WO2003063799A2 - Compositions et methodes d'inhibition systemique de la degradation du cartilage - Google Patents

Compositions et methodes d'inhibition systemique de la degradation du cartilage Download PDF

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Publication number
WO2003063799A2
WO2003063799A2 PCT/US2003/003175 US0303175W WO03063799A2 WO 2003063799 A2 WO2003063799 A2 WO 2003063799A2 US 0303175 W US0303175 W US 0303175W WO 03063799 A2 WO03063799 A2 WO 03063799A2
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WIPO (PCT)
Prior art keywords
cartilage
targeted
agents
chondroprotective
delivery system
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PCT/US2003/003175
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English (en)
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WO2003063799A3 (fr
Inventor
Gregory A. Demopulos
Pamela Pierce Palmer
Jeffrey M. Herz
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Omeros Corporation
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Priority to JP2003563495A priority Critical patent/JP2005519917A/ja
Priority to AU2003212898A priority patent/AU2003212898B2/en
Priority to MXPA04007124A priority patent/MXPA04007124A/es
Priority to EP03708941A priority patent/EP1496835A4/fr
Priority to KR10-2004-7011603A priority patent/KR20040094413A/ko
Priority to CA2474645A priority patent/CA2474645C/fr
Publication of WO2003063799A2 publication Critical patent/WO2003063799A2/fr
Publication of WO2003063799A3 publication Critical patent/WO2003063799A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
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    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
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    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
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    • A61K47/6935—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
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Definitions

  • the present invention relates to therapeutic compositions and methods for the protection of articular cartilage.
  • OA osteoarthritis
  • RA rheumatoid arthritis
  • OA osteoarthritis
  • hyaline articular cartilage The destruction of hyaline articular cartilage is the hallmark of OA and disabling RA. Although various therapeutic approaches may provide relief of symptoms, no therapeutic regimen has been proven to retard progression of articular cartilage degradation. The progressive deterioration and loss of articular cartilage leads to an irreversible impairment of joint motion. These changes in cartilage are the final pathogenic events that are common to osteoarthritis (OA) and rheumatoid arthritis (RA). Cartilage destructive processes may also be associated with or initiated by surgical procedures of the joint.
  • OA osteoarthritis
  • RA rheumatoid arthritis
  • Arthroscopy is a surgical procedure in which a camera, attached to a remote light source and video monitor, is inserted into an anatomic joint (e.g., knee, shoulder, etc.) through a small portal incision in the overlying skin and joint capsule. Through similar portal incisions, surgical instruments may be placed in the joint, their use guided by arthroscopic visualization.
  • anatomic joint e.g., knee, shoulder, etc.
  • surgical instruments may be placed in the joint, their use guided by arthroscopic visualization.
  • arthroscopists' skills have ' improved, an increasing number of operative procedures, once performed by "open" surgical technique, now can be accomplished arthroscopically. Such procedures include, for example, partial meniscectomies and ligament reconstructions in the knee, shoulder acromioplasties and rotator cuff debridements and elbow synovectomies.
  • wrist and ankle arthroscopies also have become routine.
  • physiologic irrigation fluid e.g., normal saline or lactated Ringer's
  • physiologic irrigation fluid e.g., normal saline or lactated Ringer's
  • isomolar solution of glycerol in water for a non-conductive and optically clear irrigation solution for arthroscopy.
  • Conventional physiologic irrigation fluids do not provide analgesic, anti-inflammatory or anti-cartilage degradation effects.
  • the present invention provides methods and compositions for reducing or preventing destruction of articular cartilage in a joint, by administering a combination of two or more metabolically active chondroprotective agents.
  • Metabolically active agents include, but are not limited to, compounds that act directly or indirectly to modulate or alter the biological, biochemical or biophysical state of a cell, including agents that alter the electrical potential of the plasma membrane, the ligand binding or enzymatic activity of cellular receptors, intracellular or extracellularly located enzymes, protein-protein interactions, RNA-protein interactions, or DNA-protein interactions.
  • pharmaceutical compositions of metabolically active chondroprotective agents are provided that are based upon a combination of at least two agents that act simultaneously on distinct molecular targets.
  • Representative chondroprotective agents include, for example: (1) antagonists of receptors for the interleukin-1 family of proteins, including, for example, IL-l ⁇ , IL-17 and IL-18; (2) antagonists of the tumor necrosis factor (TNF) receptor family, including, for example, TNF-R1; (3) agonists for interleukin 4, 10 and 13 receptors; (4) agonists for the TGF- ⁇ receptor superfamily, including, for example, BMP-2, BMP-4 and BMP-7; (5) inhibitors of COX-2; (6) inhibitors of the MAP kinase family, including, for example, p38 MAP kinase; (7) inhibitors of the matrix metalloproteinases (MMP) family of proteins, including, for example, MMP-3 and MMP-9; (8) inhibitors of the NF- ⁇ B family of proteins, including, for example, the p50/p65 dimer complex with I ⁇ B; (9) inhibitors of the nitric oxide synthase (NOS) family,
  • Additional chondroprotective agents include other growth factors, such as by way of example insulin-like growth factors (e.g., IGF-1) and fibroblast growth factors (e.g., bFGF).
  • IGF-1 insulin-like growth factors
  • fibroblast growth factors e.g., bFGF
  • at least one agent is a cytokine or growth factor receptor agonist that directly provides anti-inflammatory activity and/or promotes cartilage anabolic processes, also referenced herein as an "anabolic agent”
  • at least a second agent is a receptor antagonist or enzyme inhibitor that acts to inhibit cartilage catabolic processes and that may also inhibit pro-inflammatory processes, also referenced herein as an "inhibitor of cartilage catabolism” or “catabolic inhibitory agent”.
  • the term "chondroprotective agents" is intended to include both anabolic agents and inhibitors of cartilage catabolism.
  • At least a first chondroprotective agent is an anti-inflammatory/anabolic cytokine, which act functionally to suppress the role of pro-inflammatory cytokines in the joint, promote cartilage matrix synthesis and inhibit matrix degradation.
  • receptor agonists include, for example, specific anti-inflammatory and anabolic cytokines, such as the interleukin (IL) agonists (e.g., IL-4, IL-10 and IL-13) and specific members of the transforming growth factor- ⁇ superfamily (e.g., TGF ⁇ and BMP-7), insulin-like growth factors (e.g., IGF-1) and fibroblast growth factors (e.g., bFGF).
  • IL interleukin
  • IGF-1 insulin-like growth factors
  • fibroblast growth factors e.g., bFGF
  • At least a second chondroprotective agent is drawn from a class of cartilage catabolic inhibitors that include receptor antagonists or enzyme inhibitors that acts to inhibit and reduce the activity or the expression of a pro-inflammatory molecular target (e.g., the IL-1 receptor antagonists, TNF- ⁇ receptor antagonists, cyclooxygenase-2 inhibitors, MAP kinase inhibitors, nitric oxide synthase (NOS) inhibitors, and nuclear factor kappaB (NFKB) inhibitors).
  • a pro-inflammatory molecular target e.g., the IL-1 receptor antagonists, TNF- ⁇ receptor antagonists, cyclooxygenase-2 inhibitors, MAP kinase inhibitors, nitric oxide synthase (NOS) inhibitors, and nuclear factor kappaB (NFKB) inhibitors.
  • the second chondroprotective agent may also be selected from inhibitors of matrix metalloproteinases that inhibit cartilage catabolism, cell adhesion molecules, including integrin agonists and integrin antagonists, that inhibit cartilage catabolism, intracellular signaling inhibitors, including protein kinase C inhibitors and protein tyrosine kinase inhibitors, that inhibit cartilage catabolism, and inhibitors of SH2 domains that inhibit cartilage catabolism.
  • Articular cartilage is a specialized extracellular matrix that is produced and maintained by metabolically active articular chondrocytes.
  • the maintenance of a normal, healthy extracellular matrix reflects a dynamic balance between the rate of biosynthesis and incorporation of matrix components, and the rate of their degradation and subsequent loss from the cartilage into the synovial fluid.
  • Matrix homeostasis is generally regarded to represent a dynamic balance between the effects of catabolic cytokines and anabolic cytokines (including growth factors).
  • the optimal combination of therapeutic agents useful for cartilage protection shifts the dynamic matrix equilibrium through accelerating the synthetic rate and simultaneously inhibiting the rate of breakdown, thus maximizing anabolic processes and promoting repair.
  • Catabolic cytokines such as IL-l ⁇ and TNF- ⁇ act at specific receptors on chondrocytes to induce production of MMPs that induce matrix degradation while the degradation is inhibited by anabolic cytokines such as TGF- ⁇ , BMP-2 and IGF-1.
  • anabolic cytokines such as TGF- ⁇ , BMP-2 and IGF-1.
  • a plurality of chondroprotective agents are administered via a systemic route to a patient at risk of articular cartilage degradation.
  • the plurality of agents that are administered systemically include at least one agent that promotes cartilage anabolic activity and at least one agent that inhibits cartilage catabolism.
  • Each agent is included in a sufficient amount to provide a combination that is therapeutically effective when the solution is delivered to the joint of a patient to both inhibit cartilage catabolic processes and to promote cartilage anabolic processes.
  • one or more agents that act to inhibit pain and/or inflammation may be administered with the chondroprotective agents.
  • Systemic administration of the plurality of chondroprotective agents may be preferred when a patient is at risk of cartilage degradation, or suffers from degenerative disease, at multiple joints simultaneously.
