NMR inPharmaceuticalSciences

eMagRes BookseMagRes (formerly the Encyclopedia of Magnetic Resonance) publishes a wide range of online articles on all aspects ofmagnetic resonance in physics, chemistry, biology and medicine. The existence of this large number of articles, written byexperts in various fields, is enabling the publication of a series of eMagRes Books – handbooks on specific areas of NMR andMRI. The chapters of each of these handbooks will comprise a carefully chosen selection of eMagRes articles.

Published eMagRes BooksNMR Crystallography RF Coils for MRIEdited by Robin K. Harris, Roderick E. Wasylishen, Edited by John T. Vaughan, John R. Griffiths

Melinda J. Duer ISBN 978-0-470-77076-4ISBN 978-0-470-69961-4

Multidimensional NMR Methods for the Solution State MRI of Tissues with Short T2s or T2∗s

Edited by Gareth A. Morris, James W. Emsley Edited by Graeme M. Bydder, Gary D. Fullerton, Ian R. YoungISBN 978-0-470-77075-7 ISBN 978-0-470-68835-9

Solid-State NMR Studies of Biopolymers NMR Spectroscopy: A Versatile Tool for Environmental ResearchEdited by Ann E. McDermott, Tatyana Polenova Edited by Myrna J. Simpson, André J. SimpsonISBN 978-0-470-72122-3 ISBN 978-1-118-61647-5

NMR of Quadrupolar Nuclei in Solid Materials NMR in Pharmaceutical SciencesEdited by Roderick E. Wasylishen, Sharon E. Ashbrook, Edited by Jeremy R. Everett, Robin K. Harris, John C. Lindon,

Stephen Wimperis Ian D. WilsonISBN 978-0-470-97398-1 ISBN 978-1-118-66025-6

Forthcoming eMagRes BooksHandbook of in vivo MRSEdited by John R. Griffiths, Paul A. BottomleyISBN 978-1-118-99766-6

eMagResEdited by Roderick E. Wasylishen, Edwin D. Becker, Marina Carravetta, George A. Gray, John R. Griffiths, Ann E. McDermott,Tatyana Polenova, André J. Simpson, Myrna J. Simpson, Ian R. Young.

eMagRes (formerly the Encyclopedia of Magnetic Resonance) is based on the original publication of the Encyclopedia ofNuclear Magnetic Resonance, first published in 1996 with an updated volume added in 2000. The Encyclopedia of MagneticResonance was launched in 2007 online with all the existing published material, and was later relaunched as eMagRes in 2013.New updates are added on a regular basis through the year to keep content up to date with current developments. NMR andMRI have developed into two very different research and application areas with their own dynamic development. eMagResaddresses this development by presenting the content as two separate topics. Readers can browse through the extensive lists ofeither the NMR topics or the MRI topics.

For more information see: http://www.wileyonlinelibrary.com/ref/eMagRes

NMR inPharmaceuticalSciencesEditors

Jeremy R. EverettUniversity of Greenwich, Kent, UK

Robin K. HarrisUniversity of Durham, Durham, UK

John C. LindonImperial College London, UK

Ian D. WilsonImperial College London, UK

This edition first published 2015© 2015 John Wiley & Sons Ltd

Registered officeJohn Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex,PO19 8SQ, United Kingdom

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The right of the authors to be identified as the authors of this work has been asserted inaccordance with the Copyright, Designs and Patents Act 1988.

All rights reserved. No part of this publication may be reproduced, stored in aretrieval system, or transmitted, in any form or by any means, electronic, mechanical,photocopying, recording or otherwise, except as permitted by the UK Copyright, Designsand Patents Act 1988, without the prior permission of the publisher.

Wiley also publishes its books in a variety of electronic formats. Some content thatappears in print may not be available in electronic books.

Designations used by companies to distinguish their products are often claimed astrademarks. All brand names and product names used in this book are trade names,service marks, trademarks or registered trademarks of their respective owners. Thepublisher is not associated with any product or vendor mentioned in this book. Thispublication is designed to provide accurate and authoritative information in regard to thesubject matter covered. It is sold on the understanding that the publisher is not engagedin rendering professional services. If professional advice or other expert assistance isrequired, the services of a competent professional should be sought.

Library of Congress Cataloging-in-Publication Data

NMR in pharmaceutical sciences / editors Jeremy R. Everett, Robin K. Harris,John C. Lindon, Ian D. Wilson.

pages cmNuclear magnetic resonance in pharmaceutical sciencesIncludes bibliographical references and index.ISBN 978-1-118-66025-6 (cloth)1. Drug development. 2. Nuclear magnetic resonance. 3. Pharmaceutical

technology. I. Everett, Jeremy R., editor. II. Harris, Robin K. (Robin Kingsley), editor.III. Lindon, John C., editor. IV. Wilson, Ian D., editor. V. Title: Nuclear magneticresonance in pharmaceutical sciences.

RM301.25.N63 2015615.1’9--dc23

2015018210

A catalogue record for this book is available from the British Library.

ISBN-13: 978-1-118-66025-6

Cover image courtesy of the Laboratory of Neuro Imaging and Martinos Centerfor Biomedical Imaging, Consortium of the Human Connectome Project:www.humanconnectome.org

Set in 9.5∕11.5 pt Times by SPi Global, Chennai, IndiaPrinted and bound in Singapore by Markono Print Media Pte Ltd

eMagRes

Editorial Board

Editor-in-ChiefRoderick E. WasylishenUniversity of AlbertaEdmonton, AlbertaCanada

