Use of an IL-1 antagonist for treating arthritis

[0001] This application is a continuation in part of U.S. Ser. No. 10/282,162 filed 28 Oct. 2002, which is hereby incorporated by reference into this application in its entirety.

[0002] The invention relates to methods of treating arthritis, including rheumatoid arthritis and osteoarthritis, with a an interleukin-1 (IL-1) antagonist.

[0003] In a first aspect, the invention features a method of treating, inhibiting, or ameliorating arthritis, comprising administering to a subject in need an interleukin 1 (IL-1) antagonist. An IL-1 antagonist is a compound capable of blocking or inhibiting the biological action of IL-1, including fusion proteins capable of trapping and blocking IL-1. In a preferred embodiment, the IL-1-specific fusion protein comprising two IL-1 receptor components and a multimerizing component, for example, an IL-1-specific fusion protein described in U.S. Pat. No. 6,472,179 and U.S. patent publication No. 2003/0143697, published 31 Jul. 2003, herein specifically incorporated by reference in their entirety. An IL-1-specific fusion protein comprises an IL-1 binding portion of the extracellular domain of human II-1RAcP, an IL-1 binding portion of the extracellular domain of human IL-1RI, and a multimerizing component. In a specific embodiment, the IL-1-specific fusion protein is the fusion protein shown in SEQ ID NO:4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26. In one preferred embodiment, the IL-1-specific fusion protein is SEQ ID NO:10. The invention encompasses the use of an IL-1-specific fusion protein substantially identical to the protein of SEQ ID NO:4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, or 26, that is, a protein having at least 95% identity, at least 97% identity, at least 98% identity to the protein of SEQ ID NO:4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, or 26 and capable of binding and inhibiting IL-1. Further, in specific embodiments, the IL-1 antagonist is a modified IL-1-specific fusion protein comprising one or more receptor components and one or more immunoglobulin-derived components specific for IL-1 and/or an IL-1 receptor. In another embodiment, the IL-1 antagonist is a modified IL-1-specific fusion protein comprising one or more immunoglobulin-derived components specific for IL-1 and/or an IL-1 receptor.

[0004] The subject being treated is most preferably a human diagnosed as suffering from arthritis, including, but not limited to, rheumatoid arthritis, osteoarthritis, psoriatic arthritis, ankylosing spondylitis, and juvenile rheumatoid arthritis, and other inflammatory arthritides, as well as other arthritidies in which inflammatory mediators plays a role. More specifically, the subject is a human adult or child diagnosed with arthritis. Methods for diagnosing the presence of arthritis are known in the art.

[0005] In a second aspect, the invention features a method of treating, inhibiting, or ameliorating osteoarthritis, comprising administering to a subject in need an interleukin 1 (IL-1) antagonist. The IL-1 antagonist useful in the methods of the invention is described above. The subject being treated is most preferably a human diagnosed as suffering from osteoarthritis.

[0006] In a third aspect, the invention features a therapeutic method of treating rheumatoid arthritis, comprising administering a pharmaceutical composition comprising an IL-1-specific fusion protein and a pharmaceutically acceptable carrier.

[0007] In a fifth aspect, the invention features a therapeutic method of treating psoriatic arthritis, comprising administering a pharmaceutical composition comprising an IL-1-specific fusion protein and a pharmaceutically acceptable carrier.

[0008] In a sixth aspect, the invention features a therapeutic method of treating ankylosing spondylitis, comprising administering a pharmaceutical composition comprising an IL-1-specific fusion protein and a pharmaceutically acceptable carrier.

[0009] In a seventh aspect, the invention features a therapeutic method of treating juvenile rheumatoid arthritis, comprising administering a pharmaceutical composition comprising an IL-1-specific fusion protein and a pharmaceutically acceptable carrier.

[0010] The method of the invention includes administration of the IL-1 antagonist by any means known to the art, for example, subcutaneous, intramuscular, intranasal, intraarticular, intravenous, topical, transdermal administration or oral routes of administration. Preferably, administration is by subcutaneous, intraarticular, or intravenous injection or infusion.

