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Molecular and Cellular Therapeutics

Molecular and Cellular Therapeutics

David Whitehouse

Ralph Rapley

E D I T O R S

EDITORS

David WhitehouseSchool of Life Sciences, University of Hertfordshire, UK

Ralph RapleyUniversity of Hertfordshire, UK

Molecular and Cellular Therapeutics aims to bring together key developments in the areas of molecular diagnostics, therapeutics and drug discovery. The book covers topics including diagnostics, therapeutics, model systems, clinical trials and drug discovery. The developing approaches to molecular and cellular therapies, diagnostics and drug discovery are presented in the context of the pathologies they are devised to treat.

Molecular and Cellular Therapeutics is a unique collection of the key high technology advances in the area in the context of the common disorders which they counteract. Advances driven by underlying molecular pathology, represents outputs of genome projects in the understanding of the common multifactorial diseases.

• Clarifies and explains the ideas and technologies which underlie new medical research

• Bridges the gap between traditional medicine and treatments and the origin of the new approaches to therapeutics

• Systems based organisation

• Accessible to medical students, biological sciences undergraduates and post-graduates, molecular and medical research scientists and clinicians Molecular and Cellular

Therapeutics

ED

ITO

RS

WhitehouseRapley

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Molecular and CellularTherapeutics


Molecular and CellularTherapeutics

Edited by

David Whitehouse

School of Life Sciences, University of Hertfordshire, UK

Ralph Rapley

University of Hertfordshire, UK

A John Wiley & Sons, Ltd., Publication


This edition first published 2012© 2012 by John Wiley & Sons, Ltd

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Molecular and cellular therapeutics / [edited by] David B. Whitehouse and Ralph Rapley.p. ; cm.

Includes bibliographical references and index.ISBN 978-0-470-74814-5 (cloth) – ISBN 978-1-119-96729-3 (ePDF) – ISBN 978-1-119-96730-9 (Wiley Online

Library) – ISBN 978-1-119-96780-4 (ePub) – ISBN 978-1-119-96781-1 (Mobi)I. Whitehouse, David, 1946- II. Rapley, Ralph.[DNLM: 1. Biological Therapy–methods. 2. Molecular Biology–methods. 3. Molecular Targeted

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Contents

List of contributors vii

Preface xi

1 Cytochrome P450 pharmacogenetics: from bench to bedside 1Imtiaz M. Shah, Catherine J. Breslin and Simon P. Mackay

2 Cancer biomarkers for diagnosis, prognosis and therapy 18Debmalya Barh, Vaishali Agte, Dipali Dhawan, Varsha Agte and Harish Padh

3 HER2 targeted therapy-induced gastrointestinal toxicity: fromthe clinical experience to possible molecular mechanisms 69Noor Al-Dasooqi, Rachel J. Gibson, Joanne M. Bowen andDorothy M. Keefe

4 Antibody-targeted photodynamic therapy 103Mahendra Deonarain, Ioanna Stamati and Gokhan Yahioglu

5 Anti-ageing strategy of the lung for chronic inflammatoryrespiratory disease – targeting protein deacetylases 125Kazuhiro Ito and Nicolas Mercado

6 RNA interference: from basics to therapeutics 140Sunit Kumar Singh and Praveensingh B. Hajeri

7 Delivery of RNAi effectors by tkRNAi 168Hermann Lage, Andrea Kruhn and Johannes H. Fruehauf

8 Human stem cell therapy 187M. Ian Phillips, Yao-Liang Tang and Henrique Cheng

9 Gene therapy in organ transplantation 208Thomas Ritter and Matthew D. Griffin


vi CONTENTS

10 Advances in the treatment of Alzheimer’s disease 233Michael S. Rafii

11 Novel molecular therapeutics in Parkinson’s disease 245Susana Goncalves, Hugo Vicente Miranda and Tiago F. Outeiro

12 Emerging insights and therapies for human microbial disease 266Joanne L. Fothergill and Craig Winstanley

13 Vaccine design and vaccination 286Niall McMullan

Index 305

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List of contributors

Vaishali AgteAgharkar Research Institute,G. G. Agarkar Road,Pune 411004,India

Varsha AgteMITCON Institute of Management and

Technology,Balewadi,Pune 411045,India

Noor Al-DasooqiDepartment of Medical Oncology,Royal Adelaide Hospital,Level 4 East Wing,North Terrace,Adelaide 5000,South Australia,Australia

Debmalya BarhCentre for Genomics and Applied Gene

Technology,Institute of Integrative Omics and Applied

Biotechnology (IIOAB),Nonakuri,Purba Medinipur,WB-721172,India

Joanne M. BowenDiscipline of Physiology,University of Adelaide,North Terrace,Adelaide 5005,South AustraliaAustralia

Catherine J. BreslinStrathclyde Institute of Pharmacy and

Biomedical Sciences,University of Strathclyde,Glasgow G4 0RE,Scotland, UK

Henrique ChengDepartment of Comparative Biomedical

Sciences,School of Veterinary Medicine,Skip Bertman Drive,Louisiana State University,Baton Rouge LA 79803, USA

Mahendra DeonarainDivision of Cell and Molecular Biology,Faculty of Natural Sciences,Imperial College London,Exhibition Road,London SW7 2AZ, UK


viii LIST OF CONTRIBUTORS

Dipali DhawanDepartment of Cellular and Molecular

Biology,B. V. Patel Pharmaceutical Education and

Research Development (PERD) Centre,Thaltej-Gandhinagar Highway,Thaltej,Ahmedabad 380054,Gujarat, India

Joanne L. FothergillInstitute of Infection and Global Health,University of Liverpool,Liverpool L69 3GA,UK