  • a therapeutic strategy is to deliver the combination of agents in a carrier or delivery vehicle that is targeted to the joint.
  • the at least one anabolic chondroprotective agent and/or the at least one catabolic inhibitory chondroprotective agent, and preferably both the anabolic and catabolic inhibitory chondroprotective agents may be encapsulated within a delivery vehicle, such as a nano sphere.
  • a targeting antibody or antibody fragment is coupled to the nanosphere. The antibody or antibody fragment is specific to a targeted antigenic determinant that is localized within the joint.
  • a therapeutic method of the present invention includes systemically administering this targeted, encapsulated composition of one or more chondroprotective agents to a patient at risk of cartilage degradation, preferably by intravascular, intramuscular, subcutaneous or inhalational administration.
  • compositions for systemic administration include a plurality of chondroprotective agents, including at least one agent that promotes cartilage anabolic activity and at least one agent that inhibits cartilage catabolism. Additionally, one or more agents that act to inhibit pain and/or inflammation may be included in the compositions. All agents are included at a dosage sufficient to provide a cartilage protective therapeutic effect at a joint or joints when administered systemically. Methods of manufacturing a medicament including such a composition for use in treating a patient at risk of cartilage degradation are also provided.
  • the at least one anabolic chondroprotective agent and/or the at least one catabolic inhibitory chondroprotective agent, and preferably both the anabolic and catabolic inhibitory chondroprotective agents are encapsulated within a delivery vehicle, such as a nanosphere, to which is coupled an antibody or antibody fragment that is specific to an antigenic determinant that is localized within the joint.
  • a delivery vehicle such as a nanosphere
  • a method is also provided for manufacturing such a medicament including an encapsulated chondroprotective agent(s) coupled to an antibody or antibody fragment, such antibody or antibody fragment being targeted to an antigenic determinant that is localized within the joint, for use in treating a patient at risk of cartilage degradation.
  • a composition which includes one or preferably multiple metabolically active chondroprotective agents together with one or more agents for the inhibition of pain, inflammation, or the like, or more preferably a multiple agent combination of anabolic agents and inhibitors of catabolism, in a pharmaceutically effective carrier may be prepared for intra-articular delivery directly to the joint of a patient. While systemic delivery of the chondroprotective compositions of the present invention may be preferred for diseases or conditions affecting multiple joints, local delivery of the compositions of the present invention may be preferred in other instances.
  • Such instances may include the treatment of patients with a cartilage degenerative condition or diseases affecting only a single or limited number of joints, periprocedural administration associated with an operative or interventional procedure at a joint, or in instances where undesirable side effects may be associated with systemic administration.
  • such compositions are delivered locally by intra-articular injection (including for the treatment of cartilage degenerative diseases such as osteoarthritis or rheumatoid arthritis) or via infusion, including administration periprocedurally (i.e., preoperatively and/or intraoperatively and/or postoperatively) during surgical arthroscopic procedures.
  • This local delivery aspect of the present invention provides a solution constituting a mixture of multiple agents in low concentrations directed at inhibiting locally the mediators of pain, inflammation, and cartilage degradation in a physiologic electrolyte carrier fluid.
  • the invention also provides a method for perioperative delivery of the irrigation solution containing these agents directly to a surgical site, where it works locally at the receptor and enzyme levels to preemptively limit pain, inflammation, and cartilage degradation at the site. Due to the local perioperative delivery method of the present invention, a desired therapeutic effect can be achieved with lower doses of agents than are necessary when employing other methods of delivery (i.e., intravenous, intramuscular, subcutaneous and oral).
  • the anti-pain and/or anti-inflammation agents and/or anti-cartilage degradation agents in the solution include agents selected from multiple classes of receptor antagonists and agonists and enzyme activators and inhibitors, each class acting through a differing molecular mechanism of action for pain and/or inflammation inhibition and/or cartilage degradation.
  • compositions of the inventions may include anti-pain and/or anti-inflammation agents.
  • agents for the inhibition of pain and/or inflammation include, for example: (1) serotonin receptor antagonists; (2) serotonin receptor agonists; (3) histamine receptor antagonists; (4) bradykinin receptor antagonists; (5) kallikrein inhibitors; (6) tachykinin receptor antagonists, including neurokininj and neurokinin 2 receptor subtype antagonists; (7) calcitonin gene-related peptide (CGRP) receptor antagonists; (8) interleukin receptor antagonists; (9) inhibitors of enzymes active in the synthetic pathway for arachidonic acid metabolites, including (a) phospholipase inhibitors, including PLA 2 isoform inhibitors and PLC isoform inhibitors, (b) cyclooxygenase inhibitors, and (c) lipooxygenase inhibitors; (10) prostanoid receptor antagonists including eicos
  • the present invention also provides a method for manufacturing a medicament compounded in one aspect of the invention as a dilute irrigation solution for use in continuously irrigating an operative site, typically at the site of a joint of a patient, during an arthroscopic operative procedure.
  • the method entails dissolving in a physiologic electrolyte carrier fluid at least one anti-cartilage degradation agent and preferably one or more anti-pain/anti-inflammatory agents, and for some applications anti- cartilage degradation agents, each agent included at a concentration of preferably no more than about 100,000 nanomolar, 'more preferably no more than about 25,000 nanomolar, and most preferably no more than about 10,000 nanomolar.
  • a method of the local delivery aspect of the present invention provides for the delivery of a dilute combination of multiple receptor antagonists and agonists and enzyme inhibitors and activators directly to a wound or operative site, during therapeutic or diagnostic procedures for the inhibition of cartilage degradation, pain, and/or inflammation. Since the active ingredients in the solution are being locally . applied directly to the operative tissues in a continuous fashion, the drags may be used efficaciously at extremely low doses relative to those doses required for therapeutic effect when the same drugs are delivered orally, intramuscularly, subcutaneously or intravenously.
  • the term "local” encompasses application of a drug in and around a wound or other operative site, and excludes oral, subcutaneous, intravenous and intramuscular administration.
  • continuous encompasses uninterrupted application, repeated application at frequent intervals, and applications which are uninterrupted except for brief cessations such as to permit the introduction of other drugs or agents or procedural equipment, such that a substantially constant predetermined concentration is maintained locally at the wound or operative site.
  • the advantages of local administration of the agents via irrigation or other fluid application in accordance with this aspect of the invention are the following: (1) local administration guarantees a known concentration at the target site, regardless of interpatient variability in metabolism, blood flow, etc.; (2) because of the direct mode of delivery, a therapeutic concentration is obtained instantaneously and, thus, improved dosage control is provided; and (3) local administration of the active agents directly to a wound or operative site also substantially reduces degradation of the agents through systemic processes (e.g., first- and second-pass metabolism) that would otherwise occur if the agents were given orally, intravenously, subcutaneously or intramuscularly. This is particularly true for those active agents that are proteins and peptides, which are metabolized rapidly.
  • systemic processes e.g., first- and second-pass metabolism
  • agents in the following classes are peptidic: bradykinin receptor antagonists; tachykinin receptor antagonists; opioid receptor agonists; CGRP receptor antagonists; and interleukin receptor antagonists, TNF-receptor antagomsts; TGF- ⁇ receptor agonists; BMP-2 and BMP-7 receptor agonists; IL4, IL10 and IL-13 receptor agonists; and integrin receptor agonists and antagonists.
  • Local, continuous delivery to the wound or operative site minimizes drug degradation or metabolism while also providing for the continuous replacement of that portion of the agent that may be degraded, to ensure that a local therapeutic concentration, sufficient to maintain receptor occupancy or enzymatic saturation, is maintained throughout the duration of the operative procedure.
  • the term "perioperative” encompasses application intraprocedurally, pre- and mtraprocedurally, intra- and postprocedurally, and pre-, intra- and postprocedurally.
  • the solutions of the present invention are most preferably applied pre-, intra- and postoperatively.
  • the agents of the present solution modulate specific pathways to preemptively inhibit the targeted pathologic process. If inflammatory mediators and processes are preemptively inhibited in accordance with the present invention before they can exert tissue damage, the benefit is more substantial than if given after the damage has been initiated. Inhibiting more than one pain, inflammatory or cartilage degradation mediator by application of the multiple agent solutions of the present invention has been shown to dramatically reduce the degree of inflammation and pain, and theoretically should provide a cartilage protective effect.
  • the irrigation solutions of the present invention include combinations of drags, each solution acting on multiple receptors or enzymes.
  • the drag agents are thus simultaneously effective against a combination of pathologic processes, including pain and inflammation, and loss of cartilage homeostasis.
  • the action of these agents is considered to be synergistic, in that the multiple receptor antagonists and inhibitory agonists of the present invention provide a disproportionately increased efficacy in combination relative to the efficacy of the individual agents.
  • the synergistic action of several of the agents of the present invention are discussed below, by way of example.
  • the solution should result in a clinically significant decrease in operative site pain and inflammation, and of cartilage degradation, relative to currently-used irrigation fluids, thereby decreasing the patient's postoperative analgesic (i.e., opiate) requirement and, where appropriate, allowing earlier patient mobilization of the operative site. No extra effort on the part of the surgeon and operating room personnel is required to use the present solution relative to conventional irrigation fluids.
  • the solutions of the invention are administered directly to a joint prior to, during and/or after a surgical procedure.