[0011] In specific embodiments of the therapeutic method of the invention, the subject is treated with a combination of an IL-1-specific fusion protein and a second therapeutic agent. The second therapeutic agent may be a second IL-1 antagonist such as a chimeric, humanized or human antibody to IL-1α or β (such as CDP484, Celltech) or to the IL-1 receptor (for example, AMG-108, Amgen; R-1599, Roche), IL-1Ra (anakinra, Amgen; IL-1ra gene therapy, Orthogen), and ICE inhibitor, such as Vx-765 (Vertex), p38 MAP inhibitors, IKK ½ inhibitors (such as, UK436303, Pfizer; SPC-839, Serono/Signal), collagenase inhibitors (Periostat™, Collagenex), etc. The second therapeutic agent may also be selected from an anti-IL-18 compound, such as IL-18BP or a derivative, an. IL-18-specific fusion protein (trap), anti-IL-18, anti-IL-18R1, or anti-IL-18Rβ. Other co-therapies include low dose colchicine for FMF, aspirin or other NSAIDs, steroids such as prednisone, prednisolone, Depo-Medrol™ and Kenalog™; and other disease modifying anti-rheumatic drugs (DMARDs) such as methotrexate; low dose cyclosporine A, TNF inhibitors such as Enbrel®, or Humira®, other inflammatory inhibitors such as inhibitors of caspase-1, p38, IKK½, CTLA-4Ig, anti-IL-6 or anti-IL6Ra; and hyaluronic derivates such as Hyalgan™, Synvisc™, Orthovisc™, and Supartz™.

[0012] Other objects and advantages will become apparent from a review of the ensuing detailed description.

[0013] FIG. 1: Human IL-1-specific fusion protein blocks the effects of IL-1 in inflamed joints.

[0014] FIGS. 2-3: Murine IL-1-specific fusion protein reduces the severity of arthritis symptoms in a Zymosan-Accelerated Collagen-Induced Arthritis (CIA) model.

[0015] FIG. 4: Various concentrations of IL-1-specific fusion protein 1649 were incubated in the presence of 5 pM human IL-1b overnight at room temperature.

[0016] Before the present methods are described, it is to be understood that this invention is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0017] As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus for example, a reference to “a method” includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0018] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference in their entirety.

Rheumatoid Arthritis and Osteoarthritis

[0019] Rheumatoid arthritis (RA) is a chronic systemic disease characterized by progressive joint deformity and joint destruction in which cytokines play a central pathogenic role. The clinical course of RA is variable and often shows a remitting pattern. Three forms of RA can be distinguished: mild, self-limiting disease; mildly progressive disease; and aggressive disease which is difficult to control with medication, and is characterized by functional decline and radiologic deterioration of the joints, e.g., joint space narrowing and erosions. In accordance with the systemic nature of the disease, there are extra-articular manifestations which include vasculitis, alveolitis, and ocular disease. Prevalence of the disease as reported in the literature is approximately 1% of the U.S. population, with women accounting for two-thirds of all cases. The disease affects mainly adults but there is a juvenile form of rheumatoid arthritis (Chikanza et al (1998) J Pharm Pharmacol 50:357-69).

[0020] Onset of RA is often insidious with fatigue, anorexia, generalized weakness, and vague musculoskeletal symptoms. Specific symptoms appear later. Several joints, usually in a symmetrical fashion, are affected. Most often these are joints of the hands, wrists, knees, and feet. Joints are painful and swollen, and motion is limited. Morning stiffness of more than one hour is a very typical finding. With persistent inflammation, a variety of deformities develop which include most typically radial deviation of the wrist and hyperextension or flexion of the proximal interphalangeal joints; other deformities occur as well. Atrophy of skeletal muscle sets in. In approximately 20 to 30% of all patients, there is development of rheumatoid nodules on periarticular structures or sites of trauma, but they are usually of limited clinical significance. The nodules may be found in other structures such as the pleura or the meninges. Rheumatoid vasculitis can affect nearly all organ systems (lung, GI-tract, liver, spleen, pancreas, lymph nodes, testis, and the eye). Osteoporosis is common and may be aggravated by corticosteroids used in therapy (Lipsky (1998) Harrison's Textbook of Medicine 14^th Ed. pp.1880-8).

