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
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
CONTENTS
CONTENTS
ABBREVIATIONS .. 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. 1.1.3.2. 1.1.3.3. 1.1.3.4. CYP1A subfamily ... 16 CYP2C subfamily ... 17 CYP2D subfamily ... 18 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. 4.1.1.2. 4.1.1.3. SSRIs and clozapine 31 Other inhibitors of CYP enzymes and clozapine 32 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
CONTENTS
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
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ABBREVIATIONS
ABBREVIATIONS
AhR Arnt AUC AUC(0- ) b.i.d. BPRS C/D ratio CL Cmax CNS CRP CYP CV D DSM-IV EM EPS F FMO HPLC 5-HT IM Ki Km NADPH ND NS PAH P-gp aryl hydrocarbon receptor AhR nuclear translocator protein area under drug concentration-time curve area under drug concentration-time curve (time 0 to infinity) twice daily Brief Psychiatric Rating Scale ratio of serum drug concentration / daily dose of the drug clearance peak concentration central nervous system c-reactive protein cytochrome P450 coefficient of variation dopamine Diagnostic and Statistical Manual of Mental Disorders, 4th ed. extensive metabolizer extrapyramidal symptoms bioavailability flavine-containing monooxygenase high-performance liquid chromatography serotonin (5-hydroxytryptamine) intermediate metabolizer inhibition constant Michaelis-Menten constant reduced form of nicotinamide adenine dinucleotide phosphate not determined or unknown statistically non-significant polycyclic aromatic hydrocarbon P-glycoprotein
ABBREVIATIONS
PM PXR RXR SD SSRI T TDM Tmax UKU v VCFS Vd
poor metabolizer pregnane X receptor retinoid X receptor standard deviation selective serotonin reuptake inhibitor elimination half-life therapeutic drug monitoring time to peak concentration Udvalg for Kliniske Undersgelser side-effect rating scale volume velo-cardio-facial syndrome apparent volume of distribution
LIST OF ORIGINAL PUBLICATIONS
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.
Raaska K, Neuvonen PJ. Serum concentration of clozapine and Ndesmethylclozapine 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 Ndesmethylclozapine: 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
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).
ABSTRACT
ABSTRACT
ackground: Clozapine is an atypical antipsychotic drug that was first introduced into clinical use in the 1970s. 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.
M R
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
ABSTRACT
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, Ndesmethylclozapine 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
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 drugmetabolizing 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 (Seppl 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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INTRODUCTION
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 risperidonewhich 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 coffeedrinking 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)
A
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 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-3demethylation 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 (Brsen 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 isfor 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 & Anzenbacherov 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 Pglycoprotein substrate susceptible to clinically relevant drug interactions with P-gp inhibitors (Partanen et al. 1996, Fromm et al. 1999, Silverman 2000). Midazolam, not a Pgp 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).
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Table 1. Human CYP families, subfamilies, locations of functional genes, and main functions of corresponding CYP enzymes.
Subfamilies1 Main functions 2-6
CYP family1
CYP1 CYP2
CYP3 CYP4
CYP5 CYP7 CYP8 CYP11 CYP17 CYP19 CYP20 CYP21 CYP24 CYP26 CYP27 CYP39 CYP46 CYP51
1 4
1A 1B 2A, 2B, 2F, 2G, 2S, 2T 2C, 2E 2D 2J 2R 2U 2W 3A 4A, 4B 4F 4V 4X 4Z 5A 7A, 7B 8A 8B 11A 11B 17A 19 20 21A 24 26A, 26C 26B 27A, 27C 27B 39A 46 51
Location of functional genes (chromosome)1 15 2 19 10 22 1 11 4 7 7 1 19 4 1 1 7 8 20 3 15 8 10 15 2 6 20 10 2 2 12 6 14 7
Xenobiotic metabolism Estradiol 4-hydroxylation Xenobiotic metabolism (2A, 2B) Xenobiotic metabolism Xenobiotic metabolism Arachidonic acid and xenobiotic metabolism Unknown Unknown Unknown Xenobiotic metabolism Fatty acid, arachidonic acid and leukotriene metabolism Fatty acid and arachidonic acid metabolism Unknown Unknown Unknown Thromboxane A2 synthesis Bile acid synthesis Prostacyclin synthesis Bile acid synthesis Cholesterol side-chain cleavage Steroid 11 -hydroxylation, aldosterone synthesis Steroid 17 -hydroxylation Steroid aromatization (estrogen synthesis) Unknown Steroid 21-hydroxylation Vitamin D metabolism Vitamin A metabolism Vitamin A metabolism Vitamin D synthesis, bile acid synthesis Vitamin D synthesis Bile acid synthesis Cholesterol 24-hydroxylation Sterol 14-demethylation
