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

7

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

C O N T E N T S

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CONTENTSABBREVIATIONS ……………………………………………………..……6

LIST OF ORIGINAL PUBLICATIONS ………………………………… 8

ABSTRACT …………………………………………………………………… 9

INTRODUCTION ……………………………………………………………. 11

REVIEW OF THE LITERATURE ……………………………………….. 13

1. Pharmacokinetics ……………………………………………………………. 13

1.1. Drug elimination ………………………………………………………… 13

1.1.1. Metabolism ………………………………………………………..13

1.1.2. Excretion …………………………………………………………. 14

1.1.3. Cytochrome P450 (CYP) ………………………………………… 15

1.1.3.1. CYP1A subfamily ………………………………………... 16

1.1.3.2. CYP2C subfamily ………………………………………... 17

1.1.3.3. CYP2D subfamily ………………………………………... 18

1.1.3.4. CYP3A subfamily ………………………………………... 18

1.1.4. P-glycoprotein ……………………………………………………. 19

2. Metabolic drug interactions in psychiatry …………………………………….22

3. Schizophrenia, other psychotic disorders, and their drug treatment …………. 23

4. Clozapine …………………………………………………………………….. 25

4.1. Pharmacokinetics ………………………………………………………... 26

4.1.1. Pharmacokinetic interactions …………………………………….. 31

4.1.1.1. SSRIs and clozapine ………………………………………31

4.1.1.2. Other inhibitors of CYP enzymes and clozapine ………… 32

4.1.1.3. Inducers of CYP enzymes and clozapine …………………34

4.2. Pharmacodynamics ……………………………………………………… 35

4.3. Adverse effects ………………………………………………………….. 38

4.4. Therapeutic drug monitoring (TDM) …………………………………….39

5. Drugs and other factors as inhibitors of clozapine metabolism ……………... 40

5.1. Itraconazole ………………………………………………………………40

5.2. Ciprofloxacin ……………………………………………………………. 42

5.3. Risperidone ……………………………………………………………… 44

5.4. Influenza vaccination ……………………………………………………. 46

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5.5. Caffeine and coffee-drinking …………………………………………… 47

AIMS OF THE STUDY ………………………………………………........... 51

MATERIALS AND METHODS ………………………………………….. 53

1. Subjects ………………………………………………………………………. 53

2. Design of the studies …………………………………………………………. 55

3. Blood sampling ………………………………………………………………. 57

4. Determination of serum drug, caffeine, and CRP concentrations …………… 58

4.1. Clozapine and its metabolites …………………………………………… 58

4.2. Itraconazole and hydroxyitraconazole …………………………………... 59

4.3. Ciprofloxacin ……………………………………………………………. 59

4.4. Caffeine and paraxanthine ………………………………………………. 59

4.5. C-reactive protein (CRP) ………………………………………………... 60

5. Statistical analysis ……………………………………………………………. 60

6. Ethical considerations ………………………………………………………... 60

RESULTS ………………………………………..…………………………….. 63

1. Itraconazole (Study I) ………………………………………………………... 63

2. Ciprofloxacin (Study II) ……………………………………………………… 63

3. Influenza vaccination (Study III) …………………………………………….. 64

4. Risperidone (Study IV) ………………………………………………………. 64

5. Coffee-drinking (Study V) …………………………………………………… 65

DISCUSSION ………………………………………………………………… 70

1. Methodological considerations ………………………………………………. 70

2. Effect of itraconazole on serum clozapine concentration ……………………. 71

3. Effect of ciprofloxacin on serum clozapine concentration ……………………72

4. Effect of risperidone on serum clozapine concentration ……………………...73

5. Effect of influenza vaccination on serum clozapine concentration …………...75

6. Effect of coffee-drinking on serum clozapine concentration ………………… 76

7. General discussion …………………………………………………………… 78

CONCLUSIONS ……………………………………………………………… 81

ACKNOWLEDGEMENTS ………………………………………………….82

REFERENCES …………………………………………………………………85

ORIGINAL PUBLICATIONS ……………………………………………... 111

A B B R E V I A T I O N S

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ABBREVIATIONS

AhR aryl hydrocarbon receptor

Arnt AhR nuclear translocator protein

AUC area under drug concentration-time curve

AUC(0- ) area under drug concentration-time curve (time 0 to infinity)

b.i.d. twice daily

BPRS Brief Psychiatric Rating Scale

C/D ratio ratio of serum drug concentration / daily dose of the drug

CL clearance

Cmax peak concentration

CNS central nervous system

CRP c-reactive protein

CYP cytochrome P450

CV coefficient of variation

D dopamine

DSM-IV Diagnostic and Statistical Manual of Mental Disorders, 4th ed.

EM extensive metabolizer

EPS extrapyramidal symptoms

F bioavailability

FMO flavine-containing monooxygenase

HPLC high-performance liquid chromatography

5-HT serotonin (5-hydroxytryptamine)

IM intermediate metabolizer

Ki inhibition constant

Km Michaelis-Menten constant

NADPH reduced form of nicotinamide adenine dinucleotide phosphate

ND not determined or unknown

NS statistically non-significant

PAH polycyclic aromatic hydrocarbon

P-gp P-glycoprotein

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PM poor metabolizer

PXR pregnane X receptor

RXR retinoid X receptor

SD standard deviation

SSRI selective serotonin reuptake inhibitor

T½ elimination half-life

TDM therapeutic drug monitoring

Tmax time to peak concentration

UKU Udvalg for Kliniske Undersøgelser side-effect rating scale

v volume

VCFS velo-cardio-facial syndrome

Vd apparent volume of distribution

L I S T O F O R I G I N A L P U B L I C A T I O N S

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LIST OF ORIGINAL PUBLICATIONS

This thesis is based on the following publications, which will be referred to in the text by

Roman numerals I to V.

I Raaska K, Neuvonen PJ. Serum concentration of clozapine and N-

desmethylclozapine are unaffected by the potent CYP3A4 inhibitor

itraconazole. Eur J Clin Pharmacol 1998;54:167-170

II Raaska K, Neuvonen PJ. Ciprofloxacin increases serum clozapine and N-

desmethylclozapine: a study in patients with schizophrenia. Eur J Clin

Pharmacol 2000;56:585-589

III Raaska K, Raitasuo V, Neuvonen PJ. Effect of influenza vaccination on serum

clozapine and its main metabolite concentrations in patients with

schizophrenia. Eur J Clin Pharmacol 2001;57:705-708

IV Raaska K, Raitasuo V, Neuvonen PJ. Therapeutic drug monitoring data:

Risperidone does not increase serum clozapine concentration. Eur J Clin

Pharmacol 2002;58:587-591

V Raaska K, Raitasuo V, Laitila J, Neuvonen PJ. Effect of caffeine-containing

versus decaffeinated coffee on serum clozapine concentrations in hospitalised

patients. Pharmacol Toxicol. In press.

The original published articles are reprinted with permission of the copyright holder

(Springer-Verlag GmbH & Co. KG).

A B S T R A C T

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ABSTRACT

ackground: Clozapine is an atypical antipsychotic drug that was first introduced into

clinical use in the 1970’s. After a few years it was withdrawn from the market

worldwide because of fatal cases of agranulocytosis cases first reported in Finland. Because

of its superior efficacy over typical antipsychotics in treatment-refractory schizophrenia,

however, clozapine was reintroduced under a strict blood-cell-monitoring requirement.

Today, clozapine is the gold standard in treatment-refractory schizophrenia. It is eliminated

mainly by cytochrome P450 (CYP) enzyme-mediated metabolism in the liver. The CYP

enzymes involved in the metabolism include CYP1A2, CYP2C19, CYP2D6, and CYP3A4.

Despite its relatively long clinical history, understanding of its pharmacokinetic interactions

has until recently been based largely on case-reports and in vitro studies. The case-reports

suggest that erythromycin (CYP3A4 inhibitor), ciprofloxacin (CYP1A2 inhibitor),

risperidone (CYP2D6 substrate) and caffeine (CYP1A2 substrate) are able to increase serum

clozapine concentration.

ethods: Effects of itraconazole (I), ciprofloxacin (II), risperidone (IV), influenza

vaccination (III), and coffee-drinking (V) on serum concentrations of clozapine or

its main metabolites were investigated in five studies (I-V) in hospitalized clozapine-treated

patients with schizophrenia or other psychotic disorders. Three of the Studies (I, II, and V)

were randomized clinical crossover trials, Study III was an open prospective trial, and Study

IV an analysis of retrospective therapeutic drug monitoring (TDM) data.

esults and discussion: Potent CYP3A4 inhibition by itraconazole showed no effect

on serum clozapine or its metabolite N-desmethylclozapine concentration. This was

in contradiction to reported cases of apparent interactions between clozapine and another

CYP3A4-inhibitor, erythromycin. Based on these reports, CYP3A4-inhibition was widely

assumed to increase clozapine concentration. A low dose of the moderate CYP1A2-inhibitor

ciprofloxacin elevated serum clozapine (P < 0.01) and N-desmethylclozapine (P < 0.05)

concentrations by about 30%, confirming assumptions based on case-reports. Contrary to

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current belief based on case-reports, another atypical antipsychotic, risperidone, had no

effect on serum clozapine concentrations in the present retrospective analysis of TDM data.

Influenza vaccination had no effect on serum clozapine, or on its two main metabolites, N-

desmethylclozapine or clozapine-N-oxide. In Study III, a minor infection occurred during

the study period in two patients. In both of them, during the infection, serum clozapine

concentrations increased. Coffee-drinking caused a minor enhancement of effect on serum

clozapine concentrations, suggesting that “normal” amounts of coffee have a low propensity

to cause any clinically significant increase in serum clozapine concentration.

onclusions: Potent inhibition of CYP3A4 by itraconazole does not affect serum

concentrations of clozapine or N-desmethylclozapine to a clinically significant

degree. Coffee-drinking has a low interaction potential with clozapine. Even a low dose of

the CYP1A2 inhibitor ciprofloxacin, however, raises serum clozapine concentrations to a

degree which may have clinical relevance for some of the patients. Clozapine serum

concentrations seem to be left unaltered by risperidone and influenza vaccination. Although

clozapine is metabolized by both CYP1A2 and CYP3A4, it seems that, in vivo, only

CYP1A2 inhibition can significantly elevate serum clozapine concentrations.

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INTRODUCTION

lozapine and many other antipsychotic drugs are extensively metabolized by

cytochrome P450 (CYP) enzymes, whose activity can be suppressed (inhibition) or

enhanced (induction) by some drugs and environmental factors. Inhibition and induction of

CYP enzymes are the most important mechanisms for drug interactions that may raise or

reduce serum concentrations of drugs, and lead to undesirable outcomes. Genes for drug-

metabolizing CYP enzymes exist in many allelic variants, which can cause profound

differences in specific CYP activities between individuals (Coutts & Urichuk 1999). Some

of the drug-metabolizing CYPs are polymorphically expressed, meaning that individuals are

either extensive (EM) or poor (PM) metabolizers in respect to that enzyme. Both EMs and

PMs are common in all populations (Coutts & Urichuk 1999). In the case of antipsychotic

drugs, induction of the CYPs mainly responsible for their metabolism may lead to relapse or

worsening of psychotic symptoms, and inhibition of the same enzymes may lead to the

intensification of adverse effects and of toxicity. The safety of antipsychotics in

combination with other drugs is important because patients treated with these drugs often

need them for years. During that time, other psychotropic drugs may be necessary to control

their psychiatric symptoms, and patients may also need different types of drugs, for

example, to treat infections. Among other factors that could possibly affect clozapine

pharmacokinetics are age, sex, tobacco-smoking, coffee-drinking, and vaccination. For

example, tobacco-smoking induces CYP1A2 and reduces serum clozapine concentrations

(Seppälä et al. 1999).

Several case-reports suggest that clozapine concentrations may be affected by some drugs

and environmental factors. These reports, together with what is known about clozapine

metabolism, imply that the most likely mechanisms in these apparent interactions involve

CYP enzymes. This is suggested by the fact that the potent CYP1A2 inhibitor fluvoxamine

seems to elevate clozapine concentrations by up to 10-fold (Hiemke et al. 1994, Koponen et

al. 1996, Dequardo & Roberts 1996, DuMortier et al. 1996, Bender & Eap 1998, Wetzel et

al. 1998). Serious adverse effects and increase in plasma clozapine concentrations have

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occurred in co-administration with the CYP1A2 inhibitor ciprofloxacin (Markowitz et al.

1997). Another antibacterial drug, the potent CYP3A4 inhibitor erythromycin, is reported to

double clozapine concentrations (Funderburg et al. 1994, Cohen et al. 1996). A similar

degree of inhibition of clozapine metabolism is suggested by an antipsychotic drug–

risperidone–which is a substrate for and a weak inhibitor of CYP2D6 (Koreen et al. 1995,

Tyson et al. 1995). Paroxetine, a strong CYP2D6 inhibitor, apparently also elevates

clozapine concentrations (Centorrino et al. 1996, Joos et al. 1997, Spina et al. 2000). All of

these CYP enzymes are to some extent involved in the metabolism of clozapine, although

CYP2D6 seems to be far less important than CYP1A2 and CYP3A4. In vitro, CYP1A2 is

the most important enzyme in the formation of N-desmethylclozapine (Pirmohamed et al.

1995). The formation of another main metabolite, clozapine-N-oxide, is catalyzed

principally by CYP3A4 (Eierman et al. 1997).

Assumptions as to the pharmacokinetic interactions of clozapine were until recently mostly

based on sporadic case-reports, and on conclusions drawn from clozapine metabolism

studies. Administration of antimicrobial drugs like itraconazole or ciprofloxacin to treat

infections, or of risperidone to augment the antipsychotic efficacy of clozapine could

theoretically put patients at risk for adverse effects due to increasing clozapine

concentrations. The possibility that influenza vaccination may affect CYP1A2-mediated

drug metabolism (Renton et al. 1980, Meredith et al. 1985) raises the question of possible

effects of influenza vaccination on serum clozapine concentrations. Although coffee-

drinking seems to have some effect on serum clozapine concentrations, its mean effect in

patients in clinical setting remains elusive.

The purpose of the present studies was to investigate in hospitalized patients the effects of

itraconazole (CYP3A4 inhibitor), ciprofloxacin (CYP1A2 inhibitor), risperidone, influenza

vaccination, and coffee-drinking on serum concentrations of clozapine and its main

metabolites.

R E V I E W O F T H E L I T E R A T U R E

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REVIEW OF THE LITERATURE

1. Pharmacokinetics

oth pharmacokinetics and pharmacodymamics study drug-body interactions. Whereas

pharmacodynamics deals with the effects of drugs on the body, pharmacokinetics

deals with the effects of the body on drugs. Pharmacokinetic parameters describe how drugs

are absorbed from different sites of administration, how they are distributed, and how they

are eliminated from the body. Bioavailability (F) is the percentage (0-100%) of an

administered drug that reaches the systemic circulation; it is affected by the extent of

absorption and pre-systemic elimination. The apparent volume of distribution (Vd, the unit

is L/kg) of a drug describes how the drug is distributed between plasma and tissue.

Clearance (CL), which describes the elimination of a drug, is the plasma volume from

which the drug is totally removed in one minute.

1.1. Drug elimination

rugs and other foreign compounds (xenobiotics) are eliminated either by metabolism

(yielding metabolites), or by excretion from the body as unchanged parent

compounds. Metabolites are either excreted or further metabolized. Each metabolite has its

own pharmacokinetic characteristics that determine its distribution and its routes and the

rate of elimination.

1.1.1. Metabolism

ost orally administered drugs are lipid-soluble (lipophilic) compounds. This

facilitates their passage through biological membranes. Metabolism transforms

them to more water-soluble (hydrophilic) molecules that can be effectively excreted from

the body. Although metabolites are generally less toxic than their parent compounds,

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sometimes metabolism yields highly toxic compounds. Some drugs are administered as

biologically inactive prodrugs that require metabolism to convert them into active drugs.

Drugs are metabolized most importantly in the liver. In addition, the small intestine has

considerable metabolic activity towards a number of drugs. Other tissues contribute less to

overall drug metabolism, although they may have some local importance in protection from

organ-specific toxicity. Drug metabolism is often divided into phase I and phase II

enzymatic reactions. Together phase I and II reactions make up a form of protective

detoxification system that transforms lipophilic molecules into less toxic hydrophilic

compounds. Phase I (functionalization) reactions consist of oxidation, reduction, and

hydrolysis, which usually lead to metabolites that are more polar than is the parent

compound (Gibson & Skett 2001). Of phase II, glucuronidation, sulphation, acetylation, and

conjugation to glutathione and amino acids are the major conjugation reactions (Gibson &

Skett 2001). All drugs do not require phase I metabolism before they can take part in

conjugation reactions. Furthermore, as an exception to the rule, some conjugates such as

morphine-6-glucuronide possess greater biological activity than the parent drug, and some

are chemically highly reactive (Tephly & Green 2000).

1.1.2. Excretion

enal excretion of drugs and metabolites into the urine is the most important excretion

route. This may be passive, or be an active energy-consuming process. Generally,

compounds have to be hydrophilic in order to be effectively excreted. Along with some

other conjugates, most glucuronide conjugates are excreted in the bile (Roberts et al. 2002).

After they enter, with the bile, the small intestine, they can either leave the body with the

feces, or the conjugate can be cleaved by bacterial -glucuronidase enzymes back to the

parent drug (Roberts et al. 2002). The drug can be reabsorbed from the gut into the systemic

circulation. This absorption-biliary excretion-reabsorption cycle is called the enterohepatic

circulation.

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1.1.3. Cytochrome P450 (CYP)

reduced pigment of the NADPH/O2-dependent oxidation system has an absorption

band with a max at 450 nm after binding to carbon monoxide (Klingenberg 1957,

Garfinkle 1958). This pigment was first characterized as a P450 hemoprotein (Omura &

Sato 1961). Over the next 20 years, it became evident that this microsomal mixed function

monooxygenase system consists of multiple forms of P450 enzymes (CYPs). Currently 18

CYP families and 43 subfamilies have been identified in human being

(www.drnelson.utmem.edu/famcount.html, and www.imm.ki.se/CYPalleles/, Nelson 2003)

(Table 1). CYP enzymes metabolize both endogenic (fatty acids and steroids) and exogenic

(xenobiotics) compounds (Gonzalez 1988, Nebert & Russell 2002) (Table 1). Drugs are

among the exogenic compounds, as are carcinogens such as polycyclic aromatic

hydrocarbons (PAH) and arylamines (Gonzalez 1988, Nebert & Russell 2002). Only the

families CYP1, CYP2, and CYP3 seem to be important in human drug metabolism (Tables

1 and 2).

Individual CYP forms show affinity towards structurally unrelated compounds. Many

substrates can be significantly metabolized by more than one CYP form (Gonzalez 1988).

The principal function of CYP enzymes is the mono-oxygenation of various substrates

(Gonzalez 1988). This requires molecular oxygen and a supply of reducing equivalents from

the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) (Gibson &

Skett 2001).

The nomenclature of CYP enzymes is based on their identity in amino acid sequences

(Nelson et al. 1996). The CYP superfamily is divided into families and subfamilies. The

abbreviation “CYP” for the cytochrome P450 superfamily is followed by the number of that

family, e.g., CYP1. The protein sequences of the enzymes within each family are at least

40% identical (e.g., CYP2C8 and CYP2D6), and within each subfamily they are > 55%

identical (e.g., CYP2C8 and CYP2C9). An individual enzyme is identified by a number

following the letter for a subfamily (e.g., CYP1A2).

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Subfamilies CYP1A, CYP2A, CYP2C, CYP2D, CYP2E, and CYP3A include important

drug-metabolizing enzymes (Nebert & Russell 2003, Tables 1 and 2). It seems that all of

them except CYP2D6 are inducible (Table 2). For one or more isoenzymes belonging to

these subfamilies, several drugs act as inhibitors (Table 2). Inhibition of the metabolizing

enzymes may be either reversible (competitive, non-competitive or uncompetitive) or

irreversible (stable complex formation or suicide inhibition) (Thummel et al. 2000).