Section EditorsSOLID-STATE NMR & PHYSICS

Marina CarravettaUniversity of SouthamptonSouthamptonUK

SOLUTION-STATE NMR & CHEMISTRY

George A. GrayVarian Inc.Palo Alto, CAUSA

BIOCHEMICAL NMR

Ann E. McDermottColumbia UniversityNew York, NYUSA

Tatyana PolenovaUniversity of DelawareNewark, DEUSA

ENVIRONMENTAL & ECOLOGICAL NMR

André J. SimpsonUniversity of TorontoOntarioCanada

Myrna J. SimpsonUniversity of TorontoOntarioCanada

vi eMagRes

MRI & MRS

John R. GriffithsCancer Research UKCambridge Research InstituteCambridgeUK

Ian R. YoungImperial CollegeLondonUK

HISTORICAL PERSPECTIVES

Edwin D. BeckerNational Institutes of HealthBethesda, MDUSA

eMagRes vii

International Advisory Board

Robin K. Harris(Chairman)University of DurhamDurhamUK

Isao AndoTokyo Institute

of TechnologyTokyoJapan

David M. Grant(Past Chairman - deceased)University of UtahSalt Lake City, UTUSA

Adriaan BaxNational Institutes of HealthBethesda, MDUSA

Chris BoeschUniversity of BernBernSwitzerland

Paul A. BottomleyJohns Hopkins UniversityBaltimore, MDUSA

William G. BradleyUCSD Medical CenterSan Diego, CAUSA

Graeme M. BydderUCSD Medical CenterSan Diego, CAUSA

Paul T. Callaghan(deceased)Victoria University

of WellingtonWellingtonNew Zealand

Melinda J. DuerUniversity of CambridgeCambridgeUK

James W. EmsleyUniversity of SouthamptonSouthamptonUK

Richard R. ErnstEidgenössische Technische

Hochschule (ETH)ZürichSwitzerland

Ray FreemanUniversity of CambridgeCambridgeUK

Lucio FrydmanWeizmann Institute

of ScienceRehovotIsrael

Bernard C. GersteinAmes, IAUSA

Maurice GoldmanVillebon sur YvetteFrance

Harald GüntherUniversität SiegenSiegenGermany

Herbert Y. KresselHarvard Medical SchoolBoston, MAUSA

Gareth A. MorrisUniversity of ManchesterManchesterUK

C. Leon PartainVanderbilt University Medical

CenterNashville, TNUSA

Alexander PinesUniversity of California

at BerkeleyBerkeley, CAUSA

George K. RaddaUniversity of OxfordOxfordUK

viii eMagRes

Hans Wolfgang SpiessMax-Planck Institute

of Polymer ResearchMainzGermany

Charles P. SlichterUniversity of Illinois

at Urbana-ChampaignUrbana, ILUSA

John S. Waugh (deceased)Massachusetts Institute

of Technology (MIT)Cambridge, MAUSA

Bernd WrackmeyerUniversität BayreuthBayreuthGermany

Kurt WüthrichThe Scripps Research

InstituteLa Jolla, CAUSA

and

ETH ZürichZürichSwitzerland

Contents

Contributors xi

Series Preface xvii

Preface xix

Part A: Introduction 1

1 Drug Discovery and Development: The Role of NMRJeremy R. Everett 3

Part B: NMR Theory & Experimental Methods 21

2 Modern NMR Pulse Sequences in Pharmaceutical R&DJohn A. Parkinson 23

3 Experimental NMR Methods for Pharmaceutical Research and DevelopmentAnthony C. Dona 41

4 19F NMR Spectroscopy: Applications in Pharmaceutical StudiesJohn C. Lindon and Ian D. Wilson 53

5 Quantitative NMR Spectroscopy in Pharmaceutical R&DUlrike Holzgrabe 63

6 High-throughput NMR in Pharmaceutical R&DJohn C. Hollerton 79

7 Multivariate Data Analysis Methods for NMR-based Metabolic Phenotyping in Pharmaceutical and Clinical ResearchKirill A. Veselkov, James S. McKenzie, and Jeremy K. Nicholson 89

Part C: Idea to Lead 103

8 The Role of NMR in Target Identification and Validation for Pharmaceutical R&DKrishna Saxena and Harald Schwalbe 105

9 High-resolution MAS NMR of Tissues and CellsLeo L. Cheng 117

10 NMR Studies of Inborn Errors of MetabolismSarantos Kostidis and Emmanuel Mikros 131

11 NMR-based Structure Confirmation of Hits and Leads in Pharmaceutical R&DPhilip J. Sidebottom 147

12 Fragment-based Drug Design Using NMR MethodsLeonor Puchades-Carrasco and Antonio Pineda-Lucena 155

x Contents

13 Hit Discovery from Natural Products in Pharmaceutical R&DOlivia Corcoran 173

Part D: Lead to Drug Candidate 183

14 NMR-based Structure Determination of Drug Leads and CandidatesTorren M. Peakman 185

15 Mixture Analysis in Pharmaceutical R&D Using Hyphenated NMR TechniquesIan D. Wilson and John C. Lindon 197

16 Conformation and Stereochemical Analysis of Drug MoleculesGary J. Sharman 207

17 NMR Methods for the Assignment of Absolute Stereochemistry of Bioactive CompoundsJose M. Seco and Ricardo Riguera 221

18 Applications of Preclinical MRI/MRS in the Evaluation of Drug Efficacy and SafetyThomas M. Bocan, Lauren Keith, and David M. Thomasson 255

19 Practical Applications of NMR Spectroscopy in Preclinical Drug Metabolism StudiesRaman Sharma and Gregory S. Walker 267

20 Preclinical Drug Efficacy and Safety Using NMR SpectroscopyMuireann Coen and Ian D. Wilson 281

21 Characterization of Pharmaceutical Compounds by Solid-state NMRFrederick G. Vogt 297

22 Structure-based Drug Design Using NMRMark Jeeves, Lee Quill, and Michael Overduin 317

23 Pharmaceutical Technology Studied by MRIDavid G. Reid and Stephen J. Byard 331

Part E: Clinical Development 345

24 NMR-based Metabolic Phenotyping for Disease Diagnosis and StratificationBeatriz Jiménez 347

25 NMR-based Pharmacometabonomics: A New Approach to Personalized MedicineJeremy R. Everett 359

26 Clinical MRI Studies of Drug Efficacy and SafetyDavid G. Reid, Paul D. Hockings, and Nadeem Saeed 373

27 The Role of NMR in the Protection of Intellectual Property in Pharmaceutical R&DFrederick G. Vogt 385

Part F: Drug Manufacture 395

28 Analysis of Counterfeit Medicines and Adulterated Dietary Supplements by NMRMyriam Malet-Martino and Robert Martino 397