[0021] Laboratory findings may include elevation of erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) along with rheumatoid factor. Rheumatoid factor is an autoantibody against the Fc portion of IgG found in more than two-thirds of all patients. High titers of rheumatoid factor are a good indicator of disease activity. Mild anemia (normochromic, normocytic) and eosinophilia may be present as well. With progression of the disease, X-ray abnormalities such as general deformity, juxta-articular osteopenia, loss of articular cartilage, and bone erosion become more evident.

[0022] There is no curative treatment for RA. All drug regimens primarily attempt to relieve the symptoms and the inflammation. Aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) with a rapid onset of action are the first line of treatment. Selective COX-2 inhibitors such as Celebrex® and Vioxx® have been found to be better tolerated than regular NSAIDS which act both on COX-1 and COX-2. Oral glucocorticoids are added to the drug regimen if necessary. The third line of treatment includes disease modifying antirheumatic drugs (DMARDs); they have a slow onset of action, in some cases several months. DMARDs include azathioprine, sulphasalazine, gold, D-penicillamine, hydroxychloroquine, methotrexate, and cyclosporine. The most recent addition of etanercept/Enbrel® (chimeric TNF-receptor fusion protein) to the therapeutic armamentarium appears to be a successful step to improve patient treatment in a rational way. A second drug, infliximab/Remicade® (monoclonal anti-TNF antibody) has been approved for treatment of RA in combination with methotrexate. A third, IL-1ra (Antril®), a recombinant version of the naturally occurring IL-1 receptor antagonist, has been reported to have clinical benefit, as well (Bresnihan et al. (1 998) Arthritis & Rheumatism 41:2196-2204; Campion et al. (1996) Arthritis & Rheumatism 39:1092-1101; Cohen et al. (1999) Arthritis & Rheumatism Abstracts 42(Supp):S273).

[0023] Osteoarthritis is the most common form of arthritis in Western populations (Jordan et al. (2003) Ann Rheum Dis. 62(12):1145-55). Knee OA, characterized clinically by pain and functional disability, is the leading cause of chronic disability among the elderly in the US. Risk factors for OA include age, gender, race, trauma, repetitive stress/joint overload, muscle weakness, and genetic factors.

[0024] Pathologically, the most striking changes in OA are focal loss of articular cartilage and marginal and central new bone formation. However, OA is not simply a disease of articular cartilage and the subchondral bone. Rather, it is a disease of the synovial joint, with alterations also found in the synovium, capsule, ligaments, periarticular muscle, and sensory nerves.

[0025] Although OA was once considered a non-inflammatory arthropathy, patients often present with signs and symptoms consistent with local inflammation and synovitis, and recent evidence from preclinical and clinical studies supports the role of inflammation and inflammatory mediators in its pathophysiology (Pelletier et al. (2001) Arthritis Rheum 44(6):1237-47). Both chondrocytes and synovium in OA can produce proinflammatory cytokines, including IL-1β, which can alter cartilage homeostasis in favor of cartilage degradation. For example, IL-1β appears to be a major factor stimulating matrix metalloproteinase synthesis and other cartilage catabolic responses in OA. Thus, inflammation and inflammatory mediators may play a role in the joint destruction associated with OA as well as in pain.

[0026] Current treatment of osteoarthritis includes non-medicinal therapy, medicinal therapy, and surgical treatments. Non-medicinal treatments include exercise, thermal treatment, and assistive devices or bracing. For knee OA, range-of-motion and strengthening exercises are geared toward reduction of impairment, improvement of function, and joint protection. Medications include analgesics (e.g., acetaminophen), non-steroidal anti-inflammatory drugs (NSAIDS) that are either non-selective cyclooxygenase (COX) inhibitors or selective inhibitors of the COX-2 enzyme, injected intra-articular corticosteroids or viscosupplementation, and proven or putative disease-modifying osteoarthritis drugs (DMOADs). Surgical procedures include joint debridement and lavage, and lastly total knee arthroplasty.

[0027] The most commonly used medicinal treatments for knee OA typically provide less than 50% relief of pain. For example, use of acetaminophen, selective NSAIDs or non-selective NSAIDs typically results in mean improvements in knee OA pain of no more than 30 points from a baseline of ˜70 points using 100 point (100-mm) visual analog scales (Kivitz et al (2002) J Fam Pract 51(6):530-537). While this is a clinically important improvement, this indicates that there is substantial room for improvement in the pain management of knee OA. Further, no therapy has been demonstrated to retard the progression of structural degradation.