1.1.3.1. CYP1A subfamily

YP1A1 is expressed mainly in extrahepatic tissue, and CYP1A2 is liver-specific

(Raunio et al. 1995). CYP1A2 accounts for about 15% of the total hepatic CYP

content (Pelkonen et al. 1998). Although the CYP1A2-mediated caffeine N-3-demethylation

rate in 22 human livers was shown to vary 54-fold (Butler et al. 1989), CYP1A2 is not

polymorphically expressed (Smith et al. 1998, Miners & McKinnon 2000, Table 2). CYP1A

enzymes transform some procarcinogens to carcinogens such as PAH compounds and

aflatoxin B1 (Miners & McKinnon 2000). Dioxin is the prototypic inducer for CYP1A

(Pelkonen et al. 1998, Miners & McKinnon 2000). The large interindividual variation in

CYP1A1 inducibility is probably linked to genetic polymorphism in the aryl hydrocarbon

receptor (AhR) (Miners & McKinnon 2000), and the inducers of CYP1A2 are ligands of

this transcription factor (Schrenk 1998). Studies suggest an increased risk for malignancies

in human beings who belong to the high-affinity AhR phenotype (Miners & McKinnon

2000). It seems that CYP1A induction may be either AhR-mediated (ligand-dependent or

ligand-independent) or AhR-independent (Miners & McKinnon 2000). After AhR-ligand

binding in the cytoplasm, the complex binds to the AhR nuclear translocator protein (Arnt)

in the nucleus to form a dimer that acts as a nuclear transcription factor which enhances the

transcription of the CYP1A2 gene by binding to Ah-responsive element sequences (Schrenk

1998, Miners & McKinnon 2000).

CYP1A2 is an important drug-metabolizing enzyme (Table 2), for which caffeine is often

used as a probe substance (Butler et al. 1989, Miners & McKinnon 2000). The caffeine N-3-

demethylation that yields paraxanthine is a suitable index of CYP1A2 activity in vivo

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(Butler et al. 1989, Fuhr & Rost 1994, Miners & McKinnon 2000). Some of the commonly

used drugs inhibit or induce CYP1A2 activity (Table 2); fluvoxamine, in particular, is a

potent CYP1A2 inhibitor (Brøsen et al. 1993, Rasmussen et al. 1995).

1.1.3.2. CYP2C subfamily

YP2C, the most complex of the drug-metabolizing CYP subfamilies (Rettie et al.

2000), includes CYP2C8, CYP2C9, CYP2C18, and CYP2C19 (Rettie et al. 2000).

Due to pharmacogenetic polymorphism, 2% to 5% of Caucasians, but 13% to 23% of

Asians, are “poor metabolizers” (PM) of CYP2C19, and the rest are “extensive

metabolizers” (EM) (Nakamura et al. 1985, Rettie et al. 2000). In PMs, diazepam

(CYP2C19 substrate) produces prolonged sedation (Goldstein 2001). Of the other members

of this subfamily, at least CYP2C9 and CYP2C18 are polymorphically expressed (Rettie et

al. 2000, Goldstein 2001) (Table 2). The CYP2C subfamily makes up about 20% of total

hepatic CYP content (Pelkonen et al. 1998), the most abundant member is CYP2C9 (Rettie

et al. 2000).

Paclitaxel can serve as a prototypic substrate and trimetoprim as a selective inhibitor of

CYP2C8 (Dai et al. 2001, Wen et al. 2002). Sulfaphenazole is a prototypic competitive

inhibitor of CYP2C9 (Rettie et al. 2000). The clinical importance of the CYP2C18 isoform

seems to be minor, but it is–for example, with its low Michaelis-Menten constant (Km)–

capable of catalyzing diazepam N-demethylation in vitro (Rettie et al. 2000). Hydroxylation

of S-mephenytoin can serve as a prototypic reaction for CYP2C19 (Goldstein et al. 1994).

At high concentrations, S-mephenytoin is suitable as a selective CYP2C19 inhibitor (Rettie

et al. 2000).

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1.1.3.3. CYP2D subfamily

YP2D6 is polymorphically expressed, 7% of Caucasians being PMs, and thus carrying

two non-functional alleles (Coutts & Urichuk 1999). The level of CYP2D6 protein

expression in the liver varies among individuals from undetectable (PMs), low levels

(intermediate metabolizers, IMs), and “normal” (EMs), to levels of expression more than

100-fold higher than in the majority of EMs; on average, CYP2D6 expression accounts for

about 2% to 5% of total CYP content (Zanger & Eichelbaum 2000), but CYP2D6

metabolizes about 30% of all drugs (Anzenbacher & Anzenbacherová 2001). Potent

inhibitors of CYP2D6 include quinidine, ritonavir, and paroxetine (Zanger & Eichelbaum

2000, Shapiro & Shear 2002). Since CYP2D6 seems not to be inducible, gene duplications

may be one way to adapt to environmental chemical pressures (Ingelman-Sundberg et al.

1999).

1.1.3.4. CYP3A subfamily

YP3A takes part in the metabolism of more than half of all drugs (Wrighton &

Thummel 2000). The isoenzymes CYP3A4, CYP3A5, and CYP3A7 together account

for about 30% of total hepatic CYP content, making CYP3A the most prominent of the CYP

enzymes in the liver (Pelkonen et al. 1998, Wrighton & Thummel 2000). By far the most

important of the CYP3A enzymes is CYP3A4, whose expression varies 20-fold in the

human liver, but is not polymorphically expressed (Table 2). Wrighton & Thummel (2000)

discuss the following characteristics: CYP3A4 is the most prominent CYP also in the small

intestine, CYP3A5 is absent from or poorly expressed in about 70% of human livers with a

level of expression usually much lower than CYP3A4 expression, but still it is the major

CYP3A form in about 5% of the livers. CYP3A4, CYP3A5, and CYP3A7 seem to show

similar substrate specificity. CYP3A7 is poorly expressed in the adult human liver, but it

represents about 50% of the total CYP content in the human fetal liver (Wrighton &

Thummel 2000).

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Midazolam 1-hydroxylation and testosterone 6-hydroxylation reactions, among others, can

serve as probe reactions for CYP3A (Wrighton & Thummel 2000, Pelkonen et al. 1998).

Induction of CYP3A4 is mediated by nuclear receptors: pregnane X receptor (PXR),

constitutive androstane receptor (CAR), and glucocorticoid receptor (Gibson & Skett 2001).

In order to activate the CYP3A4 gene, PXR and CAR have first to bind to an inducer, and

thereafter form heterodimers with the retinoid X receptor (RXR) which binds to specific

gene regulation elements (Gibson & Skett 2001).

1.1.4. P-glycoprotein

-glycoprotein (P-gp) is a transmembrane transporter which is an ATP-dependent efflux

pump that transports a wide variety of chemically unrelated substances. A high

concentration of P-gp is located in the epithelial cells lining the luminal surface of

enterocytes in the small intestine and the kidney, in the biliary canalicular membranes of

hepatocytes, and in the luminal surface of the endothelial cells making up the blood-brain

barrier (Shapiro & Shear 2002). Although P-gp and CYP3A4 have overlapping substrate

specificity (Lin & Yamazaki 2003), important exceptions to this “rule” exist: Digoxin,

which is eliminated almost entirely by excretion (Mooradian 1988), is a prototypical P-

glycoprotein substrate susceptible to clinically relevant drug interactions with P-gp

inhibitors (Partanen et al. 1996, Fromm et al. 1999, Silverman 2000). Midazolam, not a P-

gp substrate (Kim et al. 1999), is a prototypical CYP3A4 substrate.

It seems that intact P-gp activity is of special importance in preventing central nervous

system (CNS) -adverse effects of drugs (Lin & Yamazaki 2003). Generally, P-gp inhibition

leads to a much greater increase in drug concentrations in the brain than in plasma (Lin &

Yamazaki 2003). Healthy volunteers tolerated the P-gp substrate loperamide well when it

was ingested with placebo, but when administered with the P-gp inhibitor quinidine, it

caused respiratory depression (Sadeque et al. 2000).

P


20

Table 1. Human CYP families, subfamilies, locations of functional genes, and main

functions of corresponding CYP enzymes.

CYPfamily1

Subfamilies1 Location offunctional

genes(chromosome)1

Main functions 2-6

1A 15 Xenobiotic metabolismCYP11B 2 Estradiol 4-hydroxylation

2A, 2B, 2F, 2G, 2S,2T

19 Xenobiotic metabolism (2A, 2B)

2C, 2E 10 Xenobiotic metabolism2D 22 Xenobiotic metabolism2J 1 Arachidonic acid and xenobiotic metabolism2R 11 Unknown2U 4 Unknown

CYP2

2W 7 UnknownCYP3 3A 7 Xenobiotic metabolism

4A, 4B 1 Fatty acid, arachidonic acid and leukotriene metabolism4F 19 Fatty acid and arachidonic acid metabolism4V 4 Unknown4X 1 Unknown

CYP4

4Z 1 UnknownCYP5 5A 7 Thromboxane A2 synthesisCYP7 7A, 7B 8 Bile acid synthesis

8A 20 Prostacyclin synthesisCYP88B 3 Bile acid synthesis

11A 15 Cholesterol side-chain cleavageCYP1111B 8 Steroid 11 -hydroxylation, aldosterone synthesis

CYP17 17A 10 Steroid 17 -hydroxylationCYP19 19 15 Steroid aromatization (estrogen synthesis)CYP20 20 2 UnknownCYP21 21A 6 Steroid 21-hydroxylationCYP24 24 20 Vitamin D metabolism

26A, 26C 10 Vitamin A metabolismCYP2626B 2 Vitamin A metabolism

27A, 27C 2 Vitamin D synthesis, bile acid synthesisCYP2727B 12 Vitamin D synthesis

CYP39 39A 6 Bile acid synthesisCYP46 46 14 Cholesterol 24-hydroxylationCYP51 51 7 Sterol 14-demethylation

1 Nelson:http://drnelson.utmem.edu/human.genecount.html, 2 Nebert & Russell 2002, 3 Lund et al. 1999,4Yoshida et al. 2000, 5Matsumoto et al. 2002, 6Bylund et al. 2002.

RE

VIE

W O

F T

HE

LIT

ER

AT

UR

E

21

Tab

le 2

. Gen

etic

pol

ymor

phis

m, s

elec

ted

subs

trate

s, in

hibi

tors

, and

indu

cers

of t

he m

ain

drug

-met

abol

izin

g C

YP

enzy

mes

.

GEN

ETIC

PO

LYM

OR

PHIS

M

CY

P1A

2C

YP2

C8

CY

P2C

9C

YP2

C19

CY

P2D

6C

YP3

A4

No1,

2Su

gges

ted3

Yes

3Y

es3

Yes

4N

o5, 6

SUB

STR

AT

ES

CY

P1A

2C

YP2

C8

CY

P2C

9C

YP2

C19

CY

P2D

6C

YP3

A4

Caf

fein

e2, 7

Clo

zapi

ne8,

9

Imip

ram

ine7

Lido

cain

e10

Map

rotil

ine7

Ola

nzap

ine9

Prop

rano

lol7

Rop

ivac

aine

11

R-w

arfa

rin2

Tacr

ine2

Theo

phyl

line2

Car

bam

azep

ine3

Cer

ivas

tatin

12, 1

3

Ibup

rofe

n3

Pacl

itaxe

l3

Rep

aglin

ide14

Ros

iglit

azon

e15

Tolb

utam

ide3

Dic

lofe

nac3

Fluv

asta

tin7

Ibup

rofe

n3

Losa

rtan3

Nap

roxe

n3

Phen

ytoi

n3

Piro

xica

m3

S-w

arfa

rin3

Tolb

utam

ide3

Clo

mip

ram

ine7

Dia

zepa

m3

Imip

ram

ine7

Om

epra

zole

3

Phen

ytoi

n3

Prop

rano

lol7

Prog

uani

l3

Am

itrip

tylin

e4

Clo

mip

ram

ine4

Cod

eine

4

Fluo

xetin

e7

Fluv

oxam

ine4

Hal

oper

idol

4

Met

opro

lol4

Nor

tript

ylin

e4

Paro

xetin

e4

Ris

perid

one4,

9, 1

6

Thio

ridaz

ine4

Am

ioda

rone

7

Am

itrip

tylin

e7

Alp

razo

lam

7

Car

bam

azep

ine7

Cic

losp

orin

7

Eryt

hrom

ycin

7, 1

7

Felo

dipi

ne7

Lova

stat

in7

Mid

azol

am7,

17

Nef

azod

one7

Qui

nidi

ne7

INH

IBIT

OR

S

CY

P1A

2C

YP2

C8

CY

P2C

9C

YP2

C19

CY

P2D

6C

YP3

A4

Cim

etid

ine7

Cip

roflo

xaci

n7, 1

8

Fluv

oxam

ine2,

19

Gre

paflo

xaci

n7

Gem

fibro

zil12

, 13

Trim

etho

prim

20A

mio

daro

ne3

Fluc

onaz

ole3,

7

Mic

onaz

ole3

Sulp

haph

enaz

ole3

Fluo

xetin

e3

Fluv

oxam

ine3

Om

epra

zole

3

Flec

aini

de4

Fluo

xetin

e4

Paro

xetin

e4

Qui

nidi

ne4

Eryt

hrom

ycin

17

Itrac

onaz

ole17

Nef

azod

one17

Rito

navi

r17

IND

UC

ERS

CY

P1A

2C

YP2

C8

CY

P2C

9C

YP2

C19

CY

P2D

6C

YP3

A4

Car

bam

azep

ine21

Dio

xin2

Phen

obar

bita

l7, 2

2

Rifa

mpi

cin2,

23

Toba

cco

smok

e3, 7

, 22

Phen

obar

bita

l24

Rifa

mpi

cin23

, 24

Phen

obar

bita

l7, 2

4

Rifa

mpi

cin7,

23,

24

Phen

obar

bita

l24

Rifa

mpi

cin7,

23,

24

Unk

now

n4C

arba

maz

epin

e17

Dex

amet

haso

ne17

Phen

obar

bita

l17

Phen

ytoi

n17

Rifa

mpi

cin7,

23

1 Smith

et a

l. 19

98, 2 M

iner

s & M

cKin

non

2000

, 3 Ret

tieet

al.

2000

, 4 Zang

er &

Eic

helb

aum

200

0, 5 La

mba

et a

l. 20

02, 6 G

arci

a-M

artin

et a

l. 20

02, 7

Stoc

kley

200

2, 8Ta

ylor

199

7, 9 de

Van

e &

Mar

kow

itz 2

000,

10W

ang

et a

l. 20

00, 11

Arla

nder

et a

l. 19

98, 12

Wan

g et

al.

2002

b, 13

Bac

kman

et a

l. 20

02, 14

Nie

mi e

t al.

2003

b, 15

Nie

mi e

t al.

2003

a, 1

6 Yas

ui-F

uruk

ori e

t al.

2001

, 17W

right

on &

Thu

mm

el 2

000,

18 Fu

hr e

t al.

1992

,19

Brø

sen

et a

l. 19

93, 20

Wen

et a

l. 20

02, 21

Park

eret

al.

1998

, 22 Pe

lkon

enet

al.

1998

, 23N

iem

i et a

l. 20

03c,

24G

erba

l-Cha

loin

et a

l. 20

01.


22

2. Metabolic drug interactions in psychiatry

sychiatric patients are at increased risk for metabolic drug-drug interactions, because

only a minority who are treated for such conditions as depression or schizophrenia are

on monotherapy (Rittmannsberger et al. 1999). In Austrian psychiatric clinics, patients with

schizophrenia were using on average 2.5 to 3.5 psychotropic drugs, and a mean total of 3 to

4 drugs. About half these patients with schizophrenia each received two antipsychotic drugs.

Depending on the clinic, 6% to 35% of the patients with schizophrenia were receiving

antidepressants, 29% to 74% receiving anxiolytics, and 6% to 9% receiving anticonvulsants

(Rittmannsberger et al. 1999).

Many of the newer antidepressants are potent inhibitors, while some of the anticonvulsants

are potent inducers of the drug-metabolizing CYP enzymes. Furthermore, some of the

psychotropic drugs, especially lithium and tricyclic antidepressants, have narrow therapeutic

indices. Tricyclics are, in addition, subject to considerable pharmacogenetic variability.

Nearly half of all patients with schizophrenia have a comorbic medical condition that

requires drug-treatment but is often either misdiagnosed or undiagnosed (Goldman 1999).

Liver and renal diseases may directly slow drug elimination, and like the

pharmacogenetically determined poor metabolism genotype, they put patients at risk for

high drug concentrations. It is also common for patients on antipsychotic medication not to

take their drugs as prescribed. About 40% of patients stop taking antipsychotics within one

year, and about 75% within 2 years (Perkins 1999). In addition, patients may use over-the-

counter drugs or herbal products that interact with the prescribed drugs. Psychiatric

disorders are present in almost all completed suicides, with schizophrenia and depression

demonstrating an especially a high suicide risk (Isometsä 2001, Joukamaa et al. 2001). Drug

overdose with multiple drugs, often at least one psychotropic drug, is a common method of

suicide (Baca-García et al. 2002), in which case consequences of overdose are worse due to

interaction between drugs.

P


23

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

bout 1% of the world population is affected by schizophrenia, a severe psychiatric

disorder (Norquist & Narrow 2000). The diagnostic and statistical manual of mental

disorders, 4th edition (DSM-IV) describes the characteristic symptoms as delusions,

hallucinations, disorganized speech, grossly disorganized (or catatonic) behavior, and

negative symptoms (i.e., affective flattening, alogia, or avolition) (American Psychiatric

Association 1994). The illness begins typically in early adulthood (Schultz & Andreasen

1999). Generally, the symptoms lead to marked social dysfunction, with the majority of the

affected individuals unable to continue to work or study (Schultz & Andreasen 1999,

Cancro & Lehman 2000). Susceptibility to schizophrenia is determined by both genetic and

environmental factors (Norquist & Narrow 2000), with the heritability is estimated at about

80% (Harrison & Owen 2003). Several genes are implicated as susceptibility loci for

schizophrenia, but none, so far, have been adequately confirmed (Harrison & Owen 2003).

The genetic syndrome velo-cardio-facial syndrome (VCFS, or DiGeorge syndrome) is

associated with a high rate of schizophrenia (Murphy 2002). The characteristic

microdeletion on chromosome 22 is present in about 85% of VCFS patients, and in about

1/4000 live births (Murphy 2002). The implicated early-life environmental factors

associated with later occurrence of schizophrenia include exposure to some viruses,

nutritional deficiencies, and obstetric complications (Schultz & Andreasen 1999, Norquist

& Narrow 2000). In addition, to clarify the ethiopathogenesis of schizophrenia, which is still

largely unknown, mechanisms of action of antipsychotic drugs, as well as the occurrence of

neuroanatomical abnormalities, are undergoing study (Egan & Hyde 2000, Sawa & Snyder

2002). At the level of neurotransmitters, involvement of at least dopamine, serotonin, and

glutamate is strongly implicated in schizophrenia (Olney & Farber 1995, Goff & Coyle

2001, Sawa & Snyder 2002, Meltzer 2002).

All antipsychotics are effective against positive symptoms of schizophrenia, reducing

disturbed thoughts and hallucinations (Schultz & Andreasen 1999). Older, typical

antipsychotics, or neuroleptics, although they are effective for positive symptoms, may even

worsen anhedonia, withdrawal, and other negative symptoms (Karow & Naber 2002).

A


24

Common adverse effects of typical antipsychotics include extrapyramidal and

anticholinergic symptoms. The newer atypical antipsychotics, which are also called

serotonin-dopamine antagonists, are as effective in the treatment of positive symptoms as

the typical antipsychotics, but they reduce negative symptoms of schizophrenia more

effectively (Kane 1996, Schultz & Andreasen 1999, Marder 2000). They have considerably

fewer motor adverse effects, but some of them cause weight gain, insulin resistance, and

lipid abnormalities (Henderson 2001, Lindenmeyer et al. 2003). Compared to the effect of

typical antipsychotics, atypical antipsychotics seem to lead to better subjective well-being

and quality of life (Karow & Naber 2002). Antipsychotic drugs can be administered in

maintenance treatment either as daily oral doses or as depot injections. Other medications

often used in combination with antipsychotic drugs include lithium, benzodiazepines,

anticonvulsants, and antidepressants (Kane 1996). Although antipsychotics are critical in the

acute and maintenance treatment of schizophrenia, so are various psychosocial interventions

(Bustillo et al. 2000).