Johannes H. FruehaufSkip Ackerman Center for Molecular

Therapeutics,Beth Israel Deaconess Medical Center,Boston, MA,USA

Rachel J. GibsonDiscipline of Anatomy and Pathology,School of Medical Sciences,University of Adelaide,North Terrace,Adelaide 5000,South Australia,Australia

Susana GoncalvesCell and Molecular Neuroscience Unit,Instituto de Medicina Molecular,Av. Prof. Egas Moniz,1649-028 Lisboa,Portugal

Matthew D. GriffinRegenerative Medicine Institute

(REMEDI),National Centre for Biomedical

Engineering Science (NCBES),Orbsen Building,University Road,National University of Ireland,Galway, Ireland

Praveensingh B. HajeriDepartment of Surgery,University of Minnesota,Minneapolis MN:55455,USA

Kazuhiro ItoAirway Disease,National Heart and Lung Institute,Imperial College London,Dovehouse Street,London SW3 6LY,UK

Dorothy M. KeefeDepartment of Medical Oncology,Royal Adelaide Hospital,Level 4 East Wing,North Terrace,Adelaide 5005,South AustraliaAustralia

Andrea KruhnCharite Campus Mitte,Institute of Pathology,Chariteplatz 1,10117 Berlin,Germany


LIST OF CONTRIBUTORS ix

Hermann LageCharite Campus Mitte,Institute of Pathology,Chariteplatz 1,10117 Berlin,Germany

Simon P. MackayStrathclyde Institute of Pharmacy and


Niall McMullanSchool of Life Sciences,University of Hertfordshire,College Lane,Hatfield,Herts AL10 9AB,UK

Nicolas MercadoAirway Disease,National Heart and Lung Institute,Imperial College London,Dovehouse Street,London SW3 6LY,UK

Hugo Vicente MirandaCell and Molecular Neuroscience Unit,Instituto de Medicina Molecular,Av. Prof. Egas Moniz,1649-028 Lisboa,Portugal

Tiago F. OuteiroCell and Molecular Neuroscience Unit,Instituto de Medicina Molecular,Av. Prof. Egas Moniz,1649-028 Lisboa,Portugal

Harish PadhDepartment of Cellular and Molecular

Biology,B. V. Patel Pharmaceutical Education and

Research Development (PERD) Centre,Thaltej-Gandhinagar Highway,Thaltej,Ahmedabad 380054,Gujarat, India

M. Ian PhillipsCenter for Rare Disease TherapiesKeck Graduate Institute of Applied

Life Sciences,535 Watson Drive,Claremont,CA 91711,USA

Michael S. RafiiDirector, Memory Disorders Clinic,Assistant Professor of Neurosciences,University of California,San Diego 9500 Gilman Drive,#0949 La Jolla,California 92093,USA

Thomas RitterRegenerative Medicine Institute

(REMEDI),National Centre for Biomedical

Engineering Science (NCBES),Orbsen Building,University Road,National University of Ireland,Galway, Ireland

Imtiaz M. ShahStrathclyde Institute of Pharmacy and



x LIST OF CONTRIBUTORS

Sunit Kumar SinghLaboratory of Neurovirology and

Inflammation Biology,Section of Infectious Diseases,Centre for Cellular and Molecular Biology

(CCMB),Council of Scientific and Industrial

Research (CSIR),Uppal Road,Hyderabad-500007, AP,India

Ioanna StamatiDivision of Cell and Molecular Biology,Faculty of Natural Sciences,Imperial College London,Exhibition Road,London SW7 2AZ,UK

Yao-Liang TangDivision of Cardiovascular Disease,

Internal Medicine,University of Cincinnati,231 Albert Sabin Way, ML0542,Cincinnati OH 45267,USA

Craig WinstanleyInstitute of Infection and Global Health,University of Liverpool,Liverpool L69 3GA,UK

Gokhan YahiogluPhotoBiotics Ltd,Montague House,Chancery Lane,Thrapston,Northamptonshire NN14 4LN,UK

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Preface

The inspiration for this volume emerged from a combination of the completion of theHuman Genome Project and advances in cellular and molecular medicine. Together thesedisciplines have established the basis for a new wave of translational research where theaim is for advances in basic science to impact directly on improved clinical outcomes.Although medical pathology has historically been divided into subsets dependent on theorgan system involved and disease aetiologies, it is becoming increasingly evident thatdiagnoses and treatment of disease, whether acquired or heritable, should additionally bebased on the detailed knowledge of epidemiology and genetics, lifestyle, molecular andcellular pathology. The objective of the book is to provide an exciting insight into ad-vances in key areas of molecular and cellular aspects of applied medical research. Basedon a series of authoritative chapters that provide opinion and data across a broad fieldof medicine, the aim is to enable the reader to acquire a usable platform of knowledgesufficient, for example, to gain access to the specialist literature.

The first two chapters address molecular aspects of pharmacogenetics and biomarkers.The opening chapter from Imtiaz Shah and colleagues describes some of the potential

practical outputs of the Human Genome Project. Person to person variability in responseto drugs has long been recognized and the authors summarize the recent research findingon genotype testing in relationship to variability in drug response with reference to theCYP genes. Continuing with the theme of molecular analysis in relation to physiologicalstates, Debmalya Barh and colleagues reviews the field of biomarkers. These substanceswhich can indicate disease states and treatment outcomes are of increasing importance inmedicine. The chapter provides an introductory review of biomarkers in general whilstfocusing on cancer related molecular markers, their classification, detection approachesand applications. Chapters 3 and 4 review aspects of cancer therapy. In Chapter 3, NoorAl-Dasooqi and colleagues focus on HER2 targeted therapy and the role of HER2 in can-cer. The central theme addresses the gastrointestinal toxicities associated with commonlyused HER2 targeted therapy drugs and the possible underlying mechanisms. Althoughdrugs that target HER2 and other EGF receptors have proved to be effective in man-aging a range of cancers, toxic side effects remain a significant problem. In Chapter 4,Mahendra Deonarain and colleagues sustain the theme of cancer therapy with a review ofphotodynamic therapy (PDT). The complexity of PDT targeting, lack of potency and sideeffects have limited the technology and restricted its general usage by oncologists. Theproblem of targeting disease cells is addressed and the translational research harnessingmonoclonal antibodies and antibody fragments is described.