[0028] The pain and structural alterations of osteoarthritis are associated with inflammation and with alterations in inflammatory mediators, including IL-1. Hence, there is potential utility for agents that diminish the action of IL-1 in the treatment of both OA pain and OA disease (structural) modification. Indeed, a small, uncontrolled clinical study of intra-articularly administered IL-1 receptor antagonist, IL-1ra (anakinra), in knee osteoarthritis demonstrated a prolonged reduction in knee pain, supporting the potential of IL-1 inhibition as a therapeutic approach in treating OA (Goupille et al. (2003) Arthritis Rheum 48(suppl):S696). Although IL-1-specific fusion protein has not previously been studied in osteoarthritis, it has been shown to have anti-inflammatory activity associated with clinical effect in both animal models and humans in clinical trials.

Definitions

[0029] By the term “blocker”, “inhibitor”, or “antagonist” is meant a substance that retards or prevents a chemical or physiological reaction or response. Common blockers or inhibitors include but are not limited to antisense molecules, antibodies, antagonists and their derivatives. More specifically, an example of an IL-1 blocker or inhibitor is an IL-1 antagonist including, but not limited to, IL-1-specific fusion protein, which binds and inhibits IL-1.

[0030] By the term “therapeutically effective dose” is meant a dose that produces the desired effect for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0031] By the term “substantially identical” is meant a protein sequence having at least 95% identity to an amino acid sequence selected from the group consisting of the amino acid sequences SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26, and capable of binding IL-1 and inhibiting the biological activity of IL-1.

[0032] The term “identity” or “homology” is construed to mean the percentage of amino acid residues in the candidate sequence that are identical with the residue of a corresponding sequence to which it is compared, after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent identity for the entire sequence, and not considering any conservative substitutions as part of the sequence identity. Neither N- or C-terminal extensions nor insertions will be construed as reducing identity or homology. Methods and computer programs for the alignment are well known in the art. Sequence identity may be measured using sequence analysis software (e.g., Sequence Analysis Software Package, Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Ave., Madison, Wis. 53705). This software matches similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications.

IL-1-Specific Fusion Protein Antagonists

[0033] IL-1-specific fusion proteins (sometimes referred to as “IL-1 traps”) are multimers of fusion proteins containing IL-1 receptor components and a multimerizing component capable of interacting with the multimerizing component present in another fusion protein to form a higher order structure, such as a dimer. Cytokine traps include two distinct receptor components that bind a single cytokine, resulting in the generation of antagonists with dramatically increased affinity over that offered by single component reagents. In fact, the cytokine traps that are described herein are among the most potent cytokine blockers ever described. Briefly, the cytokine traps called IL-1 traps are comprised of the extracellular domain of human IL-1R Type I (IL-1RI) or Type II (IL-1RII) followed by the extracellular domain of human IL-1 Accessory protein (II-1RAcP), followed by a multimerizing component. In one embodiment, the multimerizing component is an immunoglobulin-derived domain, such as, for example, the Fc region of human IgG, including part of the hinge region, the CH2 and CH3 domains. An immunoglobulin-derived domain may be selected from any of the major classes of immunoglobulins, including IgA, IgD, IgE, IgG and IgM, and any subclass or isotype, e.g. IgG1, IgG2, IgG3 and IgG4; IgA-1 and IgA-2. For a more detailed description of the IL-1 traps, see WO 00/18932, which publication is herein specifically incorporated by reference in its entirety. Preferred IL-1-specific fusion proteins have the amino acid sequence shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26, or a substantially identical protein at least 95% identity to a sequence of SEQ ID NO:4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26, and capable of binding and inhibiting IL-1.