  • compositions for the protection of cartilage including anabolic-promoting agents and catabolic inhibitory agents are provided.
  • Such combinations result in a state that is characterized by: cartilage anabolic activity equaling or exceeding cartilage catabolic activity; the maintenance of cartilage tissue so as to either maintain existing, or to increase, cartilage volume; or an increase in the synthesis of cartilage matrix by articular chondrocytes and in the concomitant reduction in degradation of the cartilage matrix.
  • FIGURE 1 is a schematic overview of a chondrocyte cell showing molecular targets and flow of signaling information leading to the production of mediators of inflammation and shifts in cartilage metabolism.
  • cytokine receptor such as the interleukin-1 (IL-1) receptor family and the tumor necrosis factor (TNF) receptor family
  • TGF- ⁇ receptor superfamily the TGF- ⁇ receptor superfamily and integrins is shown to converge on common intracellular signaling pathways that include major groups of protein molecules that are therapeutic targets of drugs included in the solutions of the present invention (MAP kinases, PKC, tyrosine kinases, SH2 proteins, COX, PLA2 and NF- KB.
  • Activation of these signaling pathways controls chondrocyte expression of a number of inducible gene products, including IL-1, TNF- ⁇ , IL-6, IL-8 and Stromelysin (MMP-3), and other mediators (nitric oxide (NO) and PGE2) which may lead to inflammation and/or cartilage degradation, or synthesis of matrix molecules and chondrocyte proliferation;
  • inducible gene products including IL-1, TNF- ⁇ , IL-6, IL-8 and Stromelysin (MMP-3), and other mediators (nitric oxide (NO) and PGE2)
  • FIGURE 2 provides a schematic overview of a synoviocyte cell showing molecular targets and flow of signaling information leading to the production of mediators of inflammation and shifts in cartilage metabolism.
  • cytokine receptors which include the interleukin-1 (IL-1) receptor family and the tumor necrosis factor (TNF) receptor family
  • TNF tumor necrosis factor
  • G-protein coupled receptors which include bradykinin, histamine and serotonin subtypes
  • integrins is shown to converge on common intracellular signaling pathways that include major groups of protein molecules that are therapeutic targets of drags included in the solutions of the present invention (MAP kinases, PKC, tyrosine kinases, SH2 proteins, COX, PLA2 and NF-
  • Activation of these signaling pathways controls synoviocyte expression of a number of inducible gene products, including IL-1, TNF- ⁇ , IL-6, IL-8 and
  • MMP-3 Stromelysin
  • FIGURE 3 is a diagram of common signaling pathways in both chondrocyte and synoviocyte cells, including key signaling proteins responsible for "crosstalk” between GPCR activated receptor pathways and pro-inflammatory cytokine pathways that lead to inflammation and . or cartilage degradation;
  • FIGURE 4 is a diagram of common signaling pathways in both chondrocyte and synoviocyte cells, including key signaling proteins responsible for "crosstalk" between GPCR activated receptor pathways and pro-inflammatory cytokine pathways. Specific molecular sites of action for some drags in a preferred chondroprotective solution of the present invention are identified;
  • FIGURE 5 is a diagram of molecular targets present on either chondrocytes or synoviocytes that promote an anabolic response of cartilage. Specific sites of action of some drugs in the preferred chondroprotective solution of the present are identified;
  • FIGURE 6 is a diagram .of molecular targets present on either chondrocytes or synoviocytes that promote a catabolic response in cartilage. Specific sites of action of some drugs in the preferred chondroprotective solution of the present invention are identified;
  • FIGURE 7 is a graphical representation of the production of prostaglandin E2 in synovial cultures by G-protein regulatory agonists following overnight priming with interleukin-1 (IL-1, lOU/ml).
  • the cultures were stimulated for the indicated times with histamine (100 ⁇ M, open bars), or bradykinin (1 ⁇ M, closed bars), and the prostaglandin E2 released to the culture supernatant was determined as described in Study 1 herein below.
  • the values shown are the mean ⁇ the standard deviation from a representative experiment, and are corrected for basal prostaglandin E2 production by unstimulated cultures;
  • FIGURE 8 is a graphical representation of the inhibition of prostaglandin E2 production in synovial cultures by ketoprofen.
  • the cultures were primed overnight with IL-1 (lOU/ml) in the presence (shown as “ ⁇ ”) or absence (shown as “ ⁇ ” or “V”) of the indicated concentrations of ketoprofen.
  • prostaglandin E2 was measured in the supernatants of cultures treated overnight with ketoprofen, and the remaining cultures were washed, incubated for 10 minutes with the indicated concentrations of ketoprofen, and then prostaglandin E2 production was measured in response to a subsequent 3 minute challenge with histamine (100 ⁇ M, V) or bradykinin (1 ⁇ M, ⁇ ) in the continuing presence of the indicated amounts of ketoprofen.
  • FIGURE 9 is a graphical representation of the effect of ketoprofen on IL-6 production by synovial cultures at 16 hours in the presence of the indicated concentrations of IL-1 plus the added G-protein coupled receptor ligands.
  • Cultures were incubated for 16 hours with IL-1 at the indicated concentration (o.3, 1.0 and 3.0 pg/ml) in the absence and presence of 0.75 ⁇ M ketoprofen in experimental growth medium with one of the following additional receptor ligands: 1) isoproterenol (ISO) at 1.0 ⁇ M to activate the camp pathway, or 2) histamine (HIS) at 100 ⁇ M to activate the IP3/calcium pathway.
  • ISO isoproterenol
  • HIS histamine
  • a method for locally administering to a joint a composition including at least a first agent that acts to promote cartilage anabolic activity and at least a second agent that acts to inhibit cartilage catabolism.
  • such compositions are locally delivered by injection of the composition, which may include a sustained release delivery vehicle, into a joint.
  • the composition includes a liquid irrigation carrier and is locally and perioperatively delivered to the joint during an operative or interventional procedure.
  • a method for systemically administering to a patient a composition including at least a first agent that acts to promote cartilage anabolic activity and at least a second agent that acts to inhibit cartilage catabolism.
  • a method for systemically administering to a patient a composition including at least a first agent that acts to promote cartilage anabolic activity and/or at least a second agent that acts to inhibit cartilage catabolism, in which at least one of the agents is targeted to the joint.
  • pro-inflammatory mediators that have been implicated in producing loss of cartilage in the inflamed joint are the cytokines, TNF- ⁇ , IL-1, IL-6 and IL-8. Elevated levels of a number of these pro-inflammatory cytokines appear rapidly in the synovial fluid of acutely injured knee joints and remain elevated in patients for at least 4 weeks (Cameron, M.L. et al., "Synovial fluid cytokine concentrations as possible prognostic indicators in the ACL-deficient knee," Knee Surg. Sports Traumatol Arthroscopy 2:38-44 (1994)).
  • cytokines are produced locally in the joint from several activated cell types, including synovial fibroblasts, synovial macrophages, and chondrocytes.
  • the locally produced cytokines mediate pathophysiological events in acute and chronic inflammatory conditions and are important autocrine and paracrine mediators of cartilage catabolism.
  • the actions of these cytokines are characterized by their ability to cause multiple effects on distinct cellular targets and by their ability to interact in either a positive or negative synergistic manner with other cytokines.
  • IL-1 and TNF- ⁇ are particularly important because they also initiate chondrodestractive effects by disrupting the balance between the normal turnover and destruction of cartilage matrix components by modulating the activity of endogenous proteins (e.g., matrix metalloproteinases (MMPs)) and tissue inhibitor of metalloproteinase (TIMP).
  • MMPs matrix metalloproteinases
  • TNF tissue inhibitor of metalloproteinase
  • Cytokine control of cartilage homeostasis represents a highly regulated balance between active mediators acting on chondrocytes, which determines whether matrix degradation or repair occurs. Damage to the joint frequently produces an inflammatory response within the joint space that involves the synovial tissue and may lead to degradation of articular cartilage.
  • MMPs matrix metalloproteinases
  • collagenase and stromelysin- 1 are elevated in the synovial fluid of patients after acute trauma (Lohmander, L.S. et al., "Temporal patterns of stromelysin- 1 tissue inhibitor, and proteoglycan fragments in human knee joint fluid after injury to the cruciate ligament or meniscus," J. Orthopaedic Res. 12:21-28 (1994)).
  • cytokines and cartilage matrix markers e.g., proteoglycans
  • synovial fluid which are correlated with cartilage degeneration
  • OA osteoarthritis
  • the combined effects of initial joint injury and surgical trauma may induce a sustained inflammatory state and associated changes in cartilage matrix metabolism which appear to be causative factors resulting in the subsequent development of degenerative changes in articular cartilage and early development of osteoarthritis.
  • a method for reducing or preventing destruction of articular cartilage in a joint, by administering directly to the joint of a patient a composition which includes one . or preferably multiple metabolically active chondroprotective agents together with one or more agents for the inhibition of pain and/or inflammation, as previously described, or preferably a combination of two or more metabolically active chondroprotective agents, at least one of which promotes cartilage anabolic processes and at least one of which is an inhibitor of cartilage catabolic processes, in a pharmaceutically effective carrier for intra-articular delivery.
  • Metabolically active agents include, but are not limited to, all compounds that act directly or indirectly to modulate or alter the biological, biochemical or biophysical state of a cell, including agents that alter the electrical potential of the plasma membrane, the ligand binding or enzymatic activity of cellular receptors, intracellular or exfracellularly located enzymes, protein-protein interactions, RNA-proteins interactions, or DNA-protein interactions.