At least 20% of all patients with schizophrenia are resistant to drug treatment (Conley &

Kelly 2001). The most well-accepted criteria define treatment-resistant schizophrenia as at

least moderately severe and persistent illness with no stable periods of good social or

occupational functioning within the previous 5 years, plus persistent positive psychotic

symptoms, all despite at least three adequate treatment trials with typical antipsychotics

from at least two chemical classes (Kane et al. 1988). The only drug with demonstrated

efficacy in treatment resistance is clozapine; other atypical antipsychotics than clozapine

may also be more effective than typical antipsychotics in treatment-resistant patients

(Conley & Kelly 2001).

Patients with schizoaffective disorder suffer from both schizophrenia-like symptoms and

prominent mood symptoms (major depressive, manic, or mixed episodes) (DSM-IV). Most

of them are treated with a combination of antipsychotic, mood stabilizing, or antidepressant

drugs, or a combination of these (Levinson et al. 1999). Antipsychotic drugs are also used to

treat patients with other diagnostically specified or unspecified psychoses.


25

4. Clozapine

lozapine, an orally administered antipsychotic drug, was the first of the antipsychotics

to be called “atypical.” It was synthesized in Switzerland in 1958 and subsequently

identified in 1959 (Meyer & Simpson 1997, Hippius 1999). It was introduced into clinical

practice in Finland in February 1975, but in the same summer, eight Finnish patients who

were taking clozapine died from agranulocytosis (Idänpään-Heikkilä et al. 1975), a

granulocyte count less than 500 cells/mm3. This led the manufacturer to voluntarily

withdraw clozapine from the market. After two comparative trials demonstrated the superior

efficacy of clozapine in treatment-resistant patients (Kane et al. 1988, Claghorn et al. 1987),

however, clozapine was approved by the Food and Drug Administration (FDA) in 1990 for

clinical practice in the United States. Since then, a large number of studies have been

conducted on various aspects of clozapine. Until recently, however, controlled studies on

the pharmacokinetic interactions involving clozapine have been scarce.

Today clozapine has a unique place among antipsychotics due to its superior efficacy in

reducing both positive and negative symptoms of schizophrenia, and due to an adverse-

effect profile often more favorable than those of typical antipsychotics.

Clozapine may, however, cause agranulocytosis. For this reason, its use is restricted to

treatment-resistant schizophrenia, and to patients who are intolerant of typical

antipsychotics. Clozapine’s efficacy is superior to that of chlorpromazine, and at 6 weeks it

is effective in 30% of treatment-resistant patients (Kane et al. 1988). It reduces both positive

and negative symptoms, and improves the impaired cognition (Kuoppasalmi et al. 1993,

Wagstaff & Bryson 1995, McGurk 1999). Emerging reports indicate that clozapine reduces

suicidality (Meltzer & Okayli 1995, Meltzer et al. 2003), violence, and persistent aggression

and substance abuse (Volavka 1999) among patients with schizophrenia. In 2003 clozapine

was approved by the FDA in USA for reducing the risk of suicidal behavior in patients with

schizophrenia or schizoaffective disorder (FDA Consumer 2003).

C


26

4.1. Pharmacokinetics

lozapine has a linear, or first-order, kinetics in therapeutically relevant concentrations

(Perry et al. 1998, Guitton et al. 1998, Haring et al. 1989, 1990). This means that its

rates of absorption and elimination are proportional to drug concentrations (Sjöqvist et al.

1997). The absolute bioavailability of clozapine is widely variable. After oral

administration, 27 to 47% of the clozapine dose reaches the systemic circulation unchanged

(Cheng et al. 1988, Choc et al. 1990). Peak concentration (Cmax) is reached in 1 to 3 hours,

mean elimination half-life (T½) is 10 to 17 hours (range 5-60 hours), mean volume of

distribution (Vd) is 2 to 5 L/kg (range 1-10 L/kg) and mean plasma clearance (CL) is 13 to

57 L/h (range 11-435 L/h) (Byerly & De Vane 1996) (Table 3).

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

Parameter Value

Oral bioavailability (F) 27-47%Time to peak concentration (Tmax) 1-3 hoursElimination half-life (T½) 10-17 hoursVolume of distribution (Vd) 2-5 L/kgPlasma clearance (CL) 13-57 L/hUnbound fraction 5.5%

Clozapine is principally biotransformed by N-demethylation and N-oxidation of the

piperazine ring, yielding N-desmethylclozapine and clozapine N-oxide metabolites (Jann et

al. 1993, Pirmohamed et al. 1995, Fang et al. 1998; Figure 1). In addition, hydroxylated and

chemically reactive metabolites are formed (Jann et al. 1993, Pirmohamed et al. 1995).

Clozapine-N-oxide may be reduced back to clozapine in the presence of NADPH, but the

reaction is inhibited in vitro by ascorbic acid (Pirmohamed et al. 1995). The enzyme

responsible for this conversion remains unknown. The interconversion of clozapine and

clozapine-N-oxide was demonstrated also in vivo in a randomized crossover study in which

formation of clozapine-N-oxide from clozapine was slower than the formation of clozapine

from clozapine-N-oxide (Chang et al. 1998).

C


27

Unbound fractions in serum for clozapine, N-desmethylclozapine, and clozapine-N-oxide

are approximately 5.5%, 9.7%, and 24.6% (Schaber et al. 1998). The mean ratio

clozapine:N-desmethylclozapine:clozapine-N-oxide varies greatly, but in plasma it is about

3:2.5:1, and in urine about 1:43:77 (Wagstaff & Bryson 1995, Schaber et al. 1998). At least

80% of a clozapine dose is eliminated in metabolites in the urine and feces (Baldessarini &

Frankenburg et al. 1991). Enterohepatic recycling of clozapine is suggested by the biphasic

manner of its elimination, with a second increase in concentration taking place within a few

hours after Tmax (Lin et al. 1994, Dahl et al. 1994). If a very high dose of clozapine is

ingested (intentional poisoning), the N-demethylation pathway is saturated (Reith et al.

1998). Clozapine is a poor substrate for P-gp, as suggested by the minimal effects of some

P-gp inhibitors on its plasma concentrations (Lane et al. 2001a, 2001b, Taylor et al. 1999)

and by the in vitro Km value of 58 M for P-gp ATPase activity towards clozapine (Boulton

et al. 2002).

Figure 1. Clozapine metabolism

Several in vitro studies suggest that in clozapine metabolism CYP1A2 is the main enzyme

catalyzing the formation of N-desmethylclozapine, and CYP3A4 is mostly responsible for

the formation of clozapine-N-oxide (Tugnait et al. 1999, Eiermann et al. 1997, Pirmohamed

et al. 1995, Tugnait et al. 1997, Linnet & Olesen 1997, Olesen & Linnet 2001) (Table 4).

One study found CYP3A4 to be the principal enzyme in the formation of both N-

desmethylclozapine and clozapine N-oxide (Fang et al. 1998). In addition, a few other CYP

N

N

N

NCH3

Cl

H

O

N

N

N

NCH3

Cl

H

N

N

N

N

Cl

H

H

Other metabolites

Clozapine-N-oxide Clozapine N-desmethylclozapine

CYP1A2CYP3A4CYP3A3CYP2C19

CYP1A2FMO


28

enzymes, and flavine-containing monooxygenase type 3 (FMO3), have been shown to

metabolize clozapine to N-desmethylclozapine or clozapine-N-oxide (Tugnait et al. 1997)

(Table 4). An earlier study showed that in human liver microsomal preparations and in

recombinant RT2D6 cells, clozapine was metabolized by CYP2D6 to unknown metabolites

other than N-desmethylclozapine or clozapine-N-oxide (Fischer et al. 1992). In the overall

metabolism of clozapine, CYP2D6 seems to be of only minor importance. The formation of

N-desmethylclozapine, clozapine-N-oxide, 7-hydroxyclozapine, and two unidentified

metabolites was similar in those human liver microsomes prepared from the livers of poor

or of extensive metabolizers of CYP2D6 (Pirmohamed et al. 1995).

Table 4. Studies on in vitro oxidation of clozapine to its main metabolites N-desmethylclozapine and clozapine N-oxide.

Enzymes responsible for metabolism of

clozapine to

Source Method Clozapineconcentration

N-desmethylclozapine Clozapine-N-oxide

Pirmohamed et al. 1995 Human livermicrosomes

2 M CYP1A2 CYP3A4, FMO

Eiermann et al. 1997 Human livermicrosomes

50 M CYP1A2, 3A4 CYP3A4

Linnet & Olesen 1997 cDNA-expressedhuman CYPs

0.5-50 M CYP3A4, 1A2, 2C,2C19, 2D6

CYP3A4

Fang et al. 1998 Human livermicrosomes

20 M300 M

CYP1A2, 3A4CYP3A4, 1A2

CYP3A4, 1A2CYP3A4

Human livermicrosomes

1-1000 M CYP1A2, 3A4 CYP3A4, 1A2Tugnait et al. 1999

cDNA-expressedhuman CYPs

350 M CYP2D6, 1A2, 3A4 CYP3A4, 1A2

Olesen & Linnet 2001 Human livermicrosomes

5 M50 M

CYP1A2, 2C19CYP3A4, 1A2, 2C19

Not studied

Clozapine clearance and CYP1A2 activity are similarly distributed in a Swedish population

(Jerling et al. 1997). About 70% of variance in the oral clearance of clozapine is explained

by CYP1A2 activity, determined by the caffeine N3-demethylation index (Bertilsson et al.


29

1994). Very high (ultrarapid) CYP1A2 activity, caused by homozygous alleles with C A

polymorphism in intron 1 of the CYP1A2 gene (CYP1A2*1F), is linked to exceptionally

low serum clozapine concentration and non-response to clozapine (Bender & Eap 1998,

Sachse et al. 1999, Özdemir et al. 2001). A study by Carrillo et al. (1998) showed that the

plasma concentration ratio of N-desmethylclozapine to clozapine is a fairly good estimate of

individual CYP1A2 activity. Using that as an estimate of CYP1A2 activity, Dailly et al.

(2002) found in their population pharmacokinetic analysis that CYP1A2 activity has a major

and a linear effect on clozapine clearance that could explain inter- and intraindividual

variation in clozapine plasma concentrations.

Serum clozapine concentrations were similar in healthy volunteers who each took a single

oral dose of 10 mg clozapine, irrespective of their polymorphic hydroxylation phenotype for

either debrisoquine or S-mephenytoin, indicating minimal involvement of CYP2D6 and of

CYP2C19, respectively (Dahl et al. 1994). Furthermore, serum clozapine concentrations

were similar in patients on clozapine monotherapy, and those treated with both CYP2D6

inhibitors and clozapine (Jerling et al. 1994).

The pharmacokinetics of clozapine is largely variable both inter- and intraindividually

(Byerly & De Vane 1996, Kurz et al. 1998, Schaber et al. 1998, Wagstaff & Bryson 1995,

Cheng et al. 1988, Choc et al. 1990). In a 26-week prospective study, the mean

intraindividual coefficient of variation (CV) for dose- and weight-corrected plasma

clozapine concentration was 54.9% (+ SD 23.9), even though patients (n = 41) did not use

other medications known to interact significantly with clozapine (Kurz et al. 1995). In

another study of shorter duration, the intrapatient variation was 27%, and the interpatient

weight-corrected variation 50.9%, for 44 outpatients in weekly monitoring (Centorrino et al.

1994a). In a study of 14 previously psychotropic drug-naive Chinese patients, interpatient

variability for the AUC of clozapine was over 6-fold (Lin et al. 1994). The range for Tmax

was 1.0 to 5.0 hours, for oral clearance 19 to 123 L/h, for the apparent volume of

distribution 7 to 22 L/kg, and for T½ 5.5 to 35 hours (Lin et al. 1994). The Tmax for N-

desmethylclozapine was between 1.5 and 40 hours, and the metabolite was eliminated in

parallel with clozapine, suggesting formation-rate-limited elimination of the N-


30

desmethylclozapine metabolite (Lin et al. 1994). In that case, the T½ of N-

desmethylclozapine has to be shorter than that of clozapine (Rowland & Tozer 1995).

Men seem to have higher clearance of clozapine than do women, whose dose- and weight-

corrected plasma concentrations of clozapine are 40% higher than in men (Haring et al.

1989, Centorrino et al. 1994a). In children and adolescents (aged 9-16 years, n = 6) on

clozapine monotherapy and a caffeine-free diet, N-desmethylclozapine serum

concentrations were, unlike in adults, higher than those of clozapine, but dose- and weight-

corrected serum clozapine concentrations were similar to those in adults (Frazier et al.

2003). Hepatic diseases may significantly reduce the metabolism of many drugs

metabolized by CYP enzymes (e.g., theophylline) and increase their plasma concentrations

(Gibson & Skett 2001, Kraan et al. 1988). Metastases in the liver were associated in one

report with a 10-fold increase in serum clozapine concentration (Uges et al. 2000). Renal

disease is not expected to reduce significantly the clearance of clozapine, and no

adjustments in clozapine dose are necessary (Bennett 1997). Plasma concentration of

clozapine in one newborn infant was reported to be 2-fold higher than in the maternal

plasma or amniotic fluid. In the same mother’s breast milk, during the first week after

delivery, clozapine concentration was about 3- to 4-fold higher than in her plasma (Barnas

et al. 1994).

Animal studies. In line with their differing lipophilicity, clozapine and its metabolites in

rats were not equally distributed between the brain and peripheral tissues: after repeated oral

ingestion of clozapine, concentrations of N-desmethylclozapine were 1.5- to 2.2-fold higher

in serum than those of clozapine. In these rat brains, however, clozapine concentrations

were 3- to 4-fold higher than those of N-desmethylclozapine (Weigmann et al. 1999).

Recently, fatty-acid derivatives of clozapine have been developed and tested preclinically in

the rat, and they show a more selective distribution to the brain, lower concentrations in

peripheral tissues, higher potency, and a longer duration of action than does clozapine

(Baldessarini et al. 2001).


31

4.1.1. Pharmacokinetic interactions

4.1.1.1. SSRIs and clozapine

ntil recently, knowledge of the pharmacokinetic interactions of clozapine has largely

relied on sporadic case-reports (Taylor 1997). Most impressive among the reports of

clozapine interactions are those reporting highly increased clozapine concentrations during

concomitant treatment with normal doses of the selective serotonin reuptake inhibitor

(SSRI) fluvoxamine. Such increases in serum clozapine concentrations have been high,

sometimes even more than 10-fold (Hiemke et al. 1994, Jerling et al. 1994, Dequardo &

Roberts 1996, DuMortier et al. 1996, Koponen et al. 1996, Wetzel et al. 1998, Szegedi et al.

1999, Fabrazzo et al. 2000, Özdemir et al. 2001) (Table 5). Fluvoxamine raised the serum

elimination T½ of clozapine from 17.3 to 50.6 hours (Wetzel et al. 1998). Furthermore,

adding fluvoxamine to clozapine therapy has produced extrapyramidal symptoms (Kuo et

al. 1998). Fluvoxamine is a potent inhibitor of CYP1A2 (Ki 0.12 to 0.24) and a moderate

inhibitor of CYP2C19 (Brøsen et al. 1993, Jeppesen et al. 1996a). The 2000 study by

Olesen & Linnet suggested that in vivo fluvoxamine significantly inhibits N-demethylation

of clozapine but not the N-oxidation pathway. In a clozapine-treated patient with ultrarapid

CYP1A2 activity, fluvoxamine 50 mg/day reduced his CYP1A2 activity by approximately

50% and raised serum concentration of clozapine about 4-fold (Özdemir et al. 2001).

Fluoxetine and its main metabolite norfluoxetine are both potent inhibitors of CYP2D6

(Brøsen & Skjelbo 1991). Fluoxetine seems to raise serum clozapine concentrations by 30%

to 76% (Centorrino et al. 1994b, Centorrino et al. 1996, Spina et al. 1998), but a case

showing no change in serum clozapine concentration during fluoxetine has also been

reported (Eggert et al. 1994). A fatal pharmacokinetic interaction between clozapine and

fluoxetine was suggested in the case of a patient with a high postmortem plasma clozapine

concentration who died from pulmonary edema, visceral vascular congestion, paralytic

ileus, gastroenteritis, and eosinophilia (Ferslew et al. 1998).

Studies on the effect of paroxetine and sertraline on serum clozapine concentrations are

controversial (Table 5). The potent CYP2D6 inhibitor paroxetine is reported to show a

U


32

moderate enhancement effect on clozapine concentrations of 30% to 57%, and in one case-

report an enhancement of up to 2-fold (Centorrino et al. 1996, Joos et al. 1997, Wetzel et al.

1998, Spina 2000). One study found no effect by paroxetine on serum clozapine

concentrations (Wetzel et al. 1998). One study found 30% higher serum clozapine

concentrations in patients treated with sertraline than in controls (Centorrino et al. 1996). In

another study in which patients served as their own controls, sertraline showed no effect on

clozapine concentrations (Spina et al. 2000).

A weak inhibition of clozapine metabolism by the SSRI citalopram, which is not a strong

inhibitor of any of the major drug-metabolizing enzymes, is suggested in one case-report

(Borba & Henderson 2000), but was not observable in two studies (Taylor et al. 1998).

4.1.1.2. Other inhibitors of CYP enzymes and clozapine

indings on effects of valproate on serum clozapine concentration have been

controversial (Spina & Perucca 2002). While some found a decrease of up to 51% in

serum clozapine concentration, others have found a mean increase of 39% or even no effect

at all (Finley & Warner 1994, Centorrino et al. 1994b, Longo & Salzman 1995, Facciolà et

al. 1999) (Table 5).

The potent CYP3A4 inhibitor erythromycin has been linked to increased serum clozapine

concentrations and adverse effects in two case-reports of patients with acute infections

(Funderburg et al. 1994, Cohen et al. 1996). In one randomized crossover study,

erythromycin did not affect clozapine concentrations (Hägg et al. 1999b) (Table 5).

The antidepressants nefazodone (CYP3A4 inhibitor) and reboxetine seem to have no effect

on serum clozapine concentrations, although elevated clozapine concentrations were

reported during 300 mg, but not during 200 mg of nefazodone (Taylor et al. 1999, Khan &

Preskorn 2001, Spina et al. 2001) (Table 5).

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33

Koreen et al. (1995) reported on a female patient whose serum clozapine concentration

more than doubled, and whose N-desmethylclozapine to clozapine ratio decreased when

risperidone 2 mg daily was added to her clozapine treatment. In another patient, plasma

clozapine concentration increased by 74% after addition of a 2-mg daily dose of risperidone

(Tyson et al. 1995) (Table 5).

Cimetidine, a H2-receptor antagonist that inhibiting several CYP enzymes, has raised

clozapine serum concentrations and its adverse effects (Szymanski et al. 1991). Elevated

clozapine concentrations appeared also with lamotrigine and lisinopril (Abraham et al.

2001, Kossen et al. 2001). In a study by Palego et al. (2002), those clozapine patients co-

treated with typical antipsychotics exhibited no increase in dose- and weight-corrected

plasma concentrations of clozapine and N-desmethylclozapine. In a study with only two

patients on clozapine, the anti-obesity drug orlistat had no effect on plasma concentrations

of clozapine (Hilger et al. 2002). In a retrospective analysis of clozapine patients who used

concomitant omeprazole, and whose omeprazole was later switched to pantoprazole, serum

concentrations during both drugs were similar (Mookhoek & Loonen 2002) (Table 5).

In an open study, abstinence from dietary caffeine resulted in a 47% decrease in patients’

mean concentration of serum clozapine (Carrillo et al. 1998). In healthy volunteers, oral

caffeine raised the mean clozapine AUC (0, ) by 19%, and reduced mean oral clearance of

clozapine by 14% (Hägg et al. 2000).

