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From Department of Laboratory Medicine
Division of Clinical Pharmacology
Karolinska Institutet, Stockholm, Sweden
THE ROLE OF CYP3A4/5 IN
ALPRAZOLAM METABOLISM
Annika Allqvist
Stockholm 2010
All previously published papers were reproduced with permission from the publisher.
Published by Karolinska Institutet. Printed by Rydheims tryckeri
© Annika Allqvist, 2010
ISBN 978-91-7409-950-8
ABSTRACT
Cytochrome P450 3A (CYP3A) enzyme family is involved in the metabolism of about
50 % of all drugs in clinical use. Among CYP3A, CYP3A4 and CYP3A5 are the major
enzymes in adults; CYP3A5 is polymorphic and primarily expressed in black
populations. CYP3A5 may therefore contribute significantly to the metabolism of
CYP3A substrates in African populations.
The impact of CYP3A5 expression on drug metabolism by CYP3A is fairly unknown.
Therefore, a tool for assessment of both CYP3A4 and CYP3A5 using a single probe
drug is of interest. Overlapping substrate specificities between CYP3A4 and CYP3A5
have made it difficult to differentiate the two enzymes using probe drugs. However,
experimental in vitro data have shown a preference for formation of 4hydroxyalprazolam by CYP3A4 and that of α-hydroxyalprazolam by CYP3A5.
The aim of the present thesis was to investigate the role of CYP3A4 and CYP3A5 in
alprazolam metabolism as well as to evaluate if alprazolam may serve as a probe drug
for these two enzymes.
A liquid chromatography-mass spectrometry method for determination of alprazolam
and the two metabolites 4- and α-hydroxyalprazolam was developed. When using this
method we were able to analyze samples for pharmacokinetic and metabolism studies.
The parent drug/metabolite AUC ratio has been proven to reflect the enzymatic
activity; metabolic ratio calculated from a single plasma sample can be used in
phenotyping studies.
Alprazolam metabolism was shown to be catalyzed by both CYP3A4 and CYP3A5 in
vitro. Formation rates of 4-hydroxyalprazolam in human liver microsomes and
recombinant CYP3A4 and CYP3A5 was higher than that of α-hydroxyalprazolam,
confirming that 4-hydroxylation is the most important metabolic pathway of
alprazolam. The relative formation of α-hydroxyalprazolam was 3-fold higher for
recombinant CYP3A5 compared to CYP3A4. We thus have confirmed that CYP3A5
catalyses alprazolam metabolism, in vitro. We then evaluated the role of CYP3A4 and
CYP3A5 in alprazolam metabolism in vivo in Tanzanian and Swedish healthy
subjects who express CYP3A5 and those who do not. As there were no significant
differences in MR between subjects with different CYP3A5 genotypes it shows that
CYP3A5 plays no significant role in alprazolam metabolism. Our results show that
CYP3A4 is the major enzyme in the metabolism of alprazolam in vivo.
LIST OF PUBLICATIONS
I.
Annika Allqvist, Agneta Wennerholm, Jan-Olof Svensson, Rajaa A.
Mirghani Simultaneous quantification of alprazolam, 4- and αhydroxyalprazolam in plasma sample using liquid chromatography mass
spectrometry Journal of Chromatography B, (2005) 814: 127-131
II.
Agneta Wennerholm, Annika Allqvist, Jan-Olof Svensson, Lars L.
Gustafsson, Rajaa A. Mirghani, Leif Bertilsson Alprazolam as a probe for
CYP3A using a single blood sample: pharmacokinetics of parent drug, and
of α- and 4-hydroxy metabolites in healthy subjects Eur J Clin Pharmacol,
(2005) 61: 113-118
III.
Annika Allqvist, Jun Miura, Leif Bertilsson, Rajaa A. Mirghani Inhibition
of CYP3A4 and CYP3A5 catalyzed metabolism of alprazolam and quinine
by ketoconazole as racemate and four different enantiomers Eur J Clin
Pharmacol, (2007) 63: 173-179
IV.
Annika Allqvist, Jun Miura, Eleni Aklilu, Mary Jande, Filip Josephson,
Margarita Mahindi, Rajaa A Mirghani, Agneta Wennerholm, Jane Sayi,
Jolanta Widén, Ulf Diczfalusy, Lars L. Gustafsson and Leif Bertilsson The
role of CYP3A4/5 in alprazolam metabolism in Swedes and Tanzanians
In manuscript
Related work but not included in this thesis:
Rajaa A. Mirghani, Jane Sayi, Eleni Aklillu, Annika Allqvist, Mary Jande, Agneta Wennerholm, Jaran
Eriksen, Virginie M.M. Herben, Barry C. Jones, Lars L. Gustafsson and Leif Bertilsson CYP3A5
genotype has significant effect on quinine 3-hydroxylation in Tanzanians, who have lower total
CYP3A activity than a Swedish population Pharmacogenetics and Genomics (2006) 16:637–645
F Josephson, A Allqvist, M Janabi, J Sayi, E Aklillu, M Jande, M Mahindi, J Burhenne, Y Bottiger,
LL Gustafsson, WE Haefeli and L Bertilsson CYP3A5 Genotype has an Impact on the Metabolism of
the HIV Protease Inhibitor Saquinavir (2007) 81:708-712
Ulf Diczfalusy, Jun Miura, Hyung-Keun Roh, Rajaa A. Mirghani, Jane Sayi, Hanna Larsson, Karl G.
Bodin, Annika Allqvist, Mary Jande, Jong-Wook Kim, Eleni Aklillu, Lars L. Gustafsson and Leif
Bertilsson 4b-Hydroxycholesterol is a new endogenous CYP3A marker: relationship to
CYP3A5genotype, quinine 3-hydroxylation and sex in Koreans, Swedes and Tanzanians
Pharmacogenetics and Genomics (2008) 18:201–208
CONTENTS
1 2 3 4 Introduction .................................................................................................. 1 1.1 Drug metabolism ................................................................................ 2 1.2 Cytochrome P450 enzymes................................................................ 2 1.3 UDP-glucuronosyltranseferase enzymes ........................................... 3 1.4 DRUG DISPOSITION (P-glycoprotein) ........................................... 4 1.5 Cytochrome P450 3A ......................................................................... 4 1.5.1 CYP3A4 and CYPA5 substrates ........................................... 6 1.6 Genetic polymorphism of cytochrome P450 and drug metabolism . 6 1.7 Inter-individual variation in CYP3A4 and CYP3A5 activity ........... 7 1.8 Drug interactions involving inhibition and induction of CYP3A ..... 8 1.9 Genotyping and phenotyping ........................................................... 10 1.9.1 Genotyping ........................................................................... 10 1.9.2 Phenotyping.......................................................................... 10 1.9.3 Clinical use of phenotyping and genotyping ....................... 10 1.10 Probe drugs ..................................................................................... 11 1.10.1 Phenotyping probes for CYP3A4/5 ..................................... 11 1.11 Alprazolam ..................................................................................... 13 The present study ....................................................................................... 15 2.1 Aims .................................................................................................. 15 2.2 Subjects ............................................................................................. 16 2.3 Methods and Study design ................................................................... 16 2.3 Results............................................................................................... 20 2.4 Discussion ......................................................................................... 24 2.5 conclusions and future perspectives................................................. 27 Acknowledgements .................................................................................... 29 References .................................................................................................. 31 LIST OF ABBREVIATIONS
AUC
CL
CLint
CV
CYP
DME
EM
EMBT
FDA
HIV
HPLC
Km
KTZ
LC/MS
MDR1
MR
NADPH
PCR
P-gp
PM
RFLP
SNP
TDM
TPMT
UGT
Vmax
Area under the plasma concentration versus time curve
Clearance
Intrinsic clearance
Coefficient of variation
Cytochtome P450
Drug metabolising enzyme
Extensive metaboliser
Erythromycin breath test
Food and drug administration
Human immunodefieciency virus
High performance liquid chromatography
Michaelis-Mentens constant
Ketoconazole
Liquid chromatography-mass spectorometry
Multidrug resistant gene 1
Metabolic ratio
Nicotineamide adenine dinucleotide
Polymerase chain reaction
P-glycoprotein
Poor metaboliser
Restriction fragment length polymorphism
Single nucleotide polymorphism
Therapeutic drug monitoring
Thiopurine methyltransferase
UDP-glucuronosyltranseferase enzymes
Maximum rate of formation
1 INTRODUCTION
It is well known that patients may respond differently to the same drug therapy. These
inter-individual variations can result in drug toxicity in some patients, while others will
experience therapeutic failure. The existence of large population differences with small
intra-patient variability is consistent with inheritance as determinant of drug response
and it is estimated that variation in genes encoding drug-metabolizing enzymes, drug
transporters, or drug targets can account for 20 to 95% of variability in drug response
and effects. Moreover, environmental factors such as physiologic status and diet as well
as drug interactions, concomitant therapy can influence the drug response (Kalow et
al., 1998; Evans and Relling, 1999; Evans and Johnson, 2001; McLeod and Evans,
2001).