29 Pharmaceutical Industry: Regulatory Control and Impact on NMR SpectroscopyAndrea Ruggiero and Sarah K. Branch 413

30 NMR Spectroscopy in the European and US PharmacopeiasHelen Corns and Sarah K. Branch 425

31 NMR in Pharmaceutical ManufacturingEdwin Kellenbach and Paulo Dani 441

Index 453

Contributors

Thomas M. Bocan US Army Medical Research Institute of Infectious Diseases, 1425 PorterStreet, Frederick, MD 21702, USAThe Geneva Foundation, 917 Pacific Ave, Suite 600, Tacoma, WA 98402,USAChapter 18: Applications of Preclinical MRI/MRS in the Evaluation ofDrug Efficacy and Safety

Sarah K. Branch Medicines and Healthcare Products Regulatory Agency (MHRA), 151 Buck-ingham Palace Road, Victoria, London SW1W 9SZ, UKChapter 29: Pharmaceutical Industry: Regulatory Control and Impact onNMR SpectroscopyChapter 30: NMR Spectroscopy in the European and US Pharmacopeias

Stephen J. Byard Covance Laboratories, Alnwick, UKChapter 23: Pharmaceutical Technology Studied by MRI

Leo L. Cheng Massachusetts General Hospital, Harvard Medical School, Pathology, MGH149, 13th Street Charlestown, Boston, MA 02129, USAChapter 9: High-resolution MAS NMR of Tissues and Cells

Muireann Coen Computational and Systems Medicine, Department of Surgery and Cancer,Imperial College London, Sir Alexander Fleming Building, South Kensington,London SW7 2AZ, UKChapter 20: Preclinical Drug Efficacy and Safety Using NMRSpectroscopy

Olivia Corcoran Medicines Research Group, School of Health, Sport and Bioscience, Univer-sity of East London, London E15 4LZ, UKChapter 13: Hit Discovery from Natural Products in Pharmaceutical R&D

Helen Corns Medicines and Healthcare Products Regulatory Agency (MHRA), 151 Buck-ingham Palace Road, Victoria, London SW1W 9SZ, UKChapter 30: NMR Spectroscopy in the European and US Pharmacopeias

Paulo Dani Former employee from Merck Sharp & Dohme (MSD), Oss, The NetherlandsChapter 31: NMR in Pharmaceutical Manufacturing

Anthony C. Dona MRC-NIHR National Phenome Centre, Department of Surgery & Cancer,Imperial College London, South Kensington Campus, London SW7 2AZ, UKChapter 3: Experimental NMR Methods for Pharmaceutical Research andDevelopment

xii Contributors

Jeremy R. Everett Medway Metabonomics Research Group, University of Greenwich, ChathamMaritime, Kent ME4 4TB, UKChapter 1: Drug Discovery and Development: The Role of NMRChapter 25: NMR-based Pharmacometabonomics: A New Approach toPersonalized Medicine

Paul D. Hockings Antaros Medical, BioVenture Hub, 43183 Mölndal, SwedenChapter 26: Clinical MRI Studies of Drug Efficacy and Safety

John C. Hollerton GlaxoSmithKline R&D, Gunnels Wood Road, Stevenage, Herts SG1 2NY, UKChapter 6: High-throughput NMR in Pharmaceutical R&D

Ulrike Holzgrabe Institute of Pharmacy and Food Chemistry, University of Würzburg, 97074Würzburg, GermanyChapter 5: Quantitative NMR Spectroscopy in Pharmaceutical R&D

Mark Jeeves School of Cancer Sciences, University of Birmingham, Birmingham B15 2TT,UKChapter 22: Structure-based Drug Design Using NMR

Beatriz Jiménez Clinical Phenotyping Centre, Division of Computational and SystemsMedicine, Department of Surgery and Cancer, Imperial College London,London SW7 2AZ, UKChapter 24: NMR-based Metabolic Phenotyping for Disease Diagnosisand Stratification

Lauren Keith National Institute of Allergy and Infectious Disease, 8200 Research Plaza,Frederick, MD 21702, USAChapter 18: Applications of Preclinical MRI/MRS in the Evaluation ofDrug Efficacy and Safety

Edwin Kellenbach Aspen Pharmacare, PO Box 20, 5340BH Oss, Oss, The NetherlandsChapter 31: NMR in Pharmaceutical Manufacturing

Sarantos Kostidis Center for Proteomics and Metabolomics, Leiden University Medical Center,P.O. Box 9600, 2300RC Leiden, The NetherlandsChapter 10: NMR Studies of Inborn Errors of Metabolism

John C. Lindon Computational and Systems Medicine, Department of Surgery and Cancer,Imperial College London, Sir Alexander Fleming Building, South Kensington,London SW7 2AZ, UKChapter 4: 19F NMR Spectroscopy: Applications in PharmaceuticalStudiesChapter 15: Mixture Analysis in Pharmaceutical R&D Using HyphenatedNMR Techniques

Myriam Malet-Martino Groupe de RMN Biomédicale, Laboratoire SPCMIB (UMR CNRS 5068),Université Paul Sabatier, 118 route de Narbonne, 31062 Toulouse cedex 9,FranceChapter 28: Analysis of Counterfeit Medicines and Adulterated DietarySupplements by NMR

Contributors xiii

Robert Martino Groupe de RMN Biomédicale, Laboratoire SPCMIB (UMR CNRS 5068),Université Paul Sabatier, 118 route de Narbonne, 31062 Toulouse cedex 9,FranceChapter 28: Analysis of Counterfeit Medicines and Adulterated DietarySupplements by NMR

James S. McKenzie Computational and Systems Medicine, Department of Surgery and Cancer,Faculty of Medicine, Imperial College London, London SW7 2AZ, UKChapter 7: Multivariate Data Analysis Methods for NMR-based MetabolicPhenotyping in Pharmaceutical and Clinical Research

Emmanuel Mikros Faculty of Pharmacy, University of Athens Panepistimiopolis,15771 Zografou, GreeceChapter 10: NMR Studies of Inborn Errors of Metabolism

Jeremy K. Nicholson Computational and Systems Medicine, Department of Surgery and Cancer,Faculty of Medicine, Imperial College London, London SW7 2AZ, UKChapter 7: Multivariate Data Analysis Methods for NMR-based MetabolicPhenotyping in Pharmaceutical and Clinical Research