Nelson:http://drnelson.utmem.edu/human.genecount.html, 2 Nebert & Russell 2002, 3 Lund et al. 1999, Yoshida et al. 2000, 5Matsumoto et al. 2002, 6Bylund et al. 2002.
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Table 2. Genetic polymorphism, selected substrates, inhibitors, and inducers of the main drug-metabolizing CYP enzymes.
GENETIC POLYMORPHISM CYP2C8
Suggested3 Yes3 Yes3 Yes4 No5, 6
CYP1A2 CYP2C9 CYP2C19 CYP2D6 CYP3A4
No1, 2
SUBSTRATES CYP2C8
Diclofenac3 Fluvastatin7 Ibuprofen3 Losartan3 Naproxen3 Phenytoin3 Piroxicam3 S-warfarin3 Tolbutamide3 Clomipramine7 Diazepam3 Imipramine7 Omeprazole3 Phenytoin3 Propranolol7 Proguanil3 Amitriptyline4 Clomipramine4 Codeine4 Fluoxetine7 Fluvoxamine4 Haloperidol4 Metoprolol4 Nortriptyline4 Paroxetine4 Risperidone4, 9, 16 Thioridazine4
CYP1A2
CYP2C9
CYP2C19
CYP2D6
CYP3A4
Amiodarone7 Amitriptyline7 Alprazolam7 Carbamazepine7 Ciclosporin7 Erythromycin7, 17 Felodipine7 Lovastatin7 Midazolam7, 17 Nefazodone7 Quinidine7
Caffeine2, 7 Clozapine8, 9 Imipramine7 Lidocaine10 Maprotiline7 Olanzapine9 Propranolol7 Ropivacaine11 R-warfarin2 Tacrine2 Theophylline2
Carbamazepine3 Cerivastatin12, 13 Ibuprofen3 Paclitaxel3 Repaglinide14 Rosiglitazone15 Tolbutamide3
INHIBITORS CYP2C8
Amiodarone3 Fluconazole3, 7 Miconazole3 Sulphaphenazole3
CYP1A2
CYP2C9
CYP2C19
Fluoxetine3 Fluvoxamine3 Omeprazole3
CYP2D6
Flecainide4 Fluoxetine4 Paroxetine4 Quinidine4
CYP3A4
Erythromycin17 Itraconazole17 Nefazodone17 Ritonavir17
Cimetidine7 Ciprofloxacin7, 18 Fluvoxamine2, 19 Grepafloxacin7
Gemfibrozil12, 13 Trimethoprim20
INDUCERS CYP2C8
Phenobarbital7, 24 Rifampicin7, 23, 24
CYP1A2
CYP2C9
CYP2C19
Phenobarbital24 Rifampicin7, 23, 24
CYP2D6
Unknown4
CYP3A4
Carbamazepine17 Dexamethasone17 Phenobarbital17 Phenytoin17 Rifampicin7, 23
Carbamazepine21 Dioxin2 Phenobarbital7, 22 Rifampicin2, 23 Tobacco smoke3, 7, 22
Phenobarbital24 Rifampicin23, 24
10
19
Smith et al. 1998, 2Miners & McKinnon 2000, 3Rettie et al. 2000, 4Zanger & Eichelbaum 2000, 5Lamba et al. 2002, 6Garcia-Martin et al. 2002, 7 Stockley 2002, 8Taylor 1997, 9deVane & Markowitz 2000, Wang et al. 2000, 11Arlander et al. 1998, 12Wang et al. 2002b, 13Backman et al. 2002, 14Niemi et al. 2003b, 15Niemi et al. 2003a, 16Yasui-Furukori et al. 2001, 17Wrighton & Thummel 2000, 18Fuhr et al. 1992, Brsen et al. 1993, 20Wen et al. 2002, 21Parker et al. 1998, 22Pelkonen et al. 1998, 23Niemi et al. 2003c, 24Gerbal-Chaloin et al. 2001.
21
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-thecounter 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 (Isomets 2001, Joukamaa et al. 2001). Drug overdose with multiple drugs, often at least one psychotropic drug, is a common method of suicide (Baca-Garca et al. 2002), in which case consequences of overdose are worse due to interaction between drugs.
22
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).
23
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.
24
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 (Idnpn-Heikkil 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 adverseeffect 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. Clozapines 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).
25
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 (Sjqvist 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 (T) 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 Oral bioavailability (F) Time to peak concentration (Tmax) Elimination half-life (T) Volume of distribution (Vd) Plasma clearance (CL) Unbound fraction
Value 27-47% 1-3 hours 10-17 hours 2-5 L/kg 13-57 L/h 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).
26
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).
Other metabolites
CH3 N O Cl N N
CYP3A4
CYP1A2 FMO
CH3 N N N
CYP1A2
CYP3A3 CYP2C19
H N N N
Cl
Cl
N H
Clozapine-N-oxide
N H
Clozapine
N H
N-desmethylclozapine
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 Ndesmethylclozapine and clozapine N-oxide (Fang et al. 1998). In addition, a few other CYP
27
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 Ndesmethylclozapine and clozapine N-oxide.
Source Method Clozapine concentration Enzymes responsible for metabolism of clozapine to N-desmethylclozapine Pirmohamed et al. 1995 Human liver microsomes 2 M CYP1A2 Clozapine-N-oxide CYP3A4, FMO
Eiermann et al. 1997
Human liver microsomes
50 M
CYP1A2, 3A4
CYP3A4
Linnet & Olesen 1997
cDNA-expressed human CYPs Human liver microsomes
0.5-50 M
CYP3A4, 1A2, 2C, 2C19, 2D6 CYP1A2, 3A4 CYP3A4, 1A2
CYP3A4
Fang et al. 1998
20 M 300 M
CYP3A4, 1A2 CYP3A4
Tugnait et al. 1999
Human liver microsomes cDNA-expressed human CYPs
1-1000 M 350 M
CYP1A2, 3A4 CYP2D6, 1A2, 3A4
CYP3A4, 1A2 CYP3A4, 1A2
Olesen & Linnet 2001
Human liver microsomes
5 M 50 M
CYP1A2, 2C19 CYP3A4, 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.
28
1994). Very high (ultrarapid) CYP1A2 activity, caused by homozygous alleles with C