[0034] In specific embodiments, the IL-1 antagonist comprises an antibody fragment capable of binding IL-1α, IL-1β, IL-1R1 and/or II-1RAcP, or a fragment thereof. The preferred embodiment would be an antagonist of IL-1β. One embodiment of an IL-1 antagonist comprising one or more antibody fragments, for example, single chain Fv (scFv), is described in U.S. Pat. No. 6,472,179, which publication is herein specifically incorporated by reference in its entirety. In all of the IL-1 antagonist embodiments comprising one or more antibody-derived components specific for IL-1 or an IL-1 receptor, the components may be arranged in a variety of configurations, e.g., a IL-1 receptor component(s)-scFv(s)-multimerizing component; IL-1 receptor component(s)-multimerizing component-scFv(s); scFv(s)-IL-1 receptor component(s)-multimerizing component, ScFv-ScFv-Fc, etc., so long as the molecule or multimer is capable of inhibiting the biological activity of IL-1.

Combination Therapies

[0035] In numerous embodiments, the IL-1 antagonists of the present invention may be administered in combination with one or more additional compounds or therapies. Combination therapy may be simultaneous or sequential. The IL-1-specific fusion proteins of the invention may be combined with, for example, a chimeric, humanized or human antibody to IL-1α or β (such as CDP-484, Celltech) or to the IL-1 receptor (for example, AMG-108, Amgen; R-1599, Roche), IL-1Ra (anakinra, Amgen; IL-1ra gene therapy, Orthogen), and ICE inhibitor, such as Vx-765 (Vertex), p38 MAP inhibitors, IKK ½ inhibitors (such as, UK-436303, Pfizer; SPC-839, Serono/Signal), collagenase inhibitors (Periostat™, Collagenex), etc. The second therapeutic agent may also be selected from an anti-IL-18 compound, such as IL-18BP or a derivative, an IL-18 trap, anti-IL-18, anti-IL-18R1, or anti-IL-18RAcP. Other co-therapies include low dose colchicine for FMF, aspirin or other NSAIDs, steroids such as prednisone, prednisolone, and other disease modifying anti-rheumatic drugs (DMARDs) such as methotrexate; low dose cyclosporine A, TNF inhibitors such as Enbrel®, or Humira®), other inflammatory inhibitors such as inhibitors of caspase-1, p38, IKK½, CTLA-4Ig, anti-IL-6 or anti-IL6Ra; and hyaluronic derivates such as Hyalgan™ or Synvisc™.

Pharmaceutical Compositions

[0036] The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of an active agent, and a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly, in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin.

[0037] In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[0038] The active agents of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

[0039] Other features of the invention will become apparent in the course of the following descriptions of exemplary embodiments which are given for illustration of the invention and are not intended to be limiting thereof.

[0040] The following example is put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

[0041] Zymosan is a yeast cell wall extract that when injected into the knee causes acute inflammation and upregulation of IL-1β in the joint (Joosten et al. (1994) supra). Chondrocytes will respond to the inflammation and local IL-1β by down regulating proteoglycan synthesis, a feature of human arthritis that contributes to the gradual destruction of cartilage in the joint (van den Berg W B et al. (1982) Rheum Intl 1:165-169). Antagonists to IL-1β can be used to evaluate their ability to block the effects of zymosan-induced elevations in IL-1β.

[0042] Anesthetized male C57BL/6 mice (Taconic) were given an intra-articular (i.a.) injection of Zymosan A (Sigma; 300 μg in 10 μl) into the right knee joint through the patellar ligament. Sterile PBS was injected i.a. (10 μl) into the left knee joint through the patellar ligament. Twenty-four hours prior to i.a. injections, animals were treated with either vehicle or hIL-1 trap 569 (SEQ ID NO:1-2) (19 mg/kg, s.c.). The patellae were removed 24 h after injection of zymosan in order to measure proteoglycan synthesis as described by van den Berg and colleagues (1982). Briefly, each patella and associated ligament were incubated for 3 h at 37° C., 5% CO₂ in media (RPMI with HEPES, HCO₃, glutamine & penicillin/streptomycin) containing 10 μCi/ml ³⁵S-sulfate (NEN DuPont). Following incubation, tissue was washed and fixed overnight in 10% formalin (VWR). The tissue was then placed in Decalcifing Solution (J. T. Baker) for 4 h prior to dissection of the patella from surrounding tissue. Each patella was then incubated overnight in Solvable (Packard) at 50° C. Ultima Gold liquid scintillation fluid (Packard) was added and the samples were counted in a liquid scintillation counter. Values were reported as the ratio of cpm of zymosan patella/cpm of vehicle patella for each animal.

