Inflammation, Rheumatoid Arthritis and Cytokines

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1 Inflammation, Rheumatoid Arthritis and Cytokines Rheumatoid arthritis (RA) is the commonest inflammatory joint disease with considerable morbidity and mortality. Conventional disease-modifying antirheumatic drugs like methotrexate form the cornerstone of therapy. However, they have several limitations in terms of slow onset of action, adverse effects and modest remission and retention rates. Several cytokines are involved in the pathogenesis of RA. Biological agents that specifically inhibit the effects of tumour necrosis factor-α (TNF-α) or Interleukin-1 (IL-1) represent a major advancement in the treatment of RA. By targeting molecules that are directly involved in the pathogenesis of RA, these therapies are proving to be efficacious, highly specific and better tolerated than standard therapies. The use of these agents needs to be monitored carefully for possible sideeffects, including the development of infections. Additional anticytokine agents for the treatment of RA are under further development and are the basis for this Lecture.

2 Rheumatoid Arthritis (RA) Common human autoimmune disease with a prevalence of about 1% Chronic inflammation of the synovial joints and infiltration by blood-derived cells Progressive destruction of cartilage and bone - invasion by cellular synovial tissue - cytokine induction of destructive enzymes, chiefly matrix metalloproteinases (MMP)

3 Prognosis of RA Long-term prognosis: poor - 80% of patients are disabled after 20 years - life expectancy is reduced by 3-18 years Disease modifying anti-rheumatic drug (DMARD) - limited efficacy and many side effects - do not improve long-term prognosis Efforts to develop safer and more effective treatment

4 Direct Costs of RA Other expenses 7.5% Hospital admissions 51.7% Testing 4.7% Drugs 26.1% AHP visits 1.4% Physician visits 8.6% AHP = allied health professional. Yelin E, Wanke LA. Arthritis Rheum. 1999;42:

5 Clinical Course of RA 4 2 Type 1 Type 2 Type Type 1 = Self-limited 5% to 20% Type 2 = Minimally progressive 5% to 20% Type 3 = Progressive 60% to 90% Pincus. Rheum Dis Clin North Am. 1995;21:619.

6 RA Progression Severity (arbitrary units) Early RA Intermediate Late ACR Inflammation Disability Radiographs Duration of Disease (years) Graph: Adapted from Kirwan JR. J Rheumatol. 2001;28: Photo: Copyright American College of Rheumatology.

7 Critical Window for Treating RA Radiographic progression occurs early and continues over the lifetime of a patient 70% of patients have radiographic damage within the first 3 years Severe functional decline Disease onset Early Established End Stage Radiographic damage Work disability Critical window of opportunity Premature death O Dell JR. Arthritis Rheum. 2002;46: Editorial. Landewe RBM, et al. Arthritis Rheum. 2002;46:

8 An Overview of the Inflammation Process NOXIOUS STIMULI (Injury or Infection) INATE (immunologically non-specific) MICROSCOPIC LEVEL ADAPTIVE (immunologically specific) VASCULAR EVENTS CELLULAR EVENTS ANTIBODY MEDIATED RESPONSE CELL MEDIATED RESPONSE COAGULATION CASCADE KININ CASCADE FIBRINOLYTIC CASCADE COMPLEMENT CASCADE CELLS ALREADY PRESENT 1. Vascular endothelial 2. Mast 3. Tissue macrophages CELLS ENTERING THROUGH BLOOD 1. Platelets 2. Leukocytes INDUCTION PHASE EFFECTOR PHASE PRODUCTION OF NUMBEROUS CHEMICAL MEDIATORS AUTOCOIDS (Greek: autos self and akos medical agent or remedy PARACRINE SECRETIONS OR LOCAL HORMONES Neurotransmitters MACROSCOPIC LEVEL REDDENED HOT SWOLLEN PAINFUL INTERFERENCE Hormones INFLAMMATION CASCADE