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, zdemir 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 T 5.5 to 35 hours (Lin et al. 1994). The Tmax for Ndesmethylclozapine was between 1.5 and 40 hours, and the metabolite was eliminated in parallel with clozapine, suggesting formation-rate-limited elimination of the N-
29
desmethylclozapine metabolite (Lin et al. 1994). In that case, the T of Ndesmethylclozapine 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 weightcorrected 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 weightcorrected 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 mothers 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).
30
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, zdemir et al. 2001) (Table 5). Fluvoxamine raised the serum elimination T 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 (Brsen 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 (zdemir et al. 2001).
Fluoxetine and its main metabolite norfluoxetine are both potent inhibitors of CYP2D6 (Brsen & 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
31
moderate enhancement effect on clozapine concentrations of 30% to 57%, and in one casereport 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, Facciol 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 (Hgg 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).
32
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 cotreated 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% (Hgg et al. 2000).
Grapefruit juice (CYP3A4 inhibitor) seems to have no effect on serum clozapine or its main metabolite concentrations (Vandel et al. 2000, zdemir 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
33
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). Facciol 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 (Seppl 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
34
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 (Kuoppamki 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-Noxide, is not biologically active (Kuoppamki et al. 1993).
35
Table 5. Pharmacokinetic interactions of clozapine with drugs (a) and other factors (b) a. Interacting drug Daily dose
SSRIs
Citalopram Fluoxetine 40 mg 20 mg Mean 36 mg 80 mg Mean 39 mg 20 mg 100 mg 100 to 150 mg 25 mg 100 to 200 mg 150 mg 50 mg 50 mg 100 mg 50 mg Mean 31 mg 20 mg 20 mg 20 to 40 mg Mean 92 mg 300 mg 50 to 100 mg Patients (8), sequential Patients (5), sequential Patients (6, 17 controls), parallel Patient, case-report Patients (14, 40 controls), parallel Patients (10), sequential Patient, case-report Patients (2), case-report Patient, case-report Patients (3), case-report Patients (2), case-report Patients (16), sequential Patients (16), sequential Patients (16), sequential Patient, case study Patients (16, 40 controls), parallel Patient, case-report Patients (14), sequential Patients (9), sequential Patients (10, 40 controls), parallel group Patient, case-report Patients (8), sequential No change No change Increase, 76% No change Increase, 30% Increase, 58% Increase, 8-fold Increase, 5- to 10 -fold Increase, 4- to 9 -fold Increase, 3- to 9 -fold Increase, 5- to 10 -fold Increase, 3-fold Increase, 2- to 5-fold Increase, 5-fold Increase, 3-fold Increase, 57% Increase, 30% to 2-fold No change Increase, 31% Increase, 30% Increase, 2-fold No change Avenoso et al. 1998 Taylor et al. 1998 Centorrino et al. 1994b Eggert et al. 1994 Centorrino et al. 1996 Spina et al. 1998 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 zdemir et al. 2001 Centorrino et al. 1996 Joos et al. 1997 Wetzel et al. 1998 Spina et al. 2000 Centorrino et al. 1996 Pinninti & de Leon 1997 Spina et al. 2000
Subjects (nb), Study design
Effect on serum CLZ concentration
Source
Fluvoxamine
Paroxetine
Sertraline
Other inhibitors
Cimetidine Ciprofloxacin Erythromycin 800 mg 1000 mg 1000 mg 1000 mg 1000 mg 1500 mg 400 mg 100 mg 5 to 10 mg 400 mg 200 mg 300 mg 360 mg 300 mg 8 mg 2 mg 2 mg Patient, case-report Patient, case-report Patient, case-report Patient, case-report Patient, case-report Healthy (12), randomized crossover Patients (5), sequential Patient, case-report Patient, case-report Patients (6), sequential Patient, case-report Patient, case-report Patients (2), sequential Patient, case-report Patients (7), sequential Patient, case-report Patient, case-report Increase, 60% Increase, 2-fold Increase, 60% Increase, 2-fold Increase, 3-fold No change No change Increase, 3-fold Increase, 2- to 3 -fold No change No change Increase, 75% No change No change No change Increase, 2-fold Increase, 74% Szymanski et al. 1991 Markowitz et al. 1997 Joos et al. 1998 Funderburg et al. 1994 Cohen et al. 1996 Hgg et al. 1999b Lane et al. 2001a Kossen et al. 2001 Abraham et al. 2001 Taylor et al. 1999 Khan & Preskorn 2001 Khan & Preskorn 2001 Hilger et al. 2002 Szymanski et al. 1991 Spina et al. 2001 Koreen et al. 1995 Tyson et al. 1995
Ketoconazole Lamotrigine Lisinopril Nefazodone
Orlistat Ranitidine Reboxetine Risperidone
36
Interacting drug Daily dose