  • agents may include receptor agonists that initiate signal transduction cascades, antagonists of receptors that inhibit signaling pathways, activators and inhibitors of intracellular or extracellular enzymes and agents that modulate the binding of transcription factors to DNA.
  • Suitable chondroprotective agents include, for example, (1) antagonists of receptors for the interleukin-1 family of proteins, including, for example, IL-l ⁇ , IL-17 and IL-18; (2) antagonists of the tumor necrosis factor (TNF) receptor family, including, for example, TNF-R1; (3) agonists for interleukin 4, 10 and 13 receptors; (4) agonists for the TGF- ⁇ receptor superfamily, including, for example, BMP-2, BMP-4 and BMP-7; (5) inhibitors of COX-2; (6) inhibitors of the MAP kinase family, including, for example, p38 MAP kinase; (7) inhibitors of the matrix metalloproteinases (MMP) family of proteins, including, for example, MMP-3 and MMP-9; (8) inhibitors of the NF- ⁇ B family of proteins, including, for example, the p50/p65 dimer complex with I B; (9) inhibitors of the nitric oxide synthase (NOS) family,
  • a first embodiment of the present invention provides a pharmacological method of treating the injured or operatively treated joint using a combination of cartilage protective agents delivered locally to achieve maximal therapeutic benefit.
  • a second embodiment of the present invention provides a pharmacological method of providing therapeutic treatment by systemically administering a combination of cartilage protective agents.
  • the use of a combination of chondroprotective agents overcomes the limitations of existing therapeutic approaches that rely upon on the use of a single agent to block a multifactorial cartilage destructive process in which a shift between synthesis and degradation, in favor of catabolic processes has occurred.
  • This aspect of the invention uniquely utilizes the approach of combining of agents that act simultaneously on distinct molecular targets to promote cartilage anabolism and inhibit unregulated or excess cartilage catabolic processes to achieve maximum inhibition of inflammatory processes and maintain cartilage homeostasis, thereby achieving a chondroprotective effect within the joint.
  • Inhibition of a single molecular target or biochemical mechanism known to induce cartilage destruction (catabolism), such as inhibiting interleukin-1 (IL-1) binding to the IL-1 receptor will likely not be optimal, since, for example, the actions of TNF- ⁇ mediated through its unique receptor shares many overlapping pro-inflammatory and cartilage catabolic functions with IL-1 and is also recognized as a major mediator of cartilage destruction in the joint.
  • utilizing pharmaceutical agents that only enhance cartilage anabolic processes in the absence of inhibiting catabolic processes will not optimally counteract catabolic factors present within the injured joint.
  • one aspect of the present invention provides pharmaceutical compositions of metabolically active chondroprotective agents that are based upon a combination of at least two agents that act simultaneously on distinct molecular targets.
  • at least one agent is a cytokine or growth factor receptor agonist which directly provides anti-inflammatory activity and/or promotes cartilage anabolic processes and at least a second agent is a receptor antagonist or enzyme inhibitor that acts to inhibit pro-inflammatory and/or cartilage catabolic processes.
  • a representative drag combination includes at least one agent drawn from a class of anti-inflammatory/anabolic cytokines that act functionally to suppress the role of pro-inflammatory cytokines in the joint, promote cartilage matrix synthesis and inhibit matrix degradation.
  • receptor agonists include, but are not limited to, specific anti-inflammatory and anabolic cytokines, such as the interleukin (IL) agonists (e.g., IL-4, IL-10 and IL-13) and specific members of the transforming growth factor- ⁇ superfamily (e.g., TGF ⁇ and BMP-7), insulin-like growth factors (e.g., IGF-1) and fibroblast growth factors (e.g., bFGF).
  • IL interleukin
  • TGF ⁇ and BMP-7 transforming growth factor- ⁇ superfamily
  • IGF-1 insulin-like growth factors
  • fibroblast growth factors e.g., bFGF
  • At least a second agent is drawn from a class of receptor antagonists or enzyme inhibitors that acts to inhibit and reduce the activity or the expression of a pro-inflammatory molecular target (e.g., the IL-1 receptor antagonists, TNF- ⁇ receptor antagonists, cyclooxygenase-2 inhibitors, MAP kinase inhibitors, nitric oxide synthase (NOS) inhibitors, and nuclear factor kappaB (NFKB) inhibitors).
  • the metabolically active agents include both functional agonists and antagonists of receptors located on the surfaces of cells, as well as inhibitors of membrane bound or extracellularly secreted enzymes (e.g., stromelysin and collagenase).
  • agents are directed at novel targets that are the intracellularly localized enzymes and transcription factors that transduce and integrate the signals of the surface receptors, including inhibitors of the enzymes NOS, COX-2, and mitogen-activated protein k ⁇ nases (MAPK) and inhibitors of protein-DNA interactions such as the transcription factor NFKB.
  • MAPK mitogen-activated protein k ⁇ nases
  • This method allows the integrity of cartilage to be maintained by simultaneously promoting cytokine-driven anabolic processes and inhibiting catabolic processes.
  • the compositions of preferred embodiments of the present invention constitute a novel therapeutic approach by combining multiple pharmacologic agents acting at distinct receptor and/or enzyme molecular targets.
  • the therapeutic approach of the present compositions is based on the rationale that a combination of drugs acting simultaneously on distinct molecular targets is highly effective in the inhibition of the full spectrum of events that underlie the development of a pathophysiologic state.
  • the compositions instead of targeting a specific receptor subtype alone, the compositions include drags that target common molecular mechanisms operating in different cellular physiologic processes involved in the development of pain, inflammation, and cartilage degradation (see FIGURE 1). In this way, the cascading of additional receptors and enzymes in the nociceptive, inflammatory, and cartilage degradation pathways is minimized. In these pathophysiologic pathways, the compositions inhibit the cascade effect both "upstream” and "downstream".
  • upstream inhibition is the cyclooxygenase antagonists in the setting of pain and inflammation.
  • the cyclooxygenase enzymes COX ⁇ and COX2 catalyze the conversion of arachidonic acid to prostaglandin H that is an intermediate in the biosynthesis of inflammatory and nociceptive mediators including prostaglandins, leukotrienes, and thromboxanes.
  • the cyclooxygenase inhibitors block "upstream” the formation of these inflammatory and nociceptive mediators. This strategy precludes the need to block the interactions of the seven described subtypes of prostanoid receptors with prostanoid products of the COX biochemical pathway.
  • a similar "upstream” inhibitor is aprotinin, a kallikrein inhibitor.
  • the enzyme kallikrein, a serine protease cleaves the high molecular weight kininogens in plasma to produce bradykinins, important mediators of pain and inflammation.
  • aprotinin effectively inhibits the synthesis of bradykinins, thereby providing an effective "upstream” inhibition of these inflammatory mediators.
  • compositions of the invention may also make use of "downstream" inhibitors to control the pathophysiologic pathways.
  • cytokines e.g., IL-l ⁇ and TNF ⁇
  • MAP kinase inhibitors produce a cartilage protective effect.
  • the p38 MAP kinase is a point of conveyance in signaling pathways for multiple catabolic cytokines, and its inhibition prevents upregulation of multiple cellular products mediating cartilage degradation.
  • the MAP kinase inhibitors therefore, provide a significant advantage in the setting of joint inflammation by providing "downstream" cartilage protective effects that are independent of the physiologic combination of cytokine receptor agonists initiating the shift cartilage homeostasis.
  • Specific preferred embodiments of the solution of the present invention for use in chondroprotection and surgical procedures preferably include a combination of agents that act simultaneously on distinct molecular targets to promote cartilage anabolism and inhibit unregulated or excess cartilage catabolic processes to achieve maximum inhibition of inflammatory processes and maintain cartilage homeostasis, thereby achieving a chondroprotective effect within the joint.
  • the irrigation and injectable solutions of one embodiment of the present invention are dilute solutions of one or preferably more chondroprotective agents and, optionally, one or more pain and/or inflammation inhibitory agents in a physiologic carrier.
  • the carrier is a liquid solution, which as used herein is intended to encompass biocompatible solvents, suspensions, polymerizable and non-polymerizable gels, pastes and salves, as well as sustained release delivery vehicles, such as microparticles, microspheres or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds, or inorganic compounds.
  • the carrier is an aqueous solution that may include physiologic electrolytes, such as normal saline or lactated Ringer's solution.
  • the agents are included in low concentrations in a liquid or fluid solution and are delivered locally in low doses relative to concentrations and doses required with systemic methods of drug administration to achieve the desired therapeutic effect.
  • liquid or “fluid” is intended to encompass pharmaceutically acceptable, biocompatible solvents, suspensions, polymerizable and non-polymerizable gels, pastes and salves.
  • the carrier is an aqueous solution that may include physiologic electrolytes, such as normal saline or lactated Ringer's solution.
  • each agent is determined in part based on its receptor dissociation constant, K ⁇ or enzyme inhibition constant, K j .
  • dissociation constant is intended to encompass both the equilibrium dissociation constant for its respective agonist- receptor or antagonist-receptor interaction and the equilibrium inhibitory constant for its respective activator-enzyme or inhibitor-enzyme interaction.
  • Each agent is preferably included at a low concenfration of 0.1 to 10,000 times K ⁇ or Kj.
