Diastereomeric Drug Glucuronides: Enzymatic - Helda - Helsinki.fi

84 Pages·2011·0.74 MB·English

by  Päivi Lehtonen

Division of Pharmaceutical Chemistry
Faculty of Pharmacy
University of Helsinki
Finland
Diastereomeric Drug Glucuronides:
Enzymatic Glucuronidation and Analysis by
Capillary Electrophoresis and
Liquid Chromatography–Mass Spectrometry
Päivi Lehtonen
ACADEMIC DISSERTATION
To be presented, with the permission of the Faculty of Pharmacy of the University of
Helsinki, for public examination in Auditorium 1041,
Viikki Biocenter 2 (Viikinkaari 5),
on October14th,2011, at 12 noon.
Helsinki 2011
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Supervisors:
Professor Risto Kostiainen University Lecturer Katariina Vuorensola
Division of Pharmaceutical Chemistry Division of Pharmaceutical Chemistry
Faculty of Pharmacy Faculty of Pharmacy
University of Helsinki, Finland University of Helsinki, Finland
Docent Tom Wikberg Dr.Liisa Mälkki-Laine
Finnish Medicines Agency DRAConsulting Oy
Supervision and licences laboratory Vantaa, Finland
Helsinki, Finland
Reviewers:
Professor Risto Juvonen
School of Pharmacy,
Faculty of Health Sciences,
University of Eastern Finland, Finland
DocentSusanne Wiedmer
Laboratory of Analytical Chemistry,
Department of Chemistry,
University of Helsinki, Finland
Opponent:
Professor Seppo Auriola
School of Pharmacy,
Faculty of Health Sciences,
University of Eastern Finland, Finland
© Päivi Lehtonen 2011
ISBN 978-952-10-7201-7 (paperback)
ISBN 978-952-10-7202-4 (PDF)
ISSN 1799-7372
http://ethesis.helsinki.fi
Helsinki University Print
Helsinki 2011
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CONTENTS

ACKNOWLEDGEMENTS 5
ABSTRACT 7
LIST OF ORIGINAL PUBLICATIONS 9
ABBREVIATIONS 10
SYMBOLS 12
1. INTRODUCTION 13
2. REVIEW OF THE LITERATURE 15
2.1 Drug metabolism 15
2.2 Phase I metabolism 15
2.3 Conjugation reactionsand glucuronidation 16
2.3.1 Human UGT enzymes, classification and tissue localisation 18
2.3.2 Substrate specificity of UGTs 19
2.3.3 Polymorphism and clinical significance of UGTs 22
2.4 Stereoisomers 22
2.4.1Metabolism of tramadol 24
2.4.2 Metabolismof entacapone 25
2.5 Analysis of stereoisomers 26
2.5.1 Capillary electromigration techniques 27
2.5.1.1 Capillary electrophoresis 28
2.5.1.2 Micellar electrokinetic chromatography 29
2.5.1.3 Capillary electromigration techniquesin analysisof tramadoland
entacapone metabolites 31
2.5.2 Liquid chromatography 32
2.5.2.1Glucuronidesoftramadoland entacaponeas targets of liquid
chromatographic analysis 33
3. AIMS OF THE STUDY 35
4. MATERIALS AND METHODS 36
4.1 Chemicals, standards and materials 36
4.2 Microsomes and recombinant human UGTs 36
4.3 Urine samples 38
4.4 Methods and instrumentation 39
4.4.1 Enzyme-assisted synthesis and purification of O-desmethyltramadol
glucuronides(I) 40
4.4.2 Purity and stability testsof O-desmethyltramadol glucuronides(I) 42
4.4.3 Structural characterization of O-desmethyltamadol glucuronides (I) 43
4.4.4 Enzyme activity assays (I) 43
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4.4.5 Analysis of O-desmethyltramadol glucuronides by ultra performance
liquid chromatography/ electrospray-mass spectrometry 44
4.4.6 Separation of tramadol metabolites and diastereomeric glucuronides by
capillary electromigration techniques(II) 45
4.4.7 Separation of the glucuronides of entacapone and its (Z)-isomerby micellar
electrokinetic chromatography (III,IV) 45
4.4.8 Quantitative analyses(I, IV) 46
4.4.9 Sample preparation procedures (I–IV) 46
5. RESULTS AND DISCUSSION 48
5.1 Enzyme-assisted synthesis of O-desmethyltramadol glucuronides(I) 48
5.2 Structural characterisationof O-desmethyltramadol glucuronides(I) 49