Michael Overduin School of Cancer Sciences, University of Birmingham, Birmingham B15 2TT,UKChapter 22: Structure-based Drug Design Using NMR

John A. Parkinson WestCHEM, Department of Pure and Applied Chemistry, NMR SpectroscopyFacility, University of Strathclyde, Glasgow G1 1XL, UKChapter 2: Modern NMR Pulse Sequences in Pharmaceutical R&D

Torren M. Peakman torren@nmrpeak.co.ukChapter 14: NMR-based Structure Determination of Drug Leads andCandidates

Antonio Pineda-Lucena Structural Biochemistry Laboratory, Advanced Therapies Program, Centrode Investigación Príncipe Felipe, Eduardo Primo Yúfera 3, 46012-Valencia,SpainChapter 12: Fragment-based Drug Design Using NMR Methods

Leonor Puchades-Carrasco Structural Biochemistry Laboratory, Advanced Therapies Program, Centrode Investigación Príncipe Felipe, Eduardo Primo Yúfera 3, 46012-Valencia,SpainChapter 12: Fragment-based Drug Design Using NMR Methods

Lee Quill School of Cancer Sciences, University of Birmingham, Birmingham B15 2TT,UKChapter 22: Structure-based Drug Design Using NMR

David G. Reid Department of Chemistry, University of Cambridge, Lensfield Road,Cambridge CB2 1EW, UKChapter 23: Pharmaceutical Technology Studied by MRIChapter 26: Clinical MRI Studies of Drug Efficacy and Safety

xiv Contributors

Ricardo Riguera Department of Organic Chemistry and Center for Research in BiologicalChemistry and Molecular Materials (CIQUS), University of Santiago de Com-postela, E-15782 Santiago de Compostela, SpainChapter 17: NMR Methods for the Assignment of AbsoluteStereochemistry of Bioactive Compounds

Andrea Ruggiero Medicines and Healthcare Products Regulatory Agency (MHRA), 151 Buck-ingham Palace Road, Victoria, London SW1W 9SZ, UKChapter 29: Pharmaceutical Industry: Regulatory Control and Impact onNMR Spectroscopy

Nadeem Saeed BioClinica, 72 Hammersmith Road, London W14 8UD, UKChapter 26: Clinical MRI Studies of Drug Efficacy and Safety

Krishna Saxena Institute for Organic Chemistry and Chemical Biology, Center for Biomole-cular Magnetic Resonance, Johann Wolfgang Goethe-University Frankfurt,Max-von-Laue-Str. 7, Frankfurt am Main, D-60438, GermanyGerman Cancer Consortium (DKTK), Im Neuenheimer Feld 280, 69120 Hei-delberg, GermanyChapter 8: The Role of NMR in Target Identification and Validation forPharmaceutical R&D

Harald Schwalbe Institute for Organic Chemistry and Chemical Biology, Center for Biomole-cular Magnetic Resonance, Johann Wolfgang Goethe-University Frankfurt,Max-von-Laue-Str. 7, Frankfurt am Main, D-60438, GermanyGerman Cancer Consortium (DKTK), Im Neuenheimer Feld 280, 69120 Hei-delberg, GermanyChapter 8: The Role of NMR in Target Identification and Validation forPharmaceutical R&D

Jose M. Seco Department of Organic Chemistry and Center for Research in BiologicalChemistry and Molecular Materials (CIQUS), University of Santiago de Com-postela, E-15782 Santiago de Compostela, SpainChapter 17: NMR Methods for the Assignment of AbsoluteStereochemistry of Bioactive Compounds

Raman Sharma Pfizer-PDM (Pharmacokinetics Dynamics and Metabolism), MS 8220-4374,Eastern Point Road, Groton, CT 06340, USAChapter 19: Practical Applications of NMR Spectroscopy in PreclinicalDrug Metabolism Studies

Gary J. Sharman Eli Lilly & Company, Erl Wood Manor, Sunninghill Road, Windlesham, SurreyGU20 6PH, UKChapter 16: Conformation and Stereochemical Analysis of DrugMolecules

Philip J. Sidebottom GlaxoSmithKline R&D, Chemical Sciences – Analytical Chemistry, GunnelsWood Road, Stevenage, Herts SG1 2NY, UKChapter 11: NMR-based Structure Confirmation of Hits and Leads inPharmaceutical R&D

Contributors xv

David M. Thomasson National Institute of Allergy and Infectious Disease, 8200 Research Plaza,Frederick, MD 21702, USATunnel Government Services, 6701 Democracy Boulevard, Suite 515,Bethesda, MD 20817, USAChapter 18: Applications of Preclinical MRI/MRS in the Evaluation ofDrug Efficacy and Safety

Kirill A. Veselkov Computational and Systems Medicine, Department of Surgery and Cancer,Faculty of Medicine, Imperial College London, London SW7 2AZ, UKChapter 7: Multivariate Data Analysis Methods for NMR-based MetabolicPhenotyping in Pharmaceutical and Clinical Research

Frederick G. Vogt Morgan, Lewis & Bockius, LLP, 1701 Market St., Philadelphia, PA 19103-2921, USAChapter 21: Characterization of Pharmaceutical Compounds by Solid-stateNMRChapter 27: The Role of NMR in the Protection of Intellectual Property inPharmaceutical R&D

Gregory S. Walker Pfizer-PDM (Pharmacokinetics Dynamics and Metabolism), MS 8220-4374,Eastern Point Road, Groton, CT 06340, USAChapter 19: Practical Applications of NMR Spectroscopy in PreclinicalDrug Metabolism Studies

Ian D. Wilson Computational and Systems Medicine, Department of Surgery and Cancer,Imperial College London, Sir Alexander Fleming Building, South Kensington,London SW7 2AZ, UKChapter 4: 19F NMR Spectroscopy: Applications in PharmaceuticalStudiesChapter 15: Mixture Analysis in Pharmaceutical R&D Using HyphenatedNMR TechniquesChapter 20: Preclinical Drug Efficacy and Safety Using NMRSpectroscopy

