CY TOKINE
`INHIBITORS
`
`edited by
`Gennaro Ciliberto
`Rocco Savino
`Istituto di Ricerche di Biologia Molecolare P.Angeletti
`Rome, Italy
`
`TM
`
`Marcel Dekker, Inc.
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`Copyright 2001 by Marcel Dekker, Inc. All Rights Reserved.
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`New York • Basel
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`Lassen - Exhibit 1031, p. 1
`
`

`

`ISBN: 0-0434-7
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`PRINTED IN THE UNITED STATES OF AMERICA
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`Copyright 2001 by Marcel Dekker, Inc. All Rights Reserved.
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`Lassen - Exhibit 1031, p. 2
`
`

`

`Preface
`
`Cytokines play important physiological roles in cell-to-cell communication,
`development, and differentiation. It is well known that cell differentiation in
`the hemopoietic and immune systems depends on the complex interplay and
`sequential action of several members of this heterogeneous group of polypep-
`tides, which have in common that they are produced in small quantities, often
`only upon exogenous stimuli, and in most cases act over a short distance in
`a paracrine or autocrine fashion. The differentiation-inducing activity of cyto-
`kines has opened up new avenues toward the therapy of hematological and
`immune disorders. Industrial investments on the part of biotech and pharma-
`ceutical companies have been enormous over the past years, and many recom-
`binant molecules are now marketed or used in advanced clinical trials for the
`treatment of primary or secondary anemia, granulocytopenias, thrombocyto-
`penias and bone marrow transplantation protocols. Similarly, molecules acting
`as stimulators of immune functions, such as interferons, have found a wide
`market in the therapy of chronic hepatitis, cancer, and multiple sclerosis. It
`has to be stressed that the therapeutic utilization of these recombinant mole-
`cules has so far been only partially exploited due to a combination of factors,
`which include elevated production costs, limited half-life in vivo, and lack of
`efficient and safe systems for selective delivery of the desired therapeutic
`amount to the site of action.
`Besides those cases in which exogenous cytokines can be used therapeu-
`tically either to substitute for or to potentiate physiological functions, there
`are contrasting cases in which it is desirable to block the activity of an endoge-
`nously overproduced cytokine. Research over the past ten years has clearly
`indicated that imbalanced cytokine production plays an important pathogenetic
`role in several inflammatory, chronic autoimmune diseases, and sometimes
`
`Copyright 2001 by Marcel Dekker, Inc. All Rights Reserved.
`
`Lassen - Exhibit 1031, p. 3
`
`

`

`facilitates cancer growth. Thus, blocking the activity of a central molecule
`can result in the improvement of clinical and biochemical parameters.
`While it is clear that blocking cytokine function is necessary to cure
`several pathological conditions, it is also evident that this is difficult to
`achieve. Naturally soluble cytokine antagonists, with the exception of a very
`limited number of cases, do not exist. Thus, ad hoc drug-discovery strategies
`have to be devised in order to obtain potent and specific cytokine blocking
`agents. Potency is an absolute requirement to counteract the activity of the
`endogenous cytokine, which is produced in microgram-per-day amounts in
`several pathologies and is fully active at picomolar levels. Specificity is also
`difficult to achieve, given the high degree of redundancy in the use of common
`receptors and/or signaling pathways. Finally, while several cytokine blocking
`agents in the form of recombinant proteins (monoclonal antibodies, soluble
`receptors, receptor antagonists) have been successfully generated over the
`years, they suffer from several major limitations, some of them identical to
`those encountered for wild-type cytokine delivery, i.e., the need for parenteral
`administration and the elevated costs of production. In addition, we face new
`problems, the most important being the potential immunogenicity of these
`nonnatural molecules. In order to overcome these hurdles, there is strong pres-
`sure to develop small-molecular-weight inhibitors.
`Cytokine antagonists can be obtained at least at three general levels: 1)
`blockade of cytokine production, 2) inhibition of cytokine-induced assembly
`of a functionally competent receptor complex, 3) interference with cytokine
`signaling inside target cells. All these approaches have now become more
`feasible thanks to our greater understanding of both the intracellular signaling
`pathways and the structural determinants responsible for cytokine–receptor
`interaction. In many instances the 3-D structure of cytokines and their relevant
`receptors has been solved, as was that of intracellular tyrosine kinases. This
`knowledge is of enormous help in the molecular modeling and drug design
`of selective inhibitory compounds. Once a given target is identified, the search
`for its inhibitors can be undertaken using different strategies: random screen-
`ing of libraries of compounds, molecular design, or a combination of the two.
`Furthermore, the technical improvements and the widespread diffusion of
`combinatorial chemistry are speeding up these steps enormously. We are thus
`experiencing a moment of high creativity and progress in this field, marked
`by frequent, important breakthroughs.
`The aim of this book is to provide a comprehensive and updated over-
`view of the field of cytokine inhibitors at the beginning of the third millen-
`nium. Several chapters illustrate cases of naturally produced or in vitro en-
`gineered polypeptide cytokine-blocking agents. In particular, some chapters
`
`Copyright 2001 by Marcel Dekker, Inc. All Rights Reserved.
`
`Lassen - Exhibit 1031, p. 4
`
`