Cytochrome P450 (CYP) enzymes catalyze generally most of the oxidation of
xenobiotics, in order to facilitate their excretion from the body. CYP3A is the main
CYP enzyme subfamily and catalyses the metabolism of over 50% of all clinically used
drugs. CYP3A4 and CYP3A5 are the most common forms among adults. Of these
CYP3A5 is polymorphic and expressed primarily in black populations and to a minor
extent in Caucasians. CYP3A5 may therefore contribute significantly to metabolism of
CYP3A substrates in African populations.
The aim of the present thesis was to investigate the role of CYP3A4 and CYP3A5 in
alprazolam metabolism as well as to evaluate the use of alprazolam as a probe drug for
these two enzymes.
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1.1
DRUG METABOLISM
The concentration of a drug at its site of action or the plasma concentration achieved
after administration of a drug is determined by the following processes; absorption,
distribution, metabolism and elimination. The most important factor affecting the drug
concentration is metabolism (Fukasawa et al., 2007). Drug metabolism occurs mainly
in the liver, and usually converts lipophilic drugs to more polar metabolites prior to
elimination. The drug metabolism is normally divided into phase I and phase II
reactions. In phase I, which represents many of cytochrome P450 enzymes (CYP) a
polar group is introduced to the parent drug through oxidation, reduction or hydrolysis.
The more polar intermediates from phase I reactions may be conjugated with watersoluble groups in phase II reactions through the actions of uridine diphosphate (UDP)
glucuronosyltransferases (UGTs), sulfutransferaser, etc., to further facilitate
excretion. Phase I metabolism can occur during drug absorption, either in the gut wall
or in the liver. This is called first-pass-metabolism, which determines the fraction of an
oral dose that will reach the systemic circulation i.e. the bioavailability of the drug
(Fukasawa et al., 2007)
1.2
CYTOCHROME P450 ENZYMES
Cytochrome P450, a cellular chromophore, was first named in 1961, because the
pigment (P) has a 450-nm spectral peak when reduced and bound to carbonmonoxide
(Omura and Sato, 1964). Early in the 1960s, cytochrome P450 was thought to be one
enzyme and later that decade it was associated with drug and steroid metabolism
(Nebert and Russell, 2002). Cytochrome P450s are heme-containing proteins and
their catalytic activity is integrated in the membrane of the smooth endoplasmatic
reticulum with cytosolic orientation (Nebert and Gonzalez, 1987). Cytochrome P450s
are monooxygenases, which catalyse oxidative and reductive reactions, where
commonly one of the two oxygen atoms of O2 is incorporated into the substrate (S).
Figure 1: The equation of cytochrome P450-dependent reactions
NADPH + H+ O2 + SH → NADP+ H2O + SOH
The other oxygen is reduced to H2O, NADPH or NADH function as co-substrates and
required as electron donors. The electrons are transferred via NADPH-cytochromeP450-reductase and cytochrome b5.
2
The cytochrome P450s can be functionally categorized based particular on endogenous
substances such as fatty acids, steroids and prostaglandins and those that are
responsible in the metabolism of exogenous substances such as drugs, environmental
chemicals and pollutants, and natural plant products (Gonzalez and Nebert, 1990;
Nelson et al., 1993).
The human drug metabolizing P450s belong almost exclusively to families 1, 2 and 3
(Ingelman-Sundberg et al., 2001) which are all found in the endoplasmic reticulum
(Nebert and Gonzalez, 1987) and are responsible for 70–80% of all phase I
metabolism of clinically used drugs (Bertz and Granneman, 1997; Evans and Relling,
1999). Members of individual families show at least 40% homology at protein
sequence level. These families are further divided into subfamilies whose members
are individual enzymes that show at least 55% homology to one another and are
designated by a letter, e.g. CYP3A. A second Arabic number indicates the individual
isoenzyme, e.g. CYP3A4 (Nebert et al., 1987; Nebert and Gonzalez, 1987; IngelmanSundberg et al., 2001).
CYPs are sometimes catalyzing metabolism of substrates to toxic reactive
intermediates which can contribute to increased risks of cancer, birth defects, and
other toxic effects (Dalton et al., 1999; Nebert et al., 2000; Nebert and Russell, 2002;
Rendic, 2002). Some drugs are so called prodrugs which mean that the drug is inactive
until metabolised in the liver by CYP450 enzymes. Cyclophosphamide is one example
of a prodrug which will be active after metabolism by CYP2B6 (Bray et al., 2010).
1.3
UDP-GLUCURONOSYLTRANSEFERASE ENZYMES
UDP-glucuronosyltranseferase (UGT) enzymes belong to important enzymes involved
in the deactivation and elimination of a wide range nucleophilic substances. UGT
enzymes are localized to the internal membrane of the endoplatmatic reticulum that
catalyzes the transfer of the glucuronic acid group to the functional group of e.g.
hydroxyl, carboxyl, amino, sulfur of a specific substrate. Glucuronidation increases
the polarity of the target compounds and facilitates their excretion in bile or urine
(Guillemette, 2003). Alprazolam is conjugated by glucuronidation to increase the
polarity before excretion. We were able to measure total (conjugated+unconjugated)
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amount of alprazolam and the two metabolites; it will be presented in the results part of
study IV.
1.4
DRUG DISPOSITION (P-GLYCOPROTEIN)
Transport proteins have an important role in regulating the absorption, distribution,
and excretion of many drugs. P-glycoprotein (P-gp) is a membrane transporter and
plays an important role in the defense of cells against carcinostatic drugs (Borst et al.,
2000). The gene encoding P-gp was therefore, called multidrug resistant gene 1
(MDR1 gene) and expression of this transporter is localized to the luminal surface of
the endothelial cell so the drug substrate is transported by energy-dependent backefflux into the circulating blood, which limits drug entry into the tissue. (Borst et al.,
2000; Choo et al., 2000; Brinkmann et al., 2001). The P-glycoprotein is expressed in
many normal tissues including the apical membrane of gastrointestinal tract, blood
cells, biliary tract and the kidney. This expression profile suggest that the protein has
a role in the normal secretion of xenobiotics and metabolites into bile, urine, and
directly into the lumen of the gastrointestinal tract (Thiebaut et al., 1987; Rao et al.,
1999). At the blood–brain barrier, P-glycoprotein limits the accumulation of many
drugs in the brain, e.g. digoxin and cyclosporine (Schinkel et al., 1996).