Valproate

Decrease, 23 to 51% Increase, 39% Decrease, 15%
Source
750 to 1000 mg Patients (4), sequential Mean 1060 mg Patients (11, 17 controls), parallel Mean serum Patients (7), sequential concentration 62 mg/L 900 to 1200 mg Patients (6), sequential 600 to 1500 mg Patients (15, 22 controls), parallel
Finley & Warner 1994 Centorrino et al. 1994b Longo & Salzman 1995
No change No change
Facciol et al. 1999 Facciol et al. 1999
Inducers
Carbamazepine 600 to 800 mg Not stated Patients (2), case-report Patients (17, 124 controls), retrospective, parallel 500 to 1500 mg Patients (12), sequential 60 mg Patient, case-report Concentration Patients (7, 15 controls), 12 to 31 g/mL parallel 300 mg Patients (2), case-report 600 mg Patient, case-report Decrease, 42 to 50% Decrease, 68% Decrease, 47% Decrease, 43% Decrease, 35% Decrease, 65 to 85% Decrease, 83% Raitasuo et al. 1993 Jerling et al. 1994 Tiihonen et al. 1995 Lane et al. 1998 Facciol et al. 1998 Miller 1991 Joos et al. 1998
Phenobarbital
Phenytoin Rifampicin
b. Interacting factor Daily dose

Increase, 2-fold Increase, 2-fold AUC increased 19% Decrease, 20% Decrease, 38% Decrease, 41% No change No change No change No change Increase, 2- to 10-fold Increase, 3-fold Increase, 2-fold Increase, 2-fold
Source
Caffeine + coffee Patient, case-report Coffee 150 to 1100 mg Patients (7), sequential of caffeine Caffeine 400 to 1000 mg Healthy (12), open randomized crossover Cigarette-smoking Patients (38, 18 controls ), parallel Patients (34, 10 controls), parallel Patients (11), sequential Grapefruit juice 500 mL Patients (9), sequential 250 mL Patients (21), sequential 500 mL Patients (15), sequential 1125 mL Patient, case study Infection or inflammation Patients (5), case-report Patient, case-report Patient, case-report Patients (4), case-report
Odom-White & de Leon 1996
Carrillo et al. 1998 Hgg et al. 2000

