P-glycoprotein and chiral antidepressant drugs...

Linköping University Medical Dissertations

No. 1283

P-glycoprotein and chiral antidepressant drugs:

Pharmacokinetic, pharmacogenetic and toxicological aspects

Louise Karlsson

Division of Drug Research - Clinical Pharmacology

Department of Medical and Health Sciences

Faculty of Health Sciences

Linköping University

Linköping 2012

MAIN SUPERVISOR

Fredrik C. Kugelberg, Associate Professor

Division of Drug Research – Forensic Toxicology, Department of Medical and Health

Sciences, Linköping University, Linköping, Sweden

CO-SUPERVISORS

Finn Bengtsson, Professor

Division of Drug Research – Clinical Pharmacology, Department of Medical and Health


Johan Ahlner, Professor

Division of Drug Research – Forensic Toxicology, Department of Medical and Health


FACULTY OPPONENT

Aleksander A. Mathé, Professor

Department of Clinical Neuroscience, Karolinska Institute, Stockholm, Sweden

© Louise Karlsson, 2012

Published articles have been reprinted with permission from the publishers.

Paper I © 2010

Paper II © 2011

Cover: 3D structure of P-glycoprotein obtained from Entrez´s 3D structure

database MMDB (ID70761)

Printed in Sweden by LiU-Tryck, Linköping, 2012.

ISBN: 978-91-7393-003-1

ISSN: 0345-0082

“IF YOU CAN DREAM IT, YOU CAN DO IT”

Walt Disney

TABLE OF CONTENTS

PAPERS IN THE THESIS ............................................................................. 7

ABSTRACT ..................................................................................................... 9

POPULÄRVETENSKAPLIG SAMMANFATTNING ............................. 11

ABBREVIATIONS ....................................................................................... 13

INTRODUCTION ......................................................................................... 15

PSYCHOPHARMACOLOGY....................................................................................... 15

TOXICOLOGY ........................................................................................................... 19

CHIRALITY ............................................................................................................... 20

CHIRAL ANTIDEPRESSANT DRUGS ........................................................................ 22

Venlafaxine ................................................................................................................................ 22

Citalopram and Escitalopram ................................................................................................. 25

BLOOD-BRAIN BARRIER ......................................................................................... 27

ABC transport proteins ............................................................................................................ 29

P-GLYCOPROTEIN .................................................................................................... 31

In vitro and in vivo P-gp studies ............................................................................................ 33

AIMS OF THE THESIS ............................................................................... 35

MATERIALS AND METHODS ................................................................. 37

ANIMAL STUDIES (PAPERS I-III) .......................................................................... 37

Animals ....................................................................................................................................... 37

Drug administration and sample collection ......................................................................... 37

Acute administration ........................................................................................................... 37

Chronic administration ....................................................................................................... 38

Chiral determination of drugs ................................................................................................. 38

Venlafaxine and metabolites .............................................................................................. 38

Citalopram and metabolites .............................................................................................. 39

Open-field behavior (Paper II) ............................................................................................... 40

Statistics ...................................................................................................................................... 41

Ethics committee permission .................................................................................................. 41

FORENSIC STUDY (PAPER IV)................................................................................ 42

Case selection............................................................................................................................. 42

Genotyping ................................................................................................................................. 42

Statistics ...................................................................................................................................... 43

Ethics committee permission .................................................................................................. 43

RESULTS AND DISCUSSION ................................................................... 45

ANIMAL STUDIES (PAPERS I-III) ........................................................................... 45

Pharmacokinetics ...................................................................................................................... 45

Pharmacodynamics ................................................................................................................... 51


CONCLUSIONS ............................................................................................ 59

ANIMAL STUDIES (PAPERS I-III) ........................................................................... 59


FUTURE ASPECTS ..................................................................................... 61

ACKNOWLEDGEMENTS .......................................................................... 63

REFERENCES .............................................................................................. 67

APPENDIX (PAPERS I-IV) ......................................................................... 89

7

PAPERS IN THE THESIS

This thesis is based on the following papers, which will be referred to by their

Roman numerals:

I. Blood-brain barrier penetration of the enantiomers of venlafaxine and its metabolites in mice lacking P-glycoprotein. Karlsson L, Schmitt U, Josefsson M, Carlsson B, Ahlner J,

Bengtsson F, Kugelberg FC and Hiemke C.

European Neuropsychopharmacology 20: 632-640, 2010.

II. Effects on enantiomeric drug disposition and open-field behavior after chronic treatment with venlafaxine in the P-glycoprotein knockout mice model. Karlsson L, Hiemke C, Carlsson B, Josefsson M, Ahlner J,

Bengtsson F, Schmitt U and Kugelberg FC.

Psychopharmacology 215: 367-377, 2011.

III. Altered brain concentrations of citalopram and escitalopram in P-glycoprotein deficient mice after acute and chronic treatment. Karlsson L, Carlsson B, Hiemke C, Ahlner J, Bengtsson F, Schmitt U.

and Kugelberg FC.

Submitted.

IV. ABCB1 gene polymorphisms in forensic autopsy cases positive for citalopram and venlafaxine. Karlsson L, Green H, Zackrisson AL, Bengtsson F, Jakobsen Falk I,

Carlsson B, Ahlner J and Kugelberg FC.

Submitted.

8

Other publications that are not included in the present thesis, but that are related:

1. Stereoselective determination of venlafaxine and its three demethylated metabolites in human plasma and whole blood by liquid chromatography with electrospray tandem mass spectrometric detection and solid phase extraction. Kingbäck M, Josefsson M, Karlsson L, Ahlner J, Bengtsson F,

Kugelberg FC and Carlsson B.

Journal of Pharmaceutical and Biomedical Analysis 53: 583-590, 2010.

2. Influence of CYP2D6 genotype on the disposition of the enantiomers of venlafaxine and its three major metabolites in postmortem femoral blood. Kingbäck M, Karlsson L, Zackrisson AL, Carlsson B, Josefsson M,

Bengtsson F, Ahlner J and Kugelberg FC.

Forensic Science International, 214: 124-34, 2012.

9

ABSTRACT

The blood-brain barrier (BBB) is formed by the capillary endothelial cells,

joined together by tight junctions, with transporter proteins. BBB acts to

regulate the brain concentrations of substances including many drugs.

Transport across the cells is necessary for a drug to ensure that the drug

reaches the site of action and transport proteins such as P-glycoprotein (P-gp;

ABCB1) can limit the entrance into various tissues, including the brain.

Molecules that are not superimposable on their mirror images and thus

exist in two enantiomeric forms (enantiomers) are said to be chiral. A racemic

compound is one composed of a 50:50 mixture of two enantiomers, S- and R-

enantiomers. Two examples of frequently prescribed racemic drugs are the

chiral antidepressants venlafaxine (VEN) and citalopram (CIT). The

enantiomers of VEN possess different pharmacodynamic profiles where the

R-enantiomer is a potent inhibitor of both serotonin and noradrenaline

reuptake (SNRI), while the S-enantiomer is more selective in inhibiting

serotonin reuptake (SSRI). The SSRI effect of CIT resides in the S-

enantiomer, whereas the R-enantiomer is considered to be therapeutically

inactive, or even that it counteracts the effects. The S-enantiomer of CIT is

now available as a separate SSRI (escitalopram, EsCIT). VEN and CIT are

also among the most commonly found drugs in forensic autopsy cases.

Few previous studies have examined a possible enantioselective activity of

P-gp. Thus, the general aim of this thesis was to study the enantiomeric

distribution of chiral antidepressant drugs, focusing on the role of P-gp in the

BBB. For this purpose, a mouse model disrupted of the genes coding for P-gp

(abcb1ab (-/-) mice) was used. Brain and serum concentrations of the

enantiomers of VEN and CIT, and their major metabolites, were compared to

the corresponding wild-type mice (abcb1ab (+/+) mice). The open-field

locomotor and rearing activities were examined after chronic VEN

administration. In addition to the animal studies, genetic and toxicological

aspects of P-gp were studied in a forensic autopsy material, where intoxication

cases were compared with cases that were not related to intoxications.

10

The brain to serum concentration ratios for VEN, CIT and EsCIT differed

between knockout mice and wild-type mice, with 2-3 fold higher brain

concentrations in mice with no expression of P-gp. Hence, all studied drugs,

and their major metabolites, were substrates for P-gp. There was no evidence

for a stereoselective P-gp mediated transport. The P-gp substrate properties

were reflected in the open-field behavior test where the knockout mice

displayed increased center activity compared with wild-type mice following

chronic VEN exposure. The genotype distribution of ABCB1 SNPs C1236T,

G2677T and C3435T in VEN positive cases was significantly (or borderline)

different between the intoxication cases and the non-intoxication cases. This

difference in genotype distribution was not observed for the CIT positive

cases.

To conclude, the present work has led to an increased knowledge about

how the enantiomers of VEN and CIT are affected by the BBB transporter P-

gp. Using an animal model, VEN and CIT have proved to be actively

transported out of the brain by P-gp and no difference was observed for the

enantiomers with regard to P-gp transport. Further, the ABCB1 genotype

distribution was different in intoxication cases compared with non-

intoxication cases. Taken together, these findings offer the possibility that the

expression of P-gp in humans may be a contributing factor for limited

treatment response and increased risk of side-effects following antidepressant

drug treatment.

11

POPULÄRVETENSKAPLIG SAMMANFATTNING

För att ett läkemedel ska kunna ge den önskade effekten krävs att läkemedlet

kan ta sig till sin målcell. Kroppens organ, särskilt hjärnan, skyddas av olika

barriärer, och för att passera dessa måste ett läkemedel ta sig över dessa

barriärer.

Vid aktiv transport pumpas läkemedelsmolekylerna genom cellmembranet

med hjälp av transportproteiner som sitter i membranet. I kroppen finns en

mängd olika transportproteiner som hjälper till att ta upp ämnen från blodet

och att göra sig av med nedbrytningsprodukter och gifter. Denna avhandling

fokuserar på transportproteinet P-glykoprotein som är en medlem av ABC-

transportörerna, en av de största familjerna av transportproteiner. Dessa har

en viktig roll i att påverka hur läkemedel tar sig från blodet och hur de

fördelas i kroppen, t.ex. tar sig över blod-hjärnbarriären för att kunna ge

önskad effekt.

Vissa läkemedel finns i olika former, s.k. enantiomerer vilka är spegelbilder

som förhåller sig till varandra som höger hand förhåller sig till vänster hand.

Dessa enantiomerer kan ha olika effekt i kroppen. Exempel på sådana

läkemedel är de antidepressiva medlen venlafaxin och citalopram som ges som

racemat (50:50 blandning av de två formerna). I dagsläget är det okänt hur

flera av dessa enantiomerer omsätts i kroppen, hur de transporteras över

barriärer och vilka deras separata kliniska effekter är.

Om man tar flera läkemedel samtidigt, kan dessa påverka varandras effekter

men även hämma transporten, vilket kan leda till att läkemedlet inte når sitt

mål och att effekten uteblir. Hämning av transporten kan också leda till att

läkemedlet stannar i vävnaden och att man får för hög koncentration, vilket

kan leda till allvarliga biverkningar och i värsta fall till att man avlider. Både

venlafaxin och citalopram är vanligt förekommande i rättsmedicinska

obduktionsfall. Det är därför både av kliniskt och toxikologiskt intresse att

studera dessa läkemedel.

Avhandlingen har gett värdefull information om hur olika former

(enantiomerer) av ett läkemedel påverkas av transportören P-glykoprotein.

Med hjälp av en djurmodell (mus) har de antidepressiva läkemedlen venlafaxin

12

och citalopram visat sig aktivt transporteras ut ur hjärnan av P-glykoprotein,

samt att det inte föreligger någon skillnad i hur enantiomererna transporteras.

Ett annat fynd är att fördelningen av olika genetiska varianter i genen som

kodar för P-glykoprotein, ABCB1, skilde sig åt mellan individer som avlidit till

följd av förgiftning och individer som avlidit av annan dödsorsak. Vad denna

skillnad beror på, eller betyder, har vi idag ingen förklaring till utan detta

behöver studeras ytterligare.