Of importance for drug disposition is that CYP3A and P-gp are frequently co-expressed
in the same cell or tissues and share a large number of substrates. A number of striking
overlaps between CYP3A and P-gp has been described suggesting that these have
complementary roles in the pharmacokinetics of drug absorption and elimination
(Wacher et al., 1995). However, it has been suggested that P-gp does not play a
clinically significant role in alprazolam disposition (Yasui et al., 2000) as will be
further discussed in the alprazolam section below.
1.5
CYTOCHROME P450 3A
The human CY3A subfamily is found in a cluster on chromosome 7q22 and spans
220kb. It consist of four human genes, CYP3A4, CYP3A5, CYP3A7 and CYP3A43
(Thompson et al., 2004), as well as three pseudogenes (CYP3AP1, CYP3AP2 and
CYP3AP3) (Perera, 2010). The CYP3A genes are clustered together and are therefore,
thought to be the result of gene duplication events. They are closely related with a
sequence similarity of about 95% between CYP3A4 and CYP3A7, and a lower
similarity of about 85 to 90% between CYP3A4 and the other members of the family
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(Perera, 2010). CYP3A5 share approximately 84% sequence identity with CYP3A4
(Aoyama et al., 1989), and similar substrate specificity which makes it difficult to
dissect their respective contribution to overall CYP3A-mediated drug metabolism (Xie
et al., 2004). CYP3A4 and CYP3A5 are the dominant forms in adults (Lee and
Goldstein, 2005), demonstrating the highest levels of expression in liver (30%) as
shown in Figure 2 (Shimada et al., 1994) and small intestine (82%) (Paine et al., 2006)
of total P450 content.
Figure 2: Relative abundance of different CYP isoforms in the human liver
Contents of total P450 determined spectrally and individual isoforms determined immunochemically
(percantage of total P450) in human liver microsomes Shimada et al. 1994) CYP3A consist of CYP3A4,
3A5 and 3A7: and CYP2C of CYP2C8, 2C9, 2C18 and 2C19. From (Shimada et al., 1994)
Of the two major CYP3A members, CYP3A4 is considered the major enzyme
expressed in adult livers and the small intestine in all populations examined (Lamba et
al., 2002) CYP3A7 is predominantly a foetal enzyme, with high levels of expression
in foetal liver and intestine, whereas it is only present at low levels in adults because
its expression appears to be silenced shortly after birth.(Schuetz et al., 1994; Lamba et
al., 2002; Williams et al., 2002). CYP3A7 plays an important role in the metabolism
of endogenous substances, as well as potentially toxic compounds (Ohmori et al.,
1998; Marill et al., 2002). CYP3A43 is expressed at very low levels in several tissues,
including liver, prostate, and testis (Westlind et al., 2001). Recently it was found that
expression of CYP3A43 was relatively higher in brain as compared to liver across
different ethnic populations (Agarwal et al., 2008).
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1.5.1 CYP3A4 and CYPA5 substrates
The cytochrome P450 3A (CYP3A) enzymes are responsible for phase I metabolism of
over 50% of drugs administered to humans (Li et al., 1995; Wacher et al., 1995; Evans
and Relling, 1999; Guengerich, 1999).
CYP3A4 contributes to the disposition of more than 60 therapeutically important drugs
(Ozdemir et al., 2000). A partial list of drugs metabolized by CYP3A is presented in
table 1. In addition CYP3A4 and CYP3A5 participate in the metabolism of key
endogenous substrates, such as retinoic acid, steroid hormones and bile acids
(Ingelman-Sundberg et al., 2007).
Table 1: Examples of substrates which are classified under respective drug classes (in
bold) for CYP3A
CYP3A substrates
Benzodiazepines:
midazolam
alprazolam
Steroid 6beta-OH:
testosterone
progesterone
Calcium Channel Blockers:
verapamil
HMG CoA Reductase Inhibitors:
simvastatin
Macrolide antibiotics:
erythromycin
clarithromycin
Anti- arrhythmics:
quinidine
Immune modulators:
tacrolimus
cyclosporine
HIV Antivirals:
indinavir
ritonavir
saquinavir
Other:
tamoxifen
carbemazepine
dapsone
alfentanil
quinine
Adopted from Flockhart (http://medicine.iupui.edu/clinpharm/ddis/table.asp)
1.6
GENETIC POLYMORPHISM OF CYTOCHROME P450 AND DRUG
METABOLISM
The drug metabolizing cytochrome P450 enzymes are often polymorphic and
consequently individuals are classified as poor metabolizer (PM) or extensive
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metabolizer (EM) i.e. different ability to metabolize a specific drug (Wilkinson,
2005). PMs are lacking functional enzyme due to defective or deleted genes, and
generally display higher drug concentrations and are more likely to suffer from side
effects or toxicity especially when the parent drug is metabolized exclusively by the
polymorphic enzyme. On the other hand, EM, carrying 1 or 2 functional genes may
therefore show sub-therapeutic levels at recommended doses and need higher doses to
obtain a therapeutic response. If the formation of an active metabolite is necessary for
therapeutic effects, PMs may have therapeutic failure and/or EMs may have a higher
risk of adverse effects. The variability in metabolism is mainly determined by genetic
polymorphisms, which may be a single nucleotide change in the DNA sequence, an
insertion or deletion of nucleotides, or a change in the number of gene copies (copy
number variation) (Perera, 2010). The most common types of polymorphisms are
those called single nucleotide polymorphisms (SNP). In order to define a
polymorphism the nucleotide exchange must be present in more than 1% of the
population that confer increased, decreased or no activity. As a result, variability
among people can be extremely large (Evans and McLeod, 2003; Weinshilboum,
2003). If the elimination of a drug is predominantly determined by metabolism, and a
single cytochrome P450 enzyme is primarily responsible, a functional polymorphism
may have important clinical consequences. The frequency of variant alleles and their
encoded proteins varies among populations according to race and ethnic background
(Xie et al., 2004).
1.7
INTER-INDIVIDUAL VARIATION IN CYP3A4 AND CYP3A5 ACTIVITY
Substantial inter-individual differences in CYP3A expression, exceeding 30-fold in
ethnic populations (Aoyama et al., 1989), contribute greatly to variation in oral
bioavailability and systemic clearance of CYP3A substrates, including HIV protease
inhibitors, several calcium channel blockers and some cholesterol-lowering drugs
(Table 1). Variation in CYP3A expression is particularly important for substrates
with narrow therapeutic index, such as cancer chemotherapeutics (Wrighton et al.,
1989) and immunosuppressants cyclosporin A and tacrolimus (Daly, 2006). Such
variation in CYP3A can result in clinically significant differences in drug toxicities,
nephrotoxicity and response, graft survival (Kuehl et al., 2001).
This is considerable and clinically significant inter-individual differences in CYP3A
activity, may be attributed to different factors such us variable expression, drug and
dietary interactions, and genetic polymorphisms. It has been suggested that as much as
7
70-95% of CYP3A interindividual variability maybe explained by genetic variations
(Eichelbaum et al., 2006; Oleson et al., 2010). To date, 40 CYP3A4 alleles have been
described (http://www.cypalleles.ki.se/cyp3a4.htm), but no SNPs have been discovered
that accounts for this wide variability (Oleson et al., 2010)
In contrast, CYP3A5 is highly polymorphic and expressed primarily in black
populations (60 to 73%) and to a minor extent in Caucasians (5 to 30%) (Hustert et al.,
2001; Kuehl et al., 2001). Only people with at least one CYP3A5*1 allele express
CYP3A5. SNPs in CYP3A5*3 and CYP3A5*6 cause an alternative splicing and
protein truncation, respectively. In CYP3A5*7 a frameshift, leads to severely
decreased synthesis of functional CYP3A5 and absence of CYP3A5 in tissues of
some individuals (Wojnowski et al., 2004).