  • each agent is included at a concentration of 1.0 to 1,000 times K ⁇ or Kj and most preferably at approximately 100 times K ⁇ or Kj. These concentrations are adjusted as needed to account for dilution in the absence of metabolic transformation at the local delivery site.
  • concentrations are adjusted as needed to account for dilution in the absence of metabolic transformation at the local delivery site.
  • the exact agents selected for use in the solution, and the concentration of the agents, varies in accordance with the particular application, as described below.
  • a solution in accordance with an aspect of the present invention can include just a single or multiple chondroprotective agent(s), preferably multiple chondroprotective agents at least one of which is an anabolic chondroprotective agent and at least one of which is an inhibitor of cartilage catabolism, or a combination of both chondroprotective agent(s) and pain and/or inflammation inhibitory agents, at low concentration.
  • chondroprotective agent(s) preferably multiple chondroprotective agents at least one of which is an anabolic chondroprotective agent and at least one of which is an inhibitor of cartilage catabolism, or a combination of both chondroprotective agent(s) and pain and/or inflammation inhibitory agents, at low concentration.
  • the multiple drag combination can be delivered locally by intra-articular injection or via infusion, including administration periprocedurally (i.e., preoperatively and/or intraoperatively and/or postoperatively) during surgical arthroscopic procedures, alone or coupled with postoperative sustained delivery, such as by a regulated pump or use of a sustained release delivery vehicle.
  • Sustained release delivery vehicles may include, but are not limited to, microparticles, microspheres or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds, or inorganic compounds.
  • the invention provides for a combination of agents to be delivered via injection or infusion, alone or together with analgesic and/or anti-inflammatory agents.
  • the rapid onset of action achieved by direct, local delivery of the chondroprotective agents at or closely following the time of injury has the potential to inhibit initial processes before they can trigger subsequent responses and thereby preemptively limit local tissue damage and the subsequent loss of cartilage.
  • Advantages of the present invention include: 1) a combination drug therapy directed to the multifactorial causes of cartilage destruction during acute and/or chronic conditions; 2) the combination of chondroprotective agents may be combined with anti-inflammatory and analgesic agents; 3) local delivery of the drag combination (where applicable) achieves an instantaneous therapeutic concentration of chondroprotective agents within the joint; 4) using an irrigation solution periprocedurally (where applicable) provides continuous maintenance of drug levels within the joint in a therapeutically desirable range during an arthroscopic surgical procedure; 5) local delivery (for this embodiment of the invention) permits a reduction in total drag dose and dosing frequency compared to systemic delivery; 6) local site-directed delivery to the joint (for this embodiment of the invention) avoids systemic toxicity and reduces adverse effects; and 7) direct, local delivery to the joint (for this embodiment of the invention) enables use of novel, pharmaceutically active peptides and proteins, including cytokines and growth factors, which may not be therapeutically useful if limited to systemic routes of administration.
  • these compounds are expected to exhibit chondroprotective action when applied perioperatively in an irrigation solution (in combination with other chondroprotective agents or in combination with other anti- pain and anti-inflammation agents described herein) or otherwise administered directly to the joint via infusion or injection.
  • these agents are expected to be effective drags when delivered by an irrigation solution during an arthroscopic surgical procedure.
  • Each metabolically active chondroprotective agent may preferably be delivered in combination with one or more other chondroprotective agents, including small molecule ' drags, peptides, proteins, recombinant chimeric proteins, antibodies, oligonucleotides or gene therapy vectors (viral and nonviral), to the spaces of the joint.
  • a drag such as a MAPK inhibitor can exert its actions on any cells associated with the fluid spaces of the joint and stractures comprising the joint that are involved in the normal function of the joint or are present due to a pathological condition.
  • These cells and structures include, but are not limited to: synovial cells, including both Type A fibroblast and Type B macrophage cells; the cartilaginous components of the joint such as chondroblasts and chondrocytes; cells associated with bone, including periosteal cells, osteocytes, osteoblasts, osteoclasts; inflammatory cells including lymphocytes, macrophages, mast cells, monocytes, eosinophils; and other cells including endothelial cells, smooth muscle cells, fibroblasts and neural cells; and combinations of the above.
  • This aspect of the present invention also provides for formulations of the active therapeutic agent(s) that may be delivered in a formulation useful for introduction and administration of the drag into the joint that would enhance the delivery, uptake, stability or.
  • Such a formulation may include, but is not limited to, microparticles, microspheres, nanospheres or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds, or inorganic compounds.
  • the present invention provides for the delivery of a combination of chondroprotective agents, or one or preferably multiple chondroprotective agents with one or more anti-pain and/or anti-inflammation agents present either as multiple pharmaceutically active substances within a homogeneous vehicle (e.g., a single encapsulated microsphere) or as a discrete mixture of individual delivery vehicles (e.g., a group of microspheres encapsulating one or more agents).
  • formulation molecules include, but are not limited to, hydrophilic polymers, polycations (e.g. protamine, spermidine, polylysine), peptide or synthetic ligands and antibodies capable of targeting materials to specific cell types, gels, slow release matrices (i.e., sustained delivery vehicles, soluble and insoluble particles) as well as formulation elements not listed.
  • the present invention provides for the local delivery of a combination of two or more chondroprotective agents, or one or preferably multiple chondroprotective agents in combination with one or more anti-pain and/or anti-inflammation agents, alone or in combination with one or more anti-pain and/or anti-inflammatory agents, via an irrigation solution, an infusion containing the drags whichiare present at therapeutically effective low concentrations and which enables the drags to be delivered directly to the affected tissue or joint.
  • the drug-containing infusion or irrigation solution may be employed preoperatively and/or intraoperatively and/or postoperatively in connection with a surgical procedure or may be administered at other times not related to surgical procedures.
  • Systemic methods of drug delivery have required higher concentrations of drags (and higher total dose) to be administered to the patient in order to achieve significant therapeutic concentrations in the targeted joint.
  • Systemic administration may also result in high concentrations in tissues other than the targeted joint, which is undesirable and, depending on the dose, may result in adverse side effects.
  • These systemic methods subject the drag to second-pass metabolism and rapid degradation, thereby limiting the duration of the effective therapeutic concentration.
  • chondroprotective agents with or without one or more anti-pain and/or anti-inflammatory agents
  • irrigation vascular perfusion is not required to carry the drug to the targeted tissue. This significant advantage allows for the local delivery of a lower therapeutically effective total dose for a variety of chondroprotective drugs.
  • the solutions of the present invention have application for a variety of operative/interventional procedures, including surgical, diagnostic and therapeutic techniques.
  • the combination of chondroprotective agents of the invention may be administered by injection or by irrigation.
  • the amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the patient to be treated, the nature of the active agents in the solution and the particular mode of administration. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex and diet of the patient, time of administration, route of administration, rate of excretion of the drug combination, and the severity of the particular disease undergoing therapy.
  • sterile injectable preparations for example, sterile injectable aqueous or oleagenous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in propanediol.
  • a nontoxic parenterally acceptable diluent or solvent for example, as a solution in propanediol.
  • the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any biocompatible oil may be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • the solutions for injection of the invention may be administered in connection with an arthroscopic surgical procedure or at any time determined to be desirable by a physician directing patient care.
  • the irrigation solutions of the invention may be perioperatively applied during arthroscopic surgery of anatomic joints.
  • the term "perioperative” encompasses application mtraprocedurally, pre- and mtraprocedurally, intra- and postprocedurally, and pre-, intra- and postprocedurally.
  • the solution is applied preprocedurally and/or postprocedurally as well as mtraprocedurally.
  • irrigation Such procedures conventionally utilize physiologic irrigation fluids, such as normal saline or lactated Ringer's, applied to the surgical site by techniques well known to those of ordinary skill in the art.
  • the method of the present invention involves substituting the anti-pain/anti- inflammatory/chondroprotective irrigation solutions of the present invention for conventionally applied irrigation fluids.
  • the irrigation solution is applied to the wound or surgical site prior to the initiation, of the procedure, preferably before tissue trauma, and continuously throughout the duration of the procedure, to preemptively block pain and inflammation, and cartilage degradation.
  • irrigation is intended to mean the flushing of a wound or anatomic structure with a stream of liquid.
  • the term "application” is intended to encompass irrigation and other methods of locally introducing the solution of the present invention, such as introducing a gellable version of the solution to the operative site, with the gelled solution then remaining at the site throughout the procedure.
  • the term “continuously” is intended to also include situations in which there is repeated and frequent irrigation of wounds at a frequency sufficient to maintain a predetermined therapeutic local concentration of the applied agents, and applications in which there may be intermittent cessation of irrigation fluid flow necessitated by operating technique.
  • concentrations listed above for each of the agents within the solutions of the present invention are the concentrations of the agents delivered locally, in the absence of metabolic transformation, to the operative site in order to achieve a predetermined level of effect at the operative site. It is understood that the drug concentrations in a given solution may need to be adjusted to account for local dilution upon delivery. Solution concentrations in the above embodiments are not adjusted to account for metabolic transformations or dilution by total body distribution because these circumstances are avoided by local delivery, as opposed to oral, intravenous, subcutaneous or intramuscular application.
  • Arthroscopic techniques for which the present solution may be employed include, by way of non-limiting example, partial meniscectomies and ligament reconstructions in the knee, shoulder acromioplasties, rotator cuff debridements, elbow synovectomies, and wrist and ankle arthroscopies.