xii PREFACE

In Chapter 5 Kazuhiro Ito and Nicolas Mercado review the free-radical theory of age-ing in relation to inflammatory disease. The most ageing-associated disease is recognizedas chronic inflammatory disease where oxidative stress is likely to contribute to the in-flammation. The authors describe the analysis of oxidative stress reduced anti-ageingmolecules such as those that are involved in epigenetic control of pro-inflammatory geneexpression and control of protein function.

The next two chapters are focused on RNAi, which is widely acknowledged as a po-tential basis for powerful new therapies. Sunit Singh and Praveensingh Hajeri (Chapter6) provide a sterling review of the topic. Whilst the techniques have proven potential forproviding therapeutic solutions, the authors do not shy from highlighting the hurdles to beovercome in designing strategies for knocking down specific gene expression. In Chap-ter 7 Hermann Lage and colleagues review the development and potential applicationsfor Transkingdom RNAi (TkRNAi). The tkRNAi approach described represents a newstrategy for delivery of RNAi effectors, in particular for the treatment of bowel disease.

There follow two chapters on key areas of current medical research, stem cells and genetherapy. In Chapter 8 Ian Phillips and colleagues comprehensively review the history ofstem cell biology and the current advances. The chapter addresses the key questions thatneed to be answered before new human stem cell therapies can be used routinely, includ-ing the choice of stem cells, the ease of preparing, storage and delivery of stem cells, andthe effectiveness of the therapies. In Chapter 9 Thomas Ritter and Matthew Griffin tacklegene therapy. Whilst gene therapy technologies have been successfully applied in manypreclinical models for the treatment of various diseases – including the prevention of al-logeneic organ graft rejection – mainly for safety reasons the translation into the clinichas lagged behind. The authors examine the role that gene therapy and gene transfer tech-nologies may play in the successful application of new strategies to improve the successrates and long-term, immunosuppression-free survival of organ allografts.

In Chapters 10 and 11 advances in the two most common neurodegenerative dis-eases are presented. Neurodegenerative disease, in particular Alzheimer’s disease, isan area where the burden of disease cost is set to increase significantly over the next50 years. First Michael Rafii discusses promising new treatments for Alzheimer’s disease,the most common form of progressive dementia in older people. The major therapeuticstrategies are reviewed as are the complexities of dealing with such a heterogeneous con-dition. In Chapter 11 Tiago Outeiro and colleagues review current and new therapeuticapproaches to Parkinson’s disease, the second most common neurodegenerative diseasethat is estimated to affect some 2% of the world population aged over 65.

A vital facet of twentyfirst century medicine is the control of infectious diseases. Thelast two chapters deal with approaches to bacterial infection and vaccine development.In Chapter 12 Joanne Fothergill and Craig Winstanley address the challenges for the de-velopment of new drugs in the face of increasing incidence of antibacterial resistance.In addition to traditional strategies to drug discovery, approaches based on genomic in-formation are addressed. The authors provide a snapshot of some of the approachesbeing taken to the identification of new therapeutic targets that might enable develop-ment of new and better strategies to combat infections in a post-antibiotic era. NiallMcMullan (Chapter 13) reviews advances in vaccine development. The last 30 yearshas seen promising developments in vaccinology. The integration of reverse genetics


PREFACE xiii

approaches and reverse vaccinology offer the prospect of rapid methods for developingnew vaccines. Recent successes with these new strategies in human clinical trials and thelicensing of new animal vaccines offer real hope for major breakthroughs in the controlof infectious diseases.

There can be no doubt that therapeutic and diagnostic strategies and approaches thathave emerged since the completion of the Human Genome Project have been both wideranging and highly focused. The notion of ‘bench to bedside’ which is underpinned bythe outputs of translational research has gathered momentum and credibility as evidencedby the contents of the chapters presented.

David Whitehouse and Ralph Rapley

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1Cytochrome P450pharmacogenetics: from benchto bedsideImtiaz M. Shah, Catherine J. Breslin and Simon P. Mackay

1.1 Introduction

With the elucidation of the human genome sequence over the past decade, pharmaco-genetics has evolved into an important area of translational medicine research (Inter-national Human Genome Sequencing Consortium, 2004; Grant and Hakonarson, 2007;Shurin and Nabel, 2008). Most patient populations display interindividual variabilityto drug response and efficacy, with genetic factors accounting for up to 30% in thesedifferences (Evans and McLeod, 2003). Mutations within the genetic DNA sequence(genetic polymorphism) can alter the transcribed mRNA structure and subsequent pro-tein function. This altered genotype expression can result in variability in drug activ-ity (O’Shaughnessy, 2006). Pharmacogenetics is the study of such genetic factors andits effects on drug response. The most common genetic polymorphism is a single nu-cleotide polymorphism (SNP). This results in a single nucleotide substitution within theDNA structure and accounts for 90% of human genetic variation (Eichler et al., 2007;McCarroll et al., 2006). SNPs are associated with variability in drug response betweendifferent patient populations and are an important basis for pharmacogenomics research(Twyman, 2004). This variability in patient genetic profiles can lead to potential risks ofdrug toxicity or treatment failure (Hoffman, 2007). Current pharmacogenetics researchis focusing on patient genotype testing and utilizing this genetic information to providemore ‘personalized’ drug therapy in clinical practice (Feero, Guttmacher and Collins,2010; Hoffman, 2007).