9 Inflammatory Mediators Released by Mast Cell C5a C3a F C2 RI IgE Allergen Mast Cell GRANULE CONTENT RELEASE MEMBRANE- DERIVED LIPID MEDIATORS CYTOKINE PRODUCTION (Immediate) release of preformed mediators Lipid metabolism (over minutes) New mrna and protein synthesis (over hours) Histamine Proteases heparin Tumor Necrosis Factor (TNF) Prostaglandins Leukotrienes Platelet activation factor (PAF) Interleukins IL-1 IL-3 IL-4 IL-5 IL-6 IL-8 TNF The Mast Cells are uniquely placed at the sites of possible entry into the tissues of pathogen or noxious agent: near skin surfaces, close to the mucous membranes lining body cavities and around blood vessels. They are capable of secreting or generating an array of mediators which have the ability to modify vascular and cellular reactions as well as to affect some of the plasma factors.

10 Mast Cells in Rheumatoid Arthritis

11 Damage to Joints Caused by Inflammatory Cells, Cytokines, Matrix Metalloproteinases (MMPs)) Etc.

12 Schematic Comparison of Normal and Rheumatoid Joints

13 Cytokine Signaling Pathways in RA Plasma cell RF IL-4 IL-6 IL-10 IL-4 IL-10 Th0 Th2 Macrophage IFN-γ IL-12 Interferon-γ Synovium B cell OPGL CD69 CD4 + T cell CD11 CD11 CD69 TNF-α, IL-1 IL-6 Osteoclast Fibroblast Chondrocyte Production of metalloproteinases and other effector molecules Migration of polymorphonuclear cells Erosion of bone and cartilage Adapted from Choy EHS, Panayi GS. N Engl J Med. 2001;344: Copyright 2001 Massachusetts Medical Society. All rights reserved.

14 Cytokine Sources, Targets, Levels and Damage

15 Role of Cytokines in RA(2)

16 Role of Cytokines in RA (continued)

17 Joint Damage in RA Erosion of cartilage and bone proliferation of pannus activated macrophage and synovial fibroblasts express MMP (stromelysin, collagenase) IL-1, TNF- α, activated CD4+ T cells stimulate MMP release IL-1, TNF-α stimulate expression of adhesion molecules on endothelial cell recruitment of neutrophils into the joints neutrophils release elastase and proteases degrade proteoglycans in the superficial layer of cartilage

18 Strategies for Inhibition of Cytokine Action *Current Drugs Act at this Step

19 Strategies for Inhibition of Cytokine Action (continued) Current Drug Strategies Inflammatory cytokine Cytokine receptor Inflammatory signals Monoclonal antibody Receptor antagonist No signal Normal interaction Receptor blockade Neutralization of cytokines Activation of anti-inflammatory pathways Monoclonal antibody Soluble receptor No signal Anti-inflammatory cytokine Suppression of inflammatory cytokines Adapted with permission from Choy EHS, Panayi GS. N Engl J Med. 2001;344: Copyright 2001 Massachusetts Medical Society. All rights reserved.

20 Tumour Necrosis Factor-α (TNF-α) TNFα Identified in 1970s by Lloyd Old et al., as a serum factor that caused necrosis of some murine tumours. In the 1980s, studies into the role of TNFα intensified TNFα is a multifunctional pro-inflammatory mediator a) Induction of further cytokine production b) Activation or expression of adhesion molecules c) Growth stimulation TNFα action needs to be carefully controlled by the body Figure: TNFα

21 Mediates key roles in: - acute and chronic inflammation - antitumour responses - infection TNFα Continued Overproduction of TNF α is associated with a wide range of pathological conditions. Effort to find ways to down-regulate production or inhibit its effects. The TNFα knock in mouse model: Mice produce lots of TNFα Mice develop inflamed intestines Mice have a high concentration of Th1 cytokines (TNFα, IFNγ, Il-18, and Il-12) Antibodies to TNFα prevent or attenuate disease Antibodies also result in down-regulation of Th1 cells, cytokines and adhesion molecules TNFα TNFα knock in mouse