CYP3A5 protein represents more than 50% of the total CYP3A content in one third
of Caucasian livers and over one-half of African American livers (Kuehl et al., 2001).
However, these results are contradicted by Westlind-Johnsson et al; (WestlindJohnsson et al., 2003) who were able to show that in Caucasians the proportion of
CYP3A5 of the hepatic CYP3A would only be 17% in the 11% who had quantifiable
levels. Nevertheless, CYP3A5 may contribute significantly to the metabolism of
CYP3A substrates (Xie et al., 2004) in African populations (Kuehl et al., 2001).
The lack of functional CYP3A5 may not be readily evident, because many
medications metabolized by CYP3A5 are also metabolized by the universally
expressed CYP3A4 (Evans and McLeod, 2003). For substrates metabolized by both
CYP3A4 and CYP3A5, it is important to investigate the relative importance of the two
enzymes to the in vivo clearance as it may result in different metabolic patterns in black
populations relative to Caucasians.
1.8
DRUG INTERACTIONS INVOLVING INHIBITION AND INDUCTION OF
CYP3A
Inhibition of metabolism of one drug by addition of another can cause problem, since
plasma concentration may increase which can cause adverse or toxic effects
(Wilkinson, 2005). Usually the inhibition of CYP3A is reversible, typically within
two to three days, once the interacting drug is eliminated from the body (Lin and Lu,
2001; Wilkinson, 2005) In the case that the inhibition is irreversible, the enzyme is
involved in a complex with a metabolic intermediate which made the complex unable
8
for drug metabolism. Only newly synthesized CYP3A enzyme can restore the
enzyme; for example when macrolide antibiotic, diltiazem is used (Thummel and
Wilkinson, 1998; Lin and Lu, 2001). However, inhibitors are useful in clarifying the
role of individual CYP3As in drug metabolism and in predicting the potential drugdrug interactions both in-vivo and in-vitro (Liu et al., 2007). Table 2 present
examples of inhibitors which can interact with CYP3A. In study III we investigated
the inhibition of alprazolam metabolism in vitro by ketoconazole (KTZ) and its
isomers.
Treatment with some drugs can increase CYP3A activity through transcriptional
activation (induction) that is mediated by the nuclear receptor pregnane X receptor
(PXR) and /or constitutive androstane receptor (CAR) (Liu et al., 2007). As an
example rifampin reduces the plasma concentration of for example cyclosporine and
then therapeutic failure may occur (Wilkinson, 2005).
Examples of CYP3A inhibitors and inducers are presented in table 2.
Table 2: Examples of inhibitors and inducers of CYP3A These inhibitors and inducers can interact
with any CYP3A substrate and may have important clinical consequences.
CYP3A Inhibitors
CYP3A Inducers
ketoconazole
diltiazem
verapamil
itraconazole
clarithromycin
erythromycin
troleandomycin
delavirdine
indinavir
ritonavir
saquinavir
grapefruit juice
rifabutin
rifampin
glucocorticoids
carbamazepine
phenytoin
barbiturates
efavirenz
nevirapine
St. John’s wort
Adopted from Flockhart (http://medicine.iupui.edu/clinpharm/ddis/table.asp)
9
1.9
GENOTYPING AND PHENOTYPING
1.9.1 Genotyping
The genotype refers to the inheritable genetic constitution of an individual.
Genotyping of CYPs can be difficult, due to high sequence similarity between the
different CYP genes within the same subfamily. Therefore, careful controls of the PCR
products are necessary to guarantee that the result obtained correspond to the right
target (Ingelman-Sundberg et al., 2007). There are different techniques that can be
used for genotyping different CYPs such as polymerase chain reaction (PCR) followed
by detection step, restriction fragment length polymorphism-PCR (RFLP-PCR),
Taqman and high-throughput detection methods like microarrays. The method of
choice depends on number of samples, cost and accuracy.
1.9.2 Phenotyping
Cytochrome P450 phenotyping provides variable information about real-time activity
of these important drug-metabolizing enzymes through the use of specific probe drugs.
Phenotyping provides the most clinically relevant information because it is a reflection
of the combined effects of genetic, environmental and endogenous factors on the CYP
activity (Streetman et al., 2000). The procedure includes the use of a probe drug that is
mainly metabolized by a specific CYP and estimation of a parameter that may be
clearance (CI), area under the plasma curve (AUC), both of which require multiple
sample collection, or ratio between the concentration of the parent drug and the
concentration of metabolite formed specifically by that CYP enzyme in a single plasma
or urine sample The concentration of the parent drug and the metabolite are measured
by using techniques such as HPLC (high performance liquid chromatography) and LCMS (Liquid chromatography masspecrtometry). A low metabolic ratio shows a higher
activity of the specific CYP enzyme and vice versa.
1.9.3 Clinical use of phenotyping and genotyping
Traditional therapeutic drug monitoring (TDM) can be regarded as a phenptyping
method, and used to optimise the dose for each patient and is usually used for drugs
with narrow therapeutic range e.g. nortriptyline (Dahl and Sjoqvist, 2000; Zaigler et al.,
2000). If there is an established concentration versus therapeutic effect correlation, the
drug dose can adequately be adjusted according to the TDM result. TDM is preferred if
10
there is an existing validated method. CYP3A4 does not show a relevant genetic
polymorphism and therefore the activity can only be assessed by phenotyping
(Zaigler et al., 2000).
Phenptyping procedure often requires that the patient has to be drug free, which
means that drug therapy has to be interrupted. Therefore the development of
genotyping methods, which can be used during active drug therapy have a great
potential as a complement to traditional TDM (Dahl and Sjoqvist, 2000). Food and
Drug Administration (FDA) advices that cancer treatment with tamoxifen, irinotecan
and 6-mercaptopurines should be accompanied with genetic testing of the drug
metabolizing enzyme genes CYP2D6, UGT1A4, TPMT, respectively (Schulze et al.,
2009).
1.10 PROBE DRUGS
There are validation criteria suggested for probe drugs reviewed by Watkins (Watkins,
1994). Probe drugs (or probe substrates) should be predominantly or exclusively
metabolized in vitro by an individual CYP enzyme. Drugs that are selectively
metabolized, and that can be safely administered to humans, may be used as in vivo
probe drugs for the purpose of estimating enzyme activity i.e. phenotyping (Watkins,
1994; Zaigler et al., 2000; Frye, 2004).
There are often poor correlations between different CYP3A4 probe drugs. Reasons for
this discordance among probe drugs and important information of finding a specific
CYP3A4 probe are summarized by Zaigler et al (Zaigler et al., 2000). Some of those
possible confounding factors on CYP3A4 probes are existence of other, closely related
enzymes (CYP3A5 and CYP3A7), overlapping substrate specificity with other drugmetabolizing enzymes and that dietary factors modify CYP3A activity. Grapefruit juice
can inhibit and St Johns worth may induce CYP3A enzymes.
1.10.1 Phenotyping probes for CYP3A4/5
Since, CYP3A4 activity varies 5- to 20-fold among humans and shows no evidence for
genetic polymorphisms, only phenotyping could establish CYP3A4 capacity in vivo
(Zaigler et al., 2000).
11
To assess the CYP3A activity several phenotyping probe drugs have been proposed e.g.
midazolam (Thummel et al., 1994a; Thummel et al., 1994b), erythromycin (Watkins,
1991), alfentanil (Liu et al., 2007), dapsone (Streetman et al., 2000), quinine (Mirghani
et al., 2003), as endogenous markers such as cortisol (Kovacs et al., 1998), and 4ßhydroxycholesterol (Diczfalusy et al., 2008) have been proposed.