Hasegawa et al. 1993
Seppl et al. 1999 Mayer 2001 Vandel et al. 2000 Lane et al. 2001a Lane et al. 2001b zdemir et al. 2001 van der Molen-Eijgenraam et al. 2001 Raaska et al. 2002b
de Leon & Diaz 2003 Haack et al. 2003
37
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 (Hgg 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
38
9100
g/L with a calculated peak value between 35 000
g/L and 24 000
g/L for an
assumed absorption T of 0.5 and 4 hours, respectively (Sartorius et al. 2002). For both absorption half-lives, the elimination T 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 & Brsen 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, dosecorrected 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 Parkinsons 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
39
risk for confusion, delirium, and generalized seizures (Freeman 1997, Buur-Rasmussen & Brsen 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
Cl Cl N N O H O CH2O N N N O N N CHCH2CH3 CH3
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 T of 20 to 24 hours after the usual
40
single dose, but in the steady state T 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 ones life (Meis & Verweij 2001).
Itraconazole is a potent inhibitor of CYP3A4, with Ki values < 1 M for many CYP3A4catalyzed 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 (Kivist et al. 1997). For example, itraconazole 200 mg daily will raise the AUC of oral midazolam by 10- to 15-fold, and its T 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
41
(> 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
O F N HN N COOH
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. T 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
42
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).
43
5.3. Risperidone
N N O
CH3 C C N H2 H2 N O
F
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 T 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-OHrisperidone, 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 T 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 T 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
44
slightly lower than expected, which in some patients may explain non-response to risperidone (Gzey 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).
45
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
46
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 casereports 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