  • the irrigation solution is continuously supplied intraoperatively to the joint at a flow rate sufficient to distend the joint capsule, to remove operative debris, and to enable unobstructed intra- articular visualization.
  • Suitable arthroscopic irrigation solutions for inhibition of cartilage degradation and control of pain and inflammation during such arthroscopic techniques are provided in Examples 1-3 herein below.
  • the agents are included in low concenfrations and are delivered locally in low doses relative to concentrations and doses required with systemic methods of drag administration to achieve the desired therapeutic effect. It is impossible to obtain an equivalent therapeutic effect by delivering similarly dosed agents via other (i.e., intravenous, subcutaneous, intramuscular or oral) routes of drag administration since drags given systemically are subject to first- and second-pass metabolism and are often rapidly cleared from the system circulation.
  • Practice of the present invention should be distinguished from conventional intra-articular injections of opiates and/or local anesthetics at the completion of arthroscopic or "open" joint (e.g., knee, shoulder, etc.) procedures.
  • solutions of this aspect of the present invention are used for continuous infusion throughout the surgical procedure to provide preemptive inhibition of pain and inflammation.
  • the high concentrations necessary to achieve therapeutic efficacy with a constant infusion of currently used local anesthetics can result in profound systemic toxicity.
  • chondroprotective agent(s) and/or pain and/or inflammation inhibitors as used in the irrigation solution at the operative site, as an alternative or supplement to opiates. It may also be desirable to deliver a sufficient amount of the solution to the joint after the surgical procedure so that a bolus of the solution remains in the synovial capsule of the patient following the surgical procedure.
  • compositions of the present " invention including multiple chondroprotective agents, including preferably at least one catabolic inhibitory agent and at least one anabolic-promoting agent, may also be adapted for direct injection into an atomic joint.
  • the agents are selected and each agent is included in a sufficient amount to provide a combination that is therapeutically effective when the solution is delivered locally to the joint of a patient to both inhibit cartilage catabolic processes and to promote cartilage anabolic processes.
  • Such compositions may be locally injected to provide a chondroprotective effect to a patient suffering from a chronic condition, such as osteoarthritis or rheumatoid arthritis, or an acute condition such as trauma from surgery or accidental injury.
  • a chronic condition such as osteoarthritis or rheumatoid arthritis
  • an acute condition such as trauma from surgery or accidental injury.
  • Embodiments of the present invention have thus far been described in terms of local delivery of chondroprotective compositions, such as by intra-articular injection.
  • Local administration has been described as having several advantages over systemic administration, including the avoidance of systemic side effects.
  • While local delivery of the compositions of the present invention is preferred in many instances, it may not be practical for some cartilage degenerative conditions. This is particularly the case for patients suffering from chronic cartilage degenerative diseases in which multiple sites simultaneously are at risk of cartilage degradation, such as rheumatoid arthritis, polyarticular osteoarthritis and other polyarthropathies.
  • chondroprotective compositions for such patients, injection of chondroprotective compositions into each or a majority of their diseased sites (e.g., joints) may be painful, impractical, costly or otherwise dissuasive of treatment.
  • the chondroprotective compositions described above for local delivery may, in accordance with another aspect of the present invention, be adapted for administration via systemic routes.
  • Systemic delivery of the compositions of the present invention is suitable for, but not limited to, treatment of patients with multiple sites at risk of cartilage degeneration. .
  • this aspect of the present invention may be useful for treating other noninflammatory and inflammatory arthrititides including, but not limited to, neuropathic arthropathy, acute rheumatic fever, ochronosis, systemic lupus erythematosus, juvenile rheumatic zi tFCT
  • arthritis arthritis, psoriatic arthritis, ankylosing spondylitis, and other spondyloarthropathies and crystalline arthropathies.
  • RA rheumatoid arthritis
  • OA osteoarthritis
  • RA is the most common form of inflammatory arthritis, affecting approximately 3% of women and 1% of men.
  • the majority of patients have multiple, and usually symmetrical, joint involvement, especially the small joints of the hands, the elbows, the wrists and the shoulders.
  • OA is the most common form of joint disease and is second only to cardiovascular disease as a cause of early retirement and disability.
  • OA commonly is polyarticular.
  • the destruction of hyaline articular cartilage is the hallmark of OA and disabling RA.
  • OA articular cartilage degradation
  • cytokines and inflammatory mediators have been shown to either create an imbalance in the synthetic functions of the chondrocytes or, alternatively, to increase cartilage matrix catabolism by upregulating various matrix-degrading enzymes, including the matrix metalloproteinases (including collagenases).
  • compositions including combinations of chondroprotective agents, and methods of systemic administration of such compositions.
  • Agents that target differing receptors or molecular targets are utilized for a multifactorial approach, as described previously.
  • the therapeutic compositions of the present invention include at least one chondroprotective agent that promotes cartilage anabolic activity, and at least one agent that inhibits cartilage catabolism.
  • This combination is expected to optimize conditions for homeostasis, and to be preferable over conventional therapies that address only cartilage breakdown or more recent research to develop drugs that address only cartilage synthesis.
  • Suitable anabolic-promoting agents and catabolic inhibitory agents have been described above for local administration, and are also expected to be useful for the present systemic compositions. Aspects and advantages of the compositions of the present invention described above with respect to local delivery are to be understood to also apply, to the extent applicable, to the systemic embodiments of the invention.
  • compositions of the present invention may suitably include one or more of the following anabolic-promoting agents, by way of non- limiting example: interleukin (IL) agonists (e.g., IL-4, IL-10, IL-13 agonists), members of the transforming growth factor- ⁇ superfamily, including TGF- ⁇ agonists (e.g., TGF ⁇ l, TGF ⁇ 2, TGF ⁇ 3) and bone morphogenetic protein agonists (e.g., BMP-2, BMP-4, BMP-6, BMP-7), insulin-like growth factors (e.g., IGF-1) and fibroblast growth factors (e.g., bFGF), and fragments, deletions, additions, amino acid substitutions, mutations and modifications that retain the biological characteristics of these naturally occurring agents.
  • IL interleukin
  • TGF ⁇ agonists e.g., TGF ⁇ l, TGF ⁇ 2, TGF ⁇ 3
  • bone morphogenetic protein agonists e.g., BMP-2, BMP
  • Chondroprotective compositions of the present invention may suitably include one or more of the following inhibitors of cartilage catabolism, by way of nonlimiting example: IL-1 receptor antagonists, TNF- ⁇ receptor antagonists, cyclooxygenase-2 specific inhibitors, MAP kinase inhibitors, nitric oxide synthase inhibitors, nuclear factor kB inhibitors, inhibitors of matrix metalloproteinases, cell adhesion molecules (including integrin agonists and integrin antagonists) that inhibit cartilage catabolism, intracellular signaling inhibitors (including protein kinase C inhibitors and protein tyrosine kinase inhibitors) that inhibit cartilage catabolism, and inhibitors of SH2 domains that inhibit cartilage catabolism.
  • IL-1 receptor antagonists TNF- ⁇ receptor antagonists
  • cyclooxygenase-2 specific inhibitors MAP kinase inhibitors
  • nitric oxide synthase inhibitors nuclear factor kB inhibitors
  • the at least one inhibitor of cartilage catabolism in the systemic anabolic agent/catabolic inhibitory agent ' combination may be a soluble receptor that inhibits cartilage catabolism, such as a soluble IL-1 receptor or a soluble tumor necrosis factor receptor.
  • a soluble receptor that inhibits cartilage catabolism such as a soluble IL-1 receptor or a soluble tumor necrosis factor receptor.
  • Specific examples include recombinant soluble human IL-1 receptors, soluble tumor necrosis factor receptors and chimeric rhTNFR:Fc.
  • Examples of soluble tumor necrosis factors useful for incorporation in the present invention include the functional TNF- ⁇ antagonists disclosed in U.S. Patent No. 5,605,690 issued to Jacobs et al., while examples of soluble human IL-1 receptors useful in the present invention include those disclosed in U.S. Patent No.
  • catabolic inhibitors useful for combination with anabolic agents for systemic delivery in accordance with this aspect of the present invention include IL-lra, TNFRl-IgGl fusion protein and inhibitors of matrix metalloproteinases.
  • the chondroprotective compositions may also include one or more inhibitors of pain and/or inflammation or other therapeutic agents.
  • Examples of chondroprotective compositions suitable for systemic delivery are provided in Examples 5 through 20 below.
  • systemic delivery and potentially anti-inflammatory and/or analgesic agents or other therapeutic agents, so as to provide therapeutic effect at multiple articular sites.
  • systemic delivery and “systemic administration” are intended to include but are not limited to oral, intramuscular, subcutaneous, intravenous, inhalational, sublingual, buccal, topical, transdermal, nasal, and other routes of administration that effectively result in dispersement of the delivered agent to a single or multiple sites of intended therapeutic action.
  • Preferred routes of systemic delivery for the present compositions are intravenous, intramuscular, subcutaneous and inhalational.
  • compositions of the present invention will be determined in part to account for the agent's susceptibility to metabolic transformation pathways associated with a given route of administration.
  • peptidergic agents may be most suitably administered by routes other than oral.
  • compositions of the present invention may be systemically administered on a periodic basis at intervals determined to maintain a desired level of therapeutic effect.
  • compositions may be administered, such as by subcutaneous injection, every two to four weeks.