Grapefruit juice (CYP3A4 inhibitor) seems to have no effect on serum clozapine or its main

metabolite concentrations (Vandel et al. 2000, Özdemir et al. 2001, Lane et al. 2001a,

2001b). Neither grapefruit juice nor ketoconazole 400 mg, potent inhibitors of CYP3A4,

seem to have any significant effect on plasma concentrations of clozapine or of its two main

metabolites (Lane et al. 2001a).

Clozapine as an inhibitor. Clozapine is a weak inhibitor (Ki 30-95 M) of CYP1A2-

mediated theophylline metabolism in vitro (Rasmussen et al. 1995). Fischer et al. (1992)

found in their study in human liver microsomal preparations and in recombinant RT2D6

cells that clozapine inhibits CYP2D6 (Ki 4-19 M). The serum nortripyline (CYP2D6


34

substrate) concentration was reported to increase by 2-fold after the initiation of 150 mg of

clozapine (Smith & Riskin 1994). Clozapine seems to raise serum cocaine concentration in

a dose-dependent manner, but it diminishes subjective cocaine effects (the “high” or “rush”)

(Farren et al. 2000). Cocaine is metabolized by hepatic and plasma esterases rather than by

CYP enzymes (in Farren et al. 2000).

4.1.1.3. Inducers of CYP enzymes and clozapine

everal inducers of CYP enzymes seem to enhance clozapine clearance. In a group

comparison of TDM data, patients who used both clozapine and carbamazepine had

68% lower plasma clozapine concentrations, and 43% lower clozapine concentration/dose

(C/D) ratios than did patients taking only clozapine; the C/D ratio was inversely correlated

with the daily dose of carbamazepine (Jerling et al. 1994). In the same study, all eight

patients suitable for intraindividual comparison had a lower C/D ratio when they were on

than off carbamazepine (Jerling et al. 1993). In two patients, 2 weeks after discontinuation

of carbamazepine, clozapine plasma concentrations increased by 71% and 100% (Raitasuo

et al. 1993). In a report of two cases, after phenytoin was initiated, clozapine concentrations

decreased by 65 to 85% (Miller 1991). After phenobarbital was discontinued, plasma

clozapine concentration was reported to increase by about 75% (Lane et al. 1998). Facciolà

et al. (1998) found that patients treated with clozapine and phenobarbital had lower plasma

concentrations of clozapine and higher N-desmethylclozapine to clozapine ratios than did

patients treated with clozapine alone. In one forensic patient, plasma concentration of

clozapine decreased to one-sixth 2 to 3 weeks after the initiation of rifampicin 600 mg/day,

and when ciprofloxacin was substituted for rifampicin were 60% higher than before

rifampicin (Joos et al. 1998). Another case-report also suggests that the CYP1A2 inhibitor

ciprofloxacin may raise the plasma concentration of clozapine and cause serious adverse

effects (Markowitz et al. 1997) (Table 5).

Several reports suggest that clozapine metabolism is induced by cigarette-smoking (Seppälä

et al. 1999, Hasegawa et al. 1993, Meyer 2001) (Table 5). Aspiration pneumonia occurred

in one patient who had a high clozapine concentration after smoking cessation (Meyer

S


35

2001). One clozapine patient was admitted to hospital unconscious 2 weeks after cessation

of heavy smoking: He salivated excessively and before recovery was pulseless with a

systolic blood pressure of about 40 mmHg. The authors suggested that these symptoms were

due to clozapine intoxication caused by normalization of induced clozapine metabolism

after smoking cessation (Skogh et al. 1999). In another patient, plasma clozapine

concentration increased after cessation of both tobacco- and cannabis-smoking (Zullino et

al. 2002).

4.2. Pharmacodynamics

lozapine is an antagonist, with Ki < 10 nM, for serotonin 5-HT2A, 5-HT2C, 5-HT6, and

5-HT, for dopamine D4, muscarine M1, and adrenergic 1 receptors, but unlike the

typical antipsychotics, clozapine is only a weak antagonist (Ki 172 nM) for D2 receptors

(Meltzer 1994). One of the most popular hypotheses on the mechanism of action of

clozapine is the dopamine-serotonin hypothesis, which suggests that the combination of the

weaker D2 with the more potent 5-HT2A is vital for its superior efficacy (Meltzer 1989,

Meltzer 2002). Genetic polymorphisms in dopamine and serotonin receptors are suggested

as factors influencing patients’ responsiveness to clozapine (Mancama et al. 2002).

One of the two main metabolites of clozapine, N-desmethylclozapine, is biologically active

(Kuoppamäki et al. 1993, Young et al. 1998). Its contribution to overall antipsychotic

effects of the drug seems, however, to be minor. The other main metabolite, clozapine-N-

oxide, is not biologically active (Kuoppamäki et al. 1993).

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36

Table 5. Pharmacokinetic interactions of clozapine with drugs (a) and other factors (b)

a.

Interacting drug

Daily dose

Subjects (nb),Study design

Effect on serum CLZconcentration

Source

SSRIs

Citalopram 40 mg Patients (8), sequential No change Avenoso et al. 199820 mg Patients (5), sequential No change Taylor et al. 1998

Fluoxetine Mean 36 mg Patients (6, 17 controls),parallel

Increase, 76% Centorrino et al. 1994b

80 mg Patient, case-report No change Eggert et al. 1994Mean 39 mg Patients (14, 40 controls),

parallelIncrease, 30% Centorrino et al. 1996

20 mg Patients (10), sequential Increase, 58% Spina et al. 1998Fluvoxamine 100 mg Patient, case-report Increase, 8-fold Hiemke et al. 1994

100 to 150 mg Patients (2), case-report Increase, 5- to 10 -fold Jerling et al. 199425 mg Patient, case-report Increase, 4- to 9 -fold Dequardo & Roberts 1996100 to 200 mg Patients (3), case-report Increase, 3- to 9 -fold DuMortier et al. 1996150 mg Patients (2), case-report Increase, 5- to 10 -fold Koponen et al. 199650 mg Patients (16), sequential Increase, 3-fold Wetzel et al. 199850 mg Patients (16), sequential Increase, 2- to 5-fold Szegedi et al. 1999100 mg Patients (16), sequential Increase, 5-fold Fabrazzo et al. 200050 mg Patient, case study Increase, 3-fold Özdemir et al. 2001

Paroxetine Mean 31 mg Patients (16, 40 controls),parallel

Increase, 57% Centorrino et al. 1996

20 mg Patient, case-report Increase, 30% to 2-fold Joos et al. 199720 mg Patients (14), sequential No change Wetzel et al. 199820 to 40 mg Patients (9), sequential Increase, 31% Spina et al. 2000

Sertraline Mean 92 mg Patients (10, 40 controls),parallel group

Increase, 30% Centorrino et al. 1996

300 mg Patient, case-report Increase, 2-fold Pinninti & de Leon 199750 to 100 mg Patients (8), sequential No change Spina et al. 2000

OtherinhibitorsCimetidine 800 mg Patient, case-report Increase, 60% Szymanski et al. 1991Ciprofloxacin 1000 mg Patient, case-report Increase, 2-fold Markowitz et al. 1997

1000 mg Patient, case-report Increase, 60% Joos et al. 1998Erythromycin 1000 mg Patient, case-report Increase, 2-fold Funderburg et al. 1994

1000 mg Patient, case-report Increase, 3-fold Cohen et al. 19961500 mg Healthy (12), randomized

crossoverNo change Hägg et al. 1999b

Ketoconazole 400 mg Patients (5), sequential No change Lane et al. 2001aLamotrigine 100 mg Patient, case-report Increase, 3-fold Kossen et al. 2001Lisinopril 5 to 10 mg Patient, case-report Increase, 2- to 3 -fold Abraham et al. 2001Nefazodone 400 mg Patients (6), sequential No change Taylor et al. 1999

200 mg Patient, case-report No change Khan & Preskorn 2001300 mg Patient, case-report Increase, 75% Khan & Preskorn 2001

Orlistat 360 mg Patients (2), sequential No change Hilger et al. 2002Ranitidine 300 mg Patient, case-report No change Szymanski et al. 1991Reboxetine 8 mg Patients (7), sequential No change Spina et al. 2001Risperidone 2 mg Patient, case-report Increase, 2-fold Koreen et al. 1995

2 mg Patient, case-report Increase, 74% Tyson et al. 1995


37

Interacting drug

Daily dose



Source

Valproate 750 to 1000 mg Patients (4), sequential Decrease, 23 to 51% Finley & Warner 1994Mean 1060 mg Patients (11, 17 controls),

parallelIncrease, 39% Centorrino et al. 1994b

Mean serumconcentration62 mg/L

Patients (7), sequential Decrease, 15% Longo & Salzman 1995

900 to 1200 mg Patients (6), sequential No change Facciolà et al. 1999600 to 1500 mg Patients (15, 22 controls),

parallelNo change Facciolà et al. 1999

Inducers

Carbamazepine 600 to 800 mg Patients (2), case-report Decrease, 42 to 50% Raitasuo et al. 1993Not stated Patients (17, 124 controls),

retrospective, parallelDecrease, 68% Jerling et al. 1994

500 to 1500 mg Patients (12), sequential Decrease, 47% Tiihonen et al. 1995Phenobarbital 60 mg Patient, case-report Decrease, 43% Lane et al. 1998

Concentration12 to 31 g/mL

Patients (7, 15 controls),parallel

Decrease, 35% Facciolà et al. 1998

Phenytoin 300 mg Patients (2), case-report Decrease, 65 to 85% Miller 1991Rifampicin 600 mg Patient, case-report Decrease, 83% Joos et al. 1998

b.Interacting factor

Daily dose



Source

Caffeine + coffee Patient, case-report Increase, 2-fold Odom-White & de Leon 1996Coffee 150 to 1100 mg

of caffeinePatients (7), sequential Increase, 2-fold Carrillo et al. 1998

Caffeine 400 to 1000 mg Healthy (12), open randomizedcrossover

AUC increased 19% Hägg et al. 2000

Cigarette-smoking Patients (38, 18 controls ),parallel

Decrease, 20% Hasegawa et al. 1993

Patients (34, 10 controls),parallel

Decrease, 38% Seppälä et al. 1999

Patients (11), sequential Decrease, 41% Mayer 2001Grapefruit juice 500 mL Patients (9), sequential No change Vandel et al. 2000

250 mL Patients (21), sequential No change Lane et al. 2001a500 mL Patients (15), sequential No change Lane et al. 2001b1125 mL Patient, case study No change Özdemir et al. 2001

Infection or inflammation Patients (5), case-report Increase, 2- to 10-fold van der Molen-Eijgenraamet al. 2001

Patient, case-report Increase, 3-fold Raaska et al. 2002b

Patient, case-report Increase, 2-fold de Leon & Diaz 2003

Patients (4), case-report Increase, 2-fold Haack et al. 2003


38

4.3. Adverse effects

lozapine has a low propensity to cause extrapyramidal symptoms, and it does not raise

serum prolactin or corticotrophin levels (Wagstaff & Bryson 1995). On the other

hand, common adverse effects of clozapine are sedation, hypersalivation, bodyweight gain,

constipation, tachycardia, and seizures, and most of these are dose-dependent (Wagstaff &

Bryson 1995). In one study, patients found clozapine-related adverse effect more acceptable

than neurologic symptoms they had encountered during treatments with typical

antipsychotics (Centorrino et al. 1994a).

About 1% of clozapine-treated patients develop agranulocytosis, a granulocyte count less

than 500 cells/mm3 (Lieberman & Alvir 1992), which is the reason for mandatory weekly,

and after the first 18 weeks, monthly white blood-cell count measurements as long as

patients are using the drug (Baldessarini & Frankenburg et al. 1991). As a rare but severe

acute reaction to clozapine, some patients develop myocarditis with eosinophilic infiltrates

and myocytolysis (Killian et al. 1999). A mild elevation of serum transaminases occurs in

up to 50% of patients, but toxic hepatitis is not commonly encountered (Kellner et al. 1993).

As a rare allergic manifestation, clozapine may cause acute interstitial nephritis (Elias et al.

1999). EEG abnormalities are more common with clozapine than with other antipsychotics

(Pisani et al. 2002, Centorrino et al. 2002). About 30% of clozapine recipients gain weight

(Meltzer et al. 2003). Clozapine is linked to increased risk for type 2 diabetes, and for lipid

abnormalities (Henderson 2001). Some patients suffer from nocturnal enuresis that may be

related to urinary retention caused by the anticholinergic effects of clozapine (Centorrino et

al. 1994a, Cohen et al. 1994).

Clozapine overdoses seem to be relatively rare (Reith et al. 1998, Wagstaff & Bryson 1995).

Coma, lethargy, tachycardia, agitation, and confusion are common symptoms in

intoxications (Wagstaff & Bryson 1995), with seizures and metabolic acidosis also reported

(Hägg et al. 1999a). Some patients with a lethal clozapine overdose suffer cardiac failure,

aspiration pneumonia or renal failure (Wagstaff & Bryson 1995). In one case of clozapine

intoxication, the ingestion of 12.5 g of clozapine resulted in a measured concentration of

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39

9100 g/L with a calculated peak value between 35 000 g/L and 24 000 g/L for an

assumed absorption T½ of 0.5 and 4 hours, respectively (Sartorius et al. 2002). For both

absorption half-lives, the elimination T½ was calculated to be about 24 h (Sartorius et al.

2002). Surprisingly, sometimes even with a lethal concentration of serum clozapine, typical

adverse effects of clozapine toxicity are absent or go unnoticed (Uges et al. 2000).

Clozapine should not be given to any healthy subject. In one bioequivalence study, when 17

healthy volunteers each received a single 25-mg dose of clozapine, several had serious

adverse effects uncommon in patients at similar doses. Orthostatic hypotension, 60/29

mmHg at the lowest, occurred in 10 subjects, bradycardia below a pulse rate of 40 beats per

minute in eight subjects, and in two subjects, cardiac arrest lasting 10 and 60 seconds

(Pokorny et al. 1994).

4.4. Therapeutic drug monitoring (TDM)

onitoring of serum clozapine concentrations is recommended in optimizing of

therapeutic dosage (Freeman 1997, Olesen 1998, Buur-Rasmussen & Brøsen 1999).

Based on several studies (Perry et al. 1991, Miller et al. 1994, Potkin et al. 1994, Kronig et

al. 1995, Hasegawa et al. 1993), a therapeutic threshold value of about 400 g/L has been

suggested (Freeman 1997). However, in one randomized study, serum clozapine

concentrations in the range of 350 to 450 g/L offered no advantage over concentrations in

the range of 200 to 300 g/L (VanderZwaag et al. 1996). In one prospective study with

repeated TDM of serum clozapine and its two main metabolite concentrations, dose-

corrected clozapine concentrations were significantly lower in responders (n = 21) than in

non-responders (n = 13), although the concentration data collected could not differentiate

responders from non-responders (Dettling et al. 2000). Interestingly, clozapine seems to be

effective in L-DOPA-induced psychosis in Parkinson’s disease at much lower

concentrations (< 20 g/L ) than in schizophrenia (Meltzer et al. 1995).

During clozapine treatment, TDM is also useful in avoiding unnecessarily high clozapine

concentrations. Serum clozapine concentrations above 1000 g/L (3060 nmol/L) increase

M


40

risk for confusion, delirium, and generalized seizures (Freeman 1997, Buur-Rasmussen &

Brøsen 1999). To aid in dose optimization, a dosing nomogram has been developed for

prediction of steady-state clozapine plasma concentration (Perry et al. 1998). This

nomogram is based on the effect of sex, smoking status, and dose on steady-state plasma

clozapine concentration (Perry et al. 1998).

5. Drugs and other factors as inhibitors of clozapine metabolism

5.1. Itraconazole

Figure 2. Chemical structure of itraconazole

n plasma, itraconazole is mainly bound to albumin, leaving only 0.2% of the drug

unbound (De Beule & Van Gestel 2001). It has an apparent volume of distribution of 11

L/kg (De Beule & Van Gestel 2001). Itraconazole accumulates in the kidney, liver, bone,

stomach, spleen, the female genital tract, and in muscle, and keratinous tissues (De Beule &

Van Gestel 2001). It is eliminated mostly by metabolism (De Beule & Van Gestel 2001),

with CYP3A4 the principal enzyme metabolizing it (Backman et al. 2000). Itraconazole

metabolism is induced by rifampicin, carbamazepine, and phenytoin (Backman et al. 2000).

Itraconazole has saturable elimination with a terminal T½ of 20 to 24 hours after the usual

I

N N NNNCH2O

OCHCH2CH3

CH3

H

O ON

N N

Cl

Cl


41

single dose, but in the steady state T½ raises to about 30 hours or even more (De Beule &

Van Gestel 2001). Itraconazole is a highly lipophilic and poorly soluble weak base (Gupta et

al. 1994). In order to be absorbed from the gastrointestinal tract, it has to be transformed by

acid in the stomach into a soluble hydrochloride salt. Its absorption is reduced with reduced

gastric acidity (Gupta et al. 1994). Oral bioavailability improves if the drug is taken with

food.

It is a triazole antifungal agent with a broad spectrum of activity against fungal pathogens,

including dermatophytes, Candida spp., and Aspergillus spp. (Grant & Clissold 1989, Meis

& Verweij 2001). It is primarily fungistatic (Gupta et al. 1994). Its mechanism of action is

inhibition of the fungal cytochrome P450 (CYP) enzyme 14-demethylase that is responsible

for 14 -demethylation of lanosterol in the ergosterol synthesis pathway (Meis & Verweij

2001, De Beule & Van Gestel 2001). Ergosterol is a vital component of the fungal cell

membrane (De Beule & Van Gestel 2001).

Oral itraconazole 200 to 400 mg/day for one week is used in the treatment of extensive

superficial fungal infections, or if a topical treatment has failed and is also used in treatment

of fungal infections of the nails (onychomycosis), because it accumulates in the nails and

other keratinous tissues. In the nails, therapeutic concentrations of itraconazole persist for

up to 9 months after the end of therapy (Meis & Verweij 2001). Itraconazole is also

recommended as first-choice treatment for many systemic fungal infections and duration of

itraconazole treatment may be very long: For example, in treatment of meningeal

coccidioidomycosis in immunocompromised patients, the dose is itraconazole 400 mg/day

for the rest of one’s life (Meis & Verweij 2001).

Itraconazole is a potent inhibitor of CYP3A4, with Ki values < 1 M for many CYP3A4-

catalyzed reactions (Backman et al. 2000), and strongly inhibits the metabolism of many

orally administered CYP3A4 substrates such as midazolam (Olkkola et al. 1994), triazolam

(Varhe et al. 1994), and buspirone (Kivistö et al. 1997). For example, itraconazole 200 mg

daily will raise the AUC of oral midazolam by 10- to 15-fold, and its T½ by 3- to 4-fold

(Olkkola et al. 1994). Among other CYP3A4 substrates susceptible to a considerable

increase in oral bioavailability when used in combination with itraconazole are: lovastatin


42

(> 20-fold AUC) and lovastatin acid (AUC 20-fold) (Neuvonen & Jalava 1996), felodipine

(6-fold) (Jalava et al. 1997a), simvastatin and simvastatin acid (> 10-fold) (Neuvonen et al.

1998). Oral itraconazole can also raise the AUC of the inhaled CYP3A4 substrate

budesonide by about 4-fold (Raaska et al. 2002a).

Itraconazole is also a potent inhibitor of P-glycoprotein (Backman et al. 2000, Wang et al.

2002a), and it raises serum concentration of digoxin and reduces its renal clearance

(Partanen et al. 1996, Jalava et al. 1997b).