`

`(Chapter 1, Dinarello and Fantuzzi; Chapter 2, van Deventer and van der Poll;
`Chapter 3, Bendtzen et al.; Chapter 9, Naka et al.; and Chapter 10, Starr) give
`clear examples of the homeostatic function of cytokine inhibition and indi-
`rectly substantiate a therapeutic approach oriented to potentiating a natural
`response of the organism, when required. In the group of ‘‘first-generation’’
`recombinant cytokine inhibitors the most successful clinical examples are tu-
`mor necrosis factor antagonists (Chapter 4, Hitraya and Schaible), which have
`reached the market during the past year and promise to become prominent
`tools in the therapy of chronic inflammatory conditions over the next decade.
`One case is also shown of how the detailed knowledge of the interaction be-
`tween a cytokine and a multimeric receptor complex has led to the rational
`design of potent and selective receptor antagonists (Chapter 8, Vernallis).
`Chapter 5, Murali et al.; Chapter 6, Moss et al.; Chapter 7, Proudfoot-Fichard
`et al.; Chapter 11, Lipson et al.; and Chapter 12, Gum and Young, illustrate
`various examples and approaches to the generation and clinical development
`of ‘‘second-generation,’’ small-molecular-weight, orally bioavailable cyto-
`kine inhibitors. In the future we can expect to see more and more examples
`of these inhibitors and eventually to find them on the shelves of pharmacies
`as novel and more potent anti-inflammatory, anticancer, and antiviral agents.
`The editors wish to express their gratitude to Sara Razzicchia. Without
`her invaluable editorial assistance on this project the book might not have
`been born.
`
`Gennaro Ciliberto
`Rocco Savino
`
`Copyright 2001 by Marcel Dekker, Inc. All Rights Reserved.
`
`Lassen - Exhibit 1031, p. 5
`
`

`

`Contents
`
`Preface
`Contributors
`
`1 Importance of the Interleukin-1β–Converting Enzyme in
`Disease Mediated by the Proinflammatory Cytokines IL-1β
`and IL-18
`Charles A. Dinarello and Giamila Fantuzzi
`
`2 Interleukin-10: A Cytokine with Multiple Anti-
`Inflammatory and Some Proinflammatory Activities
`Sander J. H. van Deventer and Tom van der Poll
`
`3 Natural and Induced Anticytokine Antibodies in Humans
`Klaus Bendtzen, Christian Ross, Christian Meyer, Morten
`Bagge Hansen, and Morton Svenson
`
`4 Anti-Tumor Necrosis Factor Therapies in Crohn’s Disease
`and Rheumatoid Arthritis
`Elena G. Hitraya and Thomas F. Schaible
`
`5 Design and Development of Small-Molecule Inhibitor of
`Tumor Necrosis Factor-α
`Ramachandran Murali, Kiichi Kajino, Akihiro Hasegawa,
`Alan Berezov, and Wataru Takasaki
`
`Copyright 2001 by Marcel Dekker, Inc. All Rights Reserved.
`
`Lassen - Exhibit 1031, p. 6
`
`

