Improved Non-Steroid Anti-Inflammatory Drugs COX-2 Enzyme Inhibitors The conference organisers wish to thank Boehringer Ingelheim for an educational grant to support this conference Chapter 2 Figure' A Molescript diagram of the COX-' dimer derived from the 3.' Ar efined structure. Heme groups are shown in red, Tyr385 in green and flurbiprofen in yef/ow. The bound detergent molecules are shown in orange where the two near the asterisk are involved in crystBl contacts; the other detergent molecule which is in the cyclooxygenase channel also appears in the other monomer. Helices A, B, C, 0 of the membrane binding domain as welf as the site of trypsin cleavage, Arg277, are also labeffed Chapter 5 Figure 10 NSAIO binding approach to membrane. Molecular depiction of the binding of an NSAIO to the lipid bilayer structure as a necessary and first step to binding to the COX enzyme. Improved Non-Steroid Anti-Inflammatory Drugs COX-2 Enzyme Inhibitors Edited by SIR JOHN VANE, DR JACK BOTTING and DR REGINA BOTTING The William Harvey Research Institute, Saint Bartholomew's Hospital Medical College, London, United Kingdom Proceedings of a conference held on October 10-11, 1995, at Regent's College, London KLUWER ACADEMIC PUBLISHERS WILLIAM HARVEY DORDRECHTIBOSTONILONDON PRESS Distributors for the United States and Canada: Kluwer Academic Publishers, PO Box 358, Accord Station, Hingham, MA 02018-0358, USA for all other countries: Kluwer Academic Publishers Group, Distribution Center, PO Box 322, 3300 AH Dordrecht, The Netherlands A catalogue record for this book is available from the British Library ISBN-13 :978-94-01 0-9031-5 e-ISBN-13 :978-94-01 0-9029-2 DOl: 10.1007/978-94-010-9029-2 Copyright © 1996 by Kluwer Academic Publishers and William Harvey Press Softcover reprint of the hardcover 1s t edition 1996 All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without prior permission from the publishers, Kluwer Academic Publishers BY, PO Box 17,3300 AA Dordrecht, The Netherlands. Published in the United Kingdom by Kluwer Academic Publishers, PO Box 55, Lancaster, UK, and William Harvey Press, Charterhouse Square, London, UK. Kluwer Academic Publishers BY incorporates the publishing programmes of D. Reidel, Martinus Nijhoff, Dr W. Junk and MTP Press. Typeset by Lasertext Ltd, Stretford, Manchester. Contents List of contributors vii Preface IX 1 Overview - mechanisms of action of anti-inflammatory drugs John Vane and Regina Botting 2 The three-dimensional structure of cyc100xygenases R. Michael Garavito 29 3 The dilemma of two cyc1ooxygenases: identifying the roles of COX-1 and COX-2 in inflammation and apoptosis Daniel Simmons, Xiaojun Lu, William Bradshaw and Weilin Xie 45 4 Inducible enzymes with special reference to COX-2 in the inflammatory response Derek Willoughby, Annette Tomlinson, Derek Gilroy and Dean Willis 67 5 NSAID mechanism of action: the role of intracellular pharmacokinetics Leo Berbette, Michelle Vecchiarelli and Gunther Trummlitz 85 6 Differential inhibition of COX-1 and COX-2 in vitro and pharmacological profile in vivo of NSAIDs Michel Pairet and Gunther Engelhardt 103 7 COX-2 expression and inhibition in human monocytes Carlo Patrono, Paola Patrignani, Maria Panara, Francesco Cipollone, Giovanna Santini, Maria Sciulli, Maria Rotondo, Roberto Padovano and Maria di Giamberardino 121 8 Expression and regulation of COX-2 in synovial tissues of arthritic patients Leslie Crofford 133 V vi COX-2 ENZYME INHIBITORS 9 An inhibitor of injury-induced COX-2 transcriptional activation elicits neuroprotection in a brain damage model Nicolas Bazan, Geoffrey Allan and Victor Marcheselli 145 10 COX-2 expression in labour Phillip Bennett and Donna Slater 167 11 Re-evaluation of gut toxicity of NSAIDs D. Nicholas Bateman 189 12 NSAID: can renal side effects be avoided? Jiirgen Frolich and Dirk Stichtenoth 203 13 Pharmacology, safety data and therapeutics of COX-2 inhibitors Paul Emery 229 Index 243 List of Contributors D. Nicholas Bateman Regional Drugs and Therapeutics Centre, Wolfson Unit, Claremont Place, Newcastle-upon-Tyne NEI 4LP, UK, Nicolas G. Bazan Department of Ophthalmology and LSU Neuroscience Center of Excellence, Louisiana State University Medical Center, School of Medicine, 2020 Gravier Street, Suite B, New Orleans LA 70112, USA Co-authors: Geoffrey Allan and Victor Marcheselli Phillip Bennett Royal Postgraduate Medical School, Institute of Obstetrics and Gynaecology, Queen Charlotte's and Chelsea Hospital, Goldhawk Road, London W6 OXG, UK Co-author: Donna Slater Leslie J. Crofford Department of Internal Medicine, Division of Rheumatology, University of Michigan, Ann Arbor, MI 48109, USA Paul Emery Rheumatology and Rehabilitation Research Unit, University of Leeds, 36 Clarendon Road, Leeds LS2 9JT UK Jiirgen C. Frolich Institute of Clinical Pharmacology, Hannover Medical School, 30623 Hannover, Germany Co-author: Dirk Stichtenoth R. Michael Garavito Department of Biochemistry, 522 Biochemistry Building, Michigan State University, East Lansing, MI 48824-1319, USA Leo G. Herbette Biomolecular Structure Analysis Center, University of Connecticut Health Center, Farmington, CT 06030, USA Co-authors: Michelle Vecchiarelli and Gunther Trummlitz Michel Pairet Department of Biological Research, Boehringer Ingelheim Research Laboratories, Birkendorfer Strasse 65, 88397 Biberach an der Riss, Germany Co-author: Gunther Engelhardt vii viii COX-2 ENZYME INHIBITORS Carlo Patrono University of Chieti, 'G D'Annunzio' School of Medicine, Via dei Vestini 