Series Preface

The Encyclopedia of Nuclear Magnetic Resonancewas published, in eight volumes, in 1996, in part tocelebrate the 50th anniversary of the first publicationsin NMR in January 1946. Volume 1 contained an his-torical overview and 200 articles by prominent NMRpractitioners, while the remaining 7 volumes were con-stituted by 500 articles on a wide variety of topics inNMR (including MRI). A ninth volume was broughtout in 2000 and two ‘spin-off’ volumes incorporat-ing the articles on MRI and MRS (together with somenew ones) were published in 2002. In 2006, the de-cision was taken to publish all the articles electroni-cally (i.e., on the World Wide Web) and this was car-ried out in 2007. Since then, new articles have beenplaced on the web every 3 months and some of theoriginal articles have been updated. This process iscontinuing and to recognize the fact that the Ency-clopedia of Magnetic Resonance is a true on-line re-source, the web site has been redesigned and newfunctionalities added, with a relaunch in January 2013in a new Volume and Issue format, under the newname eMagRes. In December, 2012, a print edition ofthe Encyclopedia was published in 10 volumes (6200pages). This much needed update of the 1996 editionof the Encyclopedia, encompasses the entire field ofNMR with the exception of medical imaging (MRI).

The existence of this large number of articles, writ-ten by experts in various fields, is enabling a newconcept to be implemented, namely the publicationof a series of printed handbooks on specific areasof NMR and MRI. The chapters of each of thesehandbooks will be constituted by a carefully cho-sen selection of Encyclopedia articles relevant to thearea in question. In consultation with the EditorialBoard, the handbooks are coherently planned in ad-vance by specially selected editors, and new articleswritten (together with updating of some already ex-isting articles) to give appropriate complete cover-age of the total area. The handbooks are intended tobe of value and interest to research students, post-doctoral fellows, and other researchers learning aboutthe topic in question and undertaking relevant exper-iments, whether in academia or industry. Consult theeMagRes web site (www.wileyonlinelibrary.com/ref/eMagRes) for the latest news on magnetic resonanceHandbooks.

Roderick E. Wasylishen

January 2015

Preface

During the first few years of NMR, following itsdiscovery in 1945, it was the preserve of physicists.However, the discovery of the chemical shift soonmoved it into the realm of chemistry and it hasbeen for many years a vital tool for research thathas rapidly extended to all branches of chemistry,biology, and medicine and, in particular, to researchefforts to develop new pharmaceutical drugs in bothuniversity and industrial environments. When MRIwas invented, this was also incorporated. It is nownot feasible to market a new drug system without theinvolvement of NMR at all stages of its development.While some earlier articles in the Encyclopedia ofMagnetic Resonance have addressed topics relevantto pharmaceutical applications of NMR, it was onlywhen planning started for this handbook that effortswere made to provide comprehensive coverage of thearea. We hope that this has now been achieved.

This handbook is firmly based on the key processesin drug discovery, development, and manufacturebut underpinned by an understanding of fundamentalNMR principles and the unique contribution thatNMR (including MRI) can provide. After an intro-ductory chapter, which constitutes an overview, thishandbook is organized into five sections. The firstsection is on the basics of NMR theory and relevantexperimental methods. The rest follow a sequencebased on the chronology of drug discovery and de-velopment, firstly ‘Idea to Lead’, then ‘Lead to DrugCandidate’, followed by ‘Clinical Development’, andfinally ‘Drug Manufacture’. The 31 chapters herein,written by experts, cover a vast range of topics fromanalytical chemistry, including aspects involved inregulatory matters and in the prevention of fraud, toclinical imaging studies.

While the handbook will be essential reading formany scientists based in pharmaceutical and relatedindustries, it should also be of considerable valueto a large number of academics who have research

connections with industry; for them, it will supply vi-tal understanding of the industrial concerns (whichusually prove to be the drivers of funding by in-dustry for university-based research). As with all thehandbooks in this series, the individual articles arealso to be found, with minimal differences but sub-tly changed format, in the on-line quarterly referenceeMagRes, which can be accessed at (early issues havethe title ‘Encyclopedia of Magnetic Resonance’). Theon-line versions generally contain brief autobiogra-phies of the article authors, a list of related articles,and acknowledgements. In addition, article abstractsand key words can be found on-line.

We are very grateful to all the authors involved fortheir agreements to write the on-line articles that pre-ceded this handbook, for their expert texts and for theircooperation in reaching this stage. We also thank thepeople at Wiley (especially Stacey Woods and ElkeMorice-Atkinson) and at Laserwords for all their hardwork (and patience!) in bringing this handbook to thepoint of publication.

We offer this handbook to the wider scientific com-munity in the hope that it will not only provide valuableinformation and a greater understanding of the role ofNMR in pharmaceutical R&D and the pharmaceuticalindustry but that it will also stimulate further advancesin relevant NMR procedures.

Jeremy R. EverettUniversity of Greenwich, London, UK

John C. LindonImperial College London, UK

Robin K. HarrisUniversity of Durham, UK

Ian D. WilsonImperial College London, UK

January 2015

Abbreviations and Acronyms

AA Amino AcidAAA Amino Acid AnalysisABMS Anisotropic Bulk Magnetic SusceptibilityACT Artemisin-based Combination TherapyAD Alzheimer’s DiseaseADC Apparent Diffusion CoefficientADF AutoDockFilterADME Absorption, Distribution, Metabolism, and

ExcretionAIR Ambiguous Interaction constRaintsALT ALanine TransaminaseAMIX Analysis of MIXturesANN Artificial Neural NetworksANOVA ANalysis Of VArianceAP Applicant’s PartAPI Active Pharmaceutical IngredientAPT Attached Proton TestASAP Acceleration by Sharing Adjacent

PolarizationASIS Aromatic Solvent-Induced ShiftASL Arterial Spin LabelingASMF Active Substance Master FileAST ASpartate TransaminaseASV Automated Structure Verification

BB Black-BloodBEI Binding Efficiency IndexBEST Band-Selective Excitation Short-TransientBF Blood FlowBLOKS Boston-Leeds OA Knee ScoringBML Birmingham Metabolite LibraryBMRB Biological Magnetic Resonance Data BankBOLD Blood Oxygenation Level DeterminationBP British PharmacopoeiaBPP Bipolar Pulse PairBV Blood Volume