`

`6 Screening and Design of Tumor Necrosis Factor-α:
`Converting Enzyme Inhibitors
`M. L. Moss, J. D. Becherer, J. G. Conway, J. R. Warner,
`D. M. Bickett, M. A. Leesnitzer, T. M. Seaton,
`J. L. Mitchell, R. T. McConnell, T. K. Tippin,
`L. G. Whitesell, M. C. Rizzolio, K. M. Hedeen,
`E. J. Beaudet, M. Andersen, M. H. Lambert, R. Austin,
`J. B. Stanford, D. G. Bubacz, J. H. Chan, L. T. Schaller,
`M. D. Gaul, D. J. Cowan, V. M. Boncek,
`M. H. Rabinowitz, D. L. Musso, D. L. McDougald,
`I. Kaldor, K. Glennon, R. W. Wiethe, Y. Guo, and
`R. C. Andrews
`
`7 Chemokine Receptors: Therapeutic Targets for Human
`Immunodeficiency Virus Infectivity
`Amanda E. I. Proudfoot-Fichard, Alexandra Trkola, and
`Timothy N. C. Wells
`
`8 Receptor Antagonists of gp130 Signaling Cytokines
`Ann B. Vernallis
`
`9 Negative-Feedback Regulations of Cytokine Signals
`Tetsuji Naka, Masashi Narazaki, and Tadamitsu Kishimoto
`
`10 Inhibitors of the Janus Kinase–Signal Transducers and
`Activators of Transcription (JAK/STAT) Signaling
`Pathway
`Robyn Starr
`
`11 Inhibition of Vascular Endothelial Growth Factor (VEGF)
`and Stem-Cell Factor (SCF) Receptor Kinases as
`Therapeutic Targets for the Treatment of Human Diseases
`Kenneth E. Lipson, Li Sun, Congxin Liang, and Gerald
`McMahon
`
`12 p38 Inhibition
`Rebecca J. Gum and Peter R. Young
`
`Copyright 2001 by Marcel Dekker, Inc. All Rights Reserved.
`
`Lassen - Exhibit 1031, p. 7
`
`

`

`Contributors
`
`M. Andersen, Ph.D. Department of Medicinal Chemistry, GlaxoWellcome,
`Inc., Research Triangle Park, North Carolina
`
`R. C. Andrews, Ph.D. Department of Medicinal Chemistry, GlaxoWell-
`come, Inc., Research Triangle Park, North Carolina
`
`R. Austin, Ph.D. Department of Medicinal Chemistry, GlaxoWellcome,
`Inc., Research Triangle Park, North Carolina
`
`E. J. Beaudet, M.S. Department of Research BioMet, GlaxoWellcome, Inc.,
`Research Triangle Park, North Carolina
`
`J. D. Becherer, Ph.D. Department of Molecular Biochemistry, GlaxoWell-
`come, Inc., Research Triangle Park, North Carolina
`
`Klaus Bendtzen, M.D.
`Institute for Inflammation Research, Rigshospitalet
`National University Hospital, Copenhagen, Denmark
`
`Alan Berezov, Ph.D. Department of Pathology and Laboratory Medicine,
`University of Pennsylvania, Philadelphia, Pennsylvania
`
`D. M. Bickett, M.S. Department of Molecular Biochemistry, GlaxoWell-
`come, Inc., Research Triangle Park, North Carolina
`
`V. M. Boncek, M.S. Department of Molecular Pharmacology, GlaxoWell-
`come, Inc., Research Triangle Park, North Carolina
`
`Copyright 2001 by Marcel Dekker, Inc. All Rights Reserved.
`
`Lassen - Exhibit 1031, p. 8
`
`

`

`D. G. Bubacz, M.S. Department of Medicinal Chemistry, GlaxoWellcome,
`Inc., Research Triangle Park, North Carolina
`
`J. H. Chan, Ph.D. Department of Medicinal Chemistry, GlaxoWellcome,
`Inc., Research Triangle Park, North Carolina
`
`Gennaro Ciliberto, M.D. Department of Gene Therapy, Istituto di Ricerche
`di Biologia Molecolare P. Angeletti, Rome, Italy
`
`J. G. Conway, Ph.D. Department of Molecular Pharmacology, GlaxoWell-
`come, Inc., Research Triangle Park, North Carolina
`
`D. J. Cowan, M.S. Department of Medicinal Chemistry, GlaxoWellcome,
`Inc., Research Triangle Park, North Carolina
`
`Charles A. Dinarello, M.D. Division of Infectious Diseases, Department
`of Medicine, University of Colorado Health Sciences Center, Denver, Colo-
`rado
`
`Giamila Fantuzzi, Ph.D. Department of Medicine, University of Colorado
`Health Sciences Center, Denver, Colorado
`
`M. D. Gaul, Ph.D. Department of Medicinal Chemistry, GlaxoWellcome,
`Inc., Research Triangle Park, North Carolina
`
`K. Glennon, B.S. Department of Medicinal Chemistry, GlaxoWellcome,
`Inc., Research Triangle Park, North Carolina
`
`Rebecca J. Gum, Ph.D. Department of Strategic and Exploratory Sciences,
`Abbott Laboratories, Abbott Park, Illinois
`
`Y. Guo, M.S. Department of Medicinal Chemistry, GlaxoWellcome, Inc.,
`Research Triangle Park, North Carolina
`
`Morten Bagge Hansen, M.D. D.M.Sc.
`Institute for Inflammation Research,
`Rigshospitalet National University Hospital, Copenhagen, Denmark
`
`Akihiro Hasegawa, Ph.D. Department of Pathology and Laboratory Medi-
`cine, University of Pennsylvania, Philadelphia, Pennsylvania
`
`Copyright 2001 by Marcel Dekker, Inc. All Rights Reserved.
`
`Lassen - Exhibit 1031, p. 9
`
`