[0043] Intra-articular injection of zymosan reduces proteoglycan synthesis by approximately 50% relative to vehicle injection (FIG. 1). Administration of hIL-1-trap prior to zymosan injection blocked the local action of IL-1β and proteoglycan synthesis returned to approximately 90% of control. These data demonstrate that hIL-1 trap 569 can penetrate the joints after subcutaneous injection to effectively neutralize the biological effect of IL-1 within these joints.

[0044] IL-1 has been implicated in the development of inflammation and cartilage destruction in rheumatoid arthritis (Dinarello (1996) Blood 87(6):2095-2147; Wooley et al. (1993) Arthritis & Rheumatism 36(9): 1305-1314). Collagen-induced arthritis (CIA) is a widely studied animal model of inflammatory polyarthritis with similarities to rheumatoid arthritis; common histopathological features include joint inflammation and erosion, synovial hyperplasia and inflammatory cell infiltration (Joe et al. (1999) Mol Med Today 5:367-369). Since previous studies have shown that various anti-IL-1 treatments have a positive effect on reducing arthritis symptoms in CIA animals (van den Berg et al. (1994) Clin Exp Immunol 95:237-243; Joosten et al. (1999) J Immunol 163:5049-5055.; van de Loo et al. (1992) J Rheumatol 19:348-356.), Applicants examined the effect of a murine version of the IL-1-specific fusion protein (mIL-1 trap) on the progression of arthritis symptoms in this animal model. The mIL-1 trap consists of the extracellular domain of murine II-1RAcP, followed by the extracellular domain of murine IL-1RI, followed by the hinge, CH2 and CH3 domain of murine IgG2a. The human version of the IL-1 trap is poorly cross-reactive with rodent IL-1.

[0045] Male DBA-1 mice (Jackson Laboratories) were immunized intradermally at the base of the tail with 100 μg/50 μl bovine Type II collagen (CII; Chondrex) emulsified with complete and incomplete Freund's adjuvant (2:1:1 ratio; Chondrex) and boosted intradermally with CII (100 μg/50 μl) emulsified with incomplete Freund's adjuvant on day 21. Since CIA in DBA-1 mice occurs gradually over a long time period with a low incidence (Joosten et al. (1994) supra). Applicants synchronized the onset of arthritis symptoms by injecting the animals intraperitoneally on day 30 with 3 mg zymosan (Sigma). Two hours prior to zymosan injection, the mice were randomly distributed into treatment groups and were injected with either vehicle or mIL-1 trap (31 or 10 mg/kg, 3×/week, 8 injections, s.c.). Arthritis symptoms (ASI scores, as described by Wooley et al. (1993) Arthritis & Rheumatism 36(9): 1305-1314) in the paws were evaluated 3×/week by individuals who were blinded to the treatment groups. Animals were sacrificed 24 h after the 8th injection at which time paw width along with ASI scores were measured.

[0046] Within 5 days after i.p injection of zymosan, vehicle treated animals had an significant increase in ASI score relative to those receiving mIL-1 trap (FIG. 2) with symptoms reaching a maximum 10 to 14 days after zymosan injection. Murine IL-1 trap acted in a dose-dependent fashion such that animals receiving 10 mg/kg trap had more arthritis symptoms (greater ASI score) than those receiving 31 mg/kg. However, both mIL-1 trap-treated groups had a significantly lower degree of arthritis symptoms than vehicle. This difference in ASI score is also reflected in the paw width at the time of sacrifice (FIG. 3). Animals receiving mIL-1 trap had paw widths that were similar to those of naive, non-collagen immunized animals. These data indicate that mIL-1 trap can effectively neutralize IL-1 and block the development of arthritic joints.

[0047] A Phase 1, placebo controlled, randomized, double-blind, combined single and multiple dose, dose-escalation study was conducted in subjects with rheumatoid arthritis (RA). IL-1-specific fusion protein was dosed subcutaneously. In the single-dose part, subjects in a given dose cohort received either a sing subcutaneous dose of IL-1-specific fusion protein (SEQ ID NO:10) (50, 100, 200, 400 or 800 mg/kg) or placebo. In the multi-dose phase, patients in a given cohort received six weekly doses of IL-1-specific fusion protein (50, 100, 200, 400 or 800 mg/kg) or placebo. The terminal half-life of IL-1-specific fusion protein ranged from 5.3 to 8.9 days and clearance ranged from 0.49 to 1.00 mL/hr/kg. The AUC and Cmax were dose dependent and approximately linear. No subject developed antibodies against the IL-1-specific fusion protein.