  • the dosage regimen will be determined by the physician considering various factors that may influence the action of the combination of agents. These factors will include the cartilage site intended for treatment, the size of the joint (if appropriate), the amount of cartilage tissue to be treated, the site of cartilage damage, the condition of the damaged cartilage at the time of treatment, the patient's age, sex and weight, and other clinical factors.
  • the dosage for each individual agent will vary as a function of the other anabolic and catabolic inhibitory agents that are included in the composition, as well as the presence and nature of any drug delivery vehicle (e.g., a sustained release delivery vehicle).
  • the dosage quantity may be adjusted to account for variation in the frequency of administration and the pharmacokinetic behavior of the delivered agent(s).
  • Progress in the freatment of an individual can be monitored through a variety of methods known to those in the art, including clinical assessment, radiographic and magnetic resonance imaging, computed tomography, biochemical markers and arthroscopic evaluation. D. DELIVERY VEHICLES AND TARGETING
  • compositions for various routes of systemic administration are known and may be adapted for use with the present compositions.
  • the chondroprotective agents and, if included, inflammation and pain inhibitory agents or other therapeutic agents are suitably compounded in a physiologic carrier or delivery vehicle, such as those described previously, as appropriate for a given route of systemic administration.
  • systemic delivery of these combinations of agents, or any component or components thereof may be incorporated in or combined with a drag delivery vehicle such as a sustained release delivery vehicle and/or a depot.
  • delivery vehicle is intended to include all stractures that contain, couple to or carry a therapeutic agent, such as nanospheres and other nanoparticles, microspheres and other microparticles, micelles and liposomes, including such vehicles formed of proteins, lipids, carbohydrates, synthetic organic compounds or inorganic compounds.
  • a therapeutic agent such as nanospheres and other nanoparticles, microspheres and other microparticles, micelles and liposomes, including such vehicles formed of proteins, lipids, carbohydrates, synthetic organic compounds or inorganic compounds.
  • Preferred delivery vehicles for the targeted systemic compositions of the present invention, decribed further below, are “particles,” which is intended to include nanospheres and other nanoparticles, microspheres and other microparticles, micelles, and other delivery vehicles, but excluding liposomes which are less preferred as also described below.
  • delivery system is intended to refer to a delivery vehicle and one or more contained or coupled therapeutic agents.
  • sustained release system is intended to mean a delivery system that provides for extended, enhanced or regulated delivery, duration or availability of any or all of the incorporated agents.
  • sustained release systems include but are not limited to drag-containing microparticles, microspheres, nanoparticles, proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds and injectable hydrogels such as that disclosed by U.S. Patent Application Serial Number 09/861,182 to Jun Li et al., hereby expressly incorporated by reference.
  • Suitable sustained release systems are known for other pharmaceuticals and may be adapted in accordance with the present invention to deliver chondroprotective agents at a relatively consistent therapeutic level, thereby reducing side effects and providing a longer duration of action when compared to a bolus systemic delivery of agents.
  • doctoral as used herein is intended to mean a drag delivery system, delivered at the site of intended action or remote from the site of intended action, which provides a reservoir of therapeutic agents for sustained release.
  • the individual agents may be compounded in a homogeneous mixture, may be a mixture or administration of separate microparticles, microspheres, etc., or may be concurrently and separately administered.
  • a therapeutic strategy is to deliver the combination of agents in a delivery vehicle that is preferentially targeted to a site or sites in the body that contain cartilage, in particular joints.
  • a "targeted delivery vehicle” is a delivery vehicle that can be used for the systemic delivery of a drag and that is adapted such that a greater quantity of the drag reaches the joint or desired local site of action than would otherwise reach the joint or desired local site of action using a non-adapted delivery vehicle or in the absence of a delivery vehicle (i.e., the drug is preferentially localized to the joint because the targeted delivery vehicle preferentially associates with molecules, cells or anatomic structures of the joint).
  • a “targeted delivery system” is such a targeted delivery vehicle containing one or more therapeutic drags.
  • a “targeted drag” is intended to refer to a therapeutic agent that is directly linked or coupled to a targeting stracture.
  • a systemically administered drag that has a "preferential effect" at a joint or desired local site of action exhibits greater pharmacological activity at the joint or desired local site of action than at the majority of other sites in the body.
  • TARGETING RATIONALE In OA, there may be either a suppression of normal chondrocyte function or the constitutive inability of these cells to match the rate of repair with the increased rate of degradation of the matrix.
  • Various cytokines and inflammatory mediators have been shown to either create an imbalance in the synthetic function of the chondrocytes or, alternatively, to increase cartilage matrix catabolism by upregulating various matrix-degrading enzymes, including the matrix metalloproteinases (including collagenases).
  • matrix metalloproteinases including collagenases
  • cytokines, growth factors and other bioactive molecules are often associated with serious side effects. For example, pathological effects have been correlated with the administration of systemic TGF- ⁇ 1 and other factors.
  • systemic delivery of an unprotected (naked) polypeptide, often preferable for poly-joint arthropathies as previously noted, frequently is limited by problems with stability of protein agents due to rapid degradation and inactivation of the therapeutic protein in the circulation.
  • IL-1 pro-inflammatory cytokines
  • inducible genes e.g., NO synthase, COX-2, MMPs
  • these mediators and genes are frequently expressed in OA-affected chondrocytes.
  • it is a specialized microenvironment of the joint that defines a pathophysiological milieu that critically affects the state of articular cartilage.
  • This aspect of the invention provides a mechanism for targeting a systemically administered anabolic chondroprotective agent and/or a systemically administered catabolic inhibitory chondroprotective agent, and preferably both, to the joint for cartilage protection of the joint.
  • a preferred route of delivery is thus to target these protein factors to the site of action in the joint.
  • a safe method is needed for the delivery of these agents within the joints of patients in a sustained and localized manner.
  • a biodegradable drag delivery vehicle that both protects and stabilizes systemically administered anabolic factors and/or catabolic inhibiting factors while outside the joint, and that simultaneously provides a unique method to target the drug delivery vehicle to the joint, is also desired.
  • Cartilage anabolic growth factors are potent mediators that are secreted by the body locally within the joint in minute quantities to elicit local biological responses in articular cartilage. Under normal physiologic conditions, appropriate anabolic growth factors are produced by chondrocytes within cartilage and synoviocytes within other joint stractures in sufficient concentrations to serve as the necessary signal to maintain the cartilage in a healthy, stable state by influencing cartilage matrix metabolism.
  • a preferred targeted chondroprotective composition of the present invention includes at least one anabolic-promoting chondroprotective agent and/or at least one catabolic inhibitory chondroprotective agent, and preferably both an anabolic- promoting chondroprotective agent and a catabolic inhibitory chondroprotective agent, contained within a targeted delivery vehicle.
  • the targeted delivery vehicle preferably comprises particles, and most preferably nanoparticles, that encapsulate at least one, and preferably all, of the chondroprotective agents.
  • the particles are targeted to the joint by a targeting antibody or antibody fragment that is coupled to the particle, which antibody or fragment is specific for an antigenic determinant that is localized within the joint (i.e., preferentially expressed within the joint relative to most other locations within the body, preferably highly expressed within the joint, and more preferably that is restricted in expression to the joint).
  • the antibody-targeted particles also referred to herein as "targeted immunoparticles," and the chondroprotective agent(s) encapsulated therein are systemically administered. A portion of the targeted composition is taken up by the joint. The remainder of the composition is excreted and/or metabolized. Within the joint, the targeting antibodies or fragments bind to the targeted antigen.
  • the particles degrade within the joint, delivering a therapeutic concenfration of the chondroprotective agent(s) locally within the joint in a sustained release manner over a predetermined period of time to act locally on the cells to be modulated (e.g., the primary cells of the joint) including the synoviocytes and the chondrocytes.
  • the therapeutic agents may diffuse or be released into the synovial fluid to subsequently bind to the surfaces of the cells of the joint structures, undergo uptake or entry into cells of the joint stractures, or directly act on cytokines and/or proteases that may be present within the synovial fluid.
  • the targeted compositions of this aspect of the present invention can be targeted to the joints of a patient in accordance with the present invention, without knowledge of the specific molecular pathology that underlies the joint disease.
  • the targeting antibody ensures that the encapsulated agents are preferentially localized within the joint, and more preferably are localized in close proximity to or are bound to a constituent of the articular cartilage.
  • This aspect of the present invention thus provides a method to treat patients suffering from inflammatory, non-inflammatory or other joint diseases involving onr or multiple joints by administering a pharmaceutical preparation including a targeted drug delivery vehicle, preferably containing both a cartilage anabolic agent and an anti-catabolic agent.
  • OA OA
  • RA RA
  • other diseases of the joint such as noninflammatory and inflammatory arthrititides including, but not limited to, neuropathic arthropathy, acute rheumatic fever, ochronosis, systemic lupus erythematosus, juvenile rheumatic arthritis, psoriatic arthritis, ankylosing spondylitis, and other spondyloarthropathies and crystalline arthropathies.
  • the targeted treatment methods and compositions of . this aspect of the invention are particularly well suited for patients suffering from osteoarthritis.
  • the systemic delivery of the combination of agents in a carrier that is targeted to the joint enables treatment of such conditions while minimizing adverse or unwanted systemic effects.
  • the present invention provides methods and compositions for targeting drugs to the joint, and specifies preferred targets within the joint including antigenic determinants associated with molecules, cells and tissues of articular cartilage and other joint structures.