5.2. Ciprofloxacin

Figure 3. Chemical structure of ciprofloxacin

he oral bioavailability of ciprofloxacin ranges from 55 to 85%. Tmax values range

from 47 to 90 min. The volume of distribution is between 1.7 and 2.5 L/kg. Although

tissue penetration of ciprofloxacin in most tissues is good, concentration in cerebrospinal

fluid (CSF) is less than 50% of serum concentration. Only about 20 to 40% of plasma

concentration is bound to plasma proteins. Ciprofloxacin is excreted mainly in its

unchanged form via the kidneys, in bile, and through the intestinal mucosa. T½ is between 3

and 5 hours (Aminimanizani et al. 2001).

In the gastrointestinal tract, ciprofloxacin forms insoluble cation chelates with magnesium-,

aluminium- and calcium-containing antacids and with sucralfate, which may reduce its

bioavailability to 15%; the same mechanism applies to oral iron and zinc preparations

(Aminimanizani et al. 2001). Ferrous sulphate reduced the AUC and peak plasma

T

N

OF

NNH

COOH


43

concentration of ciprofloxacin in one controlled study by more than 50% (Lehto et al.

1994). Concomitant ingestion of milk or yogurt has reduced the bioavailability of

ciprofloxacin by a respective 30% and 36% (Neuvonen et al. 1991).

Ciprofloxacin was introduced in the 1980s as the first fluoroquinolone antibacterial agent

(Aminimanizani et al. 2001). It has bactericidal activity against both Gram-negative and

Gram-positive bacteria, and its mechanism of action is in its interactions with DNA gyrase

and topoisomerase IV enzymes that lead to cleavage of bacterial DNA (Hooper 1999).

The most common adverse effects are diverse gastrointestinal symptoms, especially nausea,

with an incidence of 10%, and diarrhea (Bertino 2000). Skin reactions are common, as well

(Bertino 2000). CNS symptoms such as dizziness, headache, tremor, and restlessness occur

in about 3% of ciprofloxacin-treated patients (Bertino 2000). In spontaneously reported

adverse reactions, fluoroquinolones are linked, at a much higher percentage than are other

systemic antimicrobials, to reactions involving the musculoskeletal system (15% vs. 0.3%)

and CNS (12% vs. 4%), and involving psychiatric symptoms (9% vs. 2%) (Leone et al.

2003). Fluorokinolones as a group are associated with Achilles tendon disorders, which

seem to affect especially those who are over 60 years of age and use corticosteroids (van der

Linden et al. 2002).

Ciprofloxacin is a CYP1A2 inhibitor, although metabolism is not a major route in its

elimination (Fuhr et al. 1992). It inhibits the metabolism of CYP1A2 substrates such as

theophylline, caffeine, and ropivacaine (Batty et al. 1995, Nicolau et al. 1995, Jokinen et al.

2003).


44

5.3. Risperidone

Figure 4. Chemical structure of risperidone

isperidone is almost completely absorbed in the gastrointestinal tract. After oral

administration, time to Cmax is about 0.8 to 1 hour (Byerly & De Vane 1996). Both

in oral and intravenous administration, the T½ of risperidone is 3 to 24 hours, depending on

CYP2D6 activity (Heykants et al. 1994, Byerly & De Vane 1996). The Vd of risperidone is

1.1 L/kg (Byerly & De Vane 1996). Risperidone undergoes first-pass metabolism resulting

in the formation of the active metabolite 9-OH-risperidone, another main metabolite, 7-OH-

risperidone, and a number of less important metabolites (Byerly & De Vane 1996, Heykants

et al. 1994, DeVane & Markowitz 2000). The sum of risperidone and 9-OH-risperidone is

referred to as the antipsychotic fraction, or the active moiety (Heykants et al. 1994). The T½

and AUC (0- ) for the active moiety between intravenous, intramuscular, and oral routes of

administration do not vary (Huang et al. 1993). About 10% of risperidone is excreted

unchanged in the urine (Heykants et al. 1994).

The metabolism of risperidone is stereoselective, with CYP2D6 being mainly responsible

for (+)-9-hydroxylation, and CYP3A4 for (-)-9-hydroxylation (Yasui-Furukori et al. 2001).

The importance of these two enzymes for risperidone metabolism is supported by the

concentration of plasma risperidone and 9-OH-risperidone in patients genotyped for

CYP2D6, and the effect of drugs on the activity of these enzymes (Bork et al. 1999,

DeVane & Nemeroff 2001). In both extensive and poor metabolizers of CYP2D6, the T½ of

the active moiety is 20 hours (Heykants et al. 1994). In ultrarapid metabolizers, however,

risperidone concentration is very low, and the 9-OH-risperidone concentration may also be

R

N

N CH3

OCH2

CH2

N ON

F


45

slightly lower than expected, which in some patients may explain non-response to

risperidone (Güzey et al. 2000). The 9-OH-risperidone is eliminated mainly by renal

excretion (Huang et al. 1993). In the steady state, the concentration of 9-OH-risperidone

exceeds the concentration of the parent risperidone (Huang et al. 1993), which makes it

pharmacologically more important than risperidone in extensive metabolizers of CYP2D6.

That P-gp ATPase activity towards risperidone has an in vitro Km value of 12.4 M

(Boulton et al. 2002) suggests that P-gp may contribute to the pharmacokinetics of

risperidone.

Risperidone is the first of the new atypical antipsychotics specifically developed to have a

clozapine-like strong affinity for postsynaptic 5-HT2 and a weaker affinity for D2 receptors.

Risperidone and 9-OH-risperidone have high affinities for 5-HT2A receptors (cloned human

5-HT2A, Ki 0.4) and lower affinities for D2 receptors (Ki 3-7) (Leysen et al. 1994). In the rat

brain, the active fraction has an about 4-fold higher half-life in the frontal cortex than in

plasma, supposedly due to its binding to 5-HT2 and D2 receptors (Heykants et al. 1994).

In schizophrenia, risperidone shows efficacy for both positive and negative symptoms

(Marder et al. 1997, Marder & Meibach 1994), and is better than haloperidol in the

prevention of relapses (Csernansky et al. 2002). In schizophrenia, the optimal benefit is

usually obtained at doses of 4 to 8 mg per day (Citrome & Volavka 2002).

Risperidone is generally well tolerated. It causes fewer extrapyramidal symptoms (EPS)

than does haloperidol, but more than clozapine does (Owens 1994, Tuunainen et al. 2002,

Csernansky et al. 2002). Other adverse effects with an incidence of 5% or more include

somnolence, agitation, and hyperkinesias (Csernansky et al. 2002). Risperidone is

associated with less weight gain than are clozapine or olanzapine, but more than are

haloperidol, quetiapine, or ziprasidone (Nasrallah 2003, Csernansky et al. 2002).

Risperidone elevates plasma prolactin concentration more than do haloperidol, clozapine or

olanzapine (Kleinberg et al. 1999, Turrone et al. 2002). Its prolactin response correlates

with plasma concentration of the active moiety (Huang et al. 1993).


46

Risperidone does not seem to increase plasma concentrations of amitriptyline, lithium,

valproate or digoxin; citalopram, mirtazapine or reboxetine do not affect its plasma

concentration (DeVane & Nemeroff 2001, Spina et al. 2001). The overall interaction

potential is relatively low with risperidone, but some possibly significant interactions have

been described (DeVane & Nemeroff 2001). Fluoxetine and paroxetine, inhibitors of

CYP2D6, have a significant effect in increasing plasma concentration of risperidone in

extensive metabolizers (DeVane & Nemeroff 2001, Bork et al. 1999). Carbamazepine, an

inducer of CYP3A4 and of some other CYP enzymes, may decrease plasma concentration

of 9-OH-risperidone to one-half (de Leon & Bork 1997). Based on two case-reports,

risperidone may significantly raise plasma concentrations of clozapine (Koreen et al. 1995,

Tyson et al. 1995), but the mechanism for this apparent interaction remains unknown.

Risperidone is a weak inhibitor of CYP2D6 but does not significantly inhibit CYP1A2 or

CYP3A4 (Eap et al. 2001). A mild form of neuroleptic malignant syndrome has occurred in

a man with first-episode schizophrenia, after clozapine was combined with risperidone

(Kontaxakis et al. 2002).

5.4. Influenza vaccination

nfluenza vaccines are given to millions of people worldwide each year. As a preventive

measure, their risk-benefit ratio should be extremely favorable, and they should offer

minimal risk, and be of clear benefit to individuals and to the population in general. For

instance, they should not have clinically important effects on the pharmacokinetics of drugs.

Vaccination is a targeted immunostimulation that leads to immunization, but the

immunological response to vaccination is complex. The immunological response to a

number of factors such as infection and influenza vaccination can modulate the activity of

CYP enzymes, and the general assumption is that this effect is mediated by cytokines

(Morgan 2001). Both in vitro and in vivo studies show that administration of

proinflammatory cytokines (e.g., interleukins 1 and 6, interferon , transforming growth

factor- 1) causes a downregulation of CYP mRNA and a decrease in the corresponding

CYP protein (Haas 2001, Morgan 2001). These findings suggest that regulation of gene

I


47

transcription is a major mechanism of decreased CYP activity following administration of

cytokines (Haas 2001, Morgan 2001). Post-transcriptional mechanisms, for example, one

that requires the presence of the nuclear receptor PPAR , are also involved (Morgan 2001).

Plasma concentrations of the CYP1A2 substrate theophylline may increase due to an

influenza infection (Kraemer et al. 1982). Similarly, during an acute infection, serum

clozapine concentration may increase by several-fold (van der Molen-Eijgenraam et al.

2001, Raaska et al. 2002b, de Leon & Diaz 2003, Haack et al. 2003). In one case, a patient

who died of septicemia resulting from pneumonia with high fever (39.9 C) had at the same

time a very high serum clozapine concentration (4034 g/L); the same patient was found in

autopsy also to have a cancer with metastases to the liver (Uges et al. 2000). Several case-

reports have suggested that influenza vaccination inhibits theophylline metabolism (Renton

et al. 1980, Meredith et al. 1985, Tjia et al. 1996), but in some studies influenza vaccination

showed no effect on theophylline pharmacokinetics (Jonkman et al. 1988, Stults &

Hashisaki 1983).

5.5. Caffeine and coffee-drinking

Figure 5. Chemical structure of caffeine

affeine is completely absorbed after oral administration (Carrillo & Benítez 2000).

Time to Cmax is about 1 hour, volume of distribution is about 0.7 L/kg (Carrillo &

Benítez 2000). It is mainly eliminated through the metabolism by CYP1A2 to paraxanthine

(Butler et al. 1989, Carrillo & Benítez 1994, Jeppesen et al. 1996a). Other demethylated

metabolites, theobromine and theophylline, are produced via CYP1A2 and CYP2E1

C

N

NN

N

O

O

CH3

CH3

CH3


48

pathways. These main metabolites undergo further demethylation and oxidation (Carrillo &

Benítez 1994, Lelo et al. 1986a).

Caffeine has a T½ of about 4 hours and paraxanthine about 3 hours (Lelo et al. 1986b,

Kaplan et al. 1997). After a single 500-mg oral dose of caffeine, the T½ of caffeine was

longer (4.74 + 0.6 h vs. 3.94 + 0.5 h), and its clearance was reduced (1.64 + 0.3 mL/min/kg

vs. 2.07 + 0.4 mL/min/kg) compared to a 250-mg oral caffeine dose (Kaplan et al. 1997).

Caffeine is a central nervous system stimulant which increases alertness and improves

performance on some tasks if consumption is not excessive (Smith 2002). These desirable

effects can be explained by its inhibition of A1- and A2a-type adenosine receptors in the

brain. A1 inhibits the release of excitatory neurotransmitters, and A2a enhances the activity

of dopamine D2 receptors (Fredholm 1995). Excess consumption of caffeine, especially by

patients with anxiety disorders, may cause caffeine intoxication with such signs as

restlessness, psychomotor agitation, insomnia, increased diuresis, tachycardia, and cardiac

arrythmia (DSM-IV, Smith 2002).

The main source of caffeine in adults is coffee (Mandel 2002), and consumption varies

greatly among world regions and countries. For example, the mean consumption of raw

coffee in 1989 was 2.0 kg/person in Britain, and 12.0 kg/person in Scandinavia

(http://www.coffeeresearch.org/market/consumption.htm). It seems that patients with

schizophrenia have a higher caffeine intake than does the general population (Hughes et al.

1998b), and high caffeine consumption is associated also with institutionalization (Hughes

& Boland 1992). About 25% of psychiatric inpatients are reported to drink daily more than

5 cups of coffee (Winstead 1976). Although it is unclear whether caffeine is a drug of abuse

for some individuals, many seem to exhibit dependence-like behaviors (Hughes et al.

1998a): Abrupt cessation of caffeine intake produces withdrawal symptoms in 11% to 100%

of individuals (Dews et al. 2002). Symptoms of caffeine withdrawal may include headache,

irritability, sleepiness or insomnia, anxiety, muscle pain, and delirium (Dews et al. 2002).

Several drugs have been reported to reduce the clearance of caffeine, and for the majority of

these interactions, the apparent mechanism seems to be inhibition of CYP1A2 activity


49

(Carrillo & Benítez 2000). For example, fluvoxamine reduces the clearance of caffeine to

one-fifth (Jeppesen et al. 1996b). Although caffeine has a relatively high Km (200 M) for

the CYP1A2-mediated caffeine-3-demethylation reaction, caffeine serum concentrations in

vivo are high enough (50 M) to suggest that caffeine can have some interaction potential as

a CYP1A2 inhibitor (Pelkonen et al. 1998). In fact, in healthy subjects, caffeine-containing

instant coffee has reduced clearance of the CYP1A2 substrate theophylline by 23% (Sato et

al. 1993). Abstinence from dietary caffeine resulted in a 47% decrease in mean

concentration of serum clozapine, and caffeine reduced mean oral clearance of clozapine by

14% (Carrillo et al. 1998, Hägg et al. 2000) (Tables 5 and 6).

A pharmacodynamic interaction between clozapine and caffeine is suggested by Vainer &

Chouinard (1994) in the case of a patient who had acute psychotic exacerbations each time

he took his clozapine with two cups of coffee. The suggested mechanism was a receptor-

receptor interaction between adenosine 2 and dopamine receptors 1 and 2 (Vainer &

Chouinard 1994).

RE

VIE

W O

F T

HE

LIT

ER

AT

UR

E

50

Tab

le 6

. Exa

mpl

es o

f pha

rmac

okin

etic

dru

g in

tera

ctio

ns w

ith c

affe

ine,

cip

roflo

xaci

n, it

raco

nazo

le, a

nd ri

sper

idon

e as

inhi

bito

rs.

Inhi

bito

r

D

aily

dos

eSu

bjec

ts (n

), st

udy

desi

gnSu

bstr

ate

Eff

ect o

nph

arm

acok

inet

ics o

fsu

bstr

ate

Sour

cePr

obab

le m

echa

nism

App

roxi

mat

e m

ean

300

mg

in c

offe

ePa

tient

s (7)

, seq

uent

ial

Clo

zapi

neIn

crea

se in

seru

mco

ncen

tratio

n of

2-f

old

Car

rillo

et a

l. 19

98In

hibi

tion

of C

YP1

A2

400

to 1

000

mg

mea

n 55

0 m

g pe

r day

Hea

lthy

(12)

, ope

n ra

ndom

ized

cros

sove

rC

loza

pine

AU

C in

crea

sed

by 1

9%H

ägg

et a

l. 20

00In

hibi

tion

of C

YP1

A2

4 to

8 c

ups o

f cof

fee

Patie

nts (

11),

sequ

entia

lLi

thiu

mM

este

r et a

l. 19

95In

hibi

tion

of p

roxi

mal

tubu

lar

reab

sorp

tion

of li

thiu

m

Caf

fein

e

2 to

7 c

ups o

f ins

tant

coff

eeH

ealth

y (6

), se

quen

tial

Theo

phyl

line

Red

uced

seru

m c

once

ntra

tion

by 2

4%

Red

uced

mea

n or

al c

lear

ance

by 2

3%, T

½ in

crea

sed

32%

Sato

et a

l. 19

93In

hibi

tion

of C

YP1

A2

1000

mg

Hea

lthy

(24)

, sin

gle-

blin

d ra

ndom

ized

cros

sove

rC

affe

ine

Red

uced

AU

C 2

-fol

dN

icol

au e

t al.

1995

Inhi

bitio

n of

CY

P1A

2

1000

mg

Hea

lthy

(9),

doub

le-b

lind

rand

omiz

edcr

osso

ver

Rop

ivac

aine

Red

uced

mea

n cl

eara

nce

by31

%Jo

kine

n et

al.

2003

Inhi

bitio

n of

CY

P1A

2

Cip

roflo

xaci

n

1000

mg

Hea

lthy

(9),

sequ

entia

lTh

eoph

yllin

eR

educ

ed m

ean

oral

cle

aran

ceby

19%

Bat

ty e

t al.

1995

Inhi

bitio

n of

CY

P1A

2

200

mg

Hea

lthy

(8),

doub

le-b

lind

rand

omiz

edcr

osso

ver

Bus

piro

neR

aise

d A

UC

19-

fold

Kiv

istö

et a

l. 19

97In

hibi

tion

of C

YP3

A4

200

mg

Hea

lthy

(10)

, dou

ble-

blin

d ra

ndom

ized

cros

sove

rD

igox

inR

aise

d m

ean

seru

mco

ncen

tratio

n 80

%Pa

rtane

n et

al.

1996

Inhi

bitio

n of

P-g

p

200

mg

Hea

lthy

(9),

doub

le-b

lind

rand

omiz

edcr

osso

ver

Felo

dipi

neR

aise

d A

UC

6-f

old

Jala

va e

t al.

1997

aIn

hibi

tion

of C

YP3

A4

200

mg

Hea

lthy

(12)

, dou

ble-

blin

d ra

ndom

ized

cros

sove

rLo

vast

atin

Rai

sed

Cm

ax >

20-f

old

Neu

vone

n &

Jala

va19

96In

hibi

tion

of C

YP3

A4

Lova

stat

in a

cid

Rai

sed

Cm

ax 1

3-fo

ld a

nd A

UC

23-f

old

200

mg

Hea

lthy

(9),

doub

le-b

lind

rand

omiz

edcr

osso

ver

Mid

azol

amR

aise

d A

UC

10-

fold

Olk

kola

et a

l. 19

94In

hibi

tion

of C

YP3

A4

Itrac

onaz

ole

200

mg

Hea

lthy

(9),

doub

le-b

lind

rand

omiz

edcr

osso

ver

Tria

zola

mR

aise

d A

UC

27-

fold

Var

he e

t al.

1994

Inhi

bitio

n of

CY

P3A

4

2 m

gPa

tient

, cas

e-re

port

Clo

zapi

neR

aise

d se

rum

con

cent

ratio

n by

2-fo

ldK

oree

n et

al.

1995

Unk

now

nR

ispe

ridon

e

2 m

gPa

tient

, cas

e-re

port

Clo

zapi

neR

aise

d se

rum

con

cent

ratio

n by

74%

Tyso

n et

al.

1995

Unk

now

n

A I M S O F T H E S T U D Y

51

AIMS OF THE STUDY

lozapine is eliminated mainly through CYP enzyme-mediated metabolism, and in

vitro studies indicate that it is metabolized mainly by CYP1A2 and CYP3A4.

Knowledge of the metabolic drug interactions of clozapine has been based almost entirely

on case-reports which suggest that strong inhibition of CYP3A4 by erythromycin could

considerably elevate serum clozapine concentrations and lead to significant adverse effects.

Case-reports show strong inhibition of CYP1A2 by fluvoxamine to increase serum

clozapine concentrations by several-fold. Two open studies and some case-reports suggest

that another CYP1A2 substrate, caffeine, may competitively inhibit clozapine metabolism

and lead to elevated serum clozapine concentrations, and caffeine withdrawal may, in some

patients, considerably reduce serum clozapine concentrations.

Similarly, modest inhibition of CYP1A2 by ciprofloxacin is suggested to raise serum

clozapine concentrations and intensify its adverse effects. CYP1A2-mediated metabolism of

theophylline may be lower during influenza epidemics and lead to elevated theophylline

concentrations and adverse effects. Whether influenza or other infections, or influenza

vaccination has any effect on serum clozapine concentrations has been unknown. Case-

reports suggest that even low-dose risperidone may double the serum concentration of

clozapine.