[0048] Exploratory efficacy assessments were also obtained in the multiple dose phase of the study. Swollen and tender joint counts and serum CRP levels were improved with 800 mg/kg of the IL-1-specific fusion protein dose compared to placebo.

[0049] A Phase 2 dose range-finding study evaluated 12 weekly fixed doses of the IL-1-specific fusion protein (25, 50 or 100 mg administered subcutaneously) and placebo in RA subjects. The safety analysis indicated the drug to be well tolerated with no evidence of dose-limiting toxicities.

[0050] Unaudited efficacy results indicate that a greater proportion of subjects treated with 100 mg of the IL-1-specific fusion protein achieved American College of Rheumatology (ACR) 20, 50 and 70 scores compared with placebo, although these results were not statistically significant. Subjects on the 100 mg dose of the IL-1-specific fusion protein demonstrated significant improvement (p<0.05) relative to placebo for (1) time to onset of ACR20 and ACR50 responses, ACR-N, AUC of ACR-N, DAS28, AUC of DAS28, and ESR levels.

[0051] A study is conducted to assess the efficacy of systemic treatment (including intravenous and subcutaneous administration) with doses of up to 2000 mg IV of the IL-1-specific fusion protein (SEQ ID NO:10) versus placebo for reduction of knee pain, functional disability, and joint stiffness in subjects with osteoarthritis of the knee.

[0052] Endpoints. The primary efficacy endpoint is the change in Patient's Assessment of Arthritis Pain—visual analogue scale (VAS) score (PAAP-VAS; 100-mm scale) from Baseline to Week 4. Additional efficacy endpoints are evaluated at each time point (compared to Baseline) including: categorical reduction in Patient's Assessment of Arthritis Pain −VAS score by at least 20% (responder); change of the WOMAC™Osteoarthritis Index pain, disability, and joint stiffness dimensions; change in each of the items in the WOMAC™ Osteoarthritis Index; change in Patient's Global Assessment of Arthritis Condition; change in Physician's Global Assessment of Arthritis Condition; amount of rescue medication (acetaminophen) used; and changes in potential biomarkers of OA disease activity.

[0053] Study Design. This is a randomized, double-blind, parallel group, 12-week trial of the IL-1-specific fusion protein vs. placebo in subjects with osteoarthritis of the knee. After screening, eligible patients are washed-out of any NSAIDs taken, and are required to flare to be eligible for randomization. If NSAIDs are not being taken at Screening, patients demonstrate a clinical response to a NSAID within the previous 6 months and have discontinued treatment for reasons other than lack of efficacy. Approximately 160 subjects at approximately 12 sites are randomly allocated in a 1:1 ratio to receive single IV doses of up to 2000 mg IL-1-specific fusion protein or placebo. Subjects are then followed and assessed for efficacy, safety, and tolerability at Weeks 2, 4, 8, and 12. The primary endpoint of pain as measured by PMP-VAS will be at Week 4.

[0054] Osteoarthritis Disease Biomarkers. Serum and urine are obtained periodically during the study for possible examination of biomarkers of osteoarthritis disease activity. The urine sample must be from the first (preferable) or second morning void. Instructions and materials for collection will be provided with the laboratory kits provided by the central laboratory. Biomarkers to be examined will potentially include (but not necessarily be limited to) CTX-II (C-terminal crosslinking telopeptide of collagen type II, typically measured in urine) and COMP (cartilage oligomeric matrix protein, typically measured in serum).

[0055] Patient's Assessment of Arthritis Pain—Visual Analog Scale. The Patient's Assessment of Arthritis Pain—Visual Analog Scale (PAAP-VAS) (below) is a self-administered instrument to be used at each study visit to quantify pain in each knee. The question is to be answered using a 100 mm visual analog scale, considering the patient's status during the 24-hour period leading up to and including the time of evaluation.