`

`K. M. Hedeen, M.S. Department of Research BioMet, GlaxoWellcome,
`Inc., Research Triangle Park, North Carolina
`
`Elena G. Hitraya, M.D., Ph.D. Medical Affairs Department—Immunol-
`ogy, Centocor, Inc., Malvern, Pennsylvania
`
`Kiichi Kajino, M.D., Ph.D.* Second Department of Pathology, Shiga Uni-
`versity of Medical Sciences, Otsu, Japan
`
`I. Kaldor, M.S. Department of Medicinal Chemistry, GlaxoWellcome, Inc.,
`Research Triangle Park, North Carolina
`
`Tadamitsu Kishimoto, M.D., Ph.D. President, Osaka University, Suita
`City, Osaka, Japan
`
`M. H. Lambert, Ph.D. Department of Structural Chemistry, GlaxoWell-
`come, Inc., Research Triangle Park, North Carolina
`
`M. A. Leesnitzer, M.S. Department of Molecular Biochemistry, Glaxo-
`Wellcome, Inc., Research Triangle Park, North Carolina
`
`Congxin Liang, Ph.D. Department of Chemistry, SUGEN, Inc., South San
`Francisco, California
`
`Kenneth E. Lipson, Ph.D. Department of Drug Discovery and Cell Biol-
`ogy, SUGEN, Inc., South San Francisco, California
`
`R. T. McConnell, M.S. Department of Molecular Biochemistry, Glaxo-
`Wellcome, Inc., Research Triangle Park, North Carolina
`
`D. L. McDougald, M.S. Department of Medicinal Chemistry, GlaxoWell-
`come, Inc., Research Triangle Park, North Carolina
`
`Gerald McMahon, Ph.D. Department of Drug Discovery, SUGEN, Inc.,
`South San Francisco, California
`
`* Formerly at University of Pennsylvania, Philadelphia, Pennsylvania
`
`Copyright 2001 by Marcel Dekker, Inc. All Rights Reserved.
`
`Lassen - Exhibit 1031, p. 10
`
`

`

`Christian Meyer, M.D.
`Institute for Inflammation Research, Rigshospitalet
`National University Hospital, Copenhagen, Denmark
`
`J. L. Mitchell, B.S. Department of Molecular Biochemistry, GlaxoWell-
`come, Inc., Research Triangle Park, North Carolina
`
`M. L. Moss, Ph.D. Department of Biochemistry and Biophysics, Lineberger
`Comprehensive Cancer Center, University of North Carolina at Chapel Hill,
`Chapel Hill, North Carolina
`
`Ramachandran Murali, Ph.D. Department of Pathology and Laboratory
`Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
`
`D. L. Musso, M.S. Department of Medicinal Chemistry, GlaxoWellcome,
`Inc., Research Triangle Park, North Carolina
`
`Tetsuji Naka, M.D. Department of Molecular Medicine, Graduate School
`of Medicine, Osaka University, Suita City, Osaka, Japan
`
`Masashi Narazaki, M.D., Ph.D. Department of Molecular Medicine, Grad-
`uate School of Medicine, Osaka University, Suita City, Osaka, Japan
`
`Amanda E. I. Proudfoot-Fichard, Ph.D. Department of Biotechnology,
`Serono Pharmaceutical Research Institute S.A., Geneva, Switzerland
`
`M. H. Rabinowitz, Ph.D. Department of Medicinal Chemistry, GlaxoWell-
`come, Inc., Research Triangle Park, North Carolina
`
`M. C. Rizzolio, M.S. Department of Pharmaceutics, GlaxoWellcome, Inc.,
`Research Triangle Park, North Carolina
`
`Christian Ross, M.D.
`Institute for Inflammation Research, Rigshospitalet
`National University Hospital, Copenhagen, Denmark
`
`Rocco Savino, Ph.D. Department of Gene Therapy, Istituto di Ricerche di
`Biologia Molecolare P. Angeletti, Rome, Italy
`
`L. T. Schaller, B.S. Department of Medicinal Chemistry, GlaxoWellcome,
`Inc., Research Triangle Park, North Carolina
`
`Copyright 2001 by Marcel Dekker, Inc. All Rights Reserved.
`
`Lassen - Exhibit 1031, p. 11
`
`