Sammanfattningsvis har avhandlingen resulterat i kunskap som kan leda till

ökad förståelse för antidepressiva läkemedels kliniska och toxikologiska

effekter och varför vissa personer inte svarar tillfredställande på

läkemedelsbehandling. Detta är av största betydelse för såväl den enskilde

individen som för samhället.

13

ABBREVIATIONS

The most important abbreviations used in this thesis are listed below:

ABCB1 human P-glycoprotein gene

abcb1ab rodent P-glycoprotein genes

ATP adenosine triphosphate

BBB blood-brain barrier

CIT citalopram

CNS central nervous system

CYP cytochrome P450

DCIT demethylcitalopram

DDCIT didemethylcitalopram

DDV N,O-didesmethylvenlafaxine

EsCIT escitalopram

HPLC high performance liquid chromatography

LC-MS/MS liquid chromatography tandem mass spectrometry

LD50 drug dose that is lethal to 50% of an animal species

MAO monoamine oxidase

M/P metabolite/parent drug ratio

NDV N-desmethylvenlafaxine

ODV O-desmethylvenlafaxine

P-gp P-glycoprotein

SEM standard error of the mean

SNP single nucleotide polymorphism

S/R S-enantiomer/R-enantiomer ratio

SNRI serotonin and noradrenaline reuptake inhibitor

SPE solid-phase extraction

SSRI selective serotonin reuptake inhibitor

t½ half-life

VEN venlafaxine

INTRODUCTION | 15

INTRODUCTION

Psychopharmacology

Psychopharmacology can be described as the study of the actions of drugs and

their effects on the mind and behavior. The field of psychopharmacology

covers a wide range of substances with various psychoactive properties and

includes, for example, antidepressants, antipsychotics, benzodiazepines,

hypnotics and stimulants. Psychoactive drugs interact with specific target sites

or receptors found in the central nervous system (CNS) to induce changes in

physiological or psychological functions. Drugs are studied for their

physiochemical properties, physical and psychological side effects. Research

within this field aims to increase the knowledge of the effect of CNS drugs in

order to improve the use of commercially available drugs but also to promote

the development of new drugs.

For drugs acting on the CNS, such as antidepressants, increased awareness

regarding the very variable individual responses to treatment have been

focused on in recent years. Despite the fact that psychoactive drugs are widely

used in psychiatry, limited knowledge about the actual effects and turnover in

the body is available. This is of particular importance for antidepressants,

where as many as 3045% of patients do not respond satisfactorily to

16 | INTRODUCTION

treatment (Fava, 2000). It is of both clinical and toxicological value to increase

the knowledge about these drugs.

Pharmacodynamics can be described as the study of the effects of drugs on

the body, the mechanisms of drug action and the relationship between drug

concentration and effect. The receptors are the major sites of drug actions, to

mediate activating (agonist) or inhibiting (antagonist) effects. Psychoactive

drugs exert their effects almost entirely through by acting on

neurotransmitters and modifying one or more aspects of synaptic

transmission. Neurotransmitters are endogenous chemicals that transmit

signals from a neuron to a target cell across a synapse, and psychoactive drugs

affect this “communication”. Antidepressants are used for the treatment of

various affective psychiatric disorders, such as major depressive disorder and

panic disorder. The most commonly antidepressants include monoamine

oxidase inhibitors (MAOIs), tricyclic antidepressants (TCAs), selective

serotonin reuptake inhibitors (SSRIs) and serotonin-noradrenalin reuptake

inhibitors (SNRIs). MAOIs inhibit the degradation of the neurotransmitters

serotonin, noradrenaline and dopamine by inhibiting the enzyme MAO,

leading to increased concentrations of the neurotransmitters in the brain.

TCAs prevent the reuptake of neurotransmitters, including serotonin,

noradrenaline and, to less extent, dopamine. Nowadays the most common

antidepressants are the SSRIs, which prevents the uptake of serotonin (e.g.

citalopram; CIT), and newer SNRIs, which prevent the uptake of both

serotonin and noradrenaline (e.g. venlafaxine; VEN).

Pharmacokinetics explores what the body does to a drug. Pharmacokinetics

can be divided into four basic factors: absorption, distribution, metabolism

and excretion. In order for a drug to obtain the desired effect, the

concentration in the target organ is decisive. In other words, after oral

administration, the drug is absorbed through the digestive system, into the

bloodstream and distributed to the target organ. Before the drug reaches the

systemic circulation, the drugs undergo extensive first-pass (phase I) and

second-pass (phase II) metabolism in the liver (Figure 1). First-pass reactions

involve hydrolysis, reduction and oxidation. The second-pass reaction is a

synthetic reaction and includes glucuronidation, sulfation, acetylation,

INTRODUCTION | 17

methylation and conjugation with amino acids. Phase I reactions usually result

in more reactive, and sometimes more toxic products, whereas phase II

reactions usually result in inactive products. The drug and the products are

then excreted by either renal excretion in the kidneys, the hepatobiliary system

or by the lungs (Tozer and Rowland, 2006).

Figure 1. Schematic illustration of drug metabolism.

Pharmacogenetics has its origins in the 1950s, with discoveries that

demonstrated the influence of genetics on drug response (Hockwald et al.,

1952, Alving et al., 1956, Lehmann and Ryan, 1956, Kalow and Staron, 1957,

Hughes et al., 1954). Historically, pharmacogenetics has been used in a wide

term and refers to any influence that genetics may have on drug therapy.

Today, pharmacogenetics is the study of genetic variations and the effect on

pharmacokinetics and pharmacodynamics. The newer term pharmacogenomics

refers to the general study of genes that affect drugs. However, the terms are

often used interchangeably.

Polymorphism is defined as the presence of two or more variants (e.g.

alleles and phenotypes) in a population at a high frequency, usually 1%

(Meyer, 2000), a definition accepted by most scientists. Most genetic

polymorphisms are single nucleotide polymorphisms (SNPs) and can consist

of a base pair substitution, nucleotide insertion or deletion. SNPs may fall

Phase-I

Metabolite(s) oxidation

Urinary

excretion

DRUG

Phase-II

Metabolite(s) conjugation

18 | INTRODUCTION

within coding sequences of genes, non-coding regions of genes or in

intergenic regions. When these polymorphisms occur in genes encoding drug

metabolizing enzymes or transporters, it may change the disposition of the

drug and, as a result, its efficacy may be compromised or its toxicity altered.

The cytochrome P450 superfamily (CYP) is a large and diverse group of

enzymes. The function of most CYP enzymes is to catalyze the oxidation of

organic substances (Phase I reactions). Human CYP enzymes are the major

enzymes involved in drug metabolism and account for >75% of the total drug

metabolism. In humans, 57 CYP genes have been identified so far (Nelson et

al., 2004) and five account for >95% of the reactions. The proteins can be

divided into families (>40% amino acid identity), subfamilies (>55% amino

acid identity) and isoforms. Each gene encoding CYP enzyme, and the

enzyme itself, is designated with the abbreviation CYP, followed by an Arabic

number indicating the family, a capital letter indicating the subfamily and a

second number indicating the isoform (e.g. CYP2C19). Most of the CYPs

belonging to families 1-3 are polymorphic, with the exception of CYP1A1,

CYP2E1 and CYP3A4, and they are considered to be relatively well conserved

(Ingelman-Sundberg, 2004). Considering the metabolism of antidepressant

drugs, CYP1A2, CYP2C19, CYP2D6 and CYP3A4 are the most important

(Brosen, 1996, Kirchheiner et al., 2001, Nemeroff et al., 1996, Tanaka and

Hisawa, 1999).

When a species diverges into two separate species, the copies of a single

gene in the two resulting species are said to be orthologous. Orthologs, or

orthologous genes, are genes in different species that originated by vertical

descent from a single gene of the last common ancestor, and the term

”ortholog” was coined in 1970 by Walter Fitch (Fitch, 1970). Mouse models

are often used to study human genes because it is assumed that the expression

and function for most orthologous genes are similar between the two species.

The mouse cyp2c subfamily consists of 18 members and is considered to be

the most complex subfamily of all CYPs found in human and animals. So far,

no mouse isoform has been suggested to be the orthologue of human

CYP2C19. Few studies have been performed to characterize the cyp2d family

in mice but there are at least nine mouse cyp2d genes (Nelson et al., 2004).

INTRODUCTION | 19

The isoform cyp2d22 has been suggested to be the orthologue of human

CYP2D6 (Blume et al., 2000). In mice, there are six cyp3a isoforms identified

so far and of the mouse isoforms, cyp3a11 is the isoform most similar to

human CYP3A4 (Yanagimoto et al., 1992).

Inter-individual variability in the activity of the CYP enzymes has clinical

and toxicological implications for drug response. CYP2D6 is one of the most

important enzymes involved in the metabolism of CNS active drugs, including

antidepressants. The CYP2D6 gene is highly polymorphic. The lack of

functional activity of CYP2D6 may lead to an impaired drug metabolism (PM,

poor metabolizers) resulting in high plasma drug concentrations, lack of

therapeutic response and/or an increased risk of side-effects. Another risk

group is individuals who have a substantially increased metabolism, e.g. due to

more than two functional copies of the CYP2D6 gene, which can lead to

therapeutic failure. These so-called ultra-rapid metabolizers (UMs) may

require higher doses than usual to reach therapeutic drug levels (Dahl et al.,

1995). The genetic variability of CYP-enzymes makes the toxicity of many

compounds differ between individuals.

Toxicology

Toxicology is the study of the adverse effects of chemicals; the relationship

between dose and its effects on the exposed organism is of high significance

in toxicology. All substances are toxic under the right conditions. The term

LD50 refers to the dose of a toxic substance that kills 50% of a test population

(usually rodents or other animals). Toxicology is an inter-disciplinary science

that integrates the principles and methods of many fields: chemistry, biology,

pharmacology, molecular biology, physiology and medicine.

Forensic toxicology is the use of toxicology and other disciplines, such as

analytical chemistry, pharmacology and clinical chemistry, to aid medical or

legal investigation of death, poisoning and drug use. Determining the

substance ingested is often complicated by the body’s natural processes

(absorption, metabolism, distribution and excretion), as it is rare for a

chemical to remain in its original form once inside the body. In postmortem

toxicology, the analytical results can, in some cases, confirm a suspected

20 | INTRODUCTION

intoxication and in other cases, exclude the presence of drugs (e.g. in a driver

involved in an accident that proved to be fatal).

The interpretation of postmortem toxicological results is not always an easy

task, due to a number of pitfalls (Drummer, 2007, Pounder and Jones, 1990,

Skopp, 2010). For many substances there is no sharp dividing line between a

toxic and a lethal drug concentration (Ferner, 2008). When a drug exists as a

racemic mixture, the picture is even more complicated (Hutt, 2007, Lu, 2007,

Smith, 2009). In addition, postmortem genotyping and the analysis of

metabolite/parent drug ratios have been suggested to provide insights into the

interpretation of forensic toxicological results (Druid et al., 1999, Musshoff et

al., 2010, Sajantila et al., 2010, Wong et al., 2003). Adding information about

an individual’s metabolic capacity and ability to transport drugs across barriers

may possibly facilitate the interpretation of postmortem results.

Chirality

Chiral chemistry was discovered by Louis Pasteur when, in 1848, he separated

the two isomers of sodium ammonium tartrate for the first time. He found

that the two isomers were identical in physic-chemical properties, with the

exception of their ability to rotate plane polarized light. In 1874, van ’t Hoff

provided the explanation by hypothesizing that the carbon atom had a

tetrahedral structure and that two configurations are possible that are mirror

images of each other. Such compounds are said to be “chiral” (Brocks and

Jamali, 1995).