Molecular and Cellular Therapeutics, First Edition. Edited by David Whitehouse and Ralph Rapley.© 2012 John Wiley & Sons, Ltd. Published 2012 by John Wiley & Sons, Ltd.


2 CH01 CYTOCHROME P450 PHARMACOGENETICS: FROM BENCH TO BEDSIDE

1.1.1 The Human Genome Project

The Human Genome Project (HGP) has made a crucial contribution to research advancesin the rapidly evolving areas of pharmacogenetics and translational medicine. This ma-jor international scientific collaboration, which was completed in 2003, has elucidatedthe complete DNA sequence of the human genome (International Human Genome Se-quencing Consortium, 2004). The results of this project have started to provide importantgenotype–phenotype correlations from genome wide association studies (GWAS), andwill potentially lead to major advances in drug development and translational research(The Wellcome Trust Case Control Consortium, 2007; Chung et al., 2010). The HGP isbeing followed on by the larger 1000 Genomes Project, which will allow more detailedgenetic analysis of different ethnic populations (Gamazon et al., 2009).

The HGP analysis commenced in 1995 with the aim of sequencing three billion basepairs (bps) of DNA. The sequencing strategy involved subcloning the human genome intobacterial artificial chromosomes, which were then sequenced (shotgun method) and cor-rectly aligned (Lander et al., 2001). Once the initial sequence was determined, advancedcomputational algorithms were used to generate a final sequence map. The genome wassequenced five times to minimize any errors. The main findings from the HGP have shownthat humans have between 20 000 and 25 000 genes (International Human Genome Se-quencing Consortium, 2004). The average human gene spans between 27 000 and 29 000bases of DNA and consists of four to six exons. The main coding sequence is approx-imately 1340 bps. Genes are not evenly distributed throughout the genome, with somechromosomes containing more genetic information (chromosomes 1, 2, 11) than others(chromosomes 13, 18, 21).

The relatively small number of genes is not indicative of a similarly small numberof proteins. Genes can undergo alternative splicing, thereby increasing the number ofdifferent protein products (Barash et al., 2010; Tress et al., 2007). RNA studies haveshown that there may be an average of three different transcripts from one gene. The HGPhas also identified approximately two million SNPs, which has allowed genetic linkagestudies and location of specific diseases to their chromosome loci (Sachidanandam et al.,2001). GWAS and SNP analysis have now started to elucidate genetic associations withcommon clinical diseases (The Wellcome Trust Case Control Consortium, 2007; Chunget al., 2010). These genomic studies will potentially lead to the identification of newprotein targets for drug discovery and play an important role in translational medicineresearch (Hopkins and Groom, 2002; Schilsky, 2010).

1.2 Cytochrome P450 pharmacogenetics

Genetic polymorphisms and variation in protein structure expression can result in altereddrug–protein interactions and affect subsequent drug response. There are three main phar-macogenetic mechanisms that can influence drug activity. These molecular changes canresult in genetic polymorphisms affecting the drug metabolizing enzymes (DMEs), drugtransporter proteins and the drug receptors. This can result in altered pharmacokinetic


1.2 CYTOCHROME P450 PHARMACOGENETICS 3

properties (metabolism and transport) or pharmacodynamic properties (action) ofthe drug.

The most widely studied group of proteins displaying pharmacogenetic variability arethe DMEs and specifically the cytochrome P450 enzymes (CYP) (Sim and Ingelman-Sundberg, 2010). These enzymes are involved in phase I biotransformation reactions,which mainly result in drug substrate oxidation. Genetic polymorphism affecting theCYP enzymes can result in altered drug metabolism and efficacy (Ingelman-Sundberg andSim, 2010; Tomalik-Scharte et al., 2008). Cytochrome P450 2D6 (CYP2D6), CYP2C9and CYP2C19 have been the most extensively studied metabolic enzymes (Zhou,Liu and Chowbay, 2009). The following sections will discuss these CYP enzymes inmore detail and provide clinical examples of commonly used drugs displaying pharma-cogenetic variability.

1.2.1 CYP2D6 pharmacogenetics

CYP2D6 functions as a mono-oxygenase enzyme and is predominantly found withinthe liver. It metabolizes up to 30% of commonly used medications and important drugclasses include antidepressants, beta-blockers and analgesics. The drug substrates aremainly lipophilic bases with a protonable nitrogen atom and an aromatic ring (Costacheet al., 2007). The approval of CYP2D6 genotype testing by the FDA in 2005 has put thisenzyme at the forefront of research into personalized medicine (Frueh et al., 2008; Sunand Scott, 2010).

The CYP2D6 enzyme is a 497 amino acid protein (55.8 kDa) and contains a haemgroup (Protein Data Bank ID: 2F9Q) (Rowland et al., 2006). The gene encoding CYP2D6is located on chromosome 22. CYP2D6 has a well-defined active site structure, which islocated above the haem group. The amino acid residues that have been implicated insubstrate recognition and binding are Asp301, Glu216, Phe483 and Phe120 (Rowlandet al., 2006). The main enzyme action is drug substrate oxidation, via electron transferand substrate interaction with a dioxygen–ferrous complex. The catalysis involves theinsertion of one oxygen atom into the substrate molecule and the second oxygen atom isconverted into water. There are also alternative CYP enzyme mechanisms which result insubstrate N- and O-demethylation reactions.