`

`Thomas F. Schaible, Ph.D. Department of Medical Affairs—Immunology,
`Centocor, Inc., Malvern, Pennsylvania
`
`T. M. Seaton, B.S. Department of Molecular Biochemistry, GlaxoWell-
`come, Inc., Research Triangle Park, North Carolina
`
`J. B. Stanford, M.S. Department of Medicinal Chemistry, GlaxoWellcome,
`Inc., Research Triangle Park, North Carolina
`
`Robyn Starr, Ph.D. Division of Cancer and Haematology, The Walter and
`Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia
`
`Li Sun, Ph.D. Department of Chemistry, SUGEN, Inc., South San Fran-
`cisco, California
`
`Morten Svenson Institute for Inflammation Research, Rigshospitalet Na-
`tional University Hospital, Copenhagen, Denmark
`
`Wataru Takasaki, Ph.D.* Drug Metabolism and Pharmacokinetics Re-
`search Laboratories, Sankyo Co., Ltd., Tokyo, Japan
`
`T. K. Tippin, Ph.D. Department of Research BioMet, GlaxoWellcome, Inc.,
`Research Triangle Park, North Carolina
`
`Alexandra Trkola, Ph.D. Division of Infectious Diseases, Department of
`Internal Medicine, University Hospital Zurich, Zurich, Switzerland
`
`Sander J. H. van Deventer, M.D. Department of Experimental Internal
`Medicine, Academic Medical Center, Amsterdam, The Netherlands
`
`Tom van der Poll, M.D., Ph.D. Laboratory of Experimental Internal Medi-
`cine, Academic Medical Center, Amsterdam, The Netherlands
`
`Ann B. Vernallis, Ph.D. School of Life and Health Sciences, Aston Univer-
`sity, Birmingham, England
`
`* Formerly at University of Pennsylvania, Philadelphia, Pennsylvania
`
`Copyright 2001 by Marcel Dekker, Inc. All Rights Reserved.
`
`Lassen - Exhibit 1031, p. 12
`
`

`

`J. R. Warner, B.S. Department of Molecular Biochemistry, GlaxoWell-
`come, Inc., Research Triangle Park, North Carolina
`
`Timothy N. C. Wells Serono Pharmaceutical Research Institute, Geneva,
`Switzerland
`
`L. G. Whitesell, Ph.D. Department of Pharmaceutics, GlaxoWellcome, Inc.,
`Research Triangle Park, North Carolina
`
`R. W. Wiethe, M.S. Department of Medicinal Chemistry, GlaxoWellcome,
`Inc., Research Triangle Park, North Carolina
`
`Peter R. Young, Ph.D. Department of Cardiovascular Diseases, DuPont
`Pharmaceuticals, Wilmington, Delaware
`
`Copyright 2001 by Marcel Dekker, Inc. All Rights Reserved.
`
`Lassen - Exhibit 1031, p. 13
`
`

`

`1I
`
`mportance of the Interleukin-1␤–
`Converting Enzyme in Disease
`Mediated by the Proinflammatory
`Cytokines IL-1␤ and IL-18
`
`Charles A. Dinarello and Giamila Fantuzzi
`University of Colorado Health Sciences Center, Denver, Colorado
`
`INTRODUCTION
`
`Cytokines are small, nonstructural proteins with molecular weights ranging
`from 8,000 to 40,000 D. Originally called lymphokines and monokines to
`indicate their cellular sources, it became clear that the term cytokine is the
`best description, since nearly all nucleated cells are capable of synthesizing
`these proteins and, in turn, respond to them. There is no amino acid sequence
`motif or three-dimensional structure that links cytokines; rather their biologi-
`cal activities allow them to be grouped into different classes. For the most
`part, cytokines are primarily involved in host responses to disease or infection.
`From gene deletion studies in mice and the use of neutralizing antibodies,
`their role in any homeostatic mechanism has been nonexistent or less than
`dramatic. For example, the interleukin-1β (IL-1β)– and IL-1β–converting en-
`zyme (ICE)–deficient mouse has been bred in normal animal housing at sev-
`eral institutions without signs of increased resistance or development of spon-
`taneous disease. In the case of the IL-10–deficient mouse, which develops
`spontaneous inflammatory bowel disease in normal housing, the disease does
`not develop in germ-free housing.
`
`Copyright 2001 by Marcel Dekker, Inc. All Rights Reserved.
`
`Lassen - Exhibit 1031, p. 14
`
`