Stereoisomers are compounds made up of the same atoms, bonded by the

same sequence of chemical bonds but possess different three-dimensional

structures which are not interchangeable. Stereoisomers are divided into

enantiomers, which are mirror images of each other, and diastereomers, which are

molecules without mirror images. A molecule that is not identical to its mirror

image and does not contain a plane of symmetry is chiral. A racemate is a

racemic mixture of an equal amount of an enantiomeric pair of the chiral

molecule. The atom that carries four different substituents is called the

asymmetric, or stereogenic, centre and the individual enantiomers are

characterized by different absolute configurations around this centre, denoted

INTRODUCTION | 21

S- (sinister) or R- (rectus) configuration. The enantiomers are related to each

other as a right hand is related to a left hand (Figure 2). The enantiomers of a

chiral molecule have the same physical properties in all but their ability to

rotate the plane of polarized light in equal but opposite directions,

levorotatory rotation (-) and dextrorotatory rotation (+).

Figure 2. A pair of enantiomers. This figure illustrates how they are related to each other -

as the right hand is related to the left hand.

Even if the enantiomers have identical physical properties, the

pharmacokinetic and pharmacodynamic properties may differ (Figure 3). The

sedative drug thalidomide (Neurosedyn), which was introduced in the late

1950s, is one well-known example of a racemate where the enantiomers

display different pharmacodynamic properties (different biological effects),

where the R-enantiomer is sedative while the S-enantiomer is teratogenic

(Heger et al., 1994, Hoglund et al., 1998). The drug was withdrawn from the

market in 1961 due to teratogenicity resulting in severe birth defects. By then,

5,000-6,000 children from 46 countries had been born with various external

and internal deformities, (including phocomelia, deafness, facial and

oculomotor paralysis, and cardiac, uterine and vaginal malformation) (Mellin

and Katzenstein, 1962).

22 | INTRODUCTION

Figure 3. Enantiomeric interaction possibilities of enantiomer 1 and 2.

Chiral antidepressant drugs

A well-known problem in modern psychopharmacology is the existence of

chiral drugs composed of enantiomers which may differ in their

pharmacokinetic and pharmacodynamic properties (Baker and Prior, 2002,

Baumann et al., 2002, Howland, 2009). Many of the antidepressants, as well as

their metabolites, are chiral drugs. The most common are citalopram,

venlafaxine, fluoxetine, paroxetine and sertraline. Citalopram, venlafaxine and

fluoxetine are racemic mixtures but in the cases of paroxetine and sertraline,

only the enantiomer with the most pronounced activity is marketed as an

antidepressant.

Venlafaxine

In the early 1980s, venlafaxine (VEN) was first synthesized and found to

inhibit the uptake of serotonin and, with lower potency, noradrenaline (Muth

et al., 1986). VEN was shown to have in vivo activity in animal models of

depression and to have little affinity for muscarinic or histaminergic

postsynaptic receptors (Muth et al., 1986), and was predicted to have a better

tolerability profile than the TCAs.

Enantiomer 1

Desirable effect

Enantiomer 2

Other not wanted effects

Enantiomer 2

No effect

Enantiomer 2

Antagonist to 1

Enantiomer 2

Desirable effect

Enantiomer 2

Desirable additive effect

INTRODUCTION | 23

VEN is a bicyclic phenylethylamine compound and has a chiral centre,

which gives a racemic mixture of two enantiomers (Figure 4): S-VEN and R-

VEN (Ellingrod and Perry, 1994). VEN belongs to the pharmacodynamic

class of SNRIs, but is also a weak inhibitor of dopamine reuptake in vitro

(Muth et al., 1986). VEN is used for the treatment of psychiatric disorders

(Holliday and Benfield, 1995). Some argue that VEN is essentially a SSRI at

the lower therapeutic dosage (i.e. 75 mg/day) and that the noradrenergic

effect is recruited as the dose is increased (Kelsey, 1996). The two

enantiomers display different pharmacodynamic profiles. The R-enantiomer is

a potent inhibitor of both serotonin and noradrenaline reuptake while the S-

enantiomer is a more selective inhibitor of serotonin reuptake (Holliday and

Benfield, 1995).

Figure 4. The chemical structure of the S- and R-enantiomers of venlafaxine (VEN).

VEN undergoes extensive first-pass metabolism in the liver, mainly by the

CYP enzymes into its major active metabolite O-desmethylvenlafaxine

(ODV). Two minor metabolites are N-desmethylvenlafaxine (NDV) and

N,O-didesmethylvenlafaxine (DDV). The known major pathway for

metabolism is illustrated in Figure 5. In humans, VEN is mainly metabolized

by CYP2D6 and to a lesser extent by CYP2C19, to ODV and mainly by

CYP3A4 to NDV. The two metabolites (ODV and NDV) are then further

metabolized to DDV, possibly by CYP2D6 (Fogelman et al., 1999, McAlpine

et al., 2011, Muth et al., 1991, Muth et al., 1986, Otton et al., 1996). ODV

inhibits the reuptake of serotonin and noradrenaline with a similar potency to

that of VEN but no data are available on the relative efficiency of the two

enantiomers. NDV and DDV may also be considered as pharmacologically

active, but they are claimed to be less potent than the parent drug (Muth et al.,

1991, Muth et al., 1986).

24 | INTRODUCTION

Figure 5. Metabolism of venlafaxine to its main metabolites O-desmethylvenlafaxine, N-

desmethylvenlafaxine and N,O-didesmethylvenlafaxine by the cytochrome P450 enzymes.

In humans, ODV is present at higher plasma concentrations than VEN

itself (Howell et al., 1993). Metabolite to parent drug ratios in TDM samples

are 0.3-5.2 (ODV/VEN) (Hiemke et al., 2011). In humans, the time to peak

plasma/serum concentrations for VEN and ODV are 1-2 h and 4-5 h

respectively. The corresponding data in mice are 0.5-1 h and 0.5-1 h for VEN

and ODV respectively. The mean half life (t½) of VEN is 5 h in humans and

1 h in mice. For ODV, the t½ is 11 h in humans and 1 h in mice (Howell et

al., 1994).

Desvenlafaxine, a synthetic form of the isolated major active metabolite of

VEN (i.e. ODV), was introduced in the US in 2008 as a racemate and

approved for the treatment of depression (Perry and Cassagnol, 2009). Like

VEN, desvenlafaxine is categorized as a SNRI and was developed in hope of

improving the strengths of the parent drug. Desvenlafaxine is approximately

INTRODUCTION | 25

10 times more potent at inhibiting serotonin uptake than noradrenaline uptake

(Deecher et al., 2006).

The side-effect profile of VEN has been found to be superior to the TCAs,

although not quite as favorable as the SSRIs (Preskorn, 1995). However,

serious side effects have been observed after the administration of high doses

of VEN (Ereshefsky, 1996) and fatal overdoses have been reported for VEN

alone or in combination with other compounds (Mazur et al., 2003, Settle,

1998). In a study with Finnish postmortem data, VEN was suggested to have

a higher toxicity compared with SSRIs (Koski et al., 2005).

Citalopram and Escitalopram

Citalopram (CIT) is a bicyclic phthalane derivate and the pharmacology was

first described in 1977 (Christensen et al., 1977, Hyttel, 1977), shown to be a

very potent inhibitor of serotonin reuptake in both in vitro and in vivo models

(Hyttel, 1978, Hyttel, 1977).

CIT is a racemic mixture of the S- and R-enantiomers (Figure 6). The SSRI

activity of CIT resides in S-CIT, whereas R-CIT is practically devoid of

serotonin reuptake potency (Burke et al., 2002, Hyttel et al., 1992, Lepola et

al., 2004, Montgomery et al., 2001, Owens et al., 2001, Wade et al., 2002). In

vivo functional and behavioral studies have shown that the S-enantiomer is

responsible for almost all antidepressant activity of CIT in animal models

(Hyttel et al., 1992). Furthermore, among all available SSRIs, S-CIT is the

most selective inhibitor of serotonin reuptake and is ~30 times more potent

than R-CIT (Owens et al., 2001) Due to this fact, the therapeutically active S-

CIT has been developed and introduced onto the market as a single

enantiomer drug (escitalopram; EsCIT). In addition, using a variety of

behavioral test, it has been shown that EsCIT displays a more rapid and

efficacious effect than CIT, and that R-CIT counteracts these effects in vivo

and in vitro (Mork et al., 2003, Sanchez et al., 2003a, Sanchez et al., 2003b,

Sanchez and Kreilgaard, 2004).

26 | INTRODUCTION

Figure 6. The chemical structure of the S- and R-enantiomers of citalopram.

Racemic CIT undergoes N-demethylation by CYP enzymes to the major

metabolite demethylcitalopram (DCIT) (Figure 7). In vitro studies using human

liver microsomes indicated that CYP2C19, CYP3A4 and CYP2D6 all

contribute to the formation of DCIT (Rochat et al., 1997, von Moltke et al.,

1999). DCIT also undergoes N-demethylation to the minor metabolite

didemethylcitalopram (DDCIT) by CYP2D6 (Sindrup et al., 1993, von Moltke

et al., 2001). In human brain and liver, CIT and metabolites are oxidated by

MAO-A and MAO-B to citalopram propionic acid derivate and citalopram-N-

oxide (Kosel et al., 2001, Rochat et al., 1998). Compared to CIT, the

metabolites are weaker and have less SSRI activity, and are not considered to

play a major role in the therapeutic effect (Milne and Goa, 1991). DCIT is 4

times less potent as a SSRI and 11 times more potent as a noradrenaline

reuptake inhibitor (Hyttel, 1982).

Figure 7. The major pathway for the metabolism of citalopram (CIT) into its main

metabolites demethylcitalopram (DCIT) and didemethylcitalopram (DDCIT).

Clinical pharmacokinetic characteristics of S-CIT have recently been

studied and are similar to those described for racemic CIT (Rao, 2007). The

pharmacokinetics of S-CIT are essentially the same regardless of whether

CN

OF

N CH3

CH3

NC

OF

NCH3

CH3

O

CH2

CN

F

CH2 CH2 N

CH3

CH3

O

CH2

CN

F

CH2 CH2 N

CH3

H

O

CH2

CN

F

CH2 CH2 N

H

H

CYP3A4CYP2C19CYP2D6

CIT

*CYP2D6

* *

DCIT DDCIT

INTRODUCTION | 27

patients are given a single oral dose of 20 mg of EsCIT or 40 mg of racemic

CIT (which contains 20 mg of S-CIT) indicating that there is no

pharmacokinetic interaction or interversion between S- and R-CIT (Rao,

2007, Sogaard et al., 2005). Time to peak plasma/serum concentrations of

CIT is 2-5 h in humans and 0.5 h in mice. In mice, the mean t½ of CIT is 1.5-

3 h and 30-38 h in humans, whereas the t½ of DCIT in humans is 51 h

(Fredricson Overo, 1982). The average t½ of the enantiomers differs, as the

t½ of S-CIT (42 h) was shorter than that of R-CIT (66 h) in five hospitalized

depressed patients. Furthermore, the t½ of S-DCIT (93 h) was significantly

shorter than that of R-DCIT (228 h) (Voirol et al., 1999). The shorter

reported t½ for S-CIT is consistent with the trend towards lower steady-state

plasma concentrations of S-CIT and S-DCIT compared with R-CIT and R-

DCIT in patients (Foglia et al., 1997, Sidhu et al., 1997).

There are some in vitro results which show that S-CIT has a slightly lower

potential to interact with the CYP enzymes in the liver compared with R-CIT,

thus decreasing the likelihood of drug-drug interactions (von Moltke et al.,

2001). The SSRIs are known to have a low toxicity profile and to be safer than

the TCAs when overdosed (Henry, 1997). Overdoses with CIT have been

associated with a risk of developing serious adverse effects (such as

electrocardiogram abnormalities and convulsion) (Grundemar et al., 1997) and

fatalities with CIT occur more frequently when taken in combination with

other drugs (Dams et al., 2001).