O H3C O N N CH3 Figure 5. Chemical structure of caffeine CH3 N N
affeine is completely absorbed after oral administration (Carrillo & Bentez 2000). Time to Cmax is about 1 hour, volume of distribution is about 0.7 L/kg (Carrillo &
Bentez 2000). It is mainly eliminated through the metabolism by CYP1A2 to paraxanthine (Butler et al. 1989, Carrillo & Bentez 1994, Jeppesen et al. 1996a). Other demethylated metabolites, theobromine and theophylline, are produced via CYP1A2 and CYP2E1
47
pathways. These main metabolites undergo further demethylation and oxidation (Carrillo & Bentez 1994, Lelo et al. 1986a).
Caffeine has a T 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 T 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
48
(Carrillo & Bentez 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, Hgg 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 receptorreceptor interaction between adenosine 2 and dopamine receptors 1 and 2 (Vainer & Chouinard 1994).
49
Table 6. Examples of pharmacokinetic drug interactions with caffeine, ciprofloxacin, itraconazole, and risperidone as inhibitors.
Substrate Source Probable mechanism Effect on pharmacokinetics of substrate
Carrillo et al. 1998 Hgg et al. 2000 Mester et al. 1995 Increase in serum concentration of 2-fold AUC increased by 19% Reduced serum concentration by 24% Sato et al. 1993 Nicolau et al. 1995 Jokinen et al. 2003 Batty et al. 1995 Kivist et al. 1997 Partanen et al. 1996 Jalava et al. 1997a Neuvonen & Jalava 1996
Inhibitor
Subjects (n), study design
Daily dose
Clozapine Clozapine Lithium
Caffeine
Inhibition of CYP1A2 Inhibition of CYP1A2 Inhibition of proximal tubular reabsorption of lithium Inhibition of CYP1A2 Inhibition of CYP1A2 Inhibition of CYP1A2 Inhibition of CYP1A2 Inhibition of CYP3A4 Inhibition of P-gp Inhibition of CYP3A4 Inhibition of CYP3A4
Approximate mean 300 mg Patients (7), sequential in coffee 400 to 1000 mg Healthy (12), open randomized crossover mean 550 mg per day 4 to 8 cups of coffee Patients (11), sequential
Healthy (6), sequential Caffeine Ropivacaine Theophylline Buspirone Digoxin Felodipine Lovastatin Lovastatin acid Midazolam Triazolam Clozapine Clozapine Reduced mean oral clearance by 23%, T increased 32% Reduced AUC 2-fold
Theophylline
Ciprofloxacin
2 to 7 cups of instant coffee 1000 mg
1000 mg
1000 mg
Healthy (24), single-blind randomized crossover Healthy (9), double-blind randomized crossover Healthy (9), sequential Reduced mean clearance by 31% Reduced mean oral clearance by 19% Raised AUC 19-fold
Itraconazole
200 mg
200 mg
200 mg
Raised mean serum concentration 80% Raised AUC 6-fold Raised Cmax >20-fold
200 mg
Healthy (8), double-blind randomized crossover Healthy (10), double-blind randomized crossover Healthy (9), double-blind randomized crossover Healthy (12), double-blind randomized crossover
200 mg
Raised Cmax 13-fold and AUC 23-fold Raised AUC 10-fold Olkkola et al. 1994 Raised AUC 27-fold Varhe et al. 1994 Koreen et al. 1995 Tyson et al. 1995
Inhibition of CYP3A4 Inhibition of CYP3A4 Unknown Unknown
200 mg
Risperidone
2 mg
Healthy (9), double-blind randomized crossover Healthy (9), double-blind randomized crossover Patient, case-report
2 mg
Patient, case-report
Raised serum concentration by 2-fold Raised serum concentration by 74%
50
AIMS OF THE STUDY
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. Casereports suggest that even low-dose risperidone may double the serum concentration of clozapine.
51
AIMS OF THE STUDY
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 Ndesmethylclozapine 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.
52
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.
Study No. 1 2 3 4 5 6 7 8 9 I I I I I I I II II IV V II II II II II Female Age /male (F/M) (years) M 25 M 20 M 36 M 22 F 29 M 27 M 36 M 28 F 38 F 39 F 42 M 36 M 27 F 35 M 32 F 46 Weight* (kg) 74 66 76 70 71 64 80 94 85 ND 85 100 92 65 102 70 Smoker (yes/no) No Yes Yes No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No Yes Coffee drinker (yes/no) ND ND ND ND ND ND ND Yes Yes Yes Yes Yes Yes Yes Yes Yes Clozapine daily dose* (mg) 400 400 350 200 200 550 250 150 350 400 600 275 450 200 400 300 Clozapine monotherapy (yes/no) No No No No Yesasc Yesasc Yes No No No No Yesasc No YesIUD No No
10 11 12 13 14
53
Table 7. continued.
Study Female/ No. male (F/M) III M III M V M III F III F III M M III V M III M III F III F III F III F M III V M III M III M III M III F IV M IV M IV M IV M IV F IV M IV M IV M IV M IV F IV M IV M IV F IV F IV F IV M IV M V M V M V M V M V F V M V M V M Age (years) 48 47 48 50 45 48 35 36 26 46 43 35 34 34 35 21 22 45 34 24 53 49 49 34 53 30 43 51 45 37 35 30 32 39 48 18 39 26 41 27 22 48 18 38 Weight* (kg) 81 75 71 64 88 84 85 89 64 114 80 75 100 135 138 83 80 83 100 112/110 98/74 ND/96 ND 85 72/ND 89/90 69/ND ND/88 ND/80 ND/84 ND/113 ND 100/98 ND/70 118 96/90 92 71 85 100 75 79 92 68 Smoker (yes/no) No No No Yes No Yes No No No No Yes No Yes Yes Yes No Yes No No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No Yes Yes No Yes Yes Yes Yes Yes Yes Yes Yes Coffee drinker (yes/no) Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes ND ND Yes Yes Yes ND Yes ND No Yes Yes ND Yes Yes Yes Yes Yes Yes Yes Yes Clozapine daily dose* (mg) 275 650 650 400 400 500 300 300 750 400 450 450 450 375 375 375 550 300 800 450/425 700 600 800/700 425 450/900 300/200 450/500 500 350 600 400/600 300/225 200/250 400 450/300 100 450 350 250 750 300 500 400 450 Clozapine motherapy (yes/no) No Yes Yes No No No Yes Yes No No No No No No No Yes No Yes No No No No No No No No Yes No No No No No No No No No No No No No No No No No
15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 361 37 38 39 40 41 42 43 442 45 46 47 48 49 50 51 523 53 54 55

* asc
{ {
if the variable differs between study phases, the value is given for both phases (control phase/inhibitor phase) patient on clozapine monotherapy, but taking ascorbic acid daily IUD patient on clozapine monotherapy and wearing an intrauterine device (hormone) 1 Psychotic disorder NOS 2 Delusional disorder 3 Schizoaffective disorder ND not determined or unknown
54
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 evenings dose of clozapine and itraconazole/placebo on study days 0, 3, 7, 10, and 14. Serum was separated within one hour and stored at -20C 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 evenings 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 -20C 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 evenings 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 C until analyzed (Table 8).
55

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 patients 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
56

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 evenings 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 C 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 Undersgelser (UKU) side-effect rating scalethe 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, Ndesmethylclozapine, and clozapine-N-oxide, and concentrations of all the inhibitors studied
and of CRP were drawn 12 h after the previous evenings dose of clozapine, before the ingestion of clozapine or of the inhibitors. Serum was separated within one hour and stored at -20C until the samples were analyzed.
57
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 dayto-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.
58

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
59

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
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 Students 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. Consumers 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 Hyvink Health Care District and by the National Agency for Medicines (Finland). The study protocols of Studies
60