CYP2D6 displays extensive genetic polymorphism that influences enzyme expressionand function. More than 100 allelic variants of the CYP2D6 gene have now been identified(www.cypalleles.ki.se/cyp2d6.htm). The enzyme genetic polymorphism and metabolicactivity also shows ethnogeographic variation, with differences between Caucasian, Ori-ental and Afro-Caribbean populations (Ingelman-Sundberg, 2005). The three major al-lelic variants, which are found in the Caucasian population are CYP2D6∗3, CYP2D6∗4and CYP2D6∗5 (Table 1.1). All three variants are associated with poor metabolizer (PM)phenotypes, with CYP2D6∗4 being the most frequent allele (∼20%) (Lee et al., 2006;Mizutani, 2003). The CYP2D6∗10 allele is the most commonly found allelic variant inthe Oriental population and this enzyme displays an intermediate metabolizer (IM) phe-notype (Lee et al., 2006). This allele results in the production of an unstable enzymecaused by a double amino acid substitution (P34S, S486T) (Shen et al., 2007). The most

http://www.cypalleles.ki.se/cyp2d6.htm



Table 1.1 Common allelic variants of CYP2D6#

CYP2D6 allelicvariant Mutation Enzyme activity

CYP2D6∗3 Frameshift deletion Inactive enzymeNon-functional allele 1–3% allelic frequency in

Caucasian populationCYP2D6∗4 Defective splicing Inactive enzyme

Non-functional allele 20–25% allelic frequency inCaucasian population

CYP2D6∗5 Gene deletion No enzymeNon-functional allele ∼5% allelic frequency in

general populationCYP2D6∗10 Double amino acid mutation

(P34S, S486T)Reduced activity due to

unstable enzyme (IM)∼50% allelic frequency in

Oriental populationCYP2D6∗17 Triple amino acid mutation

(T107I, R296C, S486T)Reduced activity due to altered

substrate affinity (IM)∼30% allelic frequency in

African population

#For full information: www.cypalleles.ki.se/cyp2d6.htm.

commonly found allele in the African population is CYP2D6∗17 (Dandara et al., 2001). Itis also associated with an IM phenotype, resulting in reduced catalytic activity caused by atriple amino acid substitution (T107I, R296C, S486T) (Shen et al., 2007). The ultra-rapidmetabolizer (UM) phenotype is associated with CYP2D6 gene multiplication and enzymeover-expression. This has been most commonly associated with the Ethiopian and Mid-dle Eastern populations, with 15–30% allelic frequency (Aklillu et al., 1996; McLellanet al., 1997). Due to this extensive genetic polymorphism displayed by CYP2D6, therole of genotype and phenotype testing for this enzyme has become an important areaof research into personalized medicine and pharmacogenomics (de Leon, Armstrong andCozza, 2006).

In 2005, the FDA approved one of the first CYP2D6 genotype tests (AmpliChip-CYP450) for clinical use (de Leon et al., 2009). The introduction of this genotype testhas been a major step towards introducing personalized medicine into the clinical set-ting. The CYP AmpliChip test involves the identification of a defined genetic mutationin the CYP2D6 and CYP2C19 gene, which is associated with a specific drug metabolismphenotype. The test screens for susceptible patient genotypes and will potentially allowtailoring of drug therapy in an attempt to reduce adverse drug reactions (ADRs) or avoidtreatment failure (de Leon, Armstrong and Cozza, 2006). An example in the use of thistest has been to optimize drug therapy in patients taking antidepressant and antipsychoticmedication (de Leon et al., 2009). The AmpliChip CYP450 test is based on microarraytechnology. These DNA microarrays, also known as DNA chips, allow multiple gene ex-pression analysis. This consists of DNA oligonucleotides embedded onto tiny glass chips,

http://www.cypalleles.ki.se/cyp2d6.htm



which allow detection and analysis of the different gene variants. Patient DNA can be ex-tracted from blood or saliva samples, which is analysed via polymerase chain reaction(PCR) amplification. The PCR products are then applied to the microarray chip, whichallows binding of complementary base pairs between the patient DNA sample and themicroarray (hybridization). A laser scanner is then used to read the result, providing thephysician with information on the patient’s CYP2D6 genotype status. The AmpliChip testhas been validated against in vivo studies using CYP2D6 probe drugs and it shows goodcorrelation in detecting the different enzyme phenotypes (Heller et al., 2006). NewerPCR and microarray technologies are continuing to be developed for CYP genotyping(Deeken, 2009).

Clinical studies have demonstrated a potential increased risk of ADRs or treatment fail-ure associated with different CYP2D6 allelic variants (Ingelman-Sundberg, 2005; Zhou,2009). Most clinical studies have investigated the effect of CYP2D6 genetic polymor-phism in psychiatric patients taking antidepressant and antipsychotic medication (deLeon, Armstrong and Cozza, 2006). Larger pharmacogenetic clinical studies are ongo-ing, into evaluating the role of genotype testing in psychiatric patients (Kirchheiner andRodriguez-Antona, 2009; Uher et al., 2009). Recent interest in CYP2D6 pharmacogeneticvariability has been focusing on analgesic agents and the breast cancer drug, tamoxifen.

Tramadol and codeine are commonly used analgesic agents, which are both metab-olized by CYP2D6. Tramadol undergoes O-demethylation by CYP2D6 into the moreactive metabolite O-desmethyltramadol (Figure 1.1). A poor analgesic effect has beendemonstrated in PM phenotypes treated with tramadol (Halling, Weihe and Brosen, 2008;Stamer et al., 2007). On the other hand, UM phenotype patients taking tramadol have dis-played an increased incidence of ADRs, for example respiratory depression (Kirchheineret al., 2008; Stamer et al., 2008). Codeine is a prodrug and is metabolized into its activemetabolite, morphine. Clinical studies have shown PM phenotypes achieve a poor anal-gesic effect, caused by the decreased production of morphine (Caraco, Sheller and Wood,1996). These patient phenotypes also have a degree of protection from codeine overdose.On the other hand, UM phenotypes have been shown to be very sensitive to codeinetreatment due to its rapid conversion to morphine (Kirchheiner et al., 2007; Madadiet al., 2009). This increases the risk of developing toxic opioid side effects: drowsiness,

CYP2D6

O-desmethyltramadolTramadol

OH

O

N

OH

HO

N

Figure 1.1 Tramadol metabolism and CYP2D6. Tramadol is metabolized by CYP2D6 via O-demethy-lation into the more active metabolite O-desmethyltramadol. UMs have shown to have increasedopioid-related ADRs (Kirchheiner et al., 2008; Stamer et al., 2008).