31, 66013 Chieti, Italy Co-authors: Paola Patrignani, Maria Panara, Francesco Cipollone, Giovanna Santini, Maria Sciulli, Maria Rotondo, Roberto Padovano and Maria di Giamberardino Daniel L. Simmons Department of Chemistry and Biochemistry, E280 BNSN, Brigham Young University, Provo, UT 84602, USA Co-authors: Xiaojun Lu, William Bradshaw and Weilin Xie John R. Vane The William Harvey Research Institute, St Bartholomew's Hospital Medical College, Charterhouse Square, London ECIM 6BQ, UK Co-author: Regina M. Botting Derek Willoughby Department of Experimental Pathology, St Bartholomew's Hospital Medical College, William Harvey Research Institute, London EClM 6BQ, UK Co-authors: Annette Tomlinson, Derek Gilroy and Dean Willis Preface In 1971, Vane proposed that the mechanism of action of the aspirin-like drugs was through their inhibition of prostaglandin biosynthesis. Since then, there has been intense interest in the interaction between this diverse group of inhibitors and the enzyme known as cyclooxygenase (COX). It exists in two isoforms, COX-l and COX-2 (discovered some 5 years ago). Over the last two decades several new drugs have reached the market based on COX-l enzyme screens. Elucidation of the three-dimensional structure of COX-l has provided a new understanding for the actions of COX inhibitors. The constitutive isoform of COX, COX-l has clear physiological functions. Its activation leads, for instance, to the production of prostacyclin which when released by the endothelium is anti-thrombogenic and anti-atherosclerotic, and in the gastric mucosa is cyto protective. COX-l also generates prostaglandins in the kidney, where they help to maintain blood flow and promote natriuresis. The inducible isoform, COX-2, was discovered through its activity being increased in a number of cells by pro inflammatory stimuli. A year or so later, COX-2 was identified as a distinct isoform encoded by a different gene from COX-I. COX-2 is induced by inflammatory stimuli and by cytokines in migratory and other cells. Thus the anti-inflammatory actions of non-steroid anti-inflammatory drugs (NSAIDs) may be due to the inhibition of COX-2, whereas the unwanted side-effects such as irritation of the stomach lining and toxic effects on the kidney are due to inhibition of the constitutive enzyme, COX-I. This hypothesis has led to intense research to establish, inter alia, the relative activities of the currently used NSAIDs against COX-l and COX-2, the sites of expression of COX-2 and the therapeutic activity and toxicity profile of new, highly selective COX-2 inhibitors. The following chapters, written by leaders in the field, review the current status of this research. In the next few years, we can expect improved therapy of inflammatory disease, arising from further knowledge of the pathophysiological significance of the COX enzymes. John R. Vane ix 1 Overview - mechanisms of action of anti-inflammatory drugs J.R. VANE and R.M. BOTTING Inflammation is the response of living tissues to injury. It involves a complex battery of enzyme activation, mediator release, extravasation of fluid, cell migration, tissue breakdown and repair. This overview will concentrate on the actions of anti-inflammatory drugs, with special reference to the prostaglandin system. This system is activated by phospholipase A2, which liberates arachi donic acid, the substrate for cyclooxygenase (COX or PGH2 synthase). This leads to an increased production of prostaglandins (Figure 1). Prostaglandin E2 is the predominant eicosanoid detected in inflammatory conditions in man ranging from experimental acute oedema and sunburn through to chronic arthritis. Because inflammation is one of the few conditions in which PGE2 is a major product of COX it is possible that the process of inflammation directs the enzymic pathway towards this product. A second enzyme, 5-lipoxygenase converts arachidonic acid to the leukotrienes1, which are important mediators of asthma, but this aspect will not be discussed here. Prostaglandin E2 accounts for the characteristic vasodilatation and erythema (redness) seen in acute inflammation2. Vasodilatation increases the flow of blood through inflamed tissues and this augments the extravasation of fluid (oedema) caused by agents which increase vascular permeability such as bradykinin and histamine3• Prostaglandin E2 also acts synergistically with other mediators to produce inflammatory pain: although it has no direct pain-producing activity, PGE2 sensitizes receptors on afferent nerve endings to the actions of bradykinin and histamine4• PGE2 is also a potent pyretic agent and its production, stimulated by the release of interleukin-l (IL-l), in bacterial and viral infections contributes to the associated feverS. Many other COX products have been detected in inflammatory lesions. These include PGF2a, PGD2, prostacyclin (as 6-keto-PGF1u) and thromboxane ~ (TXAz; as TXB2), but these are usually present at less than a quarter of the con centrations of PGE2. Prostacyclin is probably the most important of these products in terms of inflammatory signs, for it is also a potent vasodilator and is a more potent hyperalgesic agent than PGEz- It is likely, therefore, that both PGE and prostacyclin contribute to the development of inflammatory erythema 2 and pain6. There was a new dawn in inflammatory research when Needleman and his colleagues found that COX activity was substantially increased by bacterial endotoxin in human monocytes in vitr07 and in mouse peritoneal macrophages