CANCOR CANonical CORrelationcap-LC capillary Liquid ChromatographyCCB Calcium Channel BlockerCD Crohn’s DiseaseCDA Chiral Derivatizing AgentCE Capillary ElectrophoresisCEC Capillary ElectrochromatographyCEST Chemical Exchange Saturation TransferCID Collision-Induced SpectraCIL Central/Core Imaging LabcLC capillary-Liquid ChromatographyCM Counterfeit MedicinesCML Chronic Myelogenous LeukemiacMRI cardiac Magnetic Resonance ImagingCNS Central Nervous SystemCOMET COnsortium for MEtabonomic ToxicologyCOPD Chronic Obstructive Pulmonary DiseaseCOW Correlation Optimized Warping

CP Cross-PolarizationCPC Clinical Phenotyping CentreCPMAS Cross-Polarization Magic Angle SpinningCPMG Carr-Purcell-Meiboom-GillCRO Contract Research OrganizationCRS Chemical Reference SubstancesCSA Chemical Shift AnisotropyCSA Chiral Solvating AgentCSF Cerebrospinal FluidCSI Chemical Shift ImagingCSP Chemical Shift PerturbationsCSTs Chemical Shift TensorsCTD Common Technical DocumentCYP3A4 Cytochrome P450 3A4CZE Capillary Zone Electrophoresis

DAD Diode Array DetectorDART Direct Analysis in Real-TimeDBS Dried Blood SpotsDCE Dynamic Contrast EnhancementDEPTQ Distortionless Enhancement by Polarization

Transfer with retention of QuaternariesDEPT Distortionless Enhancement through

Polarization TransferDESI Desorption ElectroSpray IonizationDFT Density Functional TheorydGEMRIC delayed Gadolinium Enhanced MRI of

CartilageDILI Drug-Induced Liver InjuryDMN Default Mode NetworkDMOADS Disease Modifying OsteoArthritis DrugSDMPK Distribution, Metabolism, and

PharmacoKineticsDNP Dynamic Nuclear PolarizationDOSY Diffusion-Ordered SpectroscopYDPFGSE Double-Pulsed Field Gradient Spin-EchoDQ-BABA Double-Quantum Back-to-BackDS Dietary SupplementsDSC Dynamic Susceptibility ContrastdtMRI diffusion tensor MRIDUMBO Decoupling Using Mind Boggling

OptimizationDW Diffusion-Weighted

EAPO Eurasian Patent OrganizationEBI European Institute for BioinformaticsEDQM European Directorate for Quality of

Medicines and HealthCareEE Enantiomeric ExcessEFG Electric Field GradientEGFR Epidermal Growth Factor ReceptorELS Evaporative Light ScatteringEMBL European Molecular Biology LaboratoryEPO European Patent OfficeER Estrogen Receptor

ERETIC Electronic REference To access In vivoConcentrations

ESI Electrospray Ionization

FA Factor AnalysisFABS Fluorine Atoms for Biochemical

ScreeningFAXS Fluorine chemical shift Anisotropy and

eXchange for ScreeningFBDD Fragment-Based Drug DesignFDA Food and Drug AdministrationFGFR Fibroblast-Growth-Factor-ReceptorFID Free-Induction DecayFIREMAT FIve-𝜋 REplicated Magic-Angle TurningFLAIR FLuid Attenuated Inversion RecoveryFLASH Fast Low Angle SHotfMRI functional Magnetic Resonance ImagingFOV Field Of ViewFSLG Frequency-Switched Lee-GoldburgFWHH Full Width at Half Height

GAP GTP Activating ProteinGARP Globally optimized Alternating-phase

Rectangular PulsesGC Gas ChromatographyGDM Genetically Determined MetabotypeGEF Guanine nucleotide Exchange FactorGIPAW Gauge Including Projector Augmented

WaveGIT GastroIntestinal TractGLP Good Laboratory PracticeGMP Good Manufacturing PracticeGPCR G Protein-Coupled ReceptorGSD1a Glycogen Storage Disease Type 1aGWAS Genome-Wide Association Study

H2BC Heteronuclear 2 Bond CorrelationHET-

STOCSYHETeronuclear Statistical TOtal CorrelationSpectroscopY

HILIC Hydrophobic Interaction LIquidChromatography

HLA Human Lymphocyte AntigenHMBC Heteronuclear Multiple Bond CoherenceHMBC Heteronuclear Multiple Bond CorrelationHMDB Human Metabolite DataBaseHMPAA Hydrophobically Modified PolyAcrylic

AcidHMQC Heteronulcear Multiple-Quantum

CorrelationHOBS HOmonuclear Band-Selective decoupledHOESY Heteronuclear Overhauser Effect

SpectroscopYHPLC High-Performance Liquid ChromatographyHR-MAS High-Resolution Magic Angle SpinningHR-MS High-Resolution Mass SpectrometryHSQC Heteronuclear Single Quantum

CorrelationHTS High-Throughput Screening

ICA Independent Component AnalysisICH International Conference for Harmonization

of Technical Requirements for theRegistration of Pharmaceuticals for HumanUse

ICQ Imaging Capability QuestionnaireIDP Intrinsically Disordered ProteinIEM Inborn Errors of MetabolismIGT Impaired Glucose ToleranceIMCL IntraMyoCellular LipidIR Inversion RecoveryIRMS Isotope Ratio Mass Spectrometry

J-MOD J-MODulated

KOL Key Opinion Leader

LC Liquid ChromatographyLDA Linear Discriminant AnalysisLDL Low-Density LipoproteinLE Ligand EfficiencyLLE Ligand Lipophilicity EfficiencyLMICs Low- and Middle-Income CountriesLMW Low-Molecular WeightLMWH Low Molecular Weight HeparinLOD Limit Of DetectionLOGSY Ligand Observed by Gradient

SpectroscopYLOQ Limit Of QuantitationLSR Lanthanide Shift ReagentLVEF Left Ventricular Ejection Fraction

MACS Magic-Angle Coil SpinningMALDI Matrix-Assisted Laser Desorption

IonizationMAS Magic Angle SpinningMAT Magic-Angle TurningmCRC metastatic ColoRectal CancerMDD Major Depressive DisorderMHS Mean liver Histopathology ScoreMMOA Molecular Mechanism Of ActionMPIO MicroParticles of Iron OxideMRA Magnetic Resonance AngiographyMRC Medical Research CouncilMRI Magnetic Resonance ImagingMRS Magnetic Resonance SpectroscopyMSUD Maple Syrup Urine DiseaseMS Mass SpectrometryMTC Magnetization Transfer ContrastMVDA MultiVariate Data Analysis