Tamoxifen 4-hydroxytamoxifen Endoxifen

NO

NO OH OH

HNO

Figure 1.2 Tamoxifen metabolism and CYP2D6. Tamoxifen is metabolized into the more activemetabolites, 4-hydroxytamoxifen and endoxifen, via the CYP2D6 and CYP3A4/5 enzymes.

respiratory depression and hypotension. CYP2D6 genotype testing could, therefore, po-tentially help in the prevention of ADRs or treatment failure associated with codeine andtramadol use (Foster, Mobley and Wang, 2007).

Tamoxifen is an important drug in the treatment of breast cancer. This drug targetsoestrogen receptor positive breast cancer cells and has been shown to improve long-termsurvival in these patients (Early Breast Cancer Trialists’ Collaborative Group, 2005). Ta-moxifen is metabolized by CYP2D6 and pharmacogenetic variability of this enzyme hasbeen associated with altered treatment response and patient prognosis (Kiyotani et al.,2008; Schroth et al., 2007). Tamoxifen is metabolized into the more active metabolites(Figure 1.2), 4 hydroxy-N-desmethyltamoxifen (endoxifen) and 4-hydroxytamoxifen(Ingle, 2007). The major metabolite of tamoxifen is N-desmethyltamoxifen, whichis produced via the CYP3A4/5 enzymes. This is then metabolized via CYP2D6 toendoxifen (Desta et al., 2004). CYP2D6 also directly metabolizes tamoxifen into4-hydroxytamoxifen, which is then metabolized into endoxifen via CYP3A4/5. Both4-hydroxytamoxifen and endoxifen have higher affinity for the oestrogen receptor (Limet al., 2005). CYP2D6 PMs have been shown to respond less well to tamoxifen treat-ment due to reduced production of these more active metabolites (Goetz et al., 2007; Limet al., 2007; Xu et al., 2008). This has also been associated with increased breast cancerrecurrence rates and patient mortality in CYP2D6 PM phenotypes (Goetz et al., 2007;Kiyotani et al., 2008). There has therefore been increasing interest in the role CYP2D6genotype testing for drug treatment selection in breast cancer patients (Hartman and Helft,2007; Punglia et al., 2008; Ross et al., 2008). However, some negative association studieshave also been reported and more rigorous clinical pharmacogenetic studies are requiredbefore CYP2D6 genotyping is more widely used in drug treatment selection for breastcancer patients (Limdi and Veenstra, 2010; Wegman et al., 2007).

1.2.2 CYP2C9 pharmacogenetics

Cytochrome P450 2C9 (CYP2C9) is a 490 amino acid protein (55.6 kDa) and the geneencoding this enzyme is located on chromosome 10 (Solus et al., 2004; Wang et al.,2009). CYP2C9 is one of the most abundant hepatic CYP enzymes and metabolizesapproximately 15% of commonly used drugs (Rettie and Jones, 2005). Some of theimportant drugs metabolized by CYP2C9 are the non-steroidal anti-inflammatory drugs



Table 1.2 Common allelic variants of CYP2C9#

CYP2C9 allelicvariant Mutation Enzyme activity

CYP2C9∗2 Single amino acid mutation(R144C)

Reduced enzyme activity10–20% allelic frequency in

Caucasian population

CYP2C9∗3 Single amino acid mutation(I359L)

Reduced enzyme activity5–15% allelic frequency in

Caucasian population

#For full information: www.cypalleles.ki.se/cyp2c9.htm.

(NSAIDs; diclofenac, ibuprofen), angiotensin receptor blockers (ARBs; irbesartan) andanticoagulants. The main function of CYP2C9 is drug substrate oxidation, via inter-action with the haem–oxygen complex (PDB ID: 10G5) (Williams et al., 2003). Im-portant amino acid residues implicated in active site interactions are Arg97, Arg108,Phe114 and Asp293 (Dickmann et al., 2004; Flanagan et al., 2003; Mosher et al.,2008; Ridderstrom et al., 2000). CYP2C9 displays genetic polymorphism between dif-ferent patient populations, which can result in altered enzyme activity and differentdrug pharmacokinetic profiles (Wang et al., 2009). Over 30 allelic variants have nowbeen identified (www.cypalleles.ki.se/cyp2c9.htm). The two common allelic variants ofCYP2C9 are CYP2C9∗2 and CYP2C9∗3, which result from SNPs occurring within theCYP2C9 gene (Table 2.2). This leads to amino acid substitutions within the CYP2C9∗2(R144C) and CYP2C9∗3 (I359L) enzyme structures. Both mutant enzymes result inPM phenotypes and the protein structural changes could explain the altered metabolicactivity between the enzyme variants (Takanashi et al., 2000). Both CYP2C9∗2 andCYP2C9∗3 alleles are more commonly found in the Caucasian population (Table 1.2)(Wang et al., 2009).