[0056] Physician's and Patient's Global Assessments of Arthritis Condition. Global assessments of arthritis condition (Example 7 below) are made by each of the physician and subject using 100 mm VAS scales, considering the subject's condition during the 24 hours leading up to and including the time of assessment. The physician's global assessment is made by the physician performing the focused exam of the knees, after completing this examination. Each of the physician's and patient's assessments is made without knowledge of the other's assessment score.

[0057] WOMAC™−VA Osteoarthritis Index. The WOMAC™-VA Index (Appendix K) is a self-administered questionnaire that asks 24 questions about the pain (5 questions), stiffness (2 questions), and functional limitations (17 questions) associated with arthritis. Each question is answered by making a mark along a 10.0 cm (100 mm) visual analog scale anchored by the terms “none” (left end) and “extreme” (right end). Questions are to be answered considering the patient's status during the 24-hour period leading up to and including the time of evaluation.

[0058] Knee pain+at least 1 of 3+osteophytes: (1) Age>50 years; (2) stiffness<30 minutes; (3) crepitus (Altman et al. (1986) Arthritis Rheum 29:1039-1049).

[0059] Grade 0 is defined as no OA severity with radiographic findings of no feature of OA. Grade I is defined as doubtful OA severity with minute osteophyte, with radiographic findings of doubtful significance. Grade II is defined as minimal OA severity with radiographic findings of moderate diminution of joint space. Grade II is defined as moderate OA severity with radiographic findings of moderate diminution of joint space. Grade IV is defined as severe OA with radiographic findings of joint space greatly impaired with sclerosis of subchondral bone (Kellgren et al. (1957) Ann Rhem Dis. 1957: 16: 494-501: and Department of Rheumatology and Medical Illustration, University of Manchester. The Epidemiology of Chronic Rheumatism. Atlas of Standard Radiographs of Arthritis. Philadelphia, Pa.: FA Davis Company: 1973: 1-15.

[0060] Patient's Assessment of Arthritis Pain (VAS). Patient makes a vertical mark through each of two lines which best describes the amount of pain due to arthritis in the right or left knee experienced in the last 24 hours. Range from no pain to most severe pain.

[0061] Physician's Global Assessment of Arthritis Condition. Physician is instructed to make a global assessment of the patient's disease status ranging from very good to very poor.

[0062] Patient's Global Assessment of Arthritis Condition. Patient is instructed to mark through a line ranging from very well to very poorly in response to the questions: “Considering all the ways your arthritis affects you, how are you doing today (and in the last 24 hours)?”

[0063] An initial 12-week, randomized, double blind, placebo-controlled, repeated dose, dose-escalation, safety and efficacy study is conducted with up to 60 subjects diagnosed as suffering from osteoarthritis of the knee. The subjects are randomly assigned to one of four cohorts and are treated with a single initial dose of 22 mg, 66 mg, 220 mg, 320 mg of IL-1-specific fusion protein or placebo, followed by a second dose at week 8. Observations are obtained periodically during the course of the 12-week study as described above.

[0064] A second 24-week, double blind, placebo-controlled, proof-of-concept, safety and efficacy study is conducted with about 160 patients diagnosed with osteoarthritis of the knee. Patients are randomly assigned to treatment with placebo or with the maximum tolerated dose (determined as described above) of the IL-1-specific fusion protein (SEQ ID NO:10). The primary efficacy endpoint is the change in PAAP-VAS at week 12. After week 12, but prior to week 20, contingent upon recurrence of symptoms (i.e., index knee pain), patients receive a second dose of study drug. Analyses of efficacy are based on efficacy outcome parameters as described above, and include analysis of dosing interval (“time to failure”) for the IL-1-specific fusion protein relative to placebo.

[0065] A 12-week, double blind, placebo-controlled, single dose, dose-ranging, safety and efficacy study is conducted with about 320 patients diagnosed with osteoarthritis of the knee. Patients are randomly assigned to one of four treatment groups: placebo or one of 3 doses of the IL-1-specific fusion protein. The primary efficacy endpoint is the change in PMP-VAS at week 12. Analyses of efficacy are based on efficacy outcome parameters as indicated above.

[0066] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.