NCE New Chemical EntitiesNCI National Cancer InstituteNF National FormularyNIHR National Institute for Health ResearchNMF Nonnegative Matrix FactorizationNOE Nuclear Overhauser EffectNP Natural Product

NPC National Phenome CentreNRA Nuclear Reaction AnalysisNSAID NonSteroidal Anti-Inflammatory DrugNTUs Nephelometric Turbidity UnitsNUS NonUniform Sampling

OA OsteoArthritisOMERACT Outcome Measures in Rheumatoid Arthritis

Clinical TrialsOPLS-DA Orthogonal Partial Least Squares

Discriminant AnalysisOS OverSulfated

P/HA Protons to Heavy AtomsPASS Phase-Adjusted Spinning SidebandsPAT Process Analytical TechnologyPBDS Plate-Based Diversity ScreeningPC Principal ComponentpCASL pseudo-Continuous Arterial Spin LabelingPCA Principal Component AnalysisPCR Polymerase Chain ReactionPCT Patent Cooperation TreatyPDE5i PhosphoDiEsterase-5 inhibitorsPDF Pair Distribution FunctionPEI Percentage Efficiency IndexPET Positron Emission TomographyPFG Pulse Field GradientPGSE Pulsed-field Gradient Spin EchoPh.Eur. European PharmacopoeiaPI Principal InvestigatorPII Personal Identifiable InformationPK PharmacoKineticsPLS Partial Least SquaresPLSDA Projections to Latent Structures -

Discriminant AnalysisPPI Protein-Protein InteractionPROJECT Periodic Refocusing Of J-Evolution by

Coherence TransferPROJECTED Periodic Refocusing Of J-Evolution

by Coherence Transfer Extended toDiffusion-ordered spectroscopy

PSA Polar Surface AreaPSA Prostate Specific AntigenPTK Protein Tyrosine KinasePTP Protein Tyrosine PhosphatasePTSD PostTraumatic Stress DisorderPULCON PUlse Length-based CONcentration

determinationPXRD Powder X-Ray Diffraction

QC Quality ControlQCPMG Quadrupolar Carr-Purcell-Meiboom-GillqNMR quantitative Nuclear Magnetic Resonance

RA Rheumatoid ArthritisRAAS Renin-Angiotensin-Aldosterone SystemRAMRIS Rheumatoid Arthritis Magnetic Resonance

Image scoring SystemRCDs Respiratory Chain Deficiencies

R&D Research and DevelopmentRD Relaxation DelayRDC Residual Dipolar CouplingRMMC Recursive Maximum Margin CriterionROC Receiver Operating CharacteristicRP Restricted PartRSPA Recursive Segment-wise Peak AlignmentRTK Receptor Tyrosine KinasesRTRT Real-Time Release Testing

SAR Structure-Activity RelationshipsSAX-HPLC Strong Anion-eXchange High-Performance

Liquid ChromatographySCXRD Single Crystal X-Ray DiffractionSDV Standard DeViationSE Spin EchoSEI Surface-binding Efficiency IndexSET Solubility-Enhancement TagSFC Supercritical Fluid ChromatographySGF Simulated Gastric FluidSHM Statistical Health MonitoringSHY Statistical HeterospectroscopYSIF Simulated Intestinal FluidSIMCA Soft Independent Modeling of Class

AnalogySMART SMAll Recovery TimesSMI Small Molecule InhibitorS/N Signal-to-NoiseSNIF Site-specific Natural Isotope FractionationSNP Single-Nucleotide PolymorphismSNR Signal-to-Noise RatioSOFAST band-Selective Optimized Flip-Angle

Short-TransientSOP Standard Operating ProcedureSOS Son-Of-SevenlessSPECT Single-Photon Emission Computed

TomographySPE Solid-Phase ExtractionSPINAL Small Phase INcremental ALternationSPI Single Point ImagingSPR Surface Plasmon ResonanceSR Study ReferenceSSFFC Substandard/Spurious/Falselylabeled/Falsified/

CounterfeitSSRI Selective Serotonin Reuptake InhibitorSTD Saturation Transfer DifferenceSTIR Short Tau Inversion RecoverySTOCSY Statistical TOtal Correlation SpectroscopYSTORM SubseT Optimization by Reference

MatchingSVM Support Vector Machines

T2D Type-2 DiabetesTBA Total Brain AtrophyTLC Thin-Layer ChromatographyTNF-𝛼 Tumor Necrosis Factor-𝛼TOCSY TOtal Correlation SpectroscopYToF Time of FlightTOSS TOtal Sideband SuppressionTPPM Two-Pulse Phase Modulation

trNOE transferred Nuclear Overhauser EffectTROSY Transverse Relaxation Optimized

SpectroscopY

UDEFT Uniform Driven Equilibrium FourierTransform

UF Ultra-FastUFH UnFractionated HeparinUHPLC Ultra-High-Performance Liquid

ChromatographyUSPIO Ultrasmall Superparamagnetic Particles of

Iron Oxide

USP United States PharmacopeiaUV UltraViolet

VCAM-1 Vascular Cell Adhesion Molecule-1VCD Vibrational Circular Dichroism

WATERGATE WATER suppression throughGrAdient-Tailored Excitation

WET Water suppression Enhanced Through T1Effects

WHO World Health OrganizationWORMS Whole Organ MR Scoring

PART AIntroduction

Chapter 1Drug Discovery and Development: The Role ofNMR

Jeremy R. EverettMedway Metabonomics Research Group, University of Greenwich, Chatham Maritime, Kent ME4 4TB, UK

1.1 Introduction to Drug Discoveryand Development and the Roleof NMR 3

1.2 NMR Spectroscopy in Drug Discoveryand Development 5

1.3 New Applications of NMR Spectroscopyin Pharmaceutical R&D 11

1.4 In vivo MRS and MRI 171.5 Future Developments 18

References 18

1.1 INTRODUCTION TO DRUGDISCOVERY AND DEVELOPMENTAND THE ROLE OF NMR

Drug discovery and development [also known as re-search and development (R&D)] is at a crossroads.Over the past 10 years, many major pharmaceuticalcompanies have reduced the size of their R&D organi-zations as they struggled to achieve the increased out-puts expected of them in this postgenomic era. Recent

