion. If confirmed, however, that would not necessarily mean that the two
interactions are similar in terms of clinical relevance. Infections elevate the concentration of
1 acid glycoprotein, which is the main clozapine-binding plasma protein. Any increase in
1 acid glycoprotein should raise total serum clozapine concentrations by causing an
increase in the protein-bound fraction. Three of the five patients reported by van der Molen-
Eijgenraam et al. (2001) developed delirium during elevated clozapine concentrations,
which suggests that probably not only the total serum concentration of clozapine, but the
free fraction as well, was increased. Infection itself, and not erythromycin as was suggested
in the two case-reports (Funderburg et al. 1994, Cohen et al. 1996), seems to be the more
likely explanation for the elevated clozapine concentrations during erythromycin treatment.
The enzyme that seems to be the most important in clozapine metabolism is CYP1A2
(Bertilsson et al. 1994, Jerling et al. 1997, Olesen & Linnet 2001). Its potent inhibition by
fluvoxamine leads to several-fold elevated serum clozapine concentrations (Hiemke et al.
1994, Koponen et al. 1996, Dequardo & Roberts 1996, DuMortier et al. 1996, Bender &
Eap 1998, Wetzel et al. 1998). This is clinically highly significant and may cause severe
adverse effects. Ciprofloxacin is a less potent CYP1A2 inhibitor than is fluvoxamine and
does not raise clozapine concentrations as strongly, but the effect of ciprofloxacin on serum
clozapine concentrations seems to be comparable to that of fluoxetine or paroxetine
(Centorrino et al. 1994b, 1996, Spina et al. 1998, 2000; Study II). Treatment with the low
250-mg twice daily dose of ciprofloxacin is, by itself, not likely to cause any clinically
significant increase in serum clozapine concentrations in most patients, but at a higher dose
or together with an infection, the overall increase in clozapine concentrations may be much
higher. Although the mean effect of coffee-drinking on serum clozapine concentration was
of low clinical relevance in Study V, coffee-drinking, or caffeine intake in general, may
D I S C U S S I O N
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interact significantly with clozapine pharmacokinetics in patients who are heavy caffeine
consumers. This can be predicted by the relatively high theoretical inhibition percentage of
caffeine for CYP1A2 at relevant in vivo caffeine concentrations (Pelkonen et al. 1998), by
the saturable caffeine metabolism in doses that are commonly ingested (Kaplan et al. 1997),
and by the large interindividual variability in CYP1A2 activity (Butler et al. 1989).
Taking into consideration the studies and reports of pharmacokinetic interactions involving
clozapine and clozapine metabolism, it seems that any drug or medical condition that
inhibits or induces CYP1A2 activity significantly can have clinically relevant effects on
serum clozapine concentrations. The isoenzymes CYP2C19, CYP2D6, and CYP3A4 seem
to be normally of minor clinical importance. However, if CYP1A2 activity is reduced by a
potent inhibitor such as fluvoxamine, or if its capacity is saturated by CYP1A2 substrate
overdose (e.g., clozapine or caffeine), CYP3A4, especially, may become a significant
contributor to clozapine metabolism.
Several characteristics of clozapine favor the utilization of TDM in dose optimization.
Clozapine has a relatively low therapeutic index, and it is not uncommon for patients on
therapeutic clozapine concentrations to complain of excessive sedation or salivation. Some
patients, however, tolerate well clozapine concentrations toxic to most other patients, but
maintaining an unnecessary high concentration keeps patients at increased risk for such
complications as seizures. Some patients metabolize clozapine very effectively, and their
clozapine concentrations may be too low, well below the suggested therapeutic threshold,
even if daily doses are high. For them, it may be necessary to use daily doses above 900 mg,
the recommended upper limit for a daily dose of clozapine in manufacturers’ product
labeling (Leponex®, Novartis, in the Finnish pharmacopea Pharmaca Fennica 2003). When
the optimal dose for a patient has been established, clozapine concentration should be
monitored annually, and when there is reason to suspect pharmacokinetic interaction or poor
compliance, or if a patient’s medical condition changes in such a way as to affect the
distribution or clearance of clozapine.
C O N C L U S I O N S
81
CONCLUSIONS
The following conclusions can be drawn from these five studies:
1. Itraconazole, and most likely also other CYP3A4 inhibitors, has no clinically significant
effect on serum concentrations of clozapine and its active metabolite N-
desmethylclozapine. Thus, those case-reports in which the CYP3A4 inhibitor
erythromycin seems to have caused elevated serum concentrations of clozapine have
been interpreted erroneously. Probably the infection itself, and not erythromycin, led to
the higher clozapine concentrations.
2. A low dose of the modest CYP1A2 inhibitor ciprofloxacin has a modest effect in
increasing the serum concentrations of clozapine and its metabolite N-
desmethylclozapine.
3. Influenza vaccination has no clinically significant effect on serum concentrations of
clozapine and its metabolites N-desmethylclozapine and clozapine-N-oxide.
4. Risperidone has no clinically significant effect on serum concentrations of clozapine.
5. Coffee-drinking, probably through inhibition of CYP1A2 enzyme-mediated clozapine
metabolism by caffeine, has generally a minor effect toward an increased serum
concentration of clozapine and of its metabolite N-desmethylclozapine.
A C K N O W L E D G E M E N T S
82
ACKNOWLEDGEMENTS
his work was carried out at the Department of Clinical Pharmacology, University of
Helsinki. I am grateful to all who have contributed to my work or well-being during
these years.