Blood-brain barrier

The existence of the blood-brain barrier (BBB) was first demonstrated by the

German pharmacologist and physiologist Paul Ehrlich at the end of the 19th

century and further studied by Goldman. Stern and Gautier introduced the

term BBB in 1921 (Stern and Gautier, 1921). In the 1960s, the structural basis

for the barrier was studied and established by electron microscopic studies

(Brightman and Reese, 1969, Reese and Karnovsky, 1967). Brain capillary

endothelial cells (BCECs) line the blood capillaries in the brain and form the

basis of the BBB. The barrier has cellular tight junctions between BCECs to

prevent the passive diffusion of substances through the intercellular space,

and express transport systems, which serve as an active passage into the brain,

28 | INTRODUCTION

and as an active efflux out of the brain (Ballabh et al., 2004). The passage of

compounds from the systemic blood into the CNS is limited by the presence

of the BBB. Two important functions of the BBB are the protection of the

brain against potentially toxic substances and the maintenance of a constant

internal environment, which is of primary importance to the optimal neuronal

functioning of the brain.

Several pathways affect the transport of drugs across tissue barriers,

including the BBB, and although the relative importance of the pathways

varies between tissues and for different drugs, the general mechanisms are

applicable throughout the body. The transport of drugs can be divided into

transcellular, which requires that the drug can cross the lipophilic cell

membranes, and paracellular, where diffusion occurs through the water-filled

pores of the tight junctions between the cells. In addition, both passive and

active (energy-dependent) transport can contribute to the permeability of

drugs through the transcellular pathway. Four principal pathways of drug

transport in the BBB are passive transcellular transport, paracellular transport,

active efflux and active up-take (Figure 8). All transport can occur in both

directions, depending on local drug concentrations (passive transport) and the

presence of the relevant transporters in both the luminal (apical) and the

abluminal (basolateral) membranes, which mediate transport in both

directions (active transport). Drugs of small to moderate molecular weight can

pass the BBB through pores between the cells, pores created by extracellular

tight junction proteins that connect adjacent cells (Lai et al., 2005, Ueno,

2007). In addition to passive transport, active transport via transport proteins

expressed in both luminal and abluminal membrane of capillary endothelia

cells significantly affect the entrance of substrate drugs across the BBB.

INTRODUCTION | 29

Figure 8. Mechanisms of blood-brain barrier transport include passive transcellular

transport (1), paracellular transport (2), active uptake (3) and active efflux (4). Passive

diffusion (1) can occur in both directions, depending on local drug concentration.

Correspondingly, drug transport proteins are expressed in both luminal and abluminal

membranes, and can mediate active transport in both directions.

ABC transport proteins

Transport proteins situated in the membranes of the cell significantly affect the

transport of substrates across cellular barriers. Of the more than 20,000 genes in

the human genome, there are approximately 500 encode transport proteins

(Venter et al., 2001). One of the dominating gene families among the plasma

membrane transporters is the ATP-binding cassette (ABC) transporters, with

members found in all species studied so far (Linton and Higgins, 2007). ABC

transporters with the capacity to transport drugs are expressed in tissues

throughout the body, and have been shown to affect pharmacokinetic processes

such as intestinal absorption, distribution to the CNS, and uptake into, and

subsequent excretion from the hepatocyte (Endres et al., 2006). Efflux

transporters from the ABC superfamily exert significant functional transport at

the BBB. In the human ABC transport family, 49 genes have been identified and

divided into seven sub-families: ABCA to ABCG (Choudhuri and Klaassen,

2006, Sharom, 2008). The human ABC transporters with an affinity for drugs are

mainly found in the ABCB, ABCC and ABCG sub-families.

Luminalmembrane

Abluminalmembrane

BRAIN

BRAIN

BLOOD

Transporter protein

Drug molecule

Endothelial cell

Tight junction

1 2

4

3

30 | INTRODUCTION

ABC transporters are expressed at various sites in the body, including the

liver, intestine, adrenal gland and kidney. In brain capillaries, the expression of P-

glycoprotein (P-gp, ABCB1) and multidrug resistance-associated proteins

(MRPs) (MRP1-6, e.g. ABCC1-6) has been reported for several species,

including humans (Begley, 2004). Several studies using isolated BCECs from

different species, including humans, have been performed to determine the MRP

expression at the BBB (Dombrowski et al., 2001, Miller et al., 2000, Zhang et al.,

2000). At least six MRPs are expressed at the BBB of different species. However,

the exact subcellular localization, luminal or abluminal, of most of these MRPs at

the BBB remains to be determined (Figure 9). For some of the transporters, the

localization has been determined using polarized epithelial cell lines (Schinkel

and Jonker, 2003). In the brain, the breast cancer resistance protein, BCRP,

(ABCG2) has been detected at the luminal surface (Cooray et al., 2002,

Eisenblatter et al., 2003) and has an overlap of the substrates specificity with

other ABC transporters, such as P-gp (Cascorbi, 2006). More than 18000 papers

concerning P-gp have been published up to January 2012

(www.ncbi.nlm.nih.gov), making this the most studied drug transporter.

Figure 9. Blood-brain barrier transport protein expression of selected efflux transporters: P-

glycoprotein (P-gp), multidrug resistance-associated proteins (MRPs) and breast cancer

resistance protein (BCRP). The exact subcellular localization (luminal vs. abluminal) for all

transporters shown in this figure has not been demonstrated as yet. (TJ- tight junction).

BRAIN

BLOOD

MRP5BCRPMRP4MRP1 P-gp

MRP4

TJ

MRP2

INTRODUCTION | 31

P-glycoprotein

The first ABC transporter that was discovered was P-glycoprotein (P-gp),

identified as the cause of multidrug-resistance (MDR) (Juliano and Ling,

1976). The physiological role of P-gp is still not completely understood and

suggested endogenous substrates include steroid hormones and cytokines, as

well as membrane components (phosphatidylcholine and cholesterol)

(Hoffmann and Kroemer, 2004). P-gp is highly expressed in the apical

membrane of intestinal enterocytes, in the bile canalicular membrane of the

liver and in protective tissue barriers including not only the BBB but also the

blood-testis barrier and in the placental syncytiotrophoblasts that connect

maternal and fetal blood circulation (Fojo et al., 1987, Thiebaut et al., 1987,

Thiebaut et al., 1989). This expression pattern suggests that P-gp plays an

important role in the protection of tissues and detoxification.

P-gp is a membrane-bound ATP-dependent efflux transporter present at

high concentrations in the luminal (apical) membrane of the endothelial cells

of the brain capillaries (Beaulieu et al., 1997, Biegel et al., 1995). P-gp is a

product of the multidrug resistance (MDR) gene MDR1 (also known as

ABCB1). P-gp and other drug transporters have significant effects on

absorption, distribution, metabolism and excretion of drugs (Fromm, 2003).

Most P-gp substrates are lipophilic and uncharged or weak bases, although

acidic and hydrophilic substrates, e.g. methotrexate, have also been reported

(Schinkel and Jonker, 2003). Like CYP3A4, P-gp is non-specific for a large

variety of drugs from many different drug classes. There is a substrate overlap

with other ABC transporters and with CYP3A4 (Cascorbi, 2006). The P-gp

substrates include steroid hormones, antimicrobial agents, opioids, anticancer

agents, CNS active drugs and immunosuppressants (Cascorbi and Haenisch,

2010, Fromm, 2004). Several models have been proposed to explain the

mechanism of drug efflux by P-gp. However, as of today, the exact substrate

site for interaction is unknown. The three main models are: pore model,

flippase model and hydrophobic vacuum cleaner (HVC) model. Among these,

the HVC model has gained wide acceptance in which P-gp binds its substrates

from the inner leaflet of the plasma membranes and transport through a

protein channel (Gatlik-Landwojtowicz et al., 2006, Homolya et al., 1993,

Shapiro et al., 1997). Furthermore, many drugs are known to be inducers or

32 | INTRODUCTION

inhibitors of P-gp (e.g. dexamethasone, rifampicin, verapamil, cyclosporine A)

(Narang et al., 2008, Perloff et al., 2004, Yu, 1999), as well as psychoactive

drugs, which are often co-medicated in psychiatric patients (e.g. fluoxetine, St.

John’s wort, olanzapine, risperidone and paliperidone) (Argov et al., 2009,

Hennessy et al., 2002, Moons et al., 2011, Wang et al., 2008).

The ABCB1 gene is located in the human chromosome 7 band p21-21.1

(Callen et al., 1987). This gene extends over more than 100 kb, containing 28

introns, 26 of which interrupt the protein coding sequence. The first report on

the polymorphisms of the gene was presented in 1989, gly185val and

ala893ser (Kioka et al., 1989), and the first systemic screening for

polymorphisms was performed in 2000 (Hoffmeyer et al., 2000), where they

detected 15 SNPs. To date, 28 SNPs have been found at 27 positions

(Cascorbi et al., 2001, Hoffmeyer et al., 2000, Ito et al., 2001, Kim et al., 2001,

Tanabe et al., 2001). Studies have shown that the activity of the gene coding

for P-gp, ABCB1, varies between individuals, which means that the drug

concentrations in the brain are dependent on P-gp levels (Cascorbi, 2006,

Kroetz et al., 2003).

The association of this SNP with the expression of P-gp was first reported

by Hoffmeyer et al. (Hoffmeyer et al., 2000). They found that C3435T at exon

26 influences the level of intestinal P-gp and the concentration of digoxin:

individuals homozygous for the polymorphism were associated with

decreased P-gp expression and increased digoxin concentration. This

genotype has also been shown to be a risk factor for the side effect of

orthostatic hypotension of the P-gp substrate nortriptyline (Roberts et al.,

2002). Two other SNPs, G2677T/A and C1236T, are also believed to affect

the expression level of the protein and phenotype (Hoffmeyer et al., 2000,

Kim et al., 2001, Tanabe et al., 2001). A large number of studies have

investigated the impact of polymorphisms in ABCB1 on the bioavailability of

different drugs known to be P-gp substrates (Eichelbaum et al., 2004,

Sakaeda, 2005). Some of the SNPs have also been associated with altered

pharmacokinetics and treatment response (Evans and McLeod, 2003,

Marzolini et al., 2004, Uhr et al., 2008). The clinical efficiency of

antidepressant as well as antipsychotic treatment has been positively

INTRODUCTION | 33

correlated with ABCB1 polymorphisms and substrate properties of the used

drugs (Bozina et al., 2008, Kato et al., 2008, Uhr et al., 2008).

In vitro and in vivo P-gp studies

A range of in vitro and in vivo methods can be used to study the transport of

drugs across the BBB, including the influence of transporters. Cultured brain

endothelial cell lines can be used to identify the specific transporters that act

on a drug. However, many drugs are transported by more than one

transporter, which makes it difficult to draw conclusions on the contribution

of each transporter in vivo. Also, an in vitro setting can never totally resemble

the in vivo situation with all endogenous substances present, making in vivo

experiments necessary at least for the confirmation of in vitro results.

The importance of P-gp in the BBB has been studied extensively in

cultured mammalian brain capillary endothelial cells (Tsuji et al., 1992).

However, the in vitro models cannot completely represent the complex in vivo

situation. For example, studies in Caco-2 cells have given contradicting results,

and it is still unclear whether VEN is a substrate in vitro (Ehret et al., 2007,

Oganesian et al., 2009). Furthermore, using monolayers of bovine brain

microvessel endothelial cells, Rochat and co-workers reported that CIT

crosses the BBB without the influence of P-gp (Rochat et al., 1999).

To overcome the problem seen in in vitro systems, transgenetic mice can be

used. In humans, P-gp is encoded by a single ABCB1 gene (or MDR1),

whereas two genes, abcb1a (mdr1a or mdr3) and abcb1b (mdr1b or mdr1), encode

P-gp in mice (Devault and Gros, 1990). The two mouse genes are supposed to

exhibit similar characteristics as the human isoform. The abcb1ab knockout

mouse was developed to investigate the role of P-gp in vivo and has provided a

unique and valuable pharmacological tool (Schinkel et al., 1997, Schinkel et al.,

1994, Lagas et al., 2009). So far, several antidepressant and antipsychotic drugs

have been determined to be P-gp substrates by the use of the abcb1ab

knockout mouse model. Some examples are doxepin, paroxetine,

fluvoxamine, sulpiride, risperidone, paliperidone and trimipramine (Uhr and

Grauer, 2003, Uhr et al., 2003, Doran et al., 2005, Kirschbaum et al., 2008),

whereas mirtazapine and haloperidol have not been proved to be P-gp

34 | INTRODUCTION

substrates (Kirschbaum et al., 2008, Uhr et al., 2003) in this animal model.