`

`Many scientists have made the analogy of cytokines to hormones, but
`upon closer examination, this is not an accurate comparison. Why? First, hor-
`mones tend to be constitutively expressed by highly specialized tissues but
`cytokines are synthesized by nearly every cell. Whereas hormones are the
`primary synthetic product of a cell (e.g., insulin, thyroid, ACTH), cytokines
`account for a very small amount of the synthetic output of a cell. In addition,
`hormones are expressed in response to homeostatic control signals, many of
`which are part of a daily cycle. In contrast, most cytokine genes are not ex-
`pressed (particularly at the translational level) unless specifically stimulated
`by noxious events. In fact, it has become clear that the protein kinases involved
`in triggering cytokine gene expression are activated by a variety of ‘‘cell stres-
`sors.’’ For example, ultraviolet light, heat shock, hyperosmolarity, or ad-
`herence to a foreign surface activate the mitogen-activated protein kinases
`(MAPK) which phosphorylate transcription factors for gene expression. Of
`course, infection and inflammatory products also use the MAPK pathway for
`initiating cytokine gene expression. One concludes then that cytokines them-
`selves are produced in response to ‘‘stress,’’ whereas most hormones are pro-
`duced by a daily intrinsic clock.
`Based on their primary biological activities, cytokines are often grouped
`as lymphocyte growth factors, mesenchymal growth factors,
`interferons,
`chemokines, and colony-stimulating factors. Some have been given the name
`interleukin to indicate a product of a leukocyte and a target of a leukocyte.
`But the term interleukin does not correctly connote the true pleotropic nature
`of cytokines. Nevertheless, there are presently 18 cytokines called interleu-
`kins. By convention, a newly discovered cytokine can be called an interleukin
`when a biological activity is associated with a novel human DNA sequence
`and the gene product is expressed recombinantly and shown to possess the
`same property as the natural product. Other cytokines have retained their origi-
`nal biological description such as ‘‘tumor necrosis factors’’. Another way to
`look at some cytokines is their role in inflammation, and this is particularly
`relevant to the importance to pain. Hence, some cytokines clearly promote
`inflammation and are called proinflammatory cytokines, whereas other sup-
`press the activity or production of proinflammatory cytokines and are called
`anti-inflammatory cytokines.
`Cytokines are rather promiscuous molecules with many equally promis-
`cuous cells as lovers. For example, IL-4 and IL-10 are potent activators of B
`lymphocytes. B lymphocytes are responsible for antibody formation in re-
`sponse to a foreign (or endogenous) antigen. Cytokines which ‘‘help’’ promote
`B-cell antibody formation are classified as T-lymphocyte helper cell of the
`type 2 class (Th2). The counterpart to T-lymphocyte helper cells of the type
`
`Copyright 2001 by Marcel Dekker, Inc. All Rights Reserved.
`
`Lassen - Exhibit 1031, p. 15
`
`