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.
61
Table 8. Study design. The aim was to investigate any possible effect of five factors (inhibitors or co-drugs) on serum concentration of clozapine
and its metabolites in hospitalized patients. Structure itraconazole 100 mg b.i.d (CYP3A4 inhibitor) ciprofloxacin 250 mg b.i.d (CYP1A2 inhibitor) placebo placebo Inhibitor / co-drug Control Study duration 7 days per phase
Study no. I
Patients females/males n=7 1/6
II
n=7 3/4
III
n = 16 7/9
randomized, placebo-controlled crossover study with two phases randomized, placebo-controlled crossover study with two phases open, prospective; patients as their own controls
7 days per phase + 7 days washout period between the phases 14 days
IV
n = 18 6 / 12 n = 12 2 / 10
retrospective; patients as their own controls randomized, placebo-controlled crossover study with two phases
influenza vaccination (stimulates cytokine production, cytokines inhibit CYPs) risperidone 2-4 mg daily (substrate of CYP2D6 and 3A4) caffeine-containing coffee ad libitum (caffeine is a CYP1A2 substrate)
none (before the influenza vaccination) none paired samples (without risperidone) 0.3-48 months apart decaffeinated coffee 7 days run-in period + 7 days per phase
62
RESULTS
RESULTS
Effects of itraconazole, ciprofloxacin, risperidone, influenza vaccination, and coffee-drinking on clozapine concentrations
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 Ndesmethylclozapine 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 + Ndesmethylclozapine serum concentrations and serum ciprofloxacin concentration was
63
RESULTS
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 Ndesmethylclozapine 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 Ndesmethylclozapine 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
64
RESULTS
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), Ndesmethylclozapine, 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
65
RESULTS
clozapine-N-oxide, 7% (NS, P = 0.22) higher than during the control phase. The Ndesmethylclozapine 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 Ndesmethylclozapine 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).
66
RESULTS
160
Clozapine concentration (% of control)
140 120 100

4 DAYS AFTER VAC 7 DAYS AFTER VAC 2 DAYS AFTER VAC
80 60 40
ITRA
COF 3 DAYS
0
97 129 105 102 101 92 104 120 126
II
III Study
IV
RISP
20
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.
COF 7 DAYS
COF COMB
CIPRO
67
RESULTS
Table 9. Main results of the studies. Mean effects of inhibitors or co-drugs on serum concentrations of
Study no. Clozapine Unchanged N-desmethylclozapine Unchanged
Inhibitor / co-drug
Clozapine-N-oxide Not studied Not studied Unchanged
II
III
Increase 31% (P < 0.05) Unchanged
IV Increase 6% (P < 0.05)
Not studied
Not studied Unchanged
Itraconazole 100 mg b.i.d (CYP3A4 inhibitor) Ciprofloxacin 250 mg b.i.d Increase 29% (CYP1A2 inhibitor) (P < 0.01) Unchanged Influenza vaccination (stimulates cytokine production: cytokines inhibit CYPs) Risperidone 2-4 mg daily Unchanged (substrate of CYP2D6 and 3A4) Caffeine-containing coffee ad Non-significant increase 26% (P = 0.07) libitum (caffeine is a CYP1A2 substrate)
68
RESULTS
FEMALE, age 34
Clozapine and metabolites (nmol/l) Pharyngitis
Clozapine 60 0.9 4000
40
0.6
2000
DMCLZ/CLZ-ratio CRP 0
1000
20
0.3
14
Days after vaccination
MALE, age 21
Clozapine concentration (nmol/l) Abdominal pain
1400 1200 CLOZAPINE
Sedation
80 0.5
CRP (mg/l)
1000 800 600 400 200 0 0 2 CRP 4 7 14 DMCLZ/CLZ-ratio
60
0.4 0.3 0.2 0.1
40
20
Days after vaccination
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.
DMCLZ/CLZ-ratio
69
DMCLZ/CLZ-ratio
3000
CRP (mg/l)
DISCUSSION
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
70
DISCUSSION
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 weeks 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, Kivist 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
71
DISCUSSION
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 (Kivist et al. 1997, Kivist et al. 1999).
The complete lack of effect of itraconazole on serum concentrations of clozapine and Ndesmethylclozapine in this prospective study does not support the hypothesis of a CYP3A4inhibition-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 Ndesmethylclozapine 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
72
DISCUSSION
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.
73
DISCUSSION
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 comedications. 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
74
DISCUSSION
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, Ndesmethylclozapine 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 clozapinemetabolizing 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.
75
DISCUSSION
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 & Bentez 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
76
DISCUSSION
to compete with clozapine for metabolism, or if a patients 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 (Seppl et al. 1999) and caffeine (Carrillo & Bentez 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, Hgg 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 Hgg 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 Hgg 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.
77
DISCUSSION
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, Hgg et al. 1999b, Taylor et al. 1999, Vandel et al. 2000, Lane et al. 2001a, Lane et al. 2001b, zdemir 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 3fold increase in serum clozapine concentration occurred during a probable bacterial pneumonia (Raaska et al. 2002b). During this increase, the serum concentration ratio of Ndesmethylclozapine 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
78
DISCUSSION
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 MolenEijgenraam 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
79
DISCUSSION
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 patients medical condition changes in such a way as to affect the distribution or clearance of clozapine.
80
CONCLUSIONS
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 Ndesmethylclozapine. 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.
81
ACKNOWLEDGEMENTS
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 Kivist, 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 LapattoReiniluoto, Seppo Khknen, 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.
82
ACKNOWLEDGEMENTS
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 Mrtensson, Eija Mkinen-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 Visnen, 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 Leppniemi 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 Rimn, Matti Virkkunen, Bjrn Appelberg, Acting Professor Heikki Katila, Docents Heikki Vartiainen, Antero Leppvuori, 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
83
ACKNOWLEDGEMENTS
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 sons 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
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Kari Raaska - Pharmacokinetic Interactions of Clozapine in Hospitalized Patients