C

A I M S O F T H E S T U D Y

52

The specific aims of the studies were to study whether, in clozapine-treated patients:

1. Strong inhibition of CYP3A4 by itraconazole (CYP3A4 inhibitor, antimicromic drug)

has any clinically significant effect on serum concentrations of clozapine and its

metabolite N-desmethylclozapine.

2. A low dose of ciprofloxacin (CYP1A2 inhibitor, antimicrobic drug), commonly used in

urinary tract infections, has any clinically significant effect on serum concentrations of

clozapine and its metabolite N-desmethylclozapine.

3. An influenza vaccination with the commonly used trivalent subunit vaccine has any

clinically significant effect on serum concentrations of clozapine and its metabolites N-

desmethylclozapine and clozapine-N-oxide.

4. Concomitantly used risperidone is linked to increased serum clozapine concentrations.

5. Coffee-drinking (caffeine, CYP1A2 substrate) has any clinically significant effect on

serum concentrations of clozapine and its metabolites N-desmethylclozapine and

clozapine-N-oxide.

M A T E R I A L S A N D M E T H O D S

53

MATERIALS AND METHODS

1. Subjects

ll the subjects participating in Studies I, II, III, and V were clozapine-using patients (age

range 18-50 years; weight 64-138 kg) currently hospitalized in Kellokoski Hospital. A total

of 38 (12 females, 26 males) participated in Studies I to III and V. Four of these patients

participated in two of the studies. One of the 18 patients included in Study IV also participated in

two of the other four studies. The characteristics of the patients in each Study are shown in Table 7.

The characteristics data for patients in the register-based Study IV were obtained through records in

Kellokoski Hospital

Inclusion and exclusion: Only patients that were currently being treated with clozapine could be

included. Before entering the studies all patients were required to have had stable medication for a

minimum of 2 weeks. Those who took drugs that significantly inhibit CYP enzymes were excluded.

In Studies I to III and V, patients with acute physical or mental illnesses or with unstable chronic

diseases were excluded. Their evaluation for inclusion included a psychiatric interview and

examination, blood chemistry, and an electrocardiogram. Before entering the study all patients

understood a description of the study and gave their written informed consent.

Table 7. Characteristics of the patients, all with schizophrenia unless otherwise (n = 3) noted.StudyNo.

Female/male

(F/M)

Age

(years)

Weight*

(kg)

Smoker

(yes/no)

Coffee drinker(yes/no)

Clozapinedaily dose*

(mg)

Clozapinemonotherapy

(yes/no)1 I M 25 74 No ND 400 No2 I M 20 66 Yes ND 400 No3 I M 36 76 Yes ND 350 No4 I M 22 70 No ND 200 No5 I F 29 71 Yes ND 200 Yesasc

6 I M 27 64 Yes ND 550 Yesasc

7 I M 36 80 Yes ND 250 Yes8 II M 28 94 Yes Yes 150 No9 F 38 85 Yes Yes 350 No

F 39 ND Yes Yes 400 No{ IIIVV F 42 85 Yes Yes 600 No

10 II M 36 100 Yes Yes 275 Yesasc

11 II M 27 92 Yes Yes 450 No12 II F 35 65 Yes Yes 200 YesIUD

13 II M 32 102 No Yes 400 No14 II F 46 70 Yes Yes 300 No

A


54

Table 7. continued.

StudyNo.

Female/male

(F/M)

Age

(years)

Weight*

(kg)

Smoker

(yes/no)

Coffee drinker(yes/no)

Clozapinedaily dose*

(mg)

Clozapinemotherapy(yes/no)

15 III M 48 81 No Yes 275 No16 M 47 75 No Yes 650 Yes{ III

V M 48 71 No Yes 650 Yes17 III F 50 64 Yes Yes 400 No18 III F 45 88 No Yes 400 No19 III M 48 84 Yes Yes 500 No20 M 35 85 No Yes 300 Yes{ III

V M 36 89 No Yes 300 Yes21 III M 26 64 No Yes 750 No22 III F 46 114 No Yes 400 No23 III F 43 80 Yes Yes 450 No24 III F 35 75 No No 450 No25 III F 34 100 Yes Yes 450 No26 M 34 135 Yes Yes 375 No{ III

V M 35 138 Yes Yes 375 No27 III M 21 83 No Yes 375 Yes28 III M 22 80 Yes Yes 550 No29 III M 45 83 No Yes 300 Yes30 III F 34 100 No Yes 800 No31 IV M 24 112/110 Yes Yes 450/425 No32 IV M 53 98/74 Yes Yes 700 No33 IV M 49 ND/96 Yes Yes 600 No34 IV M 49 ND Yes Yes 800/700 No35 IV F 34 85 Yes Yes 425 No361 IV M 53 72/ND Yes ND 450/900 No37 IV M 30 89/90 Yes ND 300/200 No38 IV M 43 69/ND Yes Yes 450/500 Yes39 IV M 51 ND/88 Yes Yes 500 No40 IV F 45 ND/80 Yes Yes 350 No41 IV M 37 ND/84 Yes ND 600 No42 IV M 35 ND/113 Yes Yes 400/600 No43 IV F 30 ND Yes ND 300/225 No442 IV F 32 100/98 No No 200/250 No45 IV F 39 ND/70 Yes Yes 400 No46 IV M 48 118 Yes Yes 450/300 No47 IV M 18 96/90 No ND 100 No48 V M 39 92 Yes Yes 450 No49 V M 26 71 Yes Yes 350 No50 V M 41 85 Yes Yes 250 No51 V M 27 100 Yes Yes 750 No523 V F 22 75 Yes Yes 300 No53 V M 48 79 Yes Yes 500 No54 V M 18 92 Yes Yes 400 No55 V M 38 68 Yes Yes 450 No

* if the variable differs between study phases, the value is given for both phases (control phase/inhibitor phase)asc patient on clozapine monotherapy, but taking ascorbic acid dailyIUDpatient on clozapine monotherapy and wearing an intrauterine device (hormone)1Psychotic disorder NOS2Delusional disorder3Schizoaffective disorderND – not determined or unknown


55

2. Design of the studies

tudies I, II, and V were randomized, placebo-controlled crossover studies with two periods.

Study III was a prospective open-label study, and Study IV a retrospective registered-based

study (Table 8).

Study I. Seven patients (1 female, 6 males) received either itraconazole 100 mg or placebo orally at

8 a.m. and 8 p.m. daily for 7 days. For the next 7 days itraconazole was changed to placebo and vice

versa. Venous blood samples (10 mL) for determination of serum concentrations of

clozapine, N-desmethylclozapine, itraconazole, and hydroxyitraconazole were drawn 12 h after the

previous evening’s dose of clozapine and itraconazole/placebo on study days 0, 3, 7, 10, and 14.

Serum was separated within one hour and stored at -20°C until analyzed (Table 8).

Study II. Seven patients (3 females, 4 males) received either ciprofloxacin 250 mg or placebo

orally at 8:00 h and 20:00 h daily for 7 days, followed by a 7 days’ wash-out period with no

ciprofloxacin or placebo administered. For the next 7 days the previously administered

ciprofloxacin was changed to placebo and vice versa. During both phases venous blood samples (10

mL) for determination of serum concentrations of clozapine, N-desmethylclozapine, and

ciprofloxacin were drawn 12 h after the previous evening’s dose of clozapine on Day 1, before the

first dose of ciprofloxacin or placebo and on study days 3 and 8 (i.e., the morning after the last dose

of ciprofloxacin or placebo). Serum was separated within 1 hour and stored at -20°C until analyzed

(Table 8).

Study III. Sixteen patients (7 females, 9 males) on study Day 0 received intramuscularly (into the

deltoid muscle) the recommended dose of 0.5 mL of the 2000-2001 formula of a conventional

trivalent (influenza A subtypes H1N1 and H3N2, and influenza B) influenza subunit vaccine

(Influvac ®, Solvay Pharma). Venous blood samples (10 mL) for determination of serum

concentrations of clozapine, N-desmethylclozapine, clozapine-N-oxide, and c-reactive protein

(CRP) were collected 12 h after the previous evening’s dose of clozapine at 0800 hours on

vaccination day and 2, 4, 7, and 14 days after vaccination. Serum was separated within 1 h and

stored at -20 ºC until analyzed (Table 8).

S


56

Study IV. The electronic data base in Kellokoski Hospital served for collection of therapeutic drug

monitoring (TDM) data on all serum clozapine concentration measurements in that hospital

between January 1995 and August 2001 (a total of 2490 clozapine measurements for 414 patients).

The concomitant medications at the sampling time of each clozapine concentration measurement

were recorded. For 37 patients who had used both clozapine and risperidone at the times of

sampling, patient-related data were collected concerning each 327 serum clozapine concentration

sampling date. The following variables were registered: sex, age, time of the sampling, clozapine

concentration, daily dose of clozapine and risperidone, ratio of concentration to daily dose (C/D) of

clozapine, duration of treatment with the current dose of clozapine and risperidone, and

concomitant drugs and their daily doses. Entries in follow-up charts provided data on patients’

cigarette-smoking and coffee-drinking habits around the time of the relevant clozapine

concentrations, as well as to exclude concentrations taken during a period of non-compliance with

medication. In addition, ward staff was interviewed regarding the smoking- and coffee-drinking

habits of these patients (Table 8).

Inclusion and exclusion criteria: Only morning clozapine trough concentrations were included. A

minimum of 7 days’ treatment at the current dose of clozapine and risperidone was required.

Samples were excluded if patient was concomitantly using drugs known significantly to inhibit or

induce CYP1A2 or CYP3A4 enzymes in vivo in humans. Patients whose tobacco-smoking or

coffee-drinking habits were known to differ between the phases were excluded. If more than one

serum clozapine concentration with concomitant risperidone was available for one of the patients,

the following order was used to select one sample for comparison: If the patient’s daily dose of

risperidone was higher at one date of sampling than at others, then the concentration of that

measurement was selected. If this criterion (1), the highest daily dose of risperidone, could be

applied to more than one concentration, the selection was continued between them with the second

criterion (2), the highest daily dose of clozapine, and if necessary with the third criterion (3), the

most recent date of sampling. The matching control sample without concomitant risperidone was

selected by the following criteria: 1) The closest matching daily dose of clozapine, 2) The most

similar concomitant medication, 3) The closest matching date of sampling.

Study V. The 12 patients (2 females, 10 males) who were habitual coffee-drinkers drank either

regular caffeine-containing instant coffee or decaffeinated instant coffee (Nescafe Gold Blend® or

Nescafe Gold Blend Decaffeinated®), for 7 days (Table 8). For the next 7 days caffeine-containing

coffee (caffeine period) was changed to decaffeinated coffee (control period) and vice versa. No


57

other sources of caffeine, such as any other coffee, caffeine tablets, tea, chocolate, or cola drinks

were allowed. Patients were advised to prepare their coffee servings similarly during both study

periods. Venous blood samples (10 mL) for the determination of serum concentrations of clozapine,

N-desmethylclozapine, clozapine-N-oxide, and CRP were collected 12 h after the previous

evening’s dose of clozapine before the first cup of coffee on study days 1, 4 and 8 (i.e., the morning

after the last day of each study week) of both study phases. The serum was separated within 1 h and

stored at -20 ºC until analyzed. Patients were not allowed to drink study coffee for 12 hours before

each blood sample. Other than that, the patients were free to drink study coffee at all times in

amounts of their own choosing. (Randomized coffee was always delivered to the patients in

identical packages.)

During the run-in period and study periods all patients recorded on a form the times and the number

of cups of coffee drunk and cigarettes smoked each day. After each of the 3 weeks, all patients were

rated with the Brief Psychiatric Rating Scale (BPRS) and the Udvalg for Kliniske Undersøgelser

(UKU) side-effect rating scale–the self-rating version in Finnish (UKU-SERS-Pat-Finnish)–by one

of the authors (KR). The BPRS (18 items) was scaled from 1 to 7 (1 = not present, 7 = extremely

severe) and UKU (46 items) from 0 to 3 (0 = not present, 3 = severe) for each item. Most studies

use a > 20% change in BPRS score as an indicator of treatment response (Lachar et al. 1999).

Compliance was assessed by measuring serum caffeine and paraxanthine concentrations.

Measurement of CRP concentration was done in order to ensure that patients had no infections that

could possibly inhibit clozapine metabolism.

3. Blood sampling

enous blood samples (10 mL) for determination of serum concentrations of clozapine, N-

desmethylclozapine, and clozapine-N-oxide, and concentrations of all the inhibitors studied

and of CRP were drawn 12 h after the previous evening’s dose of clozapine, before the ingestion of

clozapine or of the inhibitors. Serum was separated within one hour and stored at -20°C until the

samples were analyzed.

V


58

4. Determination of serum drug, caffeine, and CRP concentrations

erum concentrations of clozapine and N-desmethylclozapine in Studies I, II, and IV were

determined in the Laboratory of Helsinki University Central Hospital. Clozapine and its

metabolite concentrations, other drug concentrations, caffeine and paraxanthine concentrations, and

CRP concentrations were determined in the Analytical Laboratory of the Department of Clinical

Pharmacology, University of Helsinki.

4.1. Clozapine and its metabolites

erum concentrations of clozapine, N-desmethylclozapine, and clozapine-N-oxide were

determined by automated solid-phase extraction and subsequent high-performance liquid

chromatography with an electrochemical detector (Humpel et al. 1989, Lovdahl et al. 1991,

Weigmann & Hiemke 1992, Schulz et al. 1995). Analysis was performed on a Merck LiChrospher

60 RP-select B column (125 mm x 4 mm) and the electrochemical detector ESA Model 5100 A

Coulochem. The mobile phase consisted of acetonitrile-methanol-0.02 M potassiumphosphate

buffer (150:450:400, volume (v)/v/v), which was adjusted to pH 6.6. Imipramine served as an

internal standard.

In Study I, the quantitation limit was 100 nmol/L for clozapine and N-desmethylclozapine. The day-

to-day CV for clozapine at 390 nmol/L was 10.7%.

In Studies II and IV, The quantitation limit was 50 nmol/L for both compounds and the within-day

and day-to-day CVs at relevant concentrations were less than 5%.

In Study III, The quantitation limit for clozapine, N-desmethylclozapine and clozapine-N-oxide was

30 nmol/L. The day-to-day CV was for clozapine 12% and 3.3% at mean concentrations of 220

nmol/L and 1200 nmol/L, respectively; for N-desmethylclozapine 12% and 3.5% at 200 nmol/L and

1050 nmol/L; and for clozapine-N-oxide 13% and 9% at 160 nmol/L and 880 nmol/L.

S

S


59

In Study V, The quantitation limit for clozapine, N-desmethylclozapine and clozapine-N-oxide was

30 nmol/L. The day-to-day CV was for clozapine 13% and 7% at mean concentrations of 180

nmol/L and 970 nmol/L, respectively; for N-desmethylclozapine 4% and 2% at 170 nmol/L and

1040 nmol/L; and for clozapine-N-oxide 7% and 1.5% at 190 nmol/L and 1170 nmol/L.

4.2. Itraconazole and hydroxyitraconazole

oncentrations of itraconazole and hydroxyitraconazole were determined by high-performance

liquid chromatography (HPLC) with a fluorescence detector (Remmel et al. 1988, Allenmark

et al. 1990). A 15 cm x 3.9 mm I.D. reverse-phase column (Waters Nova-Pak C18) was used. The

mobile phase was water-acetonitrile (50:50), containing 0.28% of triethylamine. The mobile phase

was adjusted to pH 2.3. The quantitation limit was 10 ng/mL. The CV for itraconazole was 1.6% (at

194 ng/mL) and for hydroxyitraconazole 1.2% (at 194 ng/mL).

4.3. Ciprofloxacin

oncentrations of ciprofloxacin were determined by liquid chromatography (PERKIN ELMER

Series 200) tandem mass spectrometry using electrospray ionization (PE-SCIEX API 3000)

(Volmer et al. 1997). The chromatographic analysis was performed on a Waters Symmetry C-8

column (50 mm x 2.1 mm). The mobile phase was acetonitrile-formic acid (5:95, v/v). The

quantitation limit was 1 ng/mL. The day-to-day CV for ciprofloxacin was 3.7% at 800 ng/mL and

7.6% at 30 ng/mL.

4.4. Caffeine and paraxanthine

erum concentrations of caffeine and paraxanthine were determined by high-performance liquid

chromatography (HPLC) (Pickard et al. 1986, Holland et al. 1998). The analysis was

performed with an automated HP 1100 liquid chromatograph on a Waters Symmetry C8 column

(150 mm x 4.6 mm) and ultaviolet detector. The mobile phase consisted of methanol-15 mM

potassiumphosphate buffer (17.5:82.5, v/v), which was adjusted to pH 5.0. Beta-

hydroxyethyltheophylline served as the internal standard. The quantitation limit for caffeine and

C

C

S


60

paraxanthine was 20 g/L. The day-to-day CV was for caffeine 9.8%, 13.25%, and 2.7% at mean

concentrations of 135 g/L , 1260 g/L, and 15 400 g/L; for paraxanthine 153 g/L, 1460 g/L,

and 15 030 g/L.

4.5. C-reactive protein

erum concentration of CRP was determined with a validated high-sensitivity

immunoturbidimetric assay (Markkola et al. 2000) in the laboratory of Helsinki University

Central Hospital.

5. Statistical analysis

tudy results are given as mean values + SD, or mean + range. Statistical analysis of continuous

data was performed with Student’s two-tailed t-test for paired data. Differences were

considered statistically significant when P values were less than 0.05. The Wilcoxon Signed Ranks

Test was used for non-parametric data. Correlations between continuous data are given as the

Pearson correlation coefficient. Consumer’s risk was evaluated by taking a equivalence approach

(Steinijans et al. 1991). In testing equivalence, no interaction was assumed if the ratio test/ reference

and the corresponding 90% confidence intervals fell into the equivalence range 0.8 to 1.25. The data

are given as mean ratio test/ reference ( test = mean value during tested treatment, reference = mean

value during control treatment) and 90% confidence intervals.

6. Ethical considerations

or ethical reasons, none of these studies involved healthy volunteers, because serious adverse

effects have occurred in healthy subjects even after single low doses of clozapine (Pokorny et

al. 1994). All studies were thus carried out with the patients who were already using clozapine. All

the study protocols of these studies comply with the current laws and ethical standards of Finland.

The protocols of Studies I and II were approved by the Ethics Committee of the Hyvinkää Health

Care District and by the National Agency for Medicines (Finland). The study protocols of Studies

S

S

F


61

III and V were approved by the Ethics Committee of pediatrics and psychiatry in the hospital

district of Helsinki and Uusimaa. The study protocol of Study IV, which was a register-based study

in which the patients were not contacted, was approved by the chief physician of Kellokoski

Hospital. All the patients in Studies I to III and V received oral and written information, and after

the minimum 4 days’ reconsideration period, they gave their written informed consent before

entering the study. The ability of any patient to give an informed consent was evaluated through a

psychiatric interview by the author and through consulting the psychiatrist in charge of the

treatment of each patient.

MA

TE

RIA

LS

AN

D M

ET

HO

DS

62

Tab

le 8

. Stu

dy d

esig

n. T

he a

im w

as to

inve

stig

ate

any

poss

ible

eff

ect o

f fiv

e fa

ctor

s (in

hibi

tors

or c

o-dr

ugs)

on

seru

m c

once

ntra

tion

of c

loza

pine

and

its m

etab

olite

s in

hosp

italiz

ed p

atie

nts.