5.3 Glucuronidation of O-desmethyltramadol by UGTs and human tissue
microsomes (I) 52
5.4 Evaluation of ultra performance liquid chromatography/ electrospray–mass
spectrometryfor determination of O-desmethyltramadol glucuronides(I) 56
5.5 Separation of tramadol metabolites and diastereomeric glucuronides by capillary
electromigration techniques(II) 57
5.6 Separation of the glucuronides of entacapone and its (Z)-isomerby micellar
electrokinetic chromatography(III) 62
5.7Validation of micellar electrokinetic chromatography mehod for
quantitative analysis of glucuronides of entacapone in human urine(IV) 63
5.7.1 Identification of the glucuronides 63
5.7.2 Selectivity and limit of quantification 65
5.7.3 Linear range, within-day precision and recovery 65
5.7.4 Between-day precision and accuracy 66
5.7.5 Stability of entacapone glucuronides 67
5.8Analysis of patient urine samples 67
6. SUMMARY AND CONCLUSIONS 69
7. REFERENCES 72
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ACKNOWLEDGEMENTS
This study is based on research begun in 1996 and carried out in the Division of
Pharmaceutical Chemistry, Faculty of Pharmacy, University of Helsinki.
Professor Risto Kostiainen has been my supervisor since spring 1999. He offered me the
opportunity to work in the Division as an assistant teacher and later to carry out research. I am
most grateful to him for his encouraging guidance, constructive criticism, support and
patience, so very important during the final stages of the work. I am also grateful to Dr.
Katariina Vuorensola, my other supervisor, for many rewarding discussions and valuable
advice concerning both laboratory work and writing of the manuscripts.
Docent Tom Wikberg and Dr. Liisa Mälkki-Laine were my supervisors during early stages of
the research. I am thankful to them for their inspiring supervision and guidance and for
teaching me the basic skills of bioanalytics and capillary electrophoresis.
I wish to acknowledge all my co-authors for their contribution to this work. Warm thanks go
to Docent Moshe Finel, expert on UGTs, who introduced me to the fascinating world of UGT
enzymes, provided the isoenzymes and contributed to the metabolic part of the study.
Particular gratitude is expressed to Dr. Taina Sten and Mika Kurkela for their guidance and
help in planning the activity studies, and also to Taina for her time and trouble in carrying out
the UPLC-MS analyses. I also want to thank Olli Aitio for carrying out the important NMR
analyses. Special thanks are due to Professor Heli Siren for support and help with the
capillary electrophoretic separations and constructive comments in connection with
manuscript II. I am indebted to adjunct Professor Ilkka Ojanperä, for his donation of tramadol
metabolites for this study.
Professor Risto Juvonen and Docent Susanne Wiedmer are thanked for their careful review
and valuable comments, which led to considerable improvement of this manuscript. I am
furthermore grateful to Dr. Kathleen Ahonen for leading me to a more fluent expression in the
English language in both the original publications and this thesis.
Many people have contributed to this work. Sirkku Jäntti assisted with the LC-MS analyses
and Sanna Sistonen guided me in the operation of various HPLC instruments. Sanna Lehtinen
and Tiina Lehtonen (née Järveläinen) provided excellent assistance and pleasant collaboration
in the course of their pro gradu studies. Nenad Manevski and Kaarina Takkunen kindly
helped me with the Corel Draw pictures. To all of you I express my warm appreciation. In
addition, Waters Co., Finland is thanked for giving us the opportunity to use their UPLC
device.