Recently, there has been considerable interest in the role of CYP2C9 pharmacogenet-ics in warfarin metabolism and its therapeutic effect (Schelleman, Limdi and Kimmel,2008; Wadelius and Pirmohamed, 2007). Warfarin is a commonly used anticoagulantagent and is metabolized by CYP2C9 (PDB ID: 10G2) into its major inactive metabo-lite, 7-hydroxywarfarin (Figure 1.3) (Kaminsky and Zhang, 1997). Warfarin is used inthe treatment of venous thrombosis, for example pulmonary embolism and deep venousthrombosis (DVT). It is also used to reduce thrombo-embolic risk associated with thecardiac arrhythmia, atrial fibrillation (AF) and in patients with prosthetic heart valves(Singer et al., 2008). Warfarin has a narrow therapeutic window and requires carefulcoagulation blood test monitoring, using the International Normalized Ratio (INR). How-ever, one of the serious complications of this treatment is overcoagulation and the asso-ciated risk of bleeding, which can sometimes be fatal (Fanikos et al., 2005; Linkins,Choi and Douketis, 2003). CYP2C9 enzyme inhibition and induction by co-administereddrugs, for example antibiotics, is an important cause of altered warfarin therapeuticeffect and ADRs in the clinical setting (Lin and Lu, 1998). Warfarin related adverse

http://www.cypalleles.ki.se/cyp2c9.htm




OH

OO

O CYP2C9

Warfar in 7-hydroxywarfar in

OH

OO

O

HO

Figure 1.3 Warfarin metabolism and CYP2C9. Warfarin is metabolized by CYP2C9 via hydroxylationinto the inactive metabolite 7-hydroxywarfarin. PMs have an enhanced anticoagulation effect (Aithalet al., 1999; Higashi et al., 2002).

effects have been recognized as one of the most common ADRs among patients andhas major financial implications for the health service (Davies et al., 2009; Pirmohamedet al., 2004).

The effects of CYP2C9 pharmacogenetic variability can also alter the time to reach thetherapeutic target and increase the risk of ADRs (Caraco, Blotnick and Muszkat, 2008;Schwarz et al., 2008). GWAS have identified genetic polymorphisms of CYP2C9 andthe therapeutic target, Vitamin K epoxide reductase complex 1 (VKORC1), as importantdeterminants of warfarin activity (Cooper et al., 2008). Poor metabolism of warfarin byCYP2C9 variants can increase the risk of over-coagulation and bleeding. The two com-mon allelic variants, CYP2C9∗2 and CYP2C9∗3 have been associated with an increasedrisk of bleeding in patients taking warfarin (Aithal et al., 1999; Higashi et al., 2002). Thegenetic mutations result in amino acid changes within the protein structure (Table 2.2),which affect warfarin-CYP2C9 metabolism (Lee, Goldstein and Pieper, 2002). CYP2C9genotype testing has therefore been developed to allow more effective targeting of war-farin therapy, with the aim of reducing the risk of ADRs. The FDA approved warfaringenotype-testing in 2007 and has recently updated its label for warfarin pharmacogenetictesting (www.pharmgkb.org/clinical/warfarin.jsp). Various PCR-based CYP2C9 genotyp-ing methods are available and some have been approved by the FDA (King et al., 2008;Langley et al., 2009).

Recent clinical studies have shown that genotype-guided warfarin dosing is more ac-curate in determining the initial dosing, especially in patients requiring low or high dosewarfarin treatment (The International Warfarin Pharmacogenetics Consortium, 2009;Anderson et al., 2007). Genotype-guided dosing has also been shown to achieve betteranticoagulation control and less risk of ADRs (Caraco, Blotnick and Muszkat, 2008). Theeffects of VKORC1 genetic variants also play an important role in determining warfarinactivity (Limdi et al., 2008). Various warfarin dosing algorithms have been proposed,based on CYP2C9 and VKORC1 genotypes (Langley et al., 2009). However, further clin-ical evidence in the benefits of genotype-guided warfarin dosing is required before more

http://www.pharmgkb.org/clinical/warfarin.jsp



widespread use of this test in clinical practice (Gage and Lesko, 2008). Larger prospectivewarfarin clinical pharmacogenetic studies are ongoing (van Schie et al., 2009).

Another important area of CYP2C9 pharmacogenetics is in the treatment of diabetes.Sulphonylurea drugs are commonly used in the treatment of type 2 diabetes (Krentz andBailey, 2005). These oral hypoglycaemic agents bind to the ATP-dependent potassiumchannels on the pancreatic beta cells, which leads to the opening of the calcium channels.This results in calcium influx into the beta cell and subsequent release of insulin. Sulpho-nylureas are metabolized by CYP2C9 into inactive metabolites and clinical studies havedemonstrated pharmacogenetic variability in drug response. Patients with the CYP2C9∗2and CYP2C9∗3 genotypes have been shown to have a better treatment response to sulpho-nylureas (Zhou et al., 2010). The reduced drug metabolism in these diabetic genotypeswas also shown to lead to better glycaemic control. However, the risk of hypoglycaemiahas also been shown to increase in PM genotypes, due to the enhanced effect of sulpho-nylureas (Ragia et al., 2009b). These drugs are also playing an important role in thetreatment of some types of monogenic diabetes (single gene defects). Mutations of thebeta cell potassium channels (subunits Kir6.2 and Sur1) have been associated with defec-tive closing of these channels and subsequent development of neonatal diabetes (Sperling,2006). The sulphonylurea drugs can effectively bind to these mutated channels, resultingin insulin release and therefore avoiding the need for insulin injections in these youngdiabetic patients (Flechtner et al., 2007). Both CYP2C9 pharmacogenetics and genotyp-ing for monogenic diabetes are becoming exciting new areas in diabetes research andpersonalized medicine.