The most commonly used probe drug is midazolam (MDZ) which is a short acting
benzodiazepine. Midazolam is predominantly metabolized to 1’-hydroxy midazolam
by both CYP3A4 and CYP3A5 (Gorski et al., 1994; Wandel et al., 1994). It can be
administered both intravenously and orally (Gorski et al., 1998). However, it require
multiple blood samples for up to 6 h post dose to calculate clearance, which serves as
an index of CYP3A activity (Lee et al., 2002). Unlike other CYP3A probe drugs,
midazolam is not a P-glycoprotein substrate (Schmiedlin-Ren et al., 1997). Another
used probe is erythromycin breath test (EMBT) which is based on the observation that
demethylation of erythromycin is mediated by hepatic CYP3A4, and the carbon in the
methyl group is exhaled as CO2. The test requires intravenous administration of
radioactively labeled dose of 14C N-methylerythromycin followed by a single breath
sample collected 20 min after injection of the drug and the rate of 14CO2 production is
calculated (Franke et al., 2008)
Dapsone urinary ratio has been reported to correlate poorly with other CYP3A4 probes
(Streetman et al., 2000). Quinine 3-hydroxylation was shown to be useful as a
biomarker for activities of both CYP3A4 and CYP3A5 (Mirghani et al., 2003;
Mirghani et al., 2006). Cortisol is an endogenous marker, suggested to be used only to
detect inhibition and induction (Liu et al., 2007). Another endogenous marker, 4ßhydroxycholesterol a metabolite of cholesterol has been discussed to reflect CYP3A
activity (Diczfalusy et al., 2008).
It should be noted that several of the substrates used to measure CYP3A4 activity are
not extensively validated as accurate biomarkers for CYP3A4 activity. Among the
CYP3A4 substrates, only the erythromycin breath test and the midazolam plasma
clearance are widely accepted as in-vivo probes of CYP3A4 activity. Midazolam and
erythromycin are both extensively metabolized by CYP3A4 and meet most of the
criteria for an ideal in-vivo CYP3A4 probe (Watkins, 1994). However, erythromycin
requires intravenous administration of a radioactively labeled drug and midazolam
12
requires multiple blood samples to predict CYP3A activity (Kivisto and Kroemer,
1997).
1.11 ALPRAZOLAM
Among CYP3A, CYP3A4 and CYP3A5 are the major enzymes in adults; CYP3A5 is
polymorphic and primarily expressed in black populations. CYP3A5 may therefore
contribute significantly to the metabolism of CYP3A substrates in African populations.
The impact of CYP3A5 expression on the drug metabolism catalyzed by CYP3A is
fairly unknown (Williams et al., 2002). Therefore, a tool for assessment of both
CYP3A4 and CYP3A5 using a single probe drug is of interest. Overlapping substrate
specificities between CYP3A4 and CYP3A5 (Williams et al., 2002) have made it
difficult to differentiate the two enzymes using probe drugs. Midazolam has been
discussed as a probe drug for both CYP3A4 and CYP3A5; however the contribution
of CYP3A5 to MDZ hydroxylation in vivo has been questioned (Streetman et al.,
2000).
Alprazolam is metabolized by CYP3A producing α-hydroxyalprazolam and 4hydroxyalprazolam (figure 3). Experimental in vitro data have shown a preference for
formation of 4-hydroxyalprazolam by CYP3A4 and that of a-hydroxyalprazolam by
CYP3A5 (Gorski et al., 1999; Hirota et al., 2001; Galetin et al., 2004).
Figure 3: The metabolic pathways of alprazolam. From Hirota et al; 2001.
13
In order to evaluate CYP3A metabolic capacity it would be an advantage to use a
probe drug without affinity for P-gp to not add variability in uptake onto the
metabolic variation. It has been suggested that P-gp does not play a clinically
significant role in alprazolam disposition, which is indicated by the high oral
bioavailability of alprazolam (more than 90%) (Greenblatt and Wright, 1993; Yasui et
al., 2000).
Alprazolam has been used in healthy subjects to study the impact of CYP3A5
expression on CYP3A activity (Gashaw et al., 2003). Alprazolam might be a possible
probe drug for both CYP3A4 and CYP3A5.
14
2 THE PRESENT STUDY
2.1
AIMS
The aim of the present thesis was to investigate the role of CYP3A4 and CYP3A5 in
alprazolam metabolism as well as to evaluate if alprazolam may serve as a probe drug
for these two enzymes.
Study I
To develop a liquid chromatography mass spectrometry method for simultaneous
quantification of alprazolam, 4- and α-hydroxyalprazolam in plasma.
Study II
To study the pharmacokinetics of alprazolam and its metabolites in healthy Swedish
subjects and to investigate the use of a single plasma sample to assess CYP3A activity
using metabolic ratios of alprazolam.
Study III
To evaluate the metabolism of alprazolam and quinine in vitro by using human liver
microssomes (HLM) and recombinant CYP3A4 and CYP3A5, and to study the
inhibition of these metabolic reactions by ketoconazole (KTZ) and its isomers.
Study IV
To investigate the role of CYP3A5 in alprazolam metabolism in healthy black
Tanzanian and healthy Swedish Caucasian subjects and to study whether 4hydroxylation of alprazolam reflects the activity of CYP3A4 and α-hydroxylation
reflects the activity of CYP3A5.
15
2.2
SUBJECTS
In study II, the subjects, were healthy according to a pre study screening procedure. All
subjects were Caucasians and selected from a panel of volunteers at the Division of
Clinical Pharmacology at Karolinska University Hospital, Huddinge. Sweden. In study
IV, 144 healthy black Tanzanians were recruited from students and staffs at
Muhimbilli University College of Health Sciences, Dar es Salaam and 26 healthy
Swedish Caucasians were selected from a previous study (Diczfalusy et al., 2008) to
get as many subjects as possible with CYP3A5*1 alleles from a panel of subjects at
Division of Clinical Pharmacology at Karolinska University Hospital, Huddinge,
Sweden. All subjects were considered healthy according to a pre study screening
procedure. The subjects in study II and IV were not allowed to use medications
(including contraceptive pills and herbal drugs) during the study period. The subjects
were advised to refrain from alcohol (48 hours before study start and during study day
1) and grapefruit juice (throughout the study). All subjects were non-smokers.
2.3 METHODS AND STUDY DESIGN
Liquid chromatography-mass spectrometry method
Study I
This method was developed for simultaneous quantification of alprazolam and its two
metabolites 4-hydroxyalprazolam and alpha-hydroxyalprazolam in plasma. The work
up procedure was solid phase extraction. Alprazolam and the metabolites were eluted
with 100% acetonitrile. The samples were dried in a vacuum centrifuge and
subsequently dissolved in 50 µL 20% acetonitrile in water. Aliquots of 15 µL were
injected into the liquid chromatography–mass spectrometry (LC–MS).
Pharmacokinetic study with alprazolam
Study II
In the first part of the study, data were collected for determination of pharmacokinetic
parameters of alprazolam and its two metabolites in plasma from 12 healthy Swedish
subjects. The subjects had fasted from midnight and continued fasted 2 h after oral
ingestion of a tablet of 1 mg alprazolam at about 8 am. Blood samples were drawn
before intake and up to 72 h after intake of alprazolam.
16
In vitro study with human liver microsomes and recombinant enzymes
Study III
Incubations were performed in the linear range, regarding time and protein
concentration. NADPH was added as co-enzyme. The reactions were allowed to
proceed at 37ºC in a shaking water bath. The reaction was terminated with methanol
and rapid freezing. In vitro study for formation rates of alprazolam metabolites were
performed with both human liver rmicrosomes and recombinant CYP3A4 and
CYP3A5 enzymes and for quinine with recombinant enzymes. Inhibition experiments
were performed as incubation reactions there ketoconazole (KTZ) and four different
KTZ enantiomers at seven different concentrations were added. The concentrations of
formed 3-hydroxyquinine and 4- and α-hydroxyalprazolam were measured by HPLC
(Mirghani et al., 2001) and LC-MS (study I), respectively.