`

`2 class are cytokines that help the type 1 T lymphocytes (Th1). Th1 cytokines
`help the process of cellular immunity in which T lymphocytes attack and kill
`viral infected cells. Th1 cytokines include IL-2, IL-12, IL-18, and interferon-
`γ (IFN-γ). However, IL-4 and IL-10, although primarily considered to be
`Th2 cytokines, are also potent anti-inflammatory cytokines. They are anti-
`inflammatory cytokines by virtue of their ability to suppress genes for proin-
`flammatory cytokines such as IL-1, tumor necrosis factor (TNF), and several
`chemokines.
`IFN-γ is another example of the pleiotropic nature of cytokines. Al-
`though like IFN-α and IFN-β, IFN-γ possesses antiviral activity, IFN-γ is also
`an activator of the Th1 response which leads to cytotoxic T cells. IFN-γ is
`also considered to be a proinflammatory cytokine, because it augments TNF
`and IL-1 receptors, induces nitric oxide (NO), and upregulates endothelial
`adhesion molecules. However, listing IFN-γ as a purely proinflammatory cy-
`tokine should be done with an open mind in that depending on the biological
`process, IFN-γ may function as an anti-inflammatory cytokine. It should be
`pointed out that these same cytokines (IFN-γ, IL-6, IL-10, IL-4) are also major
`participants in directly controlling inflammation and hence serve as an exam-
`ple of the pleiotropic nature of cytokines.
`Although a vast amount of data support the concept that TNF and IL-
`1 are highly proinflammatory molecules, why the interest in whether IFN-γ
`is proinflammatory or anti-inflammatory? IFN-γ production requires the activ-
`ity of another cytokine; namely, IL-18. The biology of IL-18 and its role in
`inflammation are discussed below. However, the ability of IL-18 to induce
`IFN-γ is dependent on ICE (1,2). In addition to cleaving the IL-1β precursor
`into an active, proinflammatory cytokine, ICE also cleaves the inactive IL-18
`precursor into an active, IFN-γ–inducing factor. Hence, inhibitors of ICE must
`be considered to affect the processing and activity of two distinct cytokines,
`IL-1β and IL-18. Moreover, the effect of inhibitors of ICE cannot be regarded
`as purely anti-inflammatory, since the production of IFN-γ will affect the im-
`mune (Th1) and anti-inflammatory properties of IFN-γ. Indeed, inhibitors of
`ICE reduce endotoxin-induced IFN-γ by a non–IL-1β mechanism (3), and
`mice deficient in ICE do not produce IFN-γ following administration of endo-
`toxin, whereas mice deficient in IL-1β do.
`The concept that inhibition of ICE may not be a purely anti-inflamma-
`tory strategy is best illustrated when cells are stimulated with IL-1β to produce
`prostaglandin E2 (PGE2) via the induction of cyclooxygenase-2 (COX-2) (re-
`viewed in ref. 4). For the most part, pain is associated with inflammation.
`Nerve endings sense the swelling of local tissues which is a hallmark of in-
`flammation. The other hallmark is erythema. The injection of IL-1β locally
`
`Copyright 2001 by Marcel Dekker, Inc. All Rights Reserved.
`
`Lassen - Exhibit 1031, p. 16
`
`

`

`into humans results in pain, swelling, and erythema (4). IL-1β induces the
`genes coding for phospholipase A2 type II, COX-2, and inducible nitric oxide
`synthase (iNOS). Figure 1 illustrates the basic events of this inflammatory
`process. Whether induced by infection, trauma, ischemia, immune-activated
`T cells, or toxins, IL-1 initiates the cascade of inflammatory mediators by
`targeting the endothelium.
`However, IFN-γ is unique in that it suppresses IL-1β induction of PGE2
`(5). In fact, IFN-γ suppresses other IL-1–induced processes such as IL-1 in-
`duction of itself (6,7). IFN-γ also suppresses IL-1 induction of collagenases
`(8,9). Therefore, in blocking the processing of proIL-18 using inhibitors of
`ICE, one does not have the specificity of reducing the biological impact of a
`single cytokine.
`
`Figure 1 Proinflammatory effects of IL-1 and TNF are on the endothelium. TNF
`and IL-1 activate endothelial cells and trigger the cascade of proinflammatory small
`molecule mediators. Increased gene expression for phospholipase A 2 type II, cyclooxy-
`genase type 2 (COX-2), and inducible nitric oxide synthase (iNOS) results in elevated
`production of their products, PAF, PGE 2, and NO. Alone or in combination, these
`mediators decrease the tone of vascular smooth muscle. IL-1 and TNF also cause in-
`creased capillary leak leading to swelling and pain. Pain threshold is lowered by PGE 2.
`The upregulation of endothelial leukocyte adhesion molecules results in adherence of
`circulating neutrophils to the endothelium, and increased production of chemokines
`such as IL-8 facilitates the emigration of neutrophils into the tissues. Chemokines also
`activate degranulation of neutrophils. Activated neutrophils lead to tissue destruction
`and more inflammation.
`
`Copyright 2001 by Marcel Dekker, Inc. All Rights Reserved.
`
`Lassen - Exhibit 1031, p. 17
`
`