1.2.3 CYP2C19 pharmacogenetics

CYP2C19 metabolizes many clinically important drug classes, for example proton pumpinhibitors, antidepressants and anticonvulsants (de Leon, Armstrong and Cozza, 2006).The CYP2C19 gene is located on chromosome 10 and to date there is no crystalstructure available for this enzyme. Like the other CYPs, this also displays geneticpolymorphism, with over 20 genetic variants of CYP2C19 having now been identified(www.cypalleles.ki.se/cyp2c19.htm). CYP2C19∗2 and CYP2C19∗3 are the most commonallelic variants (Table 1.3). CYP2C19∗2 is characterized by a SNP which leads to a splic-ing defect and subsequently encodes a non-functional enzyme (de Morais et al., 1994b).This is the main inactive allelic variant found in the Caucasian population (∼15%) (Destaet al., 2002). The CYP2C19∗3 allelic variant is also associated with a PM phenotype andthis is most commonly found in the Asian population (de Morais et al., 1994a). Thesetwo CYP2C19 PM phenotypes have been associated with reduced clearance of somedrug substrates and related to ADRs (Desta et al., 2002; Jin et al., 2010). CYP2C19∗17is a newly identified allelic variant, which is associated with a specific promoter poly-morphism and more commonly found in the Caucasian and African populations (Simet al., 2006). The CYP2C19∗17 enzyme is associated with increased metabolic activityand related to altered drug efficacy (Rudberg et al., 2008; Sim et al., 2006). Due to therapid metabolism associated with this allelic variant, patients taking antidepressant drugshave been shown to have reduced plasma drug concentrations (Rudberg et al., 2008).




Table 1.3 Common allelic variants of CYP2C19#

CYP2C19allelic variant Mutation Enzyme activity

CYP2C19∗2 Splicing defectNon-functional allele

Inactive enzyme∼15% allelic frequency in

Caucasian populationCYP2C19∗3 Premature stop codon

Non-functional alleleInactive enzyme∼20% allelic frequency in

Asian populationCYP2C19∗17 Promoter polymorphism

Increased transcriptionlevels

Increased enzyme activity5–20% allelic frequency in

Caucasian and Africanpopulations

#For full information: www.cypalleles.ki.se/cyp2c19.htm.

The AmpliChip CYP450 test can be used to detect the PM phenotypes but newer PCRmethods are used for the recently identified CYP2C19∗17 allele (de Leon, Armstrong andCozza, 2006; Rudberg et al., 2008). Further analysis of these CYP2C19 genetic polymor-phisms and their pharmacogenetic effects are ongoing (Ragia et al., 2009a).

Currently, there is a lot of interest in the effect of CYP2C19 pharmacogenetics onthe antiplatelet activity of clopidogrel (Mega et al., 2009). Clopidogrel belongs to thethienopyridine drug class. It has been increasingly used in the treatment of acute coro-nary syndromes and secondary stroke prevention therapy. One of its important treatmentindications is in reducing the risk of coronary artery stent thrombosis, post-percutaneouscoronary intervention (PCI). Clopidogrel irreversibly inhibits the P2Y12 receptor onthe platelet surface, which then blocks the activation of the GpIIb/IIIa pathway that isassociated with the cross-linking of platelets via fibrin (Parikh and Beckman, 2007).Clopidogrel is a prodrug and metabolized by the CYP2C19 enzyme into the activemetabolites, which produce the antiplatelet effects (Figure 1.4). Drugs which inhibit

Figure 1.4 Clopidogrel metabolism and CYP2C19. Clopidogrel is a prodrug and metabolized byCYP2C19 into the active metabolites, which inhibits the P2Y12 platelet receptor. PMs have a re-duced anti-platelet effect, whereas UMs have a higher risk of bleeding (Mega et al., 2009; Sibbinget al., 2010).



REFERENCES 11

CYP2C19 enzyme function have been shown to reduce clopidogrel activation. One of thecommonly used proton pump inhibitors, omeprazole, has been associated with reducedclopidogrel efficacy (Cuisset et al., 2009).

CYP2C19 genetic polymorphism has also been shown to affect clopidogrel ac-tivity. CYP2C19 PMs were found to have higher plasma clopidogrel concentrationsand lower antiplatelet effect compared to EMs (Kim et al., 2008). Patients withlow CYP2C19 activity have also been shown to have increased risk of cardiovas-cular events and coronary stent thrombosis post-PCI (Mega et al., 2009; Shuldineret al., 2009). On the other hand, increased activation of clopidogrel in patients withthe CYP2C19∗17 genotype have shown an increased risk of bleeding (Sibbing et al.,2010). The FDA has recently updated its label for clopidogrel pharmacogenetic test-ing (www.pharmgkb.org/clinical/clopidogrel.jsp). Further evaluation is ongoing into howbest to implement these findings for clopidogrel use in clinical practice, and the role ofnewer P2Y12 inhibitors (Wallentin, 2009).

1.3 Conclusion

Cytochrome P450 pharmacogenomics represents an important area of translationalmedicine research. This covers the entire spectrum, from the medicinal chemistry ofCYPs to genotype testing and application to clinical practice. CYP pharmacogenetics hasbecome an important part of the drug discovery process and in lead drug candidate opti-mization (Katz et al., 2008; Roses, 2008). The approval of CYP genotype testing has beena major advance in personalized medicine. Recent clinical studies have shown a potentialrole of CYP2D6 genotyping in drug treatment selection for breast cancer patients. CYPgenotyping may also have an important role to play in determining optimal therapeuticefficacy and reducing ADRs in patients taking the antithrombotic agents, warfarin andclopidogrel. However, larger prospective clinical pharmacogenetic studies are required toprovide a more rigorous evidence-base for pharmacotyping together with well-developedgenomic services, before genotype testing is more widely used in clinical practice (Limdiand Veenstra, 2010; Ormond et al., 2010; Vizirianakis, 2007).

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