I am most grateful to Professor Pertti Neuvonen for his enthusiastic and excellent
supervision. He always had time for me whenever I needed it. I feel myself privileged and
happy to have had the opportunity to be part of his inspiring research group, for which he
has created such good facilities and a cozy atmosphere. The high standard he has set himself
as a researcher and a teacher is admirable and unparalleled.
Docents Arja Rautio and Kimmo Kuoppasalmi deserve grateful acknowledgement for their
valuable review and constructive comments on this thesis.
I am greatly indebted to my colleague Virpi Raitasuo who is a co-worker in three of these
studies. Virpi was the main figure in encouraging my interest in psychopharmacology when
I was taking my first steps as a toddler in the field of psychiatry in the fall of 1994 at
Kellokoski Hospital. During that time I finally made up my mind that I wanted to become a
psychiatrist. I am grateful for her collaboration and support.
I thank all my colleagues at the Department of Clinical Pharmacology, including Docents
Kari Kivistö, Janne Backman, and Lasse Lehtonen, plus Mikko Niemi, Jari Lilja, Laura
Juntti-Patinen, Tiina Varis, Carl Kyrklund, Kati Ahonen, Heli Malm, Harri Luurila, Maija
Kaukonen, Kirsti Villikka, Jun-Sheng Wang, Xia Wen, Eeva Lukkari-Lax, Outi Lapatto-
Reiniluoto, Seppo Kähkönen, Elina Saarenmaa, Matti Kivikko, Mika Jokinen, Vilja
Palkama, Tommi Lamberg, Teemu Kantola, Marika Granfors, Lauri Kajosaari, Samuel
Fanta, Tiina Jaakkola, Tuure Saarinen, Aleksi Tornio, Marjo Karjalainen, Aino Koskinen,
and Marja Pasanen. I express my warmest thanks to Tuija Itkonen for her help and ingenuity
in many practical matters.
T
A C K N O W L E D G E M E N T S
83
These works could not have been done without the help of the skillful technical staff of the
Department of Clinical Pharmacology. I am indebted to Jouko Laitila, Kerttu Mårtensson,
Eija Mäkinen-Pulli, Mikko Neuvonen, and Lisbet Partanen.
I express my gratitude for their support and encouragement to the former Heads of
Kellokoski Hospital Docent Ilkka Taipale and Dr. Raimo Väisänen, and to Dr. Grigori Joffe
who now heads the hospital. I thank all my colleagues in Kellokoski Hospital. Special
thanks go to Docent Eero Elomaa who introduced me to the mysteries of clozapine, and
encouraged me to do research. He also introduced me to Professor Neuvonen. I am greatly
indebted to the staff of Kellokoski Hospital. Kaija Leppäniemi and Soili Sarvaala from the
hospital laboratory, and Ilkka Raitasuo are especially warmly acknowledged for their
contributions. The list of other contributers would be almost endless.
The ones that leave me humble are the study subjects, the patients at Kellokoski Hospital
who participated in these studies. I want to express to them my deepest gratitude. They have
really taught me a great amount.
I am grateful to all colleagues and staff at the Department of Psychiatry for their support and
encouragement, especially Professors Ranan Rimón, Matti Virkkunen, Björn Appelberg,
Acting Professor Heikki Katila, Docents Heikki Vartiainen, Antero Leppävuori, Timo
Partonen, and Matti Huttunen, Dr. Teija Honkonen, and all of those on the Mieliala- and
Psykosomatiikka teams are warmly acknowledged for teaching me psychiatry.
Many thanks go to Docent Kalle Hoppu for his help in professional and private matters, and
to the staff of the Poison Information Centre. I am grateful to Professor Eija Kalso at the
Pain Clinic for stimulating discussions.
Impressively energetic Carol Norris, my neighbor and my teacher, is warmly acknowledged
for author-editing the English language of this thesis.
This work was financially supported by the Clinical Drug Research Graduate School, the
Helsinki University Central Hospital Research Fund, the National Technology Agency of
A C K N O W L E D G E M E N T S
84
Finland (TEKES), Oy H. Lundbeck Ab, and Oy Eli Lilly Finland Ab, all of which are
gratefully acknowledged.
I thank all my friends who have been invaluable to me during these years. Among them,
especially Jukka and Sirpa Sahlakari, and Tuomo and Anna-Maija Heikinheimo, have been
very important. Special thanks go to my friends Kurt Tolliver and Mike Schaeffer across the
ocean. I have enjoyed being a part of the marathon team Sankarpojat, and it has been a great
pleasure to be involved in the activities of my son’s soccer team KOPSE Kojootit P92.
My mother-in-law Suoma Takala has been most invaluable during these years taking care of
our children. We could not have managed without her helping hand. Thank you so much,
Suoma! I am grateful to my late father-in-law Aarne Takala. I thank my sister-in-law Annu
Kauppinen, her husband Juuso and their children Anni, Tuomo, Aleksi, and Joonas for their
friendship and good times spent together.
I am most grateful to my parents Marja-Liisa and Jorma Raaska for their love and support,
and I thank them for providing me a happy childhood, which is the greatest thing anyone
can receive from their parents. I thank my sister Maria Inkovaara, her husband Pekka and
their children Ronja and Rasmus for their friendship and good times spent together. My
grandparents are acknowledged with deep gratitude and respect. I also thank my other
relatives who have had an important impact on my life.
Finally, I thank my wonderful wife Hanna and our children Eveliina, Antti, and Iida with
my deepest love and gratitude. Hanna, words cannot tell how thankful I am to you. Eveliina,
Antti, and Iida, thank you for being what you are; you bring me so much joy and happiness.
I know many others deserve to be personally acknowledged. I first express my apologies
and then my thanks to all of you.
Vantaa, October 2003
Kari Raaska
R E F E R E N C E S
85
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