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I thank all my present and former colleagues in the Division of Pharmaceutical Chemistry—
Anna, Hannele, Helena, Heli, Hongbo, Inkku, Irene, JP, Katariina, Kati, Katriina, Kirsi,
Laura, Leena, Mika, Mikko, Nenad, Niina N., Niina S., Nina S. Pia, Piia, Päivi, Sanna,
Sirkku, Taina, Teemu, Tiia, Tiina K., Tiina S. and Timo S., for creating a friendly and happy
atmosphere both in the laboratory and in the coffee room. My very best friends Hannele,
Inkku and Katariina, thank you for listening and helping out in difficult times!
I am indebted to my present and former colleagues Anna-Maija, Leena, Christina, Eeva, Heli,
Jenna and Maire at the Kaivopuisto pharmacy for their understanding and friendship.
My warmest gratitude belongs to friends and family. My mother Raili Ylikylä, my sister Piia
Varila and my brother-in-law Esko Varila provided much appreciated familial support. Last,
but by no means least, my deepest gratitude is owned to Timo and our children Lasse and
Liisa for granting me the time to work with this project. Thank you, Timo, not only for
economical support of this study but for taking care of so many household chores for months
on end. Without your love this work would never have been completed.
Helsinki, April 2011
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ABSTRACT
Glucuronides are hydrophilic and polar metabolites formed from nucleophilic substrates in
phase II metabolic reactions. Glucuronidation is catalysed by UDP-glucuronosyltransferase
enzymes (UGTs). The particular regio- and stereoisomeric glucuronides formed are
determined by the structure of the substrate. Glucuronidation of (±)-O-desmethyltramadol
(M1), the active metabolite of analgesic tramadol, yields a pair of diastereomeric 3-O-
glucuronides (M1Gs), and the antiparkinson drug entacapone yields diastereomeric 3-O-
glucuronides of entacapone and its (Z)-isomer (EG and EZG). These glucuronides were
selected as testcompounds in the experimentalwork.
The investigations summarised in this thesis deal with enzymatic glucuronidation and the
development of analytical methods for drug metabolites, particularly diastereomeric
glucuronides.Liquid chromatography (LC) with ultraviolet (UV)or mass spectrometric (MS)
detection, a well-established technique in metabolite analysis, was employed, and capillary
electromigration techniques, which have good resolving power, were tested for their
suitability in the separation of diastereomeric glucuronides. Fast and selective ultra
performance liquid chromatography (UPLC) coupled with electrospray ionisation (ESI)-MS
was applied as well.
Lack of commercial glucuronide standards has led to a search forenzymaticmethods for their
production. In this study, enzyme-assisted synthesis with rat liver microsomes (RLMs) was
optimised to produce M1G diastereomers from small amounts of starting material, (±)-M1.
Glucuronide formation catalysed by RLMs wasstereoselectiveand favoured the formation of
1S,2S-diastereomer. The glucuronides were isolated by LC/UV, and UPLC/ESI-MS, while
tandem mass spectrometry (MS/MS) and proton nuclear magnetic resonance (1H NMR)
spectroscopywere employed in structural characterisation.Applicationof nuclear Overhauser
effect spectroscopy (NOESY) established that the glucuronides were phenolic O-glucuronides
of M1. When the diastereomers were isolated as a combined fraction the yield was 51% (6.5
mg).
Screening assays were carried out to identify the human UGT enzymes that catalyse (±)-M1
glucuronidation. The activity of human liver and intestinal microsomes was examined as well.
The assays revealed that several UGT enzymes are able to catalyse (±)-M1 glucuronidation.
UGTs 1A7–1A10 are strictly enantiospecific for the 1R,2R-enantiomer, while UGT2B7
glucuronidates both M1 enantiomers. Glucuronidation of (±)-M1 was also stereoselective in
human liver microsomes (HLMs), and the activity largely resembled that of UGT2B7. It
seems probable, therefore, that UGT2B7 is the main isoform involved in (±)-M1
glucuronidation in human liver.
Positive ion mode UPLC/ESI-MS was used to monitor (±)-M1 glucuronidation in incubation
samples. Formation of M1Gs was quantified with the aid of synthesised reference
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glucuronides. With its high resolution power, the UPLC technique provided a useful tool for
the analysis of diastereomeric M1Gs, which cannot be differentiated by MS alone.