Such an in vivo model has a clear advantage, since the BBB is very complex,

making it difficult to study interactions between the BBB and drugs in vitro.

Another possibility to study the BBB transport properties of a drug is to

co-administer a transporter inhibitor together with the drug. The main

disadvantage with the use of an inhibitor is a possible lack of specificity for

the studied transporter. For example, the cyclosporine analogue PSC833

(Valspodar) was, for a long period of time, considered to be an inhibitor of P-

gp only. However, BCRP and the multidrug resistant protein 2 (MRP2) are

also inhibited by PSC833 (Chen et al., 1999, Eisenblatter et al., 2003).

According to both in vitro and in vivo data, P-gp may restrict the uptake of

several antidepressants into the brain, thus contributing to the poor success

rate of current antidepressant therapies (Ejsing et al. 2007; Linnet and Ejsing,

2008; O’Brien et al., 2012) but few studies have examined a possible

stereoselective P-gp mediated transport (Choong et al., 2010). The P-gp

knockout mouse model shows that VEN and CIT are P-gp substrates (Uhr et

al., 2003, Doran et al., 2005). However, no studies have examined the role of

P-gp on possible stereoselective differences in the disposition of the

enantiomers of VEN or CIT and their main metabolites in the brain.

AIMS OF THE THESIS | 35

AIMS OF THE THESIS

The general aim of this thesis was to study the enantiomeric distribution of

various chiral antidepressant drugs and their metabolites with a particular

focus on the role of P-glycoprotein (P-gp) in the blood-brain barrier. In

addition, genetic and toxicological aspects of P-gp were studied in a forensic

autopsy material.

Specific aims:

1. To study the enantiomeric distribution in serum and brain in abcb1ab (-/-)

and abcb1ab (+/+) mice following the administration of venlafaxine,

citalopram and escitalopram. (Papers I - III)

2. To study possible P-gp dependent behavioral effects in abcb1ab (-/-) and

abcb1ab (+/+) mice following the chronic administration of venlafaxine.

(Paper II)

3. To study the genotype distributions of ABCB1 in forensic autopsy cases,

positive for citalopram and venlafaxine, with focus on deaths related to

intoxication. (Paper IV)

MATERIAL AND METHODS | 37

MATERIALS AND METHODS

Animal studies (Papers I-III)

Animals

Male abcb1ab (-/-) mice on a FVB/N background and abcb1ab (+/+) mice

were obtained from Taconic (Germantown, NY, USA). Animals had free

access to tap water ad libitum and standard laboratory pellets. The mice were

kept in groups of 2-5 in cages with sawdust bedding under climate-controlled

conditions: normal indoor temperature (22°C) and humidity (60%). The

animals were kept in a constant 12:12 h light:dark cycle.

Drug administration and sample collection

Acute administration

Racemic VEN and CIT were administered to abcb1ab (-/-) mice and abcb1ab

(+/+) wild-type mice by a single intraperitoneal (i.p.) injection of 10 mg/kg

bodyweight. EsCIT was administered to the mice by a single i.p. injection of 5

mg/kg bodyweight. Drug administration was performed during a brief

anesthesia. One to nine hours following drug administration, the mice were

decapitated under isoflurane anaesthesia, and mixed arterio-venous blood was

collected. The blood was left at room temperature to allow clotting and

38 | MATERIAL AND METHODS

thereafter centrifuged. The brain was dissected out and all serum and brain

samples were frozen and stored at -70°C until analysis.

Chronic administration

For the pharmacokinetic studies, VEN, CIT and EsCIT were administered to

the abcb1ab (-/-) mice and abcb1ab (+/+) wild-type mice by daily i.p. injections

during a brief anesthesia with isoflurane. For the pharmacodynamic study,

VEN was administered to abcb1ab (-/-) mice (n = 16) and abcb1ab (+/+) wild-

type mice (n = 15) as described above. The corresponding control mice (n =

14 abcb1ab (-/-) and n = 10 abcb1ab (+/+)) received saline in the same manner

as VEN.

Chiral determination of drugs

Venlafaxine and metabolites

The concentrations of the S- and R-enantiomers of VEN and its metabolites

in serum (nmol/l) and brain homogenate supernatant (pmol/g nmol/l) were

determined by using a liquid chromatography tandem mass spectrometry (LC-

MS/MS) method (Kingback et al., 2010).

The brain samples were weighed and homogenized in 2 ml ultra pure water

using a sonifier (Sonics VibraCell VC 130; Chemical Instruments AB, Lidingö,

Sweden) followed by centrifugation (2000g for 20 min). VEN and metabolites

were extracted from serum and brain homogenate supernatant by solid-phase

extraction (SPE) using Isolute C8 100 mg (International Sorbent Technology,

Hengoed, UK). The eluate was evaporated with nitrogen at 50°C in a block

thermostat (Grant QBT2; Grant Instruments (Cambridge) Ltd, UK) and

reconstituted in 50 µl of mobile phase (10:90 v/v; 10 mM tetrahydrofurane:

ammonium acetate, pH 6.0).

The chromatographic system consisted of an Acquity liquid

chromatography (LC) system (Waters, Milford, USA) and a Sciex API 4000

tandem mass detector equipped with an electrospray ionization (ESI) ion

source (PE Sciex, Ontario, Canada). A volume of 5 µl reconstituted sample

was injected onto a Chirobiotic-V column (5 µm particle size, 250 x 2.1 mm;

Astec, Basle, Switzerland) protected with an in-line filter (VICI AB


International, Switzerland). The temperature of the column was set to 10°C

using a Jones Chromatography Model 7955 column chiller/heater (Hengoed,

UK) and the flow rate of the mobile phase was 0.2 ml/min. Formic acid

(0.05%) in acetonitrile was delivered through a Gynkotek 480 pump (Dionex;

Sunnyvale, USA) and added post column at a flow rate of 0.2 ml/min to

increase the sensitivity. Data acquisition and peak integration, recording the

area of the peaks, were performed using Analyst 1.4 software (PE Sciex;

Ontario, Canada).

Standards were prepared with the following concentrations 1, 2.5, 10, 25,

100, 250 and 1,000 nmol/l (for each enantiomer of VEN and ODV) and 0.5,

1.25, 5, 12.5, 50, 125 and 500 nmol/l (for each enantiomer of NDV and

DDV). Two different quality controls were prepared with concentrations of 2

and 500 nmol/l (for each enantiomer of VEN and ODV) and 1 and 250

nmol/l (for each enantiomer of NDV and DDV). The mean extraction

recoveries for VEN and its metabolites in serum ranged between 75-110%.

The mean brain extraction recoveries for the enantiomers of VEN and its

metabolites ranged between 75-91% and 74-93%, respectively. The matrix

effect data for VEN and metabolites were between 99-105% and 80-88%,

respectively, indicating no severe matrix effects. The lower limit of

quantification was 0.5 nmol/l for the enantiomers of VEN and ODV and

0.25 nmol/l for NDV and DDV.

Citalopram and metabolites

The concentrations of the S- and R-enantiomers of CIT, DCIT, and DDCIT

in serum (nmol/l) and brain homogenate supernatant (pmol/g ≈ nmol/l)

were determined by using HPLC with fluorescence detection, as described

previously (Rochat et al., 1995), with some modifications (Carlsson et al.,

2001, Kugelberg et al., 2001).

The brain samples were weighed and homogenized in 2 ml ultra pure water

using a sonifier (Sonics VibraCell VC 130; Chemical Instruments AB, Lidingö,

Sweden) followed by centrifugation (2000g for 20 min). The Isolute C2 SPE

column (International Sorbent Technology, Hengoed, UK) was used for

extraction of CIT and metabolites from the serum and brain homogenate.


After elution and evaporation, the dried samples were redissolved in 100 µl of

methanol:100 mmol/l citrate triethylamine buffer, pH 6.3 (55:45; v/v). A

volume of 25 µl was injected on to a Cyclobond I 2000 Ac 250 x 4.6 mm

column (Astec, Whippany, USA) with a Gynkotek Gina 50 autosampler

(Dionex, Sunnyvale, USA). The mobile phase was delivered through a

Gynkotek 480 pump at a flowrate of 0.8 ml/min. detection was performed

using a Waters 474 fluorescence detector (Waters Corporation, Milford, USA)

at an excitation wavelength of 240 nm and an emission wavelength of 300 nm.

The column temperature was set to 30°C using a Jones Chromatography

Model 7955 column chiller/heater (Hengoed, UK). The detection signals were

recorded and processed using Chromeleon (Version 6:40; Dionex, Sunnyvale,

USA).

Standards were prepared with the following concentrations: 15-616 nmol/l

(5-200 ng/ml) for CIT, 8-322 nmol/l (2.5 -100 ng/ml) for DCIT and 1.7-67

nmol/l (0.5-20 ng/ml) for DDCIT. The limits of detection for the

enantiomers of CIT and its metabolites were 2 nmol/l respectively. The

absolute recoveries from spiked drug-free plasma were between 87 and 110%

(Carlsson et al., 2001). The brain tissue extraction recoveries for CIT and

metabolites were found to be around 40% (Wikell et al., 1999).

Open-field behavior (Paper II)

Exploratory locomotion/activity in mice was assessed in an open-field

paradigm. The test arena was made of dark-gray plastic and measured 60 x 60

x 35 cm. The arena was virtually divided into several parts: corners which

measured 15 x 15 cm, corridors near the walls which were 8 cm wide and a

centre-square measuring 15 x 15 cm (Figure 10). In addition, this allowed the

floor of the arena to be divided into two similar large areas: a peripheral

(corners and walled part) and central (centre and rest) arena. The mouse was

allowed to explore the arena for 10 min. The path of the mouse was tracked

and subsequently analyzed using the automated system for the following

parameters: distance travelled (cm), time spent in the various parts of the

arena and the number of rears. Behavioral testing was performed 30 min after

drug injection on day 7 and day 9. All behavioral tests were conducted

between 9:00 am and 2:00 pm. The hardware consisted of an IBM-type


computer combined with a video digitizer and a CCD video camera

(Panasonic, CCTV Camera WVBP330/GE, Sushou, China). The software

used was EthoVision® release 3.1 (Noldus Information Technology, Utrecht,

The Netherlands).

Figure 10. Illustration of the open-field arena divided into several parts, corners, corridors

near the wall and a centre-square (from the camera’s point of view).

Statistics

Data are presented as means ± SEM. Pharmacokinetic data were analyzed by

Student’s t-test for unpaired observations to compare differences between

abcb1ab (-/-) and abcb1ab (+/+) mice. All statistical analyses were performed

using StatView for Windows Version 5.0 (SAS Institute, Cary, USA).

Behavioral data were analyzed by analysis of variance (ANOVA) followed by

a post-hoc t-test using SPSS® for Windows Version 12.0 (SPSS Inc., Chicago,

USA). A probability of less than 5% (p < 0.05) was considered to be

statistically significant.

Ethics committee permission

The Regional Ethics Committee, Mainz, Germany, gave permission for the

animal studies (Papers I-III).


Forensic study (Paper IV)

Case selection

From the Department of Forensic Genetics and Forensic Toxicology in

Linköping, Sweden, 228 cases positive for venlafaxine and citalopram (VEN

n=116, CIT n=112) during the toxicological screening were identified and

genotyped for ABCB1 SNPs. The included samples consisted of drug

intoxications (suicides, accidents and undetermined) and cases that were not

related to drug intoxication (non-intoxications). Information related to each of

the cases (age, gender and other circumstances surrounding death) was

provided from the forensic pathology and toxicology databases at the Swedish

National Board of Forensic Medicine. Routine screening of a wide range of

prescription drugs was determined in femoral blood by capillary gas

chromatography using a nitrogen-phosphorus detector (Druid and Holmgren,

1997).