`

`WHY INHIBIT IL-1?
`
`Although the systemic effects of IL-1 have been studied in animals, there are
`now data on the effects and sensitivity to IL-1 in humans. The overwhelming
`conclusion of these studies is that IL-1 is a highly inflammatory molecule and
`reduction in its production or activity in a variety of disease states is likely a
`sensible therapeutic strategy. IL-1α or IL-1β has been injected in patients with
`various solid tumors or as part of a reconstitution strategy in bone marrow
`transplantation. Acute toxicities of either IL-1α or IL-1β were greater follow-
`ing intravenous compared to subcutaneous injection; subcutaneous injection
`was associated with significant local pain, erythema, and swelling (10,11).
`Chills and fever are observed in nearly all patients, even in the 1 ng/kg dose
`group (12). The febrile response increased in magnitude with increasing doses
`(13–17) and chills and fever were abated with indomethacin treatment (18).
`In patients receiving IL-1α (16,17) or IL-1β (13,14), nearly all subjects experi-
`enced significant hypotension at doses of 100 ng/kg or greater. Systolic blood
`pressure fell steadily and reached a nadir of 90 mm Hg or less 3–5 h after
`the infusion of IL-1. At doses of 300 ng/kg, most patients required intravenous
`pressors. By comparison, in a trial of 16 patients given IL-1β from 4 to 32
`ng/kg subcutaneously, there was only one episode of hypotension at the high-
`est dose level (10). These results suggest that the hypotension is probably due
`to induction of NO, and elevated levels of serum nitrate have been measured
`in patients with IL-1–induced hypotension (17).
`Patients given 30–100 ng/kg of IL-1β had a sharp increase in cortisol
`levels 2–3 h after the injection. Similar increases were noted in patients given
`IL-1α. In 13 of 17 patients given IL-1β, there was a fall in serum glucose
`within the first hour of administration, and in 11 patients, glucose fell to 70
`mg/100 mL or lower (14). In addition, there were increases in ACTH and
`thyroid-stimulating hormone but a decrease in testosterone (17). No changes
`were observed in coagulation parameters such as prothrombin time, partial
`thromboplastin, or fibrinogen degradation products. This latter finding is to
`be contrasted to TNF-α infusion into healthy humans which results in a distinct
`coagulopathy syndrome (19).
`Not unexpectedly, IL-1 infusion into humans significantly increased
`circulating IL-6 levels in a dose-dependent fashion (17). At a dose of 30 ng/
`kg, mean IL-6 levels were 500 pg/mL 4 h after IL-1 (baseline ⬍ 50 pg/mL)
`and 8000 pg/mL after a dose of 300 ng/kg. In another study, infusion of 30
`ng/kg of IL-1α induced elevated IL-6 levels within 2 h (20). These elevations
`in IL-6 are associated with a rise in C-reactive protein and a decrease in al-
`bumin.
`
`Copyright 2001 by Marcel Dekker, Inc. All Rights Reserved.
`
`Lassen - Exhibit 1031, p. 18
`
`

`

`BLOCKING IL-1 IN PATIENTS WITH RHEUMATOID ARTHRITIS
`WITH THE IL-1 RECEPTOR ANTAGONIST
`
`The basic concept of inhibition of ICE to reduce the processing and secretion
`of IL-1β in disease states received a great deal of support following the publi-
`cation of clinical trials of IL-1 receptor antagonist (IL-1Ra) in patients with
`rheumatoid arthritis and graft versus host disease. Also, the importance of
`endogenous IL-1Ra in patients with rheumatoid arthritis is supported by a
`study using the administration of soluble IL-1R type I to these patients. Since
`the soluble form of IL-1R type I binds IL-1Ra with a greater and near irrevers-
`ible affinity than that of IL-1α or IL-1β, the use of the soluble IL-1R type in
`humans worsened disease in these patients (21). Although IL-1Ra was used
`in three trials to reduce 28-day mortality in sepsis, the overall success of any
`anticytokine-based therapy in this patient population precludes any conclusion
`whether the anticytokine is effective (22–24). In each of these three trials,
`there was clear evidence of improved outcome in subgroups treated with IL-
`1Ra compared to placebo-treated patients. However, in each trial, the entire
`group did not reach a statistically significant reduction in mortality. On the
`other hand, the use of IL-1Ra is patients with rheumatoid arthritis and graft
`versus host disease provides convincing evidence that blocking IL-1 reduces
`disease and, moreover, blocking IL-1 is safe. Of course, inhibition of IL-1β
`is not the sole effect of ICE inhibition, and the lack of specificity of this
`strategy in humans may yield different results when it is used in the same
`diseases.
`
`IL-1Ra in Patients with Rheumatoid Arthritis
`
`IL-1Ra was initial