Phenotyping study with alprazolam
Study IV
Alprazolam (1 mg) was given to Swedish (n=26) and Tanzanians (n=144) subjects, a
blood sample for analysis was collected 10 h after drug intake at about 8 a.m. for
Swedish subjects and 8 h after drug intake at about 4 p.m. for Tanzanian subjects. The
reason for using different time points between the two populations was to observe the
drug intake of the Tanzanian subjects. Plasma was separated and analysed with LC/MS
method (study I) for quantification of alprazolam and the two metabolites. The enzyme
activity, expressed as the ratio between the substrate and its metabolite was calculated.
A low metabolic ratio shows a higher activity of the specific CYP enzyme and vice
versa.
Pharmacokinetic calculations (study II)
Pharmacokinetic parameters were determined using non-compartmental methods.
Maximum plasma concentrations (Cmax) and tmax (time to attain Cmax) were
estimated from observed plasma concentration versus time data. The elimination rate
constant (k) was determined by linear regression analysis of post-absorptive and postdistributive plasma concentration–time curve, and the apparent elimination half-life
(t1/2) of alprazolam and its metabolites was determined as 0.693/k. The area under the
plasma concentration versus time curve from 0 to infinity (AUC(0–∞) ) was obtained
by summation of AUC(0–72), as determined by the linear trapezoidal rule,
17
and AUC(72–∞). AUC(72–∞) was calculated by the ratio Ct/k where Ct was the last
measured concentration. The metabolic ratios were determined from molar
concentrations.
Enzyme kinetics (study III)
The enzyme kinetic parameters, the Michaelis-Mentens constant (Km), the maximal
formation rate (Vmax) and the intrinsic clearance (Vmax/Km) for the formation of 3hydroxyquinine were determined using Michaelis-Menten (one-enzyme) kinetic
module in the software 4.03.
Genotyping (study IV)
Tanzanian subjects were genotyped for CYP3A5*3 and *6 by Taqman and *7 by
allele-specific PCR using specific primers the Swedish subjects genotyped only for
CYP3A5*3 since, CYP3A5*6 and *7 have been reported in the literature to be absent
in Caucasians. Genotyping was performed in an earlier study as described in more
details by Mirgahni et al (Mirghani et al., 2006).
Enzymatic hydrolysis (study IV)
First, 1.5 mL of β-glucuronidase (Escherichia coli K12, was purchased from Roche)
was mixed with 5 mL of 1.0 M potassium phosphate buffer (pH 6.0) to prepare the
working solution. After thawing the samples, 0.4 ml of plasma was incubated with
0.1 mL of β-glucuronidase working solution for 15 min at 37 °C. Incubation
conditions were optimized by initial experiments using different incubation times (0,
15, 30, 60 min) and volumes of β-glucuronidase working solution (0, 50, 100, 200 µl)
at 37°C.
The concentrations of the metabolites 4- and α-hydroxyalprazolams dramatically
increased after incubation with 0.1 mL β-glucuronidase working solution for 15 min,
whereas they did not show any apparent increase with longer incubation time or with
higher enzyme volume (data not shown). Therefore, we believe that the metabolites
are hydrolyzed optimally and completely under this condition.
18
Statistical analysis (study IV)
Statistical analysis was performed on log-transformed data and presented as the antilog
of the value. The mean for each metabolic ratio (MR) was calculated from the log-MR
presented as the antilog of the value. MR is always calculated with unconjugated
concentration of alprazolam but when mentioned both with unconjugated or total
(unconjugated +conjugated) concentration of respective metabolite. The Student t-test
was used to investigate the differences in the alprazolam MR between Tanzanians and
Swedes lacking CYP3A5*1 allele, and MR within Tanzanians with different number of
functional CYP3A5 alleles. Since the Swedish subjects were selected with the regard
to CYP3A5 genotype not random, for this study we did not statistically compare the
two populations.The software Statistica version 8.0 for windows (StatSoft Inc.,
Tulsa,OK, USA) was used for the statistical calculations. P values of less than 0.05
were considered statistically significant.
19
2.3
RESULTS
Study I:
Extraction recovery of alprazolam and its two metabolites in plasma was more than
82% for all substances at three different concentration levels.
The peaks for alprazolam, the internal standard and the two metabolites were well
separated from each other. The ion chromatogram below (Fig. 4) shows alprazolam
and its two metabolites in plasma collected from a volunteer 2 h after intake o 1mg of
alprazolam. The within- and between-assay CVs of the method for alprazolam and
the two metabolites in plasma were between 1.9 and 17.8%.
Figure 4: Ion chromatogram showing alprazolam and its two metabolites in plasma collected from a
volunteer 2 h after intake 1mg of alprazolam (IS= internal standard) From (Allqvist et al., 2005).
Study II:
After a single dose of 1 mg alprazolam, the parent drug was measured for up to 72 h in
all subjects, 4-hydroxyalprazolam for up to 48 h in all subjects and αhydroxyalprazolam for up to 24 h in all subjects (figure 5, below). We found a five- and
fourfold variation in AUC (0-∞) ratio of alprazolam/4-hydroxyalprazolam (4hydroxyalprazolam ratio) and AUC (0-∞) ratio of alprazolam/α-hydroxyalprazolam (α-
20
hydroxyalprazolam ratio), respectively. All plasma concentration ratios
(alprazolam/respective metabolite) collected between 1 h and 48 h correlated
significantly to AUC (0-∞) ratios for alprazolam/4-hydroxyalprazolam and
alprazolam/α-hydroxyalprazolam.
Figure 5: Mean plasma concentration-time profiles of alprazolam (upper curve), 4hydroxyalprazolam (middle curve) and α-hydroxyalprazolam (lower curve) after oral
administration of 1 mg alprazolam (n=12) The plasma concentration of α-hydroxyalprazolam at 48
h was set to half lower limit of quantification (0.025 ng/ml) for four subjects as their levels were
below the limit of quantification From (Wennerholm et al., 2005).
Study III:
The formation rate of 4-hydroxyalprazolam in vitro was higher than that of α
hydroxyalprazolam by 3.5-, 8.4- and 2.0-fold in HLM, CYP3A4 and CYP3A5,
respectively. The relative formation of alpha-hydroxyalprazolam was 3-fold higher for
CYP3A5 compared to CYP3A4. The intrinsic clearance by CYP3A4 and CYP3A5
were similar for the formation of 3-hydroxyquinine. Alprazolam and quinine
metabolism were shown to be catalyzed by both CYP3A4 and CYP3A5. The different
KTZ enantiomers inhibited CYP3A4 and CYP3A5 to different degrees, indicating
structural differences among the enantiomers.
Study IV:
For subjects without functional alleles of CYP3A5, Tanzanians had significantly higher
metabolic ratio for 4-hydroxylation than Swedes (figure 6, lower p=0.015), which
means that Tanzanians have lower CYP3A4 activity than Swedes. The tendency was
21
the same for MR of alp/α-OH but no significant difference was observed between the
two populations (figure 6, upper).
Contrary to expected among Tanzanians without functional alleles of CYP3A5 the MR
for alp/α-OH was significantly lower than corresponding mean MR in the group of
subjects with expression of one CYP3A5 allele (figure 6, upper p=0.0084).
Figure 6: MR for unconjugated alprazolam/α-hydroxyalprazolam (upper) and unconjugated
alprazolam/4-hydroxyalprazolam (lower) in Tanzanians and Swedish subjects with none, one or two
CYP3A5*1 alleles. The MR along the y-axis are antilog values From Allqvist et al. manuscript.