Genotyping

The following ABCB1 SNPs: G1199A (exon 11, rs2229109), C1236T (exon

12, rs1128503), G2677T/A (exon 21, rs2032582) and C3435T (exon 26,

rs1045642) were identified by PCR and pyrosequncing. In brief, genomic

DNA was extracted using King Fischer ML (Thermo Scientific, Vantaa,

Finland). Extracted DNA was stored frozen at -20˚C until analyzed. PCR and

sequencing primers were designed using the PSQAssay Design program

(Qiagen, Uppsala, Sweden). PCR primers, sequencing primers and

dispensation order for the four SNPs are shown in Table 1. For PCR

amplification, the reactions were optimized for MgCl2 concentration (1.5 mM)

and annealing temperature (58˚C), and each primer was used at a final

concentration of 0.4 µM. HotStarTaq master mixture (VWR International,

Stockholm, Sweden) was used and all reactions were performed on a

Mastercycler gradient instrument (Eppendorf, Hamburg, Germany) in a total

volume of 12.5 µl. The four SNPs were analyzed using a PyroMark Q96MD

instrument (Qiagen, Uppsala, Sweden) according to the manufacturer’s

protocol.


Table 1. Primers, sequencing primers and the dispensation order for the ABCB1 SNPs.

(bio: biotinylated)

Statistics

The ABCB1 genotype frequencies for each material were tested for Hardy

Weinberg equilibrium. Statistical differences between groups were calculated

using the Fischer’s exact test. The distributions of individuals homozygous

variant was compared with individuals with the wild-type or heterozygous

genotype. A p value <0.05 was regarded as significant. All statistical analyses

were performed using StatView for Windows Version 5.0 (SAS Institute,

Cary, USA).

Ethics committee permission

The Regional Ethics Committee, Faculty of Health Sciences, Linköping

University, Sweden, gave permission for the forensic study (Paper IV)

included in this thesis.

PCR primers

SNP Forward primer Reverse primer

G1199Abio

ATTGACAGCTATTCGAAGAGTG CCTTAACTTCTTTTCGAGATGG

C1236Tbio

GTCTGTGAATTGCCTTGAAGT CAGCCACTGTTTCCAACC

G2677T/Abio

GGACAAGCACTGAAAGATAAG AGGGAGTAACAAAATAACACTGAT

C3435Tbio

ACATTGCCTATGGAGACAAC TAGGCAGTGACTCGATGAAG

Sequencing primers

SNP Sequencing primers Dispensation order

G1199A CTTTTCGAGATGGGTAA GACTCGAGTGAC

C1236T TGCACCTTCAGGTTCA TGAGCTCAGAT

G2677T/A TTAGTTTGACTCACCTTCC GCCAGTCAGCTC

C3435T CTTTGCTGCCCTCAC CAGAGTCTCT

RESULTS AND DISCUSSION | 45

RESULTS AND DISCUSSION


The present thesis includes novel data on the distribution of venlafaxine

(VEN), citalopram (CIT), escitalopram (EsCIT), and their metabolites in

serum and brain after acute and chronic drug administration in the abcb1ab

knockout mouse model. In addition, this model was used for a

pharmacodynamic investigation in the open-field behavior test.

Pharmacokinetics

The pharmacokinetic results demonstrate that P-gp decreases the brain uptake

of the S- and R-enantiomers of VEN and CIT as well as the main metabolites

ODV, NDV and DCIT. This was confirmed from the observation of higher

brain concentrations in the abcb1ab (-/-) mice compared with the abcb1ab

(+/+) mice.

The acute and chronic treatment regimens resulted in similar

pharmacokinetic profiles for CIT (wild-type brain/serum ratio 2.0 and 1.6,

acute vs chronic), indicating that the chronic treatment has no, or very little,

effect on the expression levels of P-gp, at least up to 10 days of drug

administration (Figure 11). However, VEN has been reported to induce the

expression of P-gp (Doran et al., 2005, Ehret et al., 2007), an effect not seen

46 | RESULTS AND DISCUSSION

in the acute and chronic VEN studies in the wild-type mouse (wild-type

brain/serum ratio 4.8 and 4.1, acute vs. chronic) (Figure 12). In another

recently published study, VEN was found to induce the expression of P-gp

but no effect on induction was seen for desvenlafaxine (i.e. ODV) (Bachmeier

et al., 2011).

Figure 11. Serum and brain citalopram enantiomer concentrations after acute (1 h) and

chronic (10 days) treatment with racemic citalopram (10 mg/kg). (n=5 in each group).

Figure 12. Serum and brain venlafaxine enantiomer concentrations after acute (1 h) and

chronic (10 days) treatment with racemic venlafaxine (10 mg/kg). (n=10 in each group).

Acute

0

1000

2000

3000

4000

5000

6000S-enantiomer

R-enantiomer

Serum Brain

ko wtko wt

**

***

Co

ncen

trati

on

(nm

ol/

L)

Chronic

0

1000

2000

3000

4000

5000

6000S-enantiomer

R-enantiomer

Serum Brain

ko wtko wt

***

***

Co

ncen

trati

on

(nm

ol/

L)

Acute

0

500

1000

1500

2000

2500

3000

3500

Serum Brain

ko wtko wt

***

*

***S-enantiomer

R-enantiomer

Co

ncen

trati

on

(nm

ol/l)

Chronic

0

500

1000

1500

2000

2500

3000

3500S-enantiomer

R-enantiomer

***

**

Serum Brain

wtwtko ko

Co

ncen

trati

on

(nm

ol/l

)


The present serum and brain knockout/wild-type ratios of VEN were 1.1

and 2.4, respectively, compared to 1.3 and 2.3 in the study by Uhr and co-

workers (Uhr et al., 2003) after acute administration. The VEN results in the

acute study are also in line with the results reported by Doran and co-workers

(Doran et al., 2005). One hour after drug administration, the brain/serum

concentrations of VEN in knockout mice were 10 vs 7.7 and the

corresponding ratios in wild-type mice were 4.3 vs 4.2 (present study vs

Doran et al., 2005).

For CIT, the present results are in line with the results reported by Uhr and

Grauer (2003) using the animal model. They reported a brain abcb1ab (-/-) to

wild-type concentration ratio of 3.0 1 h after s.c. treatment (present study 3.1).

The chronic results are also in line with their sub-chronic data (Uhr et al.,

2008), with a brain abcb1ab (-/-) to wild-type concentration ratio of 2.8

compared to 3.0 in Uhr et al. (2008).

Comparing the CIT treatment with the EsCIT treatment resulted in similar

serum and brain concentrations (Figure 13). The R-enantiomer does not seem

to interfere with the S-enantiomer transport mediated by P-gp. However, the

behavioral effects were not investigated and there might be pharmacodynamic

differences between the treatments.

Figure 13. S-citalopram in serum and brain after chronic treatment (10 days) with

racemic citalopram (rac-CIT) and escitalopram (EsCIT) in mice. (n = 5 in each group)

Serum

0

200

400

600

800

1000

1200abcb1ab (-/-)

abcb1ab (+/+)

rac-CIT EsCIT

Co

ncen

trati

on

(nm

ol/l)

Brain

0

500

1000

1500

2000

2500

3000

3500

rac-CIT EsCIT

Co

ncen

trati

on

(nm

ol/l)

**

***


Stereoselective metabolism of chiral drugs can additionally influence

pharmacokinetics, pharmacodynamics and toxicity (Smith, 2009). To our

knowledge, no published data in mice are available to compare with the

present results. Kingbäck (2011) reported the following S/R ratios in Sprague-

Dawley rats three hours after a single dose of VEN (15 mg/kg): VEN 0.96,

ODV 9.11 and NDV 0.12 (Kingbäck, 2011). These results can be compared

with the S/R ratios found in Paper I. For wild‐type mice (3 h after

administration): VEN 1.24, ODV 1.69 and NDV 2.77 (Figure 14). The mean

plasma S/R concentration ratios of VEN were 1.5 in dogs (chronic

administration of 22 mg/kg/day) and 5.5 in rats (chronic administration of

120 mg/kg/day) (Wang et al., 1992). Furthermore, in humans, a S/R ratio of

1.3 in plasma was reported following a single dose of racemic VEN (Wang et

al., 1992). Gex‐Fabry and co‐workers have reported S/R ratios of 0.4‐2.5 after

chronic administration of VEN (Gex-Fabry et al., 2004, Gex-Fabry et al.,

2002). A case report published by Eap et al. (2000) reported a S/R VEN ratio

of 1.9 in serum (Eap et al., 2000).

Figure 14. Serum S/R concentration ratios after acute (1 h) and chronic (10 days)

treatment with racemic VEN (10 mg/kg). (n = 10 in each group).

The present data on the serum S/R enantiomeric ratios of CIT and its main

metabolites DCIT and DDCIT in Paper III can be compared to the following

S/R ratios in Sprague-Dawley rats after a single dose of CIT (10 mg/kg, 1 and

3 h): CIT 0.91 and 0.90, respectively, was reported (Kugelberg et al., 2003).

These results can be compared with the S/R ratios found in Paper III. For

wild‐type mice (acute 1 h, 3 h and chronic 10 days): CIT 2.5-5.2, DCIT 4.6-8.8

Acute

0.0

0.5

1.0

1.5

2.0

2.5

3.0abcb1ab(-/-)

abcb1ab(+/+)

VEN NDVODV

**

S/R

rati

o

Chronic

0.0

0.5

1.0

1.5

2.0

2.5

3.0

VEN NDVODV

***

***

S/R

rati

o


and DDCIT 0.38-3.9 (Figure 15). The S/R ratios of CIT can also be

compared with S/R CIT ratios from human studies (Foglia et al., 1997,

Rochat et al., 1995, Sidhu et al., 1997, Zheng et al., 2000). The present data

were not in agreement with these clinical studies, where the mean S/R CIT

ratios were found to be between 0.5-0.7.

Figure 15. Serum S/R concentration ratios after acute (1h) and chronic (10 days)

treatment with racemic CIT (10 mg/kg). (n = 5 in each group).

Stereoselective transport of racemic drugs mediated by P-gp has been

shown for several drugs (Choong et al., 2010). A stereoselectivity for R/S-

methadone in the abcb1ab (-/-) mouse model was reported in which the brain

knockout/wild-type ratios of R- and S-methadone were 15.3 and 23.5

respectively (Wang et al., 2004). The pharmacokinetics of fexofenadine was

also shown to display stereoselectivity with a higher P-gp affinity for the S-

enantiomer than for the R-enantiomer (Miura et al., 2007). For S/R-

verapamil, an absence of stereoselective P-gp mediated transport has been

reported (Luurtsema et al., 2003, Sandstrom et al., 1998). Furthermore, no

evidence of a stereoselective P-gp mediated transport of VEN and CIT over

the BBB was obtained in Papers I-III.

When comparing S/R ratios, there can be a stereoselective metabolism in

mice not seen in humans or vice versa. In humans, stereoselective metabolism

of VEN by CYP2D6 was reported, but there was no evidence that this was

the case for CYP3A4 (Eap et al., 2003). In vitro studies in human liver

microsomes have shown that CYP2C19, CYP2D6 and CYP3A4 favor a more

Chronic

0

1

2

3

4

5

6

7abcb1ab(-/-)

abcb1ab(+/+)

CIT DCIT DDCIT

S/R

rati

os

Acute

0

1

2

3

4

5

6

7

**

*

**

S/R

rati

os

DDCITDCITCIT


rapid demethylation of S‐CIT compared with R‐CIT (Olesen and Linnet,

1999, von Moltke et al., 2001). In contrast, CYP2D6 has been shown to favor

R-DCIT over S‐DCIT in the second demethylation step (Olesen and Linnet,

1999). The metabolite/parent drug (M/P) ratios in serum for ODV/VEN

and NDV/VEN were 0.16 and 1.9 in wild-type mice (Paper II) and the M/P

ratios in serum for DCIT/CIT were 0.6 in wild-type mice after chronic

treatment (Paper III). Comparing these ratios to patients, the mean serum

ODV/VEN and NDV/VEN ratios of 5.5 and 0.6, respectively, were reported

(Reis et al., 2002). For CIT patients, a M/P ratio of 0.45 was seen for

DCIT/CIT (Reis et al., 2007). Taken together, there seems to be species

differences in the metabolic pattern, shown by M/P ratios, that needs to be

considered when results from different studies are compared. The differences

in metabolite pattern for VEN and CIT in mice compared to humans are

probably due to differences in the CYP-enzyme expression between species.