22 1. Membrane-bound TNFα (26kDa) 2. Soluble TNFα (17kDa) The Two Forms of TNFα TACE = TNFα Converting Enzyme 2 Extracellular 1 TACE Intracellular

23 The Two TNFα Receptors Extracellular TNFR-1 TNFR-2 Intracellular Share structural similarity in extra-cellular domain Intracellular domain differences Different signaling pathway

24 TNFR1 TNFR-1 TRADD Apoptosis kda TNF receptor type 1 (TNFR1) constitutively expressed in nearly all tissue Capable of inducing cell death through a death domain of the intracellular portion of the receptor Binds TNF, is internalized, recruits TRADD (a death domaincontaining signal transduction adapter molecule), which leads to DNA degradation and cell death (apoptosis).

25 TNFR2 Extracellular TNFR-2 Intracellular TRAF2 NFκB kda protein. No death domain cannot induce apoptosis Recruitment of TRAF2 leads to activation of the NFκB pathway, production of cytokines, inhibitors of apoptosis Differential expression of TNFR1 and TNFR2

26 TNFα Signal Transduction & Effector Mechanisms Extracellular TNFR-2 TNFR-1 Intracellular TRAF2 Apoptosis Nucleus IκB NFκB NFκB transcription

27 Transcription Products of TNFα Signaling: Pro-inflammatory TNFα Il-1β Upregulation of adhesion molecules (ICAM-1, VCAM-1) Cytokines that further enhance the immune response activators of inflammatory pathways (arachidonic acid metabolites, superoxides and nitric oxide) NFκB transcription Nucleus TNFα Il-1β

28 Potential Targets for the Inhibition of TNFα 1. TNFα molecule 2. TNFα receptors 3. TACE 4. Molecules in the TNFα signaling pathway 5. NFκB

29 Structure of an Antibody Variable region Constant region Three globular regions of the protein form a Y-shape Region of the protein at the tip of the arms is variable and the remainder of the protein is constant The two antigen-binding sites are at the tips of the arms Composed of two types of protein chain: heavy chains and light chains

30 Structure of Infliximab (Remicade ) Native (mouse) Antibody Chimeric Humanized (Primatized ) Infliximab - a chimeric antibody (25% mouse derived, 75% human protein) Human Protein Mouse Protein

31 Infliximab: : Mechanism of Action Binds and neutralizes both soluble and membrane bound TNFα - inhibits further activity = TNFα TNFR2 No Signal NFκB Nucleus transcription

32 Safety and Side Effects of Infliximab Use Most common: infusion reactions (itching, flushing, nausea), headache & abdominal pain. Increased risk of serious infection due to immunosuppression Upper respiratory tract infections (tuberculosis) Increased risk of non-hodgkins lymphoma Lupus Immunogenicity: patient develops HAMA (human anti-mouse antibodies) towards Infliximab

33 Immunogenicity of Infliximab Patients develop antibodies to Infliximab (a mouse protein) Human anti-mouse Ab 61% of patients had detectable antibodies after the fifth infusion of Infliximab (Baert et al., Incidence of the formation of antibodies to Infliximab is reduced with immunosuppressive therapy Human/mouse chimeric Ab (Infliximab)

34 Other Biological or Protein-Based DMARDs on the Market for RA Etanercept Enbrel Infliximab Remicade Anakinra Kineret Adalimumab Humira Class stnfr construct TNF-α mab IL-1Ra TNF-α mab Recombinant construct Human Chimeric Human Human Half-life 4.3 days 8 10 days 4 6 hours days Primary binding target TNF-α TNF-α Type I IL-1R TNF-α Administration 25 mg sc 2x/wk 3 10 mg/kg q4 8 wk iv + MTX 100 mg/d sc 40 mg sc eow* * Proposed dosing; eow = every other week. stnfr = soluble TNF-Receptor, mab = monoclono antibody, Ra = Receptor Antagonist Enbrel (etanercept) [package insert]. 2002; Remicade (infliximab) [package insert]. 2002; Kineret (anakinra) [package insert]. 2002; Salfeld J et al. Arthritis Rheum. 1998;41(suppl):S57.