In Tanzanian subjects without CYP3A5*1 (i.e. only CYP3A4 expressed) there was a
significant correlation between the MR of alprazolam/4-hydroxyalprazolam and MR
22
of quinine (figure 7, upper) and a negative significant correlation with 4ßhydroxycholesterol (endogenous marker for CYP3A) (figure 7, lower).
This strongly indicates and confirms that CYP3A4 is involved in alprazolam
metabolism.
0 CYP3A5*1 alleles
alp/4-OH MR (log10)
2,5
y = -1,1843x + 2,8478
R = 0,5731
p<0.001
n=35
2
1,5
1
0,5
0
0,00
0,50
1,00
1,50
2,00
4β-hydroxycholesterol (log10)
Figure 7: Correlation between the metabolic ratio for unconjugated alprazolam/4-hydroxyalprazolam
and two other markers of CYP3A, i.e. 4β-hydroxycholesteol (upper) and quinine (lower) in Tanzanians
without functional CYP3A5*1 alleles ( One subject had extremely low metabolic ratio for quinine and
was excluded from the linear regression analysis). Log10 values are presented From Allqvist et al.
Manuscript.
23
2.4
DISCUSSION
Study I:
A number of methods for quantification of alprazolam and the metabolites was reported
(Smith and Kroboth, 1987; Schmith et al., 1991; Jin and Lau, 1994; Hold et al., 1996;
Crouch et al., 1999; Toyo'oka et al., 2003), but none of these really met our needs to
have a simple and sensitive method for simultaneous quantification of alprazolam and
metabolites in plasma. We were able to develop a novel LC/MS method for
simultaneous quantification of low concentrations of alprazolam and the two
metabolites 4- and α-hydroxyalprazolam. When using this method we were able to
analyze samples for pharmacokinetic and metabolism studies.
Study II:
Plasma concentrations ratios in samples collected between 1 h and 48 h after drug
intake for both alprazolam/ respective metabolites correlated significantly to the
corresponding AUC (0-∞) ratios. It was therefore possible to estimate alprazolam, 4and α-hydroxylations in subjects taking a single plasma sample. Our data show
significant correlations between the AUC of alprazolam and single point
concentrations of alprazolam at 6, 8, 10 and 24 h, which are in agreement with those
of Schmider et al (Schmider et al., 1999).
If the AUC ratio has been proven to reflect the enzymatic activity, metabolic ratio in a
single plasma sample can be used in phenotyping studies. The present study indicates
that alprazolam is a candidate probe drug for further investigations in vivo. Next step
was to evaluate the role of CYP3A4 and CYP3A5 in alprazolam metabolism in vitro
(Study III) and subsequently in vivo in subjects who express CYP3A5 and those who
do not. (Study IV).
Study III:
The enzyme kinetic parameters for the formation of 3-hydroxyquinine indicated that
both CYP3A4 and CYP3A5 are involved in this metabolic pathway, which is in
agreement with Parimoo et al (Parimoo et al., 2003). The involvement of CYP3A4 in
quinine metabolism was earlier shown by our group (Mirghani et al., 1999; Mirghani et
al., 2002; Mirghani et al., 2003). The involvement of CYP3A5 in addition to CYP3A4
in the 3-hydroxylation of quinine fits well with our later finding in vivo in Tanzanians
24
(Mirghani et al., 2006). We found a clear relationship between the CYP3A5 genotype
and the rate of 3-hydroxylation of quinine.
We were not able to do any enzyme kinetic parameters with alprazolam in vitro as this
drug is fairly insoluble in water and needs to be partly dissolved in a more lipopilic
solvent. This may however decrease the catalytic activity. However, methanol
concentrations of 0.1– 1.0% in the incubation mixture showed negligible effects on
4- and α-hydroxylation at a concentration of 15 µM alprazolam. Therefore, all the
incubations with alprazolam were performed at a methanol concentration of 0.5%.
Nevertheless, formation rates of 4-hydroxyalprazolam in HLM, CYP3A4 and
CYP3A5 were higher than that of α-hydroxyalprazolam, showing that 4-hydroxylation
is the most important metabolic pathway of alprazolam which is in agreement with
Williams et al (Williams et al., 2002). The relative formation of α-hydroxyalprazolam
is 3-fold higher for recombinant CYP3A5 compared to CYP3A4. Our result is in
agreement with pervious findings by Hirota and Gorski et al (Gorski et al., 1999; Hirota
et al., 2001), who showed that CYP3A5 efficiently catalyzes α-hydroxylation of
alprazolam. Now when we have confirmed in vitro that CYP3A5 catalyses alprazolam
metabolism, it would be interesting to go further with an in vivo study to confirm the
role of CYP3A5 (Study IV).
It was shown that KTZ and enantiomers have different inhibition potency when studied
in HLM, CYP3A4 and CYP3A5. Further investigation will be needed to elucidate the
structural characteristics of specific inhibitors for CYP3A4 and CYP3A5. One of the
criteria (Watkins, 1994) which should be fulfilled when validating a probe drug is
dramatically reducing of the activity of the enzyme of interest; therefore it is still of
interest to find a specific inhibitor of in this case CYP3A4 and CYP3A5.
Study IV:
We investigated whether CYP3A5 has an influence on alprazolam metabolism in
subjects who express CYP3A5 compared to those who do not. We designed the study
based on in vitro finding that reported a preference for formation of 4hydroxyalprazolam by CYP3A4 and formation of α-hydroxyalprazolam by CYP3A5,
study III (Gorski et al., 1999; Hirota et al., 2001). However our in vivo results indicate
that CYP3A5 plays no significant role in alprazolam metabolism and CYP3A4 is the
major enzyme involved in the metabolism of alprazolam in vivo. This is based on the
25
observation that there were no significant differences in MR between subjects with
different CYP3A5 genotypes. Our result is in agreement with Gashaw et al (Gashaw et
al., 2003), who also conclude that CYP3A5 genetic polymorphism does not appear
relevant for alprazolam kinetics. Midazolam, another substrate for CYP3A, is also
suggested as a probe drug for both CYP3A4 and CYP3A5. However, Shih and Huang
(Shih and Huang, 2002) did not find any significant difference in midazolam AUC
between subjects who express CYP3A5 or not. Their results suggest that the
CYP3A5 genotypes might not be an important factor for metabolism of midazolam.
We found higher CYP3A4 activity in Swedish subjects than in Tanzanian subjects,
which is in agreement with results from earlier studies by (Wandel et al., 2000;
Mirghani et al., 2003; Mirghani et al., 2006; Diczfalusy et al., 2008). Such an
interethnic difference in activity of different drug metabolizing cytochrome P450
enzymes is well documented (Droll et al., 1998; Bathum et al., 1999; Bertilsson et al.,
2002). In Tanzanian subjects without expression of CYP3A5 (i.e. only CYP3A4
activity) a significant correlation between MR of alprazolam/4-hydroxyalprazolam and
other CYP3A markers i.e. MR of quinine as well as 4ß-hydroxycholesterol
demonstrated and shown in figure 7.
This strongly indicates and confirms that CYP3A4, but not CYP3A5, is involved in
alprazolam metabolism. The role of alprazolam as a potential probe drug for CYP3A4
and CYP3A5 will be discussed further in conclusions and future perspective part.
26
2.5
CONCLUSIONS AND FUTURE PERSPECTIVES
We were able to show in vitro a preference for the formation of 4-hydroxyalprazolam
by recombinant CYP3A4 and formation of α-hydroxyalprazolam by recombinant
CYP3A5, which is in agreement with earlier performed studies (Gorski et al., 1999;
Hirota et al., 2001; Galetin et al., 2004). However, the in vitro results did not reflect
the in vivo situation where both the 4- and α-hydroxylations are mainly performed by
CYP3A4 (study IV). Maybe the reason for this absence of in vivo-in vitro
correlations is that the condition in vitro is artificial and far from the in vivo situation.