One also has to consider how the drug was administered and the time of

blood sampling in relation to the time when the drug was given. In

therapeutic drug monitoring (TDM) trough levels are utilized which requires

blood sampling within a drug specific time interval after dosing and steady

state. It has to be taken into account that we only tested one dose. P-gp might

be a saturable transporter, and lower or higher doses of the drug could lead to

results that would differ between the knockout mice and the controls. By

comparing the data for mice and rats with the human S/R ratios in plasma, it

can be concluded that mice, compared with rats, seem to be more similar to

humans with respect to the metabolism of VEN. The reverse situation seems

to be true for the metabolism of CIT, where rats mimic the human situation

better than the mice.


Pharmacodynamics

In the literature, only limited data are available addressing the influence of P-

gp on the pharmacodynamics using P-gp knockout mice (Kalvass et al., 2007a,

Kalvass et al., 2007b, Kirschbaum et al., 2008). In Paper II, the relevance of

being a substrate to P-gp was investigated in abcb1ab knockout mice and wild-

type mice in an open-field behavior test. This test indicated an impact on

exploration-related behaviors in knockout mice as centre activity was

increased (peripheral activity decreased) and rears were decreased by chronic

VEN treatment (Figures 16 and 17).

Figure 16. Open-field peripheral locomotion: the distanced covered in the periphery of

the arena, and rears made in the periphery of the arena by abcb1ab (-/-) and abcb1ab

(+/+) mice after 9 days of chronic venlafaxine treatment (10 mg/kg i.p. twice daily).

Mice were investigated for 10 min.

The open-field test was developed by Calvin S. Hall to test the emotionality

of rodents and is used to assay general locomotor activity and anxiety

(Denenberg, 1969). The field is a box, divided into areas and crossings, rearing

and time spent moving are used to assess the activity, and time spent in

different areas (e.g. centre and corners) is used to assess the anxiety (Prut and

Belzung, 2003). The open-field test is the method of choice for analysing an

animal’s spontaneous behavior in response to a novel environment

(Avgustinovich et al., 2000, Gershenfeld and Paul, 1997). Following a drug

challenge, this test in rodents gives hints for main clinical effects (Apelqvist et

al., 1999, Kennett et al., 1987, Kugelberg et al., 2006).

0

50

100

150

200

250

300

abcb1ab (-/-) abcb1ab (+/+)

* *

9 days

Rears

(n)

0

500

1000

1500

2000

2500

3000

3500


**

9 days

saline venlafaxine

Peri

ph

era

l lo

co

mo

tio

n(c

m)


In a chronic study, rats were administered VEN (10 mg/kg per day) for 14

days by using subcutaneously implanted osmotic minipumps and the

spontaneous open-field behavior was recorded for four consecutive nights

(Wikell et al., 2001). They reported for the first night that the VEN-treated

rats displayed a higher level of locomotor activity in the central area than the

control rats. However, no differences in locomotor activity in the central area

were evident between the groups during the subsequent second, third and

fourth night (Wikell et al., 2001). This is in line with the results obtained in the

present study, where centre activity was increased in VEN-treated mice

compared with control mice (Figure 17). In another study, VEN-treated mice

showed an increase in locomotor activity when exposed to a novel

environment, in a dose-dependent manner, whereas the mice pre-exposed to

the arena showed no increase (Brocco et al., 2002).

Figure 17. Open-field total locomotion: total distance moved, and time spent in the centre

of the arena of abcb1ab (-/-) and abcb1ab (+/+) mice after 9 days of chronic venlafaxine

treatment (10 mg/kg i.p. twice daily). Mice were investigated for 10 min.

Taken together, Papers I, II and III emphasize the importance of

investigating the pharmacokinetic and pharmacodynamic profiles of racemic

drugs. Thus, the present results, together with those from other laboratories,

confirm the validity of the abcb1ab knockout mouse model and its usefulness

for the investigation of the function of BBB transporters in relation to the

pharmacokinetics of different drugs. Furthermore, by comparing the results

with similar studies in rats, significant differences concerning the

pharmacokinetics can be seen between rats and mice.

0

1000

2000

3000

4000

5000

6000

7000


*

saline venlafaxine

9 days

To

tal

loco

mo

tio

n(c

m)

0

20

40

60

80

100

120

140

160


*

9 days

Tim

e i

n c

en

ter

(s)


Since P-gp and other ABC transporters can limit the transport of drugs into

the brain (Deeley et al., 2006), all CNS drugs needs to be tested, on an

individual basis, with regard to interactions with these transport proteins.


Several studies to investigate the significance of genotyping postmortem cases

have been performed for a variety of drugs and the conclusions from these

studies have been that genotyping was a valuable tool in interpreting the

toxicological results (Jannetto et al., 2002, Kingback et al., 2012, Koski et al.,

2006, Levo et al., 2003, Wong et al., 2003). VEN and CIT have been shown to

be actively transported by P-gp using a mouse model (Papers I-III). These

drugs are also common findings in forensic autopsy cases, making the

investigation of ABCB1 genotype distribution between intoxication cases and

non-intoxication cases very interesting to provide valuable additional

information to the pathologist when understanding the cause and manner of

deaths.

The present study included 116 cases positive for VEN and 112 cases

positive for CIT. The material comprised 85 women (37%) and 143 men

(63%) in total, with ages ranging between 18 and 90 years (median 63 years).

All the 228 cases, with exception of three cases for C1236T, were successfully

genotyped for four ABCB1 SNPs and did not significantly deviate from the

Hardy-Weinberg (HW) equilibrium. The included cases were divided into

intoxications (n = 73: comprising suicides, accidents and undetermined

intoxication cases) and non-intoxications (n = 155: comprising all other

causes of deaths not related to intoxication). Very few cases were found to

carry the 2677A variant, and they were therefore excluded from further

analysis.

The genotype distribution of the ABCB1 SNPs C1236T, G2677T and

C3435T in VEN positive cases was significantly (or borderline) different

between the intoxication cases and the non-intoxication cases (Table 2). This


cases. For the SNP G1199A, a low frequency of the A variant was found and

the genotype distribution was not significantly different between intoxications


and non-intoxications. Very few individuals positive for VEN were

homozygote variant for the SNPs (i.e. individuals 1236TT, 2677TT or

3435TT) in the intoxication cases compared with the non-intoxication cases.

It seems that the carrier homozygous variant for these SNPs is more likely to

have died by suicide not associated with drug intoxication and 7/10

individuals with the TT-TT-TT haplotype (1236-2677-3435) were suicides

(hanging or drowning). If these cases are associated with low therapeutic

effects, associated with severe side effects or a behavioral phenotype leading

to the individuals being more prone to suicide, this remains to be clarified.

Table 2. Postmortem cases divided into intoxications and non-intoxications.

P-gp, which participates not only in the transport of drugs over the BBB is

also involved in drug absorption, distribution to various tissues and excretion

(Fromm, 2003). In the postmortem cases, it cannot be determined whether

the effects are due to changes in the transport over the BBB, intestinal

absorption or drug elimination. The effect of the ABCB1 SNPs on

postmortem digoxin concentration has been studied in 112 subjects. The T-T-

T haplotype allele frequency was higher in the group with high digoxin

concentration (≥7nmol/l) and females might have a higher risk of digoxin

intoxication (Neuvonen et al., 2011). This is in line with a study in volunteers,

SNP Genotype

C1236T C/C + C/T 35 100% 68 86% 31 84% 56 76%

T/T 0 - 11 14% 6 16% 18 24%

total (n) 35 79 37 74

p-value

G2677T/A G/G + G/T 32 97% 61 84% 28 80% 51 76%

T/T 1 3% 12 16% 7 20% 16 24%

total (n) 33 73 35 67

p-value

C3435T C/C + C/T 32 89% 52 65% 28 76% 51 68%

T/T 4 11% 28 35% 9 24% 24 32%

total (n) 36 80 37 75

p-value

0.0597 0.8042

0.0074 0.5100

0.0173 0.4638

venlafaxine citalopram

Intoxications Non-intoxication Intoxications Non-intoxication


where individuals homozygous for C3435T (TT) had significantly lower P-gp

expression and high digoxin plasma concentrations (Hoffmeyer et al., 2000).

In a study with methadone in postmortem cases, no association between the

ABCB1 SNPs and methadone or metabolite concentrations was found

(Buchard et al., 2010). Taken together, these studies, along with the present

study, might conclude that the effect of genetic variants in the ABCB1 gene is

substrate specific.

P-gp is responsible for transporting a substrate back into the blood thereby

inhibiting the transport of certain substances, including drugs, to enter the

brain. This means that P-gp may prevent certain drugs from reaching the CNS

in therapeutic doses. Studies have shown that the activity of the gene coding

for P-gp, ABCB1, varies between individuals, which means that drug

concentrations in the brain are dependent on P-gp levels (Kroetz et al., 2003).

A high level of gene activity may be the reason why a patient does not reach

sufficiently high concentrations in the brain, and therefore may not get the

correct effect of the treatment. Several SNPs identified in the human ABCB1

gene have been associated with altered P-gp expression and phenotype

(Hoffmeyer et al., 2000, Kim et al., 2001, Tanabe et al., 2001), and some of the

polymorphisms have been connected with antidepressant response (Gex-

Fabry et al., 2008, Kato et al., 2008, Kroetz et al., 2003, Nikisch et al., 2008,

Uhr et al., 2008). Uhr and co-workers were the first to suggest a relationship

between genetic variants in the ABCB1 gene and the clinical efficacy of

antidepressants, most likely by influencing their access to the brain (Uhr et al.,

2008).

In most of the cases in Paper IV, a mixture of several drugs was found with

an average of 4 drugs/case (range 1-11). 91 different drugs were found and

approximately 50% of the drugs observed are known to be either substrates

or inhibitor sof CYP2D6. These interactions may affect the pharmacokinetics

and pharmacodynamics of VEN and CIT. The most frequently found

prescription drugs, in addition to VEN and CIT, were zopiclone (n=43),

paracetamol (n=36), propiomazine/dihydropropiomazine (n=35), diazepam

(n=28), alimemazine (n=26), nordiazepam (n=24) and mirtazapine (n=22).

The most frequently found drugs, in addition to VEN and CIT, after the cases


were divided into intoxication cases and non-intoxication cases are shown in

Table 3. Ethanol was found in 78 cases (34%).

Table 3. Most frequently found drugs (n) in addition to VEN and CIT in postmortem

cases divided into intoxication cases and non-intoxication cases. * propiomazine +

dihydropropiomazine.

According to several studies, VEN has a relatively higher fatal toxicity

compared with SSRIs and other newer antidepressants (Buckley and

McManus, 2002, Jonsson et al., 2004, Kelly et al., 2004, Whyte et al., 2003).

Different explanations regarding this finding have been suggested. Launiainen

et al. recently reported that fatal VEN poisonings were associated with a high

prevalence of drug interactions (Launiainen et al., 2011). The clinical relevance

for P-gp substrates and co-medication is the possibility of drug interactions.