Further inthe present work,several methods relyingon capillary electromigration techniques
with UV detection were developed and optimised for qualitative and quantitative analysis of
drug metabolites, particularly glucuronides. A capillary electrophoretic (CE) method was
optimised for the separation of free and glucuronidated metabolites of tramadol, and its
suitability was tested with an authentic urine sample after tramadol administration. In
comparison with standards, tramadol and its metabolites M1, M1G, (±)-N,O-
didesmethyltramadol (M5) and its glucuronide (M5G) were easily detected in the
electropherogram. Furthermore, diastereomeric separation of tramadol glucuronides—M1Gs,
(±)-N,N,O-tridesmethyltramadol glucuronide(M4Gs)and M5Gs—was achieved in acidic (pH
2.75) background electrolyte (BGE) by CE, and in basic (pH 10.45) BGE by micellar
electrokinetic chromatography (MEKC).
A MEKC/UV method was developed and optimised for direct quantitative determination of
EG and EZG in urine. Urine samples were purified before analysis by solid phase extraction
(SPE). The validity of the method was assessed by investigating several important analytical
parameters. The limit of quantification (LOQ) for EG and EZG was 2 μg/ml (ca. 4 μM), and
the method was confirmed to be reproducible and accurate. Determination of EG and EZG
concentrations in urine samples of 34 patients treated with entacapone showed the method to
be suitable for monitoring concentrations of EG after both oral and intravenous
administration and concentrationsof EZG after oral administration. The long-term stability of
the system is limited, however, and frequent recalibration is requiredin applications involving
long sample series.
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LIST OF ORIGINAL PUBLICATIONS
This doctoral thesis is based on the following fourpublications, which will be referred to in
the text by their Roman numerals (I-IV):
I Päivi Lehtonen, Taina Sten, Olli Aitio, Mika Kurkela, Katariina Vuorensola,
Moshe Finel and Risto Kostiainen; Glucuronidation of racemic O-
desmethyltramadol,the active metabolite of tramadol. Eur.J.Pharm.Sci.41
(2010)523-530
II Päivi Lehtonen, Heli Siren, Ilkka Ojanperä and Risto Kostiainen; Migration
behaviour and separation of tramadol glucuronides by capillary electrophoresis. J.
Chromatogr.A1041 (2004) 227-234.
III Päivi Lehtonen, Liisa Mälkki-Laine and Tom Wikberg; Separation of the
glucuronidesofentacapone and its (Z)-isomer in urine by micellar electrokinetic
capillary chromatography. J. Chromatogr. B721 (1999) 127-134.
IV Päivi Lehtonen, Sanna Lehtinen, Liisa Mälkki-Laine and Tom Wikberg;
Micellar electrokinetic capillary chromatography method fordirect
determination of glucuronides of entacapone and its (Z)-isomer in human urine.
J. Chromatogr. A836(1999)173-188.
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ABBREVIATIONS
A area
ACN acetonitrile
API atmospheric pressure ionisation
BGE background electrolyte
C18 octadecyl silane(ODS)
C8 octyl silane
CAPS 3-(cyclohexylamino)-1-propenesulfonic acid
CE capillary electrophoresis
CD cyclodextrin
CIP Cahn-Ingold-Prelog
CMC critical micelle concentration
CN-I Crigler-Najjar syndrome type I
CN-II Crigler-Najjar syndrome type II
COMT catechol-O-methyl transferase
CSP chiral stationary phase
CYP cytochrome P450
CZE capillary zone electrophoresis
DAD diode array detector
DMSO dimethylsulfoxide
EG glucuronide of entacapone
EOF electroosmotic flow
ER endoplasmic reticulum
ESI electrospray ionisation
EZG glucuronide of the (Z)-isomer of entacapone
GA glucuronic acid
GC gas chromatography
HDCA hyodeoxycholic acid
HIMs human intestinal microsomes
HLMs human liver microsomes
1H NMR proton nuclear magnetic resonance
HPLC high performance liquid chromatography
i.d. internal diameter
IS internal standard
IUPAC International Union of Pure and Applied Chemistry
i.v. intravenous
LC liquid chromatography
LIF laser-induced fluorescence
LLE liquid–liquid extraction
LOD limit of detection
LOQ limit of quantification
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