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Kari Raaska - Pharmacokinetic Interactions of Clozapine in Hospitalized Patients

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Department of Clinical Pharmacology University of Helsinki Finland

PHARMACOKINETIC INTERACTIONS OF CLOZAPINE

Professor Esa Leinonen Medical School University of Tampere Tampere, Finland

To Hanna, Eveliina, Antti, and Iida

AIMS OF THE STUDY ........... 51 MATERIALS AND METHODS .. 53

CONCLUSIONS 81 ACKNOWLEDGEMENTS . 82 REFERENCES 85 ORIGINAL PUBLICATIONS ... 111

PM PXR RXR SD SSRI T TDM Tmax UKU v VCFS Vd

LIST OF ORIGINAL PUBLICATIONS

LIST OF ORIGINAL PUBLICATIONS

REVIEW OF THE LITERATURE

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1.1. Drug elimination

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1.1.3. Cytochrome P450 (CYP)

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1.1.3.1. CYP1A subfamily

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1.1.3.2. CYP2C subfamily

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1.1.3.3. CYP2D subfamily

1.1.3.4. CYP3A subfamily

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Location of functional genes (chromosome)1 15 2 19 10 22 1 11 4 7 7 1 19 4 1 1 7 8 20 3 15 8 10 15 2 6 20 10 2 2 12 6 14 7

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CYP1A2 CYP2C9 CYP2C19 CYP2D6 CYP3A4

Carbamazepine3 Cerivastatin12, 13 Ibuprofen3 Paclitaxel3 Repaglinide14 Rosiglitazone15 Tolbutamide3

Cimetidine7 Ciprofloxacin7, 18 Fluvoxamine2, 19 Grepafloxacin7

Carbamazepine21 Dioxin2 Phenobarbital7, 22 Rifampicin2, 23 Tobacco smoke3, 7, 22

REVIEW OF THE LITERATURE

2. Metabolic drug interactions in psychiatry

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3. Schizophrenia, other psychotic disorders, and their drug treatment

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Table 3. Pharmacokinetic parameters of clozapine (references in text).

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Figure 1. Clozapine metabolism

REVIEW OF THE LITERATURE

Eiermann et al. 1997

Human liver microsomes

Linnet & Olesen 1997

cDNA-expressed human CYPs Human liver microsomes

CYP3A4, 1A2, 2C, 2C19, 2D6 CYP1A2, 3A4 CYP3A4, 1A2

Fang et al. 1998

CYP3A4, 1A2 CYP3A4

Tugnait et al. 1999

Human liver microsomes cDNA-expressed human CYPs

CYP1A2, 3A4 CYP2D6, 1A2, 3A4

CYP3A4, 1A2 CYP3A4, 1A2

Olesen & Linnet 2001

Human liver microsomes

CYP1A2, 2C19 CYP3A4, 1A2, 2C19

REVIEW OF THE LITERATURE

REVIEW OF THE LITERATURE

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4.1.1. Pharmacokinetic interactions 4.1.1.1. SSRIs and clozapine

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4.1.1.2. Other inhibitors of CYP enzymes and clozapine

REVIEW OF THE LITERATURE

Clozapine as an inhibitor. Clozapine is a weak inhibitor (Ki 30-95