NMR in Pharmaceutical Sciences. Edited by Jeremy R. Everett,John C. Lindon, Ian D. Wilson, and Robin K. Harris© 2015 John Wiley & Sons, Ltd. ISBN: 978-1-118-66025-6Also published in eMagRes (online edition)DOI: 10.1002/9780470034590.emrstm1389

analyses have suggested that the common strategy ofcompanies merging and acquiring other companieshas not led to the improvements in output that wereexpected.1 Indeed, until recently, the number of newdrugs being launched per year was falling in spite ofincreasing expenditure on R&D.2 The key reasons forthe low productivity across the industry have been thevery significant project attrition at all stages of theR&D process due to lack of efficacy and lack of safetyin new drug candidates. This attrition has been particu-larly acute in the critical Phase II clinical trials, wheredrugs are tested in patients for the first time. We arenow in a new era of drug discovery and development,with far greater involvement from academic labora-tories and better cooperation between companies andacademia, as well as between companies themselves,especially in precompetitive areas such as the develop-ment of new safety testing methodologies.

Before moving on to consider the role of NMR in thischallenging environment, it is worthwhile to considerthe different stages of the drug R&D process. Thesestages can be represented as follows:

1. Disease selection2. Target selection and validation (the target is the

name for the biological macromolecule in the hu-man body that the drug is designed to interactwith to produce clinical benefit: this is usually aprotein)

3. Hit discovery against that target4. Hit-to-lead optimization

4 Introduction

5. Lead-to-drug optimization6. Phase I drug safety studies in human volunteers7. Phase II drug efficacy and safety studies in small

cohorts of patients8. Phase III efficacy and safety studies in larger pa-

tient populations9. Drug launch

10. Postlaunch support

The most critical stage (and the most difficult) isstage 2: target selection and validation. For most smallmolecule drug discovery projects, the choice of whichprotein target to prosecute (via its inhibition, agonism,antagonism, partial agonism, etc.) is absolutely vital.Many projects fail right from this stage (although theproject team will not know this for some years!) be-cause the protein target chosen will not produce suffi-cient clinical benefit in the patient, or will only produceclinical benefit together with unacceptable side effects.The reason for these problems is that many drug dis-covery targets are unprecedented; that is, at the time oflaunch of the project, there are no Phase II data show-ing that modulation of that target gives clinical benefitin the absence of side effects. Drug discovery projectteams therefore have to make key go/no-go decisionson target selection using less-reliable genomics or an-imal data.

If drug discovery and development are in difficulties,NMR, by contrast, is not. In the 70 years or so since itsdiscovery, NMR spectroscopy has progressed quicklyto become the most powerful method of molecularstructure elucidation in both the solution state and theamorphous or heterogeneous solid state, and an indis-pensable tool in chemistry and biochemistry. In addi-tion, the more recent developments of whole body MRIand magnetic resonance spectroscopy (MRS) have en-abled the influence of NMR to spread from chem-istry and biochemistry to biology and medicine, sothat NMR is now pivotal to drug R&D, and the arrayof NMR applications stretches across each of the 10stages of the R&D process listed earlier, up to and be-yond drug launch.

The reason that NMR spectroscopy has become sodominant in solution-state molecular structure eluci-dation is that the information it provides is rich, co-herent, and highly stable and reproducible over time.Information from proton, carbon-13, and fluorine-19NMR chemical shifts, for instance, can be interpretedin terms of the chemical environment of the atoms giv-ing rise to those NMR signals. In addition, the phe-nomenon of spin–spin coupling enables the establish-ment of through-bond connectivities between different

atoms in a molecule in a way that is unobtainable withany other technique. Finally, the relaxation propertiesof the nuclear spins under study give information onthe motions, dynamics, and through-space connectivi-ties in molecules that is critical for a thorough under-standing of their properties and behavior in solution.

The drawbacks to the use of NMR principally arisefrom its lack of sensitivity relative to some other tech-niques such as mass spectrometry (MS). It is actuallyappropriate to tackle this principal issue with NMRspectroscopy straight away, as tremendous progresshas been made in this area. First of all, superconduct-ing magnets of increasing field strength have beendeveloped over the past 20 years. In the 1980s, NMRspectrometers operating at 9.4 T (resonance frequencyof 400 MHz for protons) were common. Magnetsoperating at 18.8 T (800 MHz for protons) emergedin the 1990s, and the first 1 GHz system was installedin France in 2009. As the signal-to-noise ratio of aspectrum increases approximately with the 3/2 powerof the field strength used, these increases in fieldstrength have resulted in significant gains in sensitiv-ity (a doubling in sensitivity from 600 to 900 MHz forprotons) but, unfortunately, at the expense of a con-siderable rise in spectrometer costs. Therefore, otherdevelopments have been pursued. One of these is thedevelopment of cryocooled probes, where the receivercoil and preamplifier are cooled down to temperaturesas low as about 20 K using liquid helium. This has re-sulted in sensitivity gains of about three- to four-fold,relative to running the same sample on a conventionalprobe at room temperature. Although technicallydemanding, switching to cryoprobes is much morecost effective in terms of sensitivity increase thanmoving to higher magnetic field strengths. A furtherdevelopment has been in the area of microcoil probes.Reducing the size of the receiver coils in NMR probesis particularly important for samples that have limitedmass or volume, as, for constant coil length-to-volumeratio, sensitivity is inversely proportional to coil di-ameter. A standard 5 mm NMR probe may require asample volume of about 600 μl, whereas a microcoilprobe may allow measurements on volumes as lowas about 1 μl. This approach has also been exploitedfor the coils in flow-detection experiments such asliquid chromatography–nuclear magnetic resonance(LC–NMR), capillary electrophoresis–nuclear mag-netic resonance (CE–NMR), or even lab-on-a-chip.3

Significant improvements in spectrometer perfor-mance have also ensued from the development of pulsefield gradient technologies that allow for the selection