35 Strategies for Inhibition of Cytokine Action (continued) Current Drug Strategies Inflammatory cytokine Cytokine receptor Inflammatory signals Normal interaction Receptor blockade Neutralization of cytokines Monoclonal antibody Remicade, Humira Soluble receptor Enbrel Activation of No signal anti-inflammatory pathways Monoclonal antibody Receptor antagonist Kineret No signal Adapted with permission from Choy EHS, Panayi GS. N Engl J Med. 2001;344: Copyright 2001 Massachusetts Medical Society. All rights reserved. Anti-inflammatory cytokine Suppression of inflammatory cytokines

36 IL-1-Receptor Antagonist (IL-1ra) Anakinra -Kineret Recombinant form of human IL-1ra phase II placebo-controlled trial patients with active RA - received daily, subcutaneous injections of placebo or one of three doses of IL-1ra: 30, 75, or 150 mg - moderate clinical improvement - retardation of radiological changes after 24 weeks of treatment Bresnihan B et al 1998 Arthritis Rheum 41:

37 Clinical Implications of Use of Anti- Cytokine Drugs

38 Health Economic Implications of Anti-Cytokine Drugs Due to production and development issues, these drugs are expensive to prescribe. The American College of Rheumatologists (ACR) estimates that treatment with Infliximab or Etanercept costs between US $25,000 $30,000 per patient per year. Therefore, it is not surprising that these drugs are only given to patients with severe disease. Trials are ongoing with these products in less serious cases of rheumatoid arthritis. They have been shown to inhibit the progressive destruction caused by disease which means better long term treatment. However, due to cost it is difficult to envisage that these drugs will ever become first line treatments, especially in Europe where medical provision tends to be funded from central government.

39 Emerging Themes in Our Understanding of RA Early diagnosis Early treatment + Disease control of signs and symptoms Damage prevention Maintain structural integrity Preserve function AND Quality of Life? = Remission

40 Early and Aggressive Intervention in RA: Why is it important? Damage occurs early and is progressive Damage and symptoms do not correlate Early intervention leads to better outcomes Aggressive therapy (combo) is better Early and aggressive therapy leads to the best outcomes

41 Small Molecule Approaches to Anti-TNF TNF-α Therapy Potential Advantages of Small-Molecule, Oral TNF-α Inhibitors: Convenient, non-injectable with greater patient compliance Small molecule might facilitate tissue penetration Possibility for once a day dosing Reduced immunosuppression required Non-immunogenic Easier manufacturing and lower cost Potential use in combination with other anti-inflammatory therapies.

42 Small Molecule Anti-TNF TNFα Agents in Development Class of Inhibitor Product Company Palladino et al., 2003 Clinical Status p38 Kinase BIRB796 Boehringer Ingelheim Phase 2 TACE BMS Bristol Myers Phase 2 Thalomid Thalidomide Celgene Phase 3 Rationally Designed L-amino acid peptide RDP58 Sangstat Medical Phase 2 Maskos et al. PNAS Vol. 95, Issue 7, , March 31, 1998 Crystal structure of the catalytic domain of human tumor necrosis factor- -converting enzyme

43 Other Uses for Anti-TNF TNFα * Conditions associated with overproduction of TNFα* Autoimmunity Infectious Agents Tumours Other Crohn s Disease HIV infection Angiogenesis Asthma Insulin-dependent diabetes mellitus Septic shock Ovarian cancer Graft Versus Host Disease (GVHD) Multiple Schlerosis Hepatitis C Lymphoma Glomerulonephritis Rheumatoid Arthritis Pancreatitis Marriot et al., 1997