If possible, it would be interesting to do in vitro experiments with microsomes from
livers which only express CYP3A5 and see if the pattern is more like the in vivo
situation. However, it is not easy to perform pharmacokinetic studies in vitro with
alprazolam since it is difficult to dissolve in water and that the presence of methanol
in the incubation environment might change the conditions. We kept the
concentration of methanol as low as we could i.e. 0.5%. We tried to dissolve
alprazolam in other solvents without improvement compared to methanol.
In study III, inhibition studies demonstrated that each KTZ enantiomer was a potent
inhibitor of quinine and alprazolam metabolism in HLM, CYP3A4 and CYP3A5.
This inhibition implicating that the structural interactions between KTZ enantiomers
and CYPs would be related to their inhibitory potencies. Further investigations will
be needed to elucidate the structural characteristics of the inhibitors and CYPs, so that
development of specific inhibitors for CYP3A4 or CYP3A5 might be promoted.
Identification of a potent and selective CYP3A4 and CYP3A5 inhibitors are
important in the development of safe therapeutic drugs (O'Donnell et al., 2007).
There are considerable and clinically significant inter-individual differences in CYP3A
activity, due to variable expression, drug and dietary interactions, and genetic
polymorphisms. The underlying genetic mutations affecting expression of CYP3A4
and clearance variations are not fully understood, indicating that further research should
be performed to identify additional genetic variations of CYP3A4. Additional
sequencing of the intron and regulatory areas of CYP3A4, using phenotyped
individuals who have been genotyped for CYP3A5 alleles, could reveal new important
CYP3A4 haplotypes. To combine genotype analysis for defective CYP3A4 and
CYP3A5 SNPs may contribute more significantly to CYP3A variations than single
CYP3A SNP (Lee and Goldstein, 2005).
27
Our data indicate that CYP3A5 does not have a significant role to catalyze alprazolam
metabolism in vivo. However, CYP3A4 seems to have a more pronounced role,
indicating that alprazolam is a probe drug only for measuring CYP3A4 activity in
subjects. Alprazolam metabolic ratio as a measure of CYP3A4 activity fulfils some of
the criteria suggested by Watkins (Watkins, 1994) for validation of probe drugs. The
4- and α-hydroxylations decreased in our in vitro study study III (Allqvist et al.,
2007) when using a known CYP3A inhibitor, ketoconazole and studies in vivo have
shown increased AUC when concomitant treatment with inhibitor were used (Ohno et
al., 2007). When pretreatment with rifampin was used, it significantly decreased the
AUC of alprazolam (Schmider et al., 1999). Otherwise more research is needed both
in vitro and in vivo on alprazolam metabolism.
This is a disadvantage of using midazolam as a probe drug of CYP3A4 is that it
requires multiple blood samples collection for up to 6 h post dose to calculate clearance
(Lee et al., 2002). This makes alprazolam a better probe drug to estimate CYP3A
activity since one plasma sample at a single time point is required.
The impact of CYP3A5 expression on the drug metabolism by CYP3A is fairly
unknown and overlapping substrate specificities between CYP3A4 and CYP3A5 have
made it difficult to differentiate the two enzymes using probe drugs. However, our
group has found three different substrates metabolized by CYP3A5. Saquinavir is a
HIV-protease-inhibitor and the distribution of urinary ratios (saquinavir/metabolites)
between subjects who express CYP3A5*1 or whose how do not, are statistically
different (Josephson et al., 2007). Quinine is a anti-malarial drug and the quinine 3hydroxylation is catalyzed by CYP3A4 (Mirghani et al., 2003) but also CYP3A5
(Mirghani et al., 2006; Diczfalusy et al., 2008). 4ß-hydroxycholesterol an endogenous
marker of CYP3A activity. Subjects who express at least one CYP3A5*1 allele have
significantly higher concentrations than those who do not express CYP3A5 (Diczfalusy
et al., 2008). These three substrates, cholesterol, quinine and saquinavir are potential
phenotyping markers for both CYP3A4 and CYP3A5 while alprazolam is a specific
marker of CYP3A4.
28
3 ACKNOWLEDGEMENTS
There are a lot of people who have supported me throughout the work with this thesis,
but first of all I want to express my sincere gratitude to my three supervisors:
Leif Bertilsson, my main supervisor, Thank you for sharing your extensive knowledge
in drug metabolism, and for your support and encouragement. You have always had
your door open for me whenever I needed to talk. Thank you!
Rajaa Mirghani, my co-supervisor, Thank you for sharing your HPLC knowledge and
for your support during these years. And for nice dinner times we had at your former
apartment.
Eleni Aklillu, my co-supervisor, Thank you for helping me with statistics and for your
support.
A special thanks to:
Lena Ekström, my sincere gratitude to you for taking your time and for your support
during my work with the thesis.
Special thanks to all of you volunteers in Sweden and Tanzania who have participated
in my studies.
Thank you all for nice collaboration at the Department of Clinical Pharmacology,
Muhimbili University College of Health Sciences, Dar es Salaam, Tanzania, Jane Sayi,
M Janabi, Amos Massele, Mary Jande, and Omary Minzi
Walter Msangi and James Fulgence for excellent clinical assistance
Margarita Mahindi, for introducing me to the African world and that you took care of
the volunteers and me during the studies in Tanzania
Jaran Eriksen, Thanks for your help in Tanzania as “our doctor”
Lars L Gustafsson for always being kind and supporting
In addition I want to thank:
Anders Rane, head of Clinical Pharmacology, for your support
Gunnel Thybring Thank you for introducing me to the Department of Clinical
Pharmacology and the world of HPLC
Jan-Olov Svensson for your help with the alprazolam method
Jun Miura (for your lab-work and for being such a nice co-auther)
29
My former room-mates at “plan 8”
Agneta Wennerholm (for introducing me to the alprazolam project), Jolanta Widén
(for your help with LC/MS analyses of alprazolam), Lilleba Bohman (for your help
with genotyping), late Stefan (Taqman expert), Ulla, Birgitta, Gabriella, Eva, Anna
(for shairing your knowledge about in vitro experiments), Mia, Han-jing and Ümit.
Jenny, Sara, Cristine, Maja and Roza for all nice dinner discussions we had.
All colleagues at ”plan 6” thanks for fun, serious and interesting discussions during
lunch and coffee brakes; Jonathan (for your help with statistics), Filip, Magnus,
Annica B, Marie-Louise, Magdalena, Staffan, Erik E, Ylva, Erik S, Karin, Linda,
Anders and Tobias. And my latest room-mates Johan and Ilse
The girls at LIC Annika A, Marine, Åsa and Fadiea
Humanlab Susann, Karin, Marta and Peter for doing a good work with our
volunteers
I also want to thank all colleagues at rutinlab for sharing your lab with me during our
work with the alprazolam method
Margit Ekström I cannot thank you enough for what you have done for me during my
PhD time.
Georgious Panagiotidis for giving me the opportunity to teach
Fredrik Tingstedt for your IT-support
Kurskompisarna Anna B, Anna C, Christina, Jenny, Henrik, Linda, Malin och
Maria för alla roliga fika träffar och våffelkvällar vi haft.
Tack alla nära och kära som gör livet roligt!
Mamma och Pappa tack för all hjälp (med barnen) och allt stöd
Agnes, Moltas och Saga ni är bäst! Vad vore livet utan er?
Ingemar, min hustomte Tack för ditt stöd!
30
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