Stud

yno

.Pa

tient

sfe

mal

es/m

ales

Stru

ctur

eIn

hibi

tor /

co-

drug

Con

trol

Stud

y du

ratio

n

In

= 7

1 / 6

rand

omiz

ed, p

lace

bo-c

ontro

lled

cros

sove

r stu

dy w

ith tw

oph

ases

itrac

onaz

ole

100

mg

b.i.d

(CY

P3A

4 in

hibi

tor)

plac

ebo

7 da

ys p

er p

hase

IIn

= 7

3 / 4

rand

omiz

ed, p

lace

bo-c

ontro

lled

cros

sove

r stu

dy w

ith tw

oph

ases

cipr

oflo

xaci

n 25

0 m

g b.

i.d(C

YP1

A2

inhi

bito

r)pl

aceb

o7

days

per

pha

se +

7da

ys w

asho

ut p

erio

dbe

twee

n th

e ph

ases

III

n =

167

/ 9op

en, p

rosp

ectiv

e; p

atie

nts a

sth

eir o

wn

cont

rols

influ

enza

vac

cina

tion

(stim

ulat

es c

ytok

ine

prod

uctio

n,cy

toki

nes i

nhib

it C

YPs

)

none

(bef

ore

the

influ

enza

vacc

inat

ion)

14 d

ays

IVn

= 18

6 / 1

2re

trosp

ectiv

e; p

atie

nts a

s the

irow

n co

ntro

lsris

perid

one

2-4

mg

daily

(sub

stra

te o

f CY

P2D

6 an

d 3A

4)no

ne(w

ithou

t ris

perid

one)

paire

d sa

mpl

es0.

3-48

mon

ths a

part

Vn

= 12

2 / 1

0ra

ndom

ized

, pla

cebo

-con

trolle

dcr

osso

ver s

tudy

with

two

phas

es

caff

eine

-con

tain

ing

coff

eead

libi

tum

(caf

fein

e is

a C

YP1

A2

subs

trate

)

deca

ffei

nate

d co

ffee

7 da

ys ru

n-in

per

iod

+ 7

days

per

pha

se

R E S U L T S

63

RESULTS

Effects of itraconazole, ciprofloxacin, risperidone,influenza vaccination, and coffee-drinking on clozapineconcentrations

1. Itraconazole (Study I)

7-day treatment with itraconazole 100 mg twice daily had no statistically significant

effect on serum concentrations of clozapine or N-desmethylclozapine. During the

itraconazole phase, the mean clozapine concentration was 3% lower (non-significant, NS, P

= 0.79) (Figure 6) and N-desmethylclozapine 7% higher (NS, P = 0.43) than during the

placebo phase. The N-desmethylclozapine to clozapine ratio was 8% higher (P < 0.05)

during the itraconazole phase (Table 9).

Compliance with itraconazole ingestion was good in each patient as shown by serum

itraconazole concentrations. No change was reported in patients’ symptoms or in adverse

effects during the study.

2. Ciprofloxacin (Study II)

7-day treatment with ciprofloxacin 250 mg twice daily raised mean serum clozapine

concentration by 29% (P < 0.01; range 6-57%) (Figure 6) and that of N-

desmethylclozapine by 31% (P < 0.05; range 0-63%). The mean N-desmethylclozapine to

clozapine concentration ratio remained unchanged (NS, P = 0.83). The correlations between

serum ciprofloxacin concentrations and the increase (%) in serum clozapine or N-

desmethylclozapine concentrations were non-significant (r = 0.73; P < 0.10 and r = 0.74; P

< 0.10, respectively). The correlation between the increase (%) in the sum of clozapine + N-

desmethylclozapine serum concentrations and serum ciprofloxacin concentration was

A

A

R E S U L T S

64

significant (r = 0.90; P < 0.01). The correlation was significant between the increase (%) in

serum clozapine concentrations during the ciprofloxacin phase and the ratios of N-

desmethylclozapine to clozapine concentrations determined on the 8th day of the placebo

phase (r = 0.89; P < 0.01) (Table 9).

Compliance with ciprofloxacin ingestion was good in each patient, as shown by serum

ciprofloxacin concentrations.

3. Influenza vaccination (Study III)

erum concentrations of clozapine, N-desmethylclozapine or clozapine-N-oxide did not

significantly differ from baseline concentrations at any time-points during the study.

Mean serum concentration of clozapine tended to increase by 5% (NS, P = 0.22), 2% (NS, P

= 0.65), and 1% (NS, P = 0.87), 2, 4, and 7 days after vaccination (Figure 6). No significant

increase in CRP appeared after the vaccination (Table 9).

Two patients (a male and a female) were excluded from statistical analysis because both had

suffered an acute infection during the study period after the vaccination. Both had elevated

serum clozapine concentrations, and a decreased serum concentration ratio N-

desmethylclozapine to clozapine during the period of increased CRP (Figure 7).

The clinical efficacy of clozapine remained unchanged throughout the study for all 16

patients.

4. Risperidone (Study IV)

nalysis was based on the results from 18 patients who met the inclusion criteria, of

the total 37 patients who took both clozapine and risperidone in their daily

medication. Characteristics of the patients are shown in Table 7.

Neither the clozapine concentration to dose ratio nor clozapine concentration significantly

differed during risperidone from the control value. During risperidone, the mean serum

S

A

R E S U L T S

65

clozapine concentration was 3% lower (NS, P = 0.75) (Figure 6), and the clozapine

concentration to dose ratio was 8% lower (NS, P = 0.46) than during control. The ratio

test reference for clozapine concentration to dose ratios indicated equivalence with 90% CI

0.82-1.15 within limits of statistical significance (90% CI 0.80-1.25) (Table 9).

The time-range between the individual paired samples was 0.3 to 48 months (median 15.2

months). During risperidone, at the time of clozapine TDM sampling, the mean durations of

unchanged clozapine and risperidone treatments were 320 + 743 days (range 7 to 3120) and

52 + 59 days (range 7 to 193), respectively. The mean dose of risperidone was 2.9 + 0.9

mg/day (range 2 to 4 mg/day). During the control phase (without risperidone), at the time of

clozapine sampling, the mean duration of unchanged clozapine treatment was 209 + 564

days (range 7 to 2409).

5. Coffee-drinking (Study V)

erum paraxanthine concentrations indicated that of the 12 patients, six were compliant

in caffeine use throughout this study, and four up to the fourth study day (partially

compliant patients). Thus, 10 of 12 patients were included in the statistical analysis. Day 4

data were analyzed for 10 patients, and Day 8 data for six patients. In addition, the Day 4

data from 4 partially compliant patients (who were non-compliant on Day 8) and the Day 8

data from 6 fully compliant patients were pooled to increase statistical power in analysis of

the combined data.

Analysis of Day 4 data. Serum concentrations of clozapine (Figure 6), N-

desmethylclozapine, or clozapine-N-oxide were not significantly changed between the

caffeine and control phases. During the control phase, the mean serum concentration of

caffeine was 12%, and that of paraxanthine 13% of the corresponding concentrations during

the caffeine phase.

Analysis of Day 8 data. During the caffeine phase the mean serum concentration of

clozapine was 26% (NS, P = 0.07) (Figure 6) , N-desmethylclozapine, 6% (P = 0.03), and

S

R E S U L T S

66

clozapine-N-oxide, 7% (NS, P = 0.22) higher than during the control phase. The N-

desmethylclozapine to clozapine ratio was 13% (NS, P = 0.06) and the clozapine-N-oxide to

clozapine, 7% (NS, P = 0.19) lower than during the caffeine phase. Mean serum caffeine

and paraxanthine concentrations during the control phase were 5% and 8% of the

corresponding concentrations during the caffeine phase (Table 9).

Analysis of the combined data. The mean serum concentration of clozapine was 20% (P =

0.03) (Figure 6), and N-desmethylclozapine, 7% (P = 0.02) higher during the caffeine phase

than during the control phase, but the mean serum clozapine-N-oxide concentration was not

significantly increased (P = 0.11). During the caffeine phase, the ratio of N-

desmethylclozapine to clozapine was 9% (NS, P = 0.06) lower than during the control

phase.

Patients’ consumption of cigarettes did not significantly differ between study phases. Mean

coffee consumption, BPRS, and UKU ratings were non-significantly higher during the

control than caffeine phase (P > 0.05).

R E S U L T S

67

Figure 6. Serum clozapine concentration (% of control) during itraconazole (ITRA) and

ciprofloxacin (CIPRO), 2, 4, and 7 days after influenza vaccination (VAC), and after

caffeine-containing coffee for 3 and 7 days, and serum clozapine concentration to clozapine

daily dose ratio during risperidone (RISP). Medians ( ) are shown, and 95% confidence

intervals are indicated by short horizontal lines.

0

20

40

60

80

100

120

140

160

C

loza

pine

con

cent

ratio

n

(%

of c

ontr

ol)

I II III IV V1261201049210110210512997

Study

ITR

A

CIP

RO

2 D

AYS

AFTE

R V

AC

RIS

P

CO

F 3

DAY

S

CO

F C

OM

B

CO

F 7

DAY

S

4 D

AYS

AFTE

R V

AC

7 D

AYS

AFTE

R V

AC

RE

SU

LT

S

68

Tab

le 9

. Mai

n re

sults

of t

he st

udie

s.

Stud

yno

.In

hibi

tor

/ co-

drug

Mea

n ef

fect

s of i

nhib

itors

or

co-d

rugs

on

seru

m c

once

ntra

tions

of

C

loza

pine

N

-des

met

hylc

loza

pine

Clo

zapi

ne-N

-oxi

deI

Itra

cona

zole

100

mg

b.i.d

(CY

P3A

4 in

hibi

tor)

Unc

hang

edU

ncha

nged

Not

stud

ied

IIC

ipro

floxa

cin

250

mg

b.i.d

(CY

P1A

2 in

hibi

tor)

Incr

ease

29%

(P <

0.0

1)In

crea

se 3

1%(P

< 0

.05)

Not

stud

ied

III

Influ

enza

vac

cina

tion

(stim

ulat

es c

ytok

ine

prod

uctio

n:cy

toki

nes i

nhib

it C

YPs

)

Unc

hang

edU

ncha

nged

Unc

hang

ed

IVR

ispe

rido

ne 2

-4 m

g da

ily(s

ubst

rate

of C

YP2

D6

and

3A4)

Unc

hang

edN

ot st

udie

dN

ot st

udie

d

VC

affe

ine-

cont

aini

ng c

offe

ead

libitu

m(c

affe

ine

is a

CY

P1A

2su

bstra

te)

Non

-sig

nific

ant i

ncre

ase

26%

(P =

0.0

7)In

crea

se 6

%(P

< 0

.05)

Unc

hang

ed

R E S U L T S

69

Figure 7. Serum concentrations of clozapine ( ), CRP ( ), and N-desmethylclozapine to

clozapine ratios ( ) of the two excluded patients in Study III on Day 0 before vaccination

and 2, 4, 7, and 14 days after vaccination.

DM

CLZ

/CLZ

-rat

io

0 2 4 7 140

1000

2000

3000

4000C

loza

pine

and

met

abol

ites

(nm

ol/l)

20

40

60

CR

P (m

g/l)

Days after vaccination

0.3

0.6

0.9

CRP

DMCLZ/CLZ-ratio

Clozapine

Pharyngitis

FEMALE, age 34

DM

CLZ

/CLZ

-rat

io

0 2 4 7 140

200

400

600

800

1000

1200

1400

Clo

zapi

ne c

once

ntra

tion

(nm

ol/l)

0

20

40

60

80

CR

P (m

g/l)

0.1

0.2

0.3

0.4

0.5

CRP

DMCLZ/CLZ-ratio

Abdominal pain

CLOZAPINE

Days after vaccination

Sedation

MALE, age 21

D I S C U S S I O N

70

DISCUSSION

1. Methodological considerations

hese studies were conducted only with patients, because healthy volunteers should

never take clozapine (Pokorny et al. 1994). Between these patients concomitant

medication and the daily dose of clozapine varied. Patients who had medications known (or

suspected) to affect the clozapine disposition were excluded. In statistical analysis, a similar

percentage change in patients’ clozapine concentrations thus had more variable effects on

the mean concentration results than if each patient within each study had taken an identical

dosage. Within each subject, medication remained unchanged during the study phases,

making comparisons of effect for each patient relevant.

All study subjects were currently residing in one hospital under regular close surveillance.

The majority took their medication at fixed times of day under the surveillance of staff

members, and the minority resided on wards where taking medication was mostly the

patients’ own responsibility. Ward staff was informed about the study protocols, and, if

needed, they helped patients follow the protocols as planned. This ensured prompt reporting

of any significant change in subjects’ well-being.

In all of the studies, subjects served as their own controls, which reduces variability between

paired samples and reduces the number of subjects needed to attain sufficient statistical

power. The number of patients in Studies I, II, and V was relatively small (7,7, and 12). This

increases the probability of error (i.e., reduces the power to detect a true difference), but

this number of patients is sufficient to reveal an about 35% to < 30% change with 80%

power in serum concentrations between study phases ( = 0.05 and = 0.20 and within-

patient SD < 20%). The number of patients in Studies III and IV (16 and 18) was sufficient

to reveal a 20% change with 80% power between the study phases.

Much effort was put into the informed consent process, not only to ensure the ethics, but to

include only patients who were well motivated and educated about the study protocol in

T

D I S C U S S I O N

71

order to maximize compliance. Compliance with the supposed inhibitor of clozapine

metabolism was assessed by measurement of its serum concentration in Studies I

(itraconazole and metabolite), II (ciprofloxacin), and V (caffeine and metabolite).

Compliance was generally good, perhaps because of careful patient selection. Only in Study

V were some of the patients found to be non-compliant. That study required one week’s

abstinence from caffeine, which often produces withdrawal symptoms and the desire to

resume coffee-drinking.

Only a few study subjects had a diagnosis other than schizophrenia. There is, however, no

reason to suspect any profound differences in basic pharmacokinetic interaction

mechanisms, such as CYP-mediated ones, between different diagnostic categories of mental

disorders. Results of these studies can therefore be applied not only to patients with

schizophrenia, but also to clozapine-treated patients in general.

2. Effect of itraconazole on serum clozapine concentration

n this study the potent CYP3A4 inhibitor itraconazole 200 mg a day had no significant

effects on serum clozapine or N-desmethylclozapine concentrations, although

itraconazole 200 mg a day has greatly increased the AUC of many substrates of CYP3A4

(Neuvonen & Jalava 1996, Varhe et al. 1994, Olkkola et al. 1994, Kivistö et al. 1997).

Compliance in the use of itraconazole was good, confirmed by determination of its serum

concentrations. It thus seems that inhibition of CYP3A4 (or P-gp) in general does not

significantly elevate serum concentrations of clozapine or N-desmethylclozapine.

Two case-reports suggest that concomitant use of erythromycin with clozapine can elevate

serum concentrations of clozapine, leading to signs of clozapine toxicity (Funderburg et al.

1994, Cohen et al. 1996). Erythromycin is a relatively specific CYP3A4 inhibitor. Authors

of both reports suggest a pharmacokinetic interaction caused by erythromycin as the most

likely explanation for high clozapine concentrations. On the other hand, itraconazole clearly

I

D I S C U S S I O N

72

causes a more pronounced increase in plasma concentrations of various CYP3A4 substrates

than does erythromycin, e.g., of midazolam (Olkkola et al. 1994, Olkkola et al. 1993),

triazolam (Varhe et al. 1994), and buspirone (Kivistö et al. 1997, Kivistö et al. 1999).

The complete lack of effect of itraconazole on serum concentrations of clozapine and N-

desmethylclozapine in this prospective study does not support the hypothesis of a CYP3A4-

inhibition-mediated interaction between clozapine and erythromycin. Influenza (Kraemer et

al. 1982) and influenza vaccines (Renton et al. 1980) may inhibit CYP1A2-mediated drug

metabolism. Possibly infection could explain elevated clozapine concentrations in the two

cases of Funderburg et al. (1994) and Cohen et al. (1996).

It appears that CYP3A4 inhibition normally has little effect on serum clozapine

concentrations. If the CYP1A2 pathway is inhibited, however, CYP3A4 may become a

major pathway in clozapine metabolism. In that case, additional CYP3A4 inhibition could

lead to highly elevated clozapine concentrations.

3. Effect of ciprofloxacin on serum clozapine concentration

he low dose of ciprofloxacin (250 mg b.i.d.) elevated mean concentrations of both

clozapine and N-desmethylclozapine by about 30%. An increase of that magnitude in

serum clozapine concentration is generally well tolerated, but an increase of about 50% is

enough to cause significant adverse effects in some sensitive patients. The smoking and

coffee-drinking habits of the study patients remained unchanged during this study, so those

factors could not have influenced results.

The extent of the interaction was related to the serum ciprofloxacin concentration, which

varied considerably between patients. It is likely that higher doses of ciprofloxacin (500 mg

or 750 mg b.i.d.) than used in this study would cause a greater increase in clozapine and N-

desmethylclozapine concentrations. Probably due to the small sample size, the percentage

increase in clozapine or N-desmethylclozapine concentrations did not quite correlate with

ciprofloxacin concentrations, but the correlation nearly reached significance. The pooled

T

D I S C U S S I O N

73

concentration of clozapine and N-desmethylclozapine correlated well, however, with

ciprofloxacin concentrations, despite the small number of patients. Although an increase in

serum clozapine is clinically more important, an increase in N-desmethylclozapine

concentration may also be significant for some of the patients. It, too, has biological

activity, demonstrated by its clozapine-like effect in elevating fos-protein in rat brain

(Young et al. 1998).

The ratio of serum N-desmethylclozapine to clozapine correlates with CYP1A2 activity

measured by the urinary caffeine test (Carrillo et al. 1998). In this study, the individual

ratios of N-desmethylclozapine to clozapine at Day 8 of the placebo phase correlated well

with the percentage increase in clozapine concentrations at Day 8 of the ciprofloxacin phase

(r = 0.90; P < 0.01). That is, this interaction was strongest in patients whose CYP1A2

activity was highest. This suggests that inhibition of CYP1A2 is the most likely mechanism

behind this interaction. This is in line with the fact that ciprofloxacin is a CYP1A2 inhibitor

(Fuhr et al. 1992).

The suggested mechanism in this interaction is inhibition of the CYP1A2 enzyme by

ciprofloxacin. Those patients who have both a high ratio of serum N-desmethylclozapine to

clozapine concentration and a high serum concentration of ciprofloxacin seem to comprise a

risk group for a clinically relevant ciprofloxacin-clozapine interaction.

4. Effect of risperidone on serum clozapine concentration

isperidone (2-4 mg/day) had no effect on the mean serum clozapine concentration, or

on the ratio of serum clozapine concentration to clozapine dose. In fact, the mean

serum clozapine concentration was slightly lower (NS, P = 0.75) during risperidone than

during the control phase, despite the fact that the mean clozapine dose was higher (451 +

207 mg vs. 438 + 168 mg; P = 0.66), and the patients were, on average, 0.4 years older

during the risperidone phase. Because clozapine concentrations tend to increase with age

(Haring et al. 1989), this insignificant age-difference does not impair the conclusions.

R

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74

The results do not support the assumption that risperidone added to clozapine treatment

would increase serum clozapine concentrations. That assumption of a significant

risperidone-clozapine interaction is based on two case-reports (Koreen et al. 1995, Tyson et

al. 1995), which suggest that even low 2-mg daily doses of risperidone may double serum

clozapine concentrations. In the light of the present study, it seems unlikely that in those

cases the increase in clozapine concentrations was caused by risperidone or the other co-

medications. On the other hand, the results of this study are supported by a study

demonstrating that risperidone is neither a CYP1A2 nor CYP2C19 inhibitor, and that it is

only a weak CYP2D6 inhibitor (Eap et al. 2001).

Of the 18 patients in the present study, 16 were smokers, and at least 13 drank coffee daily

during both phases. No entries in the follow-up charts of any of the 18 patients suggested

any significant changes in their smoking or coffee-drinking habits during that time period.

Thus, it is unlikely that changes in these habits would have impaired the results.

Furthermore, to ensure that the results were not compromised by concomitant medication,

all patients who used clinically significant inhibitors or inducers of CYP1A2 or CYP3A4

were excluded.

Although the difference between mean clozapine concentrations was insignificant, in four of

the patients clozapine concentration to dose ratios showed an over 50% change from the

control to the risperidone phase. Differences between their co-medication, age, smoking, or

coffee-drinking seem not to explain this variability. Possibly, the explanation involves

minor changes in the co-medication, compliance or dietary factors. Although entries in the

follow-up charts did not suggest it, infections could have changed their serum clozapine

concentration to dose ratios (Raaska et al. 2002b).