Drug interactions based on transporters in the clinic may be inhibitory,

inductive or both, and may involve influx or efflux transporters. Such

interactions may alter both the efficacy and/or toxicity of the affected drug

(the substrate). These interactions can be clinically significant if the

elimination of the substrate drug or the distribution into a target tissue is

mediated primarily by the transporter. Another situation is if the interaction

results in the concentration of the affected drug at the site of action or toxicity

to fall outside of the therapeutic window. The transporter interactions may

also result in changes in concentration of the substrate in a particular tissue

without affecting the blood or plasma concentration of the substrate. One

example is the interaction of verapamil and cyclosporine A, where the

distribution of the drug in the brain is affected without significantly affecting

the plasma or blood concentration (Sasongko et al., 2005). This is possible

Intoxication Non-intoxication Intoxication Non-intoxication

Propiomazine* (18) Ethanol (31) Ethanol (18) Paracetamol (19)

Zopiclone (15) Mirtazapin (13) Propiomazine* (17) Ethanol (16)

Ethanol (13) Zopiclone (12) Zopiclone (11) Mirtazapine (7)

Diazepam (12) Alimemazine (10) Nordiazepam (6) Alimemazine (6)

Nordiazepam (8) Paracetamol (10) Paracetamol (5) Diazepam (5)

Venlafaxine positive cases Citalopram positive cases


because the amount of drug distributed into the brain is only a small fraction

of the total amount of the drug in the body.

Many of the other drugs found in the postmortem cases are metabolized by

the same enzymes as VEN and CIT, and some of them are also known

inhibitors. These interactions may affect the pharmacokinetics of the drugs

and the transport. Hence, drug interactions involving VEN and CIT may have

occurred in several of the cases. One of the possible interactions is the

combination of VEN and tramadol, which increases the risk of serotonin

syndrome. Another drug found in the cases was carbamazapine, a known

inducer of CYP3A4, which has also been found to be both a substrate and an

inhibitor of P-gp (Potschka et al., 2001, Weiss et al., 2003b). Other P-gp

inhibitors among the most frequently found drugs in the cases were

lamotrigin, methadone, promethazine, sertraline and hydroxyzine (Kan et al.,

2001, Litman et al., 2001, Stormer et al., 2001, Weiss et al., 2003a, Weiss et al.,

2003b).

An overlap of substrates for CYP enzymes and P-gp occurs; some

substances are known to be inducers not only for CYP but also for P-gp. In

studies using rifampin and St. John’s wort, inductive P-gp drug interactions

have been demonstrated (Greiner et al., 1999). PXR is a nuclear receptor

which transcriptionally up-regulates ABCB1 and CYP3A4 expression after

binding with certain ligands (Jones et al., 2000, Moore et al., 2000). Rifampin

and St. John’s wort are ligands of PXR and can induce expression and activity

of P-gp and CYP3A4 (Geick et al., 2001, Synold et al., 2001). Therefore, drugs

that induce CYP3A4 expression via the PXR activation pathway can also

induce Pgp expression. Consequently, such inducers will produce drug

interactions at the level of P-gp and CYP3A4.

Another explanation for VEN’s high toxicity might be the possible

differences in prescribing practice of VEN compared with SSRIs. VEN may

be prescribed to patients who have been previously prescribed other

antidepressant drugs (Egberts et al., 1997). These patients may not respond

satisfactorily to previous pharmacological treatment and may have a relatively

higher suicide risk compared with other patients. In a study from UK, even


after taking into consideration the fact that VEN was generally prescribed for

more severe depression, VEN was associated with 1.6-1.7 times more suicides

than fluoxetine or CIT (Rubino et al., 2007). Furthermore, it has been

suggested that VEN may be more toxic in PMs of CYP2D6 (Langford et al.,

2002, Lessard et al., 1999, Shams et al., 2006). Consequently, therapeutic doses

prescribed to such individuals may result in drug concentrations similar to

those found in overdoses, especially if concomitant drugs with CYP enzyme

inhibition properties are also taken.

Taken together, the high number of fatalities associated with VEN and CIT

underlines the importance of the correct interpretation of toxicological results

in forensic cases.

CONCLUSIONS | 59

CONCLUSIONS

The strength of this thesis is the combination of pharmacokinetic,

pharmacodynamic and pharmacogenetic studies of antidepressant treatment with

respect to the impact of P-gp expression (mouse model) and ABCB1 genotype

frequencies (forensic autopsy material).


Significantly higher brain concentrations were observed for VEN, CIT and

their metabolites in the abcb1ab knockout mice compared with wild-type

mice. These results show that VEN and CIT are substrates of P-gp.

The S/R ratios in knockout mice compared with wild-type mice indicated no

stereoselective P-gp mediated transport over the BBB for VEN and CIT.

The P-gp substrate properties were reflected in the open-field behavior test

where the knockout mice displayed increased centre activity compared with

wild-type mice following chronic VEN exposure.

The three animal studies highlight the importance of choosing the right

species for the specific drug of interest.

60 | CONCLUSIONS


All cases, with the exception of three cases for C1236T, were successfully

genotyped and did not significantly deviate from the Hardy-Weinberg

equilibrium.

The genotype distribution of the ABCB1 SNPs C1236T, G2677T and

C3435T in VEN positive cases was significantly (or borderline) different

between the intoxication cases and the non-intoxication cases. This


cases.

Only a few individuals positive for VEN were homozygote variant for the

SNPs (i.e. 1236TT, 2677TT or 3435TT individuals) in the intoxication cases

compared with the non-intoxication cases.

The haplotype TT-TT-TT (1236-2677-3435) seems to be associated with

suicide not associated with drug intoxication.

FUTURE ASPECTS | 61

FUTURE ASPECTS

A drug can be a substrate of several different transporters and can also

influence the disposition of co-administered drugs by inhibiting one or more

transporters, resulting in clinically important drug-drug interactions.

Furthermore, transporter inhibition can lead to the accumulation of toxic

drugs and metabolites and subsequent cellular toxicity. A major challenge for

future investigations in this field is, therefore, to clarify the different transport

mechanisms or the interplay with CYP enzymes.

Further animal studies are needed to investigate possible P-gp substrate

properties of CNS active drugs. By using both pharmacokinetic and

pharmacodynamis studies in the abcb1ab (-/-) mouse model, the relationship

between drug concentrations and the effect of antidepressant drugs can be

investigated. The findings in Paper I and II, that the metabolites of VEN,

ODV (desvenlafaxine) and NDV are P-gp substrates, and the absence of

published data, makes the metabolites interesting to study separately in the

mouse model. In order to assess the pharmacokinetic and pharmacodynamic

profiles of the separate enantiomers, an animal study could give valuable

information. The route of administration and different doses are also

interesting elements that can be studied further. Oral administration of the

antidepressants in the mouse model might give S/R ratios and M/P ratios

more closely resembling the human situation. Different doses (low-high-toxic)

62 | FUTURE ASPECTS

could also be interesting to study in the present mouse model in order to

investigate if P-gp is saturable.

The huge variation in the response of psychiatric patients to drug treatment

might be due not only to polymorphisms in drug transporter ABCB1, but also

in other drug transporters, as well as in receptors and CYP enzymes. The

finding in Paper IV, that the ABCB1 genotype distribution differs between

intoxication cases and non-intoxication cases, makes it of great interest to

perform studies on the disposition of the enantiomers of CIT and VEN in

relation to ABCB1 genotypes in forensic autopsy cases as well as in a clinical

material. The influence of polymorphisms in the drug transporter ABCB1 in

combination with CYP enzymes on drug concentrations in postmortem cases

is another interesting study. The concentrations of VEN and ODV were

found to be associated with CYP2C19, and it has been suggested that

polymorphisms in the CYP2C19 gene have an impact on the clinical outcome

(McAlpine et al., 2011). It would be very interesting to further elucidate this

important issue in patients as well as in forensic autopsy cases. An increased

knowledge of pharmacogenetics might contribute to a safer use of drugs in

the future.

Today, polytherapy is very common, and as a result, drug-drug interactions

are often a clinical problem. Drug-drug interactions can be pharmacokinetic

or pharmacodynamic, and it is therefore of high importance to study any

possible interactions. The co-administration of drugs might influence the drug

concentrations by inducing or inhibiting metabolizing enzymes, but it can also

influence the drug concentration in a specific tissue by interaction with

transporters. The substrate over-lap of CYP3A4 and P-gp makes this

interaction highly important. Transgenic mice expressing human CYP3A4 or

CYP2D6 (Corchero et al., 2001, Granvil et al., 2003) in addition to the abcb1ab

knockout mice can be used to study interactions. A transgenic mouse

expressing human P-gp exists (not yet commercially available), and it would

be of very interesting to study VEN and CIT in this model to confirm the

finding that both drugs are P-gp substrates.

ACKNOWLEDGEMENTS | 63

ACKNOWLEDGEMENTS

The studies in this thesis were conducted at the Department of Drug

Research, Linköping University: the National Board of Forensic Medicine,

Linköping: and the Johannes Gutenberg University of Mainz, Germany.

I would like to express my sincere gratitude to all my colleagues and friends

who, in one way or another, have made this thesis possible. I especially want

to thank:

My supervisors: Fredrik Kugelberg, for your endless support and

encouragement, always taking the time to discuss things with me and guiding

me. Johan Ahlner, for sharing your enthusiasm for science and for your

support. Finn Bengtsson, for giving me the scientific freedom and for your

trust in my ability to succeed with this thesis.

My German colleagues: Christoph Hiemke, for welcoming me into your lab,

for many valuable discussions and a fruitful collaboration. Ulrich Schmitt,

for introducing me to pharmacodynamics, sharing your knowledge about

animal research and the valuable discussions and comments.

My co-authors for good collaboration. Björn Carlsson, for your huge amount

of patience and always taking the time to discuss scientific problems: Martin

64 | ACKNOWLEDGEMENTS

Josefsson, for helping me with venlafaxine analysis, even when it meant

working evenings and weekends: Anna-Lena Zackrisson, for introducing me

to pharmacogenetics and being great company at conferences: Henrik Gréen,

for genetic and statistical discussions and always making me smile: and Ingrid

Jakobsen Falk, for helping me with genotyping and being a good friend

during and after working hours.

Maria Kingbäck, my former office mate and colleague, for good

collaboration, interesting discussions (scientific and non-scientific) and lots of

laughter.

The psychopharmacology research group for support and fruitful discussions,

especially Margareta Reis and Maria Cherma, for your enthusiasm and

encouragement, and for making me laugh. Thanks for many nice meetings!

Anita Holmgren for complementing the information about the forensic

cases from the databases and being good company at conferences.

My colleagues and friends, past and present, at the Division of Drug Research

for creating a stimulating working atmosphere, interesting discussions and

laughter during coffee breaks. I would especially like to thank Eva Ekerfeldt-

Hultin, for your support and giving me a helping hand in the lab (sharing

your knowledge and stock solutions), Andreas Ärlemalm, my new office

mate, for scientific and non-scientific discussions. Anna Jönsson, Svante

Vikingsson, Karin Skoglund and Simon Jönsson, for friendship and good

times during and after working hours.

Anita Thunberg, Erika Linden and Zdenka Petrovic, for all the help with

economic and administrative matters.

Wayne Jones, for reading my manuscripts and giving valuable comments

about the English language.

My biology friends Annelie and Camilla, for being good fellow students

during my basic education: Ann-Charlotte, Beatrice and Josefin, for all

ACKNOWLEDGEMENTS | 65

good memories, discussions about life, research and everything in between

and for being good friends.

All my chemistry friends, Amanda, Karin S, Karin A, Anngelica, Laila and

Ina, how you have inspired me during the years and showed me that science

is fun. Thank you for all the wonderful memories and fun parties.

My good friends, Martina, Frida, Emma, Ida and Ylva, for all the

wonderful memories we share. Seeing you is always great fun.

Emma and Daniel, for your support and friendship and Axel and Elias, for

letting me play with you and making me smile.

Britt-Marie and Janne, for welcoming me into your family, for your support

and providing a relaxing atmosphere outside work.

My family, Anne-Sofie and Sören, for your never-ending love,

encouragement and for always being there for me and supporting me in every

situation. My brother Christian, for your support and encouragement.

Erik, my love, just for being the person you are and for bringing so much

happiness into my life. For introducing me to the wonderful world of jazz. ♥

To those I have not mentioned by name in this list, you are not forgotten, you

are in my thoughts. Thank you!

This work was supported by grants from The National Board of Forensic

Medicine, the Swedish Research Council, and the Östergötland County

Council.

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