Two open trials of 13 and 12 patients suggest that some patients may benefit from a

combination of clozapine and risperidone (Taylor et al. 2001, Henderson & Goff 1996). In

one of these studies, serum clozapine concentrations (measured in 7 patients) increased only

insignificantly, by 2.2%, during 4 weeks of risperidone treatment (Henderson & Goff 1996).

Together with the fact that risperidone does not inhibit the main clozapine-metabolizing

D I S C U S S I O N

75

enzymes (Eap et al. 2001), it is unlikely that risperidone would cause any significant

increase in serum clozapine concentrations.

5. Effect of influenza vaccination on serum clozapine concentration

n the present study, influenza vaccination had no effect on the serum clozapine, N-

desmethylclozapine or clozapine-N-oxide concentrations. The vaccination did not

elevate CRP. However, two of the patients were excluded from the statistical analysis

because of infections during the study period after the vaccination. Interestingly, during

their high CRP levels both had higher clozapine concentrations than at baseline. It is

probable that the concentrations increased at least in part due to the reduced volume of

distribution of clozapine, because elevated acute-phase 1 acid glycoprotein during

infections is expected to bind more clozapine in plasma. But these elevated serum clozapine

concentrations may, in part, have been caused by infections inhibiting clozapine-

metabolizing enzymes.

Because several of the factors that stimulate a cytokine response may inhibit CYP enzymes

(Morgan 2001), it is important to know whether influenza vaccination with the trivalent

vaccine currently used can cause drug interactions. Influenza virus vaccination stimulates

cytokine production (Meredith et al. 1985, Saurwein-Teissl et al. 1998, Bernstein et al.

1998), but the cytokine response is, however, dependent on the type of influenza vaccine

(Saurwein-Teissl et al. 1998). Whole-virus vaccines stimulate a more pronounced response

than the type of subunit vaccines used in this trial (Saurwein-Teissl et al. 1998). It appears

that these trivalent subunit influenza vaccines cause no significant alterations in the

pharmacokinetics of clozapine.

I

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76

6. Effect of coffee-drinking on serum clozapine concentration

n this study, caffeine-containing coffee elevated mean serum clozapine concentration by

20% to 26%. Although non-significantly, the N-desmethylclozapine to clozapine ratio

fell by 9% to 13%, which suggests that caffeine or other ingredients in the coffee reduced

CYP1A2 activity.

Clozapine has higher affinity for CYP1A2 than does caffeine (Km values 60 mol/L vs.

200-500 mol/L) (Pelkonen et al. 1998, Carrillo & Benítez 2000), suggesting that in order

to inhibit CYP1A2-mediated clozapine metabolism, caffeine has to be at much higher molar

concentrations than clozapine in hepatocytes where the metabolism takes place. It is

important to keep in mind, however, that their relative concentrations in serum may differ

from those in hepatocytes. In the present study, patient 26 in Table 7 had the largest

percentage increase (+41%) in serum clozapine concentration, and his caffeine and

paraxanthine concentrations at baseline were the highest among the study patients. His

caffeine concentrations during the caffeine phase (about 13 000 nmol/L) and at baseline

(over 30 000 nmol/L) exceeded his clozapine trough concentrations by 10- to 30-fold,

although caffeine concentrations were measured 14 hours after the previous cup of coffee.

The volumes of coffee servings and their caffeine contents probably varied considerably

between the patients, making it difficult to approximate caffeine intake. Serum paraxanthine

concentration, rather than caffeine concentration, served as an indicator of caffeine intake,

because it fluctuates less during the day (Kaplan et al. 1997). Serum paraxanthine

concentrations suggest that mean caffeine intake during the caffeine phase was about 60%

of that at baseline. The mean serum concentrations of clozapine at baseline (1287 + 749

nmol/L) and during the caffeine phase (1310 + 724 nmol/L) were, however, very similar.

Mean caffeine and paraxanthine concentrations during the control phase were 3.5% to 8%

of the corresponding concentrations at baseline or during the caffeine phase, as is expected

if caffeine-containing coffee is switched to identically prepared decaffeinated coffee.

The pharmacokinetics of caffeine is non-linear with increasing doses (Kaplan et 1997).

Probably non-linearity is reached with lower caffeine concentrations than normally, if it has

I

D I S C U S S I O N

77

to compete with clozapine for metabolism, or if a patient’s CYP1A2 activity is low. Those

patients with higher clozapine concentration to dose ratios at baseline, suggesting lower

clozapine clearance, seemed to be more sensitive to the caffeine-clozapine interaction. On

the other hand, cigarette-smoking induces CYP1A2, which elevates the clearances of

clozapine (Seppälä et al. 1999) and caffeine (Carrillo & Benítez 1996). In theory, if

CYP1A2 is induced, higher caffeine concentrations are needed to saturate the clozapine

metabolism by this pathway. But if caffeine concentrations are high enough to inhibit the

induced CYP1A2 significantly, the highest possible increase in serum clozapine

concentration should be higher than in a non-induced state of clozapine metabolism. A

likely example of this is the patient in a caffeine withdrawal study (Carrillo et al. 1998),

who had the highest daily consumption of both coffee (1100 mg per day) and cigarettes (40

per day), and whose clozapine concentrations fell to one-fifth after the caffeine withdrawal,

being the largest decrease seen in that study.

Two open trials have studied the effect of either coffee-drinking or caffeine intake on serum

clozapine concentrations (Carrillo et al. 1998, Hägg et al. 2000). In the study by Carrillo et

al. (1998) in 7 hospitalized patients on clozapine monotherapy, caffeine withdrawal reduced

the mean serum concentration of clozapine in 5 days by 47%. Clozapine concentration

decreased in each one of the patients (range -29 to -80%). In the study by Hägg et al. (2000)

with 12 non-smoking healthy male volunteers, caffeine increased the mean clozapine AUC

(0, ) by 19% (range -14% to +97%, P = 0.05), and reduced the mean oral clearance of

clozapine by 14% (range -49% to +7%, P = 0.05). These healthy subjects ingested a single

12.5-mg dose of clozapine with caffeine (mean 550 mg per day). The effect of caffeine on

serum clozapine concentrations in the present study was more modest than in the Carrillo et

al. (1998) study, but quite similar to the effect in Hägg et al. (2000).

This interaction is best explained by the inhibitory effect of caffeine on the CYP1A2

enzyme. Although coffee-drinking has the minor effect of raising serum clozapine

concentrations in most of the patients, genetic factors, for example, may make some

individuals more sensitive to this interaction.

D I S C U S S I O N

78

7. General discussion

roblems due to drug interactions occur in about 10% of all patients, and in almost 40%

of elderly patients (Stockley 2002). Clinically the most important mechanisms of

pharmacokinetic drug interactions are those involving CYP enzymes that eliminate the

majority of drugs (Bertz & Granneman 1997). In this respect, the most important of them is

CYP3A4, followed by CYP2D6, the CYP2C family, and CYP1A2 (Bertz & Granneman

1997). All of these are involved in many clinically significant drug interactions. Problems

may arise, especially if a drug meant, for example, to treat psychosis, is metabolized mainly

by only one of these enzymes. The usual dose of a drug may lead to toxic drug

concentrations if the patient who takes the drug is either a poor metabolizer of that enzyme

(CYP2D6, CYP2C family), or the normally functional enzyme is inhibited by another drug

(or factor). Often important interactions are recognized first through case-reports when the

drug is already on the market.

These studies demonstrate that some of the generally accepted conclusions (Taylor 1997) on

the pharmacokinetic interactions of clozapine that were drawn from case-reports

(Funderburg et al. 1994, Koreen et al. 1995, Tyson et al. 1995, Cohen et al. 1996), were

incorrect. Although CYP3A4 is involved in clozapine metabolism, its contribution to overall

clozapine elimination seems to be minimal in vivo. When studied under controlled

conditions, several potent CYP3A4 inhibitors have no clinically significant effects on serum

clozapine concentrations (Raaska & Neuvonen 1998, Hägg et al. 1999b, Taylor et al. 1999,

Vandel et al. 2000, Lane et al. 2001a, Lane et al. 2001b, Özdemir et al. 2001).

Recent reports support the hypothesis presented in Studies I and III that infections may

elevate serum clozapine concentrations (van der Molen-Eijgenraam et al. 2001, Raaska et

al. 2002b, de Leon & Diaz 2003, Haack et al. 2003). In one female patient a more than 3-

fold increase in serum clozapine concentration occurred during a probable bacterial

pneumonia (Raaska et al. 2002b). During this increase, the serum concentration ratio of N-

desmethylclozapine to clozapine had decreased, implying CYP1A2 inhibition by the

infection (Raaska et al. 2002b). The Netherlands Pharmacovigilance Foundation has

received five reports of elevated serum clozapine concentrations (3- to 10-fold) during

P

D I S C U S S I O N

79

inflammation, and followed by normalization of the elevated clozapine concentrations after

recovery (van der Molen-Eijgenraam et al. 2001). In addition, five other patients with

inflammation had elevated serum clozapine concentrations (de Leon & Diaz 2003, Haack et

al. 2003).

These reports suggest that infections may raise total serum clozapine concentrations as

much as does fluvoxamine, but more reports and studies are needed to either confirm or

reject this conclusion. If confirmed, however, that would not necessarily mean that the two

interactions are similar in terms of clinical relevance. Infections elevate the concentration of

1 acid glycoprotein, which is the main clozapine-binding plasma protein. Any increase in

1 acid glycoprotein should raise total serum clozapine concentrations by causing an

increase in the protein-bound fraction. Three of the five patients reported by van der Molen-

Eijgenraam et al. (2001) developed delirium during elevated clozapine concentrations,

which suggests that probably not only the total serum concentration of clozapine, but the

free fraction as well, was increased. Infection itself, and not erythromycin as was suggested

in the two case-reports (Funderburg et al. 1994, Cohen et al. 1996), seems to be the more

likely explanation for the elevated clozapine concentrations during erythromycin treatment.

The enzyme that seems to be the most important in clozapine metabolism is CYP1A2

(Bertilsson et al. 1994, Jerling et al. 1997, Olesen & Linnet 2001). Its potent inhibition by

fluvoxamine leads to several-fold elevated serum clozapine concentrations (Hiemke et al.

1994, Koponen et al. 1996, Dequardo & Roberts 1996, DuMortier et al. 1996, Bender &

Eap 1998, Wetzel et al. 1998). This is clinically highly significant and may cause severe

adverse effects. Ciprofloxacin is a less potent CYP1A2 inhibitor than is fluvoxamine and

does not raise clozapine concentrations as strongly, but the effect of ciprofloxacin on serum

clozapine concentrations seems to be comparable to that of fluoxetine or paroxetine

(Centorrino et al. 1994b, 1996, Spina et al. 1998, 2000; Study II). Treatment with the low

250-mg twice daily dose of ciprofloxacin is, by itself, not likely to cause any clinically

significant increase in serum clozapine concentrations in most patients, but at a higher dose

or together with an infection, the overall increase in clozapine concentrations may be much

higher. Although the mean effect of coffee-drinking on serum clozapine concentration was

of low clinical relevance in Study V, coffee-drinking, or caffeine intake in general, may

D I S C U S S I O N

80

interact significantly with clozapine pharmacokinetics in patients who are heavy caffeine

consumers. This can be predicted by the relatively high theoretical inhibition percentage of

caffeine for CYP1A2 at relevant in vivo caffeine concentrations (Pelkonen et al. 1998), by

the saturable caffeine metabolism in doses that are commonly ingested (Kaplan et al. 1997),

and by the large interindividual variability in CYP1A2 activity (Butler et al. 1989).

Taking into consideration the studies and reports of pharmacokinetic interactions involving

clozapine and clozapine metabolism, it seems that any drug or medical condition that

inhibits or induces CYP1A2 activity significantly can have clinically relevant effects on

serum clozapine concentrations. The isoenzymes CYP2C19, CYP2D6, and CYP3A4 seem

to be normally of minor clinical importance. However, if CYP1A2 activity is reduced by a

potent inhibitor such as fluvoxamine, or if its capacity is saturated by CYP1A2 substrate

overdose (e.g., clozapine or caffeine), CYP3A4, especially, may become a significant

contributor to clozapine metabolism.

Several characteristics of clozapine favor the utilization of TDM in dose optimization.

Clozapine has a relatively low therapeutic index, and it is not uncommon for patients on

therapeutic clozapine concentrations to complain of excessive sedation or salivation. Some

patients, however, tolerate well clozapine concentrations toxic to most other patients, but

maintaining an unnecessary high concentration keeps patients at increased risk for such

complications as seizures. Some patients metabolize clozapine very effectively, and their

clozapine concentrations may be too low, well below the suggested therapeutic threshold,

even if daily doses are high. For them, it may be necessary to use daily doses above 900 mg,

the recommended upper limit for a daily dose of clozapine in manufacturers’ product

labeling (Leponex®, Novartis, in the Finnish pharmacopea Pharmaca Fennica 2003). When

the optimal dose for a patient has been established, clozapine concentration should be

monitored annually, and when there is reason to suspect pharmacokinetic interaction or poor

compliance, or if a patient’s medical condition changes in such a way as to affect the

distribution or clearance of clozapine.

C O N C L U S I O N S

81

CONCLUSIONS

The following conclusions can be drawn from these five studies:

1. Itraconazole, and most likely also other CYP3A4 inhibitors, has no clinically significant

effect on serum concentrations of clozapine and its active metabolite N-

desmethylclozapine. Thus, those case-reports in which the CYP3A4 inhibitor

erythromycin seems to have caused elevated serum concentrations of clozapine have

been interpreted erroneously. Probably the infection itself, and not erythromycin, led to

the higher clozapine concentrations.

2. A low dose of the modest CYP1A2 inhibitor ciprofloxacin has a modest effect in

increasing the serum concentrations of clozapine and its metabolite N-

desmethylclozapine.

3. Influenza vaccination has no clinically significant effect on serum concentrations of

clozapine and its metabolites N-desmethylclozapine and clozapine-N-oxide.

4. Risperidone has no clinically significant effect on serum concentrations of clozapine.

5. Coffee-drinking, probably through inhibition of CYP1A2 enzyme-mediated clozapine

metabolism by caffeine, has generally a minor effect toward an increased serum

concentration of clozapine and of its metabolite N-desmethylclozapine.

A C K N O W L E D G E M E N T S

82

ACKNOWLEDGEMENTS

his work was carried out at the Department of Clinical Pharmacology, University of

Helsinki. I am grateful to all who have contributed to my work or well-being during

these years.

I am most grateful to Professor Pertti Neuvonen for his enthusiastic and excellent

supervision. He always had time for me whenever I needed it. I feel myself privileged and

happy to have had the opportunity to be part of his inspiring research group, for which he

has created such good facilities and a cozy atmosphere. The high standard he has set himself

as a researcher and a teacher is admirable and unparalleled.

Docents Arja Rautio and Kimmo Kuoppasalmi deserve grateful acknowledgement for their

valuable review and constructive comments on this thesis.

I am greatly indebted to my colleague Virpi Raitasuo who is a co-worker in three of these

studies. Virpi was the main figure in encouraging my interest in psychopharmacology when

I was taking my first steps as a toddler in the field of psychiatry in the fall of 1994 at

Kellokoski Hospital. During that time I finally made up my mind that I wanted to become a

psychiatrist. I am grateful for her collaboration and support.

I thank all my colleagues at the Department of Clinical Pharmacology, including Docents

Kari Kivistö, Janne Backman, and Lasse Lehtonen, plus Mikko Niemi, Jari Lilja, Laura

Juntti-Patinen, Tiina Varis, Carl Kyrklund, Kati Ahonen, Heli Malm, Harri Luurila, Maija

Kaukonen, Kirsti Villikka, Jun-Sheng Wang, Xia Wen, Eeva Lukkari-Lax, Outi Lapatto-

Reiniluoto, Seppo Kähkönen, Elina Saarenmaa, Matti Kivikko, Mika Jokinen, Vilja

Palkama, Tommi Lamberg, Teemu Kantola, Marika Granfors, Lauri Kajosaari, Samuel

Fanta, Tiina Jaakkola, Tuure Saarinen, Aleksi Tornio, Marjo Karjalainen, Aino Koskinen,

and Marja Pasanen. I express my warmest thanks to Tuija Itkonen for her help and ingenuity

in many practical matters.

T


83

These works could not have been done without the help of the skillful technical staff of the

Department of Clinical Pharmacology. I am indebted to Jouko Laitila, Kerttu Mårtensson,

Eija Mäkinen-Pulli, Mikko Neuvonen, and Lisbet Partanen.

I express my gratitude for their support and encouragement to the former Heads of

Kellokoski Hospital Docent Ilkka Taipale and Dr. Raimo Väisänen, and to Dr. Grigori Joffe

who now heads the hospital. I thank all my colleagues in Kellokoski Hospital. Special

thanks go to Docent Eero Elomaa who introduced me to the mysteries of clozapine, and

encouraged me to do research. He also introduced me to Professor Neuvonen. I am greatly

indebted to the staff of Kellokoski Hospital. Kaija Leppäniemi and Soili Sarvaala from the

hospital laboratory, and Ilkka Raitasuo are especially warmly acknowledged for their

contributions. The list of other contributers would be almost endless.

The ones that leave me humble are the study subjects, the patients at Kellokoski Hospital

who participated in these studies. I want to express to them my deepest gratitude. They have

really taught me a great amount.

I am grateful to all colleagues and staff at the Department of Psychiatry for their support and

encouragement, especially Professors Ranan Rimón, Matti Virkkunen, Björn Appelberg,

Acting Professor Heikki Katila, Docents Heikki Vartiainen, Antero Leppävuori, Timo

Partonen, and Matti Huttunen, Dr. Teija Honkonen, and all of those on the Mieliala- and

Psykosomatiikka teams are warmly acknowledged for teaching me psychiatry.

Many thanks go to Docent Kalle Hoppu for his help in professional and private matters, and

to the staff of the Poison Information Centre. I am grateful to Professor Eija Kalso at the

Pain Clinic for stimulating discussions.

Impressively energetic Carol Norris, my neighbor and my teacher, is warmly acknowledged

for author-editing the English language of this thesis.

This work was financially supported by the Clinical Drug Research Graduate School, the

Helsinki University Central Hospital Research Fund, the National Technology Agency of


84

Finland (TEKES), Oy H. Lundbeck Ab, and Oy Eli Lilly Finland Ab, all of which are

gratefully acknowledged.

I thank all my friends who have been invaluable to me during these years. Among them,

especially Jukka and Sirpa Sahlakari, and Tuomo and Anna-Maija Heikinheimo, have been

very important. Special thanks go to my friends Kurt Tolliver and Mike Schaeffer across the

ocean. I have enjoyed being a part of the marathon team Sankarpojat, and it has been a great

pleasure to be involved in the activities of my son’s soccer team KOPSE Kojootit P92.

My mother-in-law Suoma Takala has been most invaluable during these years taking care of

our children. We could not have managed without her helping hand. Thank you so much,

Suoma! I am grateful to my late father-in-law Aarne Takala. I thank my sister-in-law Annu

Kauppinen, her husband Juuso and their children Anni, Tuomo, Aleksi, and Joonas for their

friendship and good times spent together.

I am most grateful to my parents Marja-Liisa and Jorma Raaska for their love and support,

and I thank them for providing me a happy childhood, which is the greatest thing anyone

can receive from their parents. I thank my sister Maria Inkovaara, her husband Pekka and

their children Ronja and Rasmus for their friendship and good times spent together. My

grandparents are acknowledged with deep gratitude and respect. I also thank my other

relatives who have had an important impact on my life.

Finally, I thank my wonderful wife Hanna and our children Eveliina, Antti, and Iida with

my deepest love and gratitude. Hanna, words cannot tell how thankful I am to you. Eveliina,

Antti, and Iida, thank you for being what you are; you bring me so much joy and happiness.

I know many others deserve to be personally acknowledged. I first express my apologies

and then my thanks to all of you.

Vantaa, October 2003

Kari Raaska

R E F E R E N C E S

85

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