  • targets are selected from: collagens, including collagen Type II and the minor collagens Type V, VI, IX, X and XI; proteoglycans including large aggregating proteoglycans, aggrecan, decorin, biglycan, fibromodulin and lumican; Cartilage oligomeric matrix protein, glycoprotein-39; proteoglycan chondroitin-sulfate and glycosaminoglycans; macrophage synoviocytes and fibroblast synoviocytes; and chondrocytes.
  • collagens including collagen Type II and the minor collagens Type V, VI, IX, X and XI
  • proteoglycans including large aggregating proteoglycans, aggrecan, decorin, biglycan, fibromodulin and lumican
  • Cartilage oligomeric matrix protein glycoprotein-39
  • proteoglycan chondroitin-sulfate and glycosaminoglycans macrophage synoviocytes and fibroblast synoviocytes
  • the targeted immunoparticles react irreversibly, bind in a reversible manner, or associate with specific components of the articular cartilage (also known as hyaline cartilage) within the joint.
  • Other molecular targets within the joint may include components of the articular cartilage extracellular matrix, such as cartilage specific collagens, including collagen Type II, V, VI, IX, X, and XI, aggrecan and other small leucine-rich proteoglycans including decorin, biglycan, fibromodulin and lumican.
  • the proteoglycans are high molecular weight complexes of protein and polysaccharide and are found throughout the stractural tissues of vertebrates, such as cartilage, but also are present on cell surfaces.
  • Glycosaminoglycans are polymers of acidic disaccharides containing derivatives of the amino sugars glucosamine or galactosamine, and are useful targets.
  • Articular cartilage contains several genetically distinct types of collagen that are useful in the present invention as molecular targets to which immunoparticles, including corresponding antibodies, . can attach, thus permitting delivery of the encapsulated therapeutic agents to the site of antibody binding.
  • Type II collagen the primary collagen of articular cartilage, accounts for 90%) to 95% of the total collagen content of articular or hyaline cartilage and forms the cross-banded fibrillar structure noted by electron microscopy.
  • Type II collagen is also a unique and specific marker of hyaline cartilage. Hollander, et al., J. Clin. Invest. 93:1722 (1994); Freed, L et al., Exp. Cell Res. 240:58 (1998).
  • a major extracellular modification of the collagen molecules, which occurs after fibril formation, is the development of covalent interfibrillar cross-links. Antibodies that bind to epitopes specific to Type II collagen have been described. Kafienah, W.
  • Type II collagen and its associated epitopes in articular cartilage represent preferred targets for the present invention.
  • clone 6B3 a monoclonal antibody to Type II collagen, isotype IgG 1 ⁇ designated clone 6B3 (Linsenmayer, T.F. et al., Biochem. Biophys. Res. Commun. 92(2):440-6 (1980)) recognizes both ⁇ l(II) and ⁇ 3(XI) chains that have identical primary structures.
  • this mAb reacts with the TC A fragment of lathyritic Type II collagen after digestion with mammalian collagenase. It also reacts with pepsin-digested Type II collagen. Its epitope is localized in the triple helix of Type II collagen and it shows no cross-reaction with Type I or Type III collagen.
  • Immunoblotting of cyanogen bromide (CNBr) peptides of collagen II shows that this mAb reacts with the CB11 fragment, which is the site of immunogenic epitopes along the intact Type II molecule.
  • CBr cyanogen
  • a monoclonal antibody to Type II collagen was developed using human cartilage specific CNBr-cleaved collagen II as the immunogen.
  • This mAb available commercially through Chemicon International (Temecula, CA), reacts with both pepsin-solubilized and CNBr-cleaved human and bovine Type II collagen. No cross-reactivity is observed with collagen Types I, III, V and IX. .
  • the antibody to Type II collagen binds to the target epitope with a dissociation constant in the range of 0.1-10 nM.
  • the quantitatively minor collagens of articular cartilage also contribute to the stracture of the matrix and serve as useful targets for the present invention.
  • Type IX collagen a short nonfibrillar collagen (which contains a glycosaminoglycan chain and is therefore also considered a proteoglycan) binds covalently to Type II collagen fibrils and may help link fibrils together or bind fibrils to other matrix molecules.
  • Type XI collagen, a minor fibrillar collagen may be involved in controlling the diameter of the Type II fibrils.
  • Other collagens, including Type V and Type VI may also form part of the matrix.
  • These minor collagens of articular cartilage may also function as targets for antibody-directed immunoparticle binding with appropriate antibodies.
  • proteoglycans contained in articular cartilage are useful in the present invention as molecular targets for the binding of immunoparticles, thus permitting delivery of the therapeutic agents to the site of antibody binding.
  • proteoglycans constitute the second largest portion of the solid phase, accounting for 5%> to 10%> of the wet weight.
  • the proteoglycans of the cartilage matrix consist primarily of large aggregating (50% to 85%) and large nonaggregating (10% to 40%) proteoglycans. Distinct small proteoglycans are also present.
  • the proteoglycans of cartilage that contribute most significantly to the material properties of the tissue are the large, high-molecular weight monomers (molecular weight, 1-4 X 10 6 ).
  • the large proteoglycans consist of an extended protein core with several distinct regions: an N-terminal region with two globular domains (GI and G2), a domain rich in keratan sulfate; a longer domain rich in chondroitin sulfate that may also contain some interspersed keratan sulfate and neutral oligosaccharide chains; and a C-terminal globular domain, G3.
  • Aggregates are formed by many proteoglycan monomers binding to a chain of hyaluronate at the GI globular domain. Each proteoglycan-hyaluronate bond is stabilized by a separate globular link protein (molecular weight, 41,000 to 48,000).
  • the large size of the chondroitin sulfate-rich region (200-400 nm) and abundance of chains of the chondroitin-sulfate proteoglycan aggregate make this a preferred target for targeted immunoparticles of this aspect of the invention.
  • HC gp-39 An additional target for antibodies or fragments thereof that are coupled to immunoparticles of the present invention is provided by HC gp-39.
  • fragments of HC gp-39 that have appropriate antigenic properties also are sufficient for targeting the drag delivery vehicle.
  • Immunoparticles may be targeted with antibodies or fragments thereof to react irreversibly, bind in a reversible manner (most commonly), or associate with specific structures of the synovial membrane of the joint.
  • the specialized cells of the joint that are preferred targets include the two principal cell types of the synovium, macrophage synoviocytes (Type A) and fibroblast synovioctyes (Type B).
  • chondrocytes Additional targets for antibodies or fragments thereof that are coupled to immunoparticles of the present invention are chondrocytes. These cells are known to express a variety of proteins which are present on their surfaces and which can serve as epitopes for cellular targeting.
  • the chondroitin-sulfate proteoglycan associated with articular cartilage represents a preferred target for the targeted drag delivery system.
  • Monoclonal antibodies useful for the present mvention that bind to epitopes specific to chondroitin-sulfate proteoglycan have been described. Morgan Jr., A. et al., Hybridoma 1:27-36 (1981); Schrappe, M. et al., Cancer Res.
  • clone 9.2.27 mouse-anti-human chondroitin-sulfate proteoglycan monoclonal antibody
  • the 9.2.27 antibody recognizes the mature chondroitin sulfate proteoglycan core glycoprotein of 250 kDa as well as precursor polypeptides of 210, 220 and 240 kDa.
  • a mouse anti-human aggregan monoclonal antibody, clone 2A2.1 is also suitable for the present invention and is commercially available from United States Biological (Swampscott, MA).
  • Biomolecular constituents of cartilage that may be either absent from normal adult cartilage or present at very low levels, but that are elevated or more highly expressed in certain stages of either RA or OA, also may serve as -targets for the targeted drug delivery systems of the present invention.
  • Preferred targets associated with cartilage degenerative conditions are neoepitopes that appear on articular cartilage of patients diagnosed with OA, RA or other degenerative joint diseases, such as neoepitopes of aggrecan or other cartilage proteoglycans, and specifically neoepitopes that are immunolocalized in the superficial layer of articular cartilage of such patients.
  • the targeting antibodies or antibody fragments specifically bind to neoepitopes or cleavage sites of Type II collagen or Type II collagen fragments, particularly such neoepitopes or cleavage sites generated by the individual or combined action of matrix metalloproteinase (MMP)-l, 3, 8 or 13, or other members of the MMP protein family, or a member of the A Disintegrin And Metalloproteinase with Thrombospondin motifs' (ADMATS) protein family.
  • ADMATS are further described in Patent Applications WO 00/04917, EP 0 823 478 and U.S. 5,811,535 and in Tang, B. et al, FEBS Lett. 445 (2-3):223-225 (1999).
  • Antibodies directed at specific epitopes that are defined by specified regions of the Type II collagen stracture are useful for targeting compositions of this invention. These stractural regions are, in part, important in cartilage affected by degradation of Type II collagen that occurs in OA and RA. Degradation of Type II collagen results in fragments of the collagen protein that are released from the cartilage matrix and appear in the synovial fluid, move into the circulation, and are eliminated through the urine. To have maximal utility, compositions of the present invention target epitopes that remain physically associated with the cartilage matrix rather than on released fragments.
  • Each of the collagenases, MMP-1 (EC 3.4.24.7), MMP-8 (EC 3.4.24.34), and MMP- 13 (EC 3.4.24.-) has the capacity to cleave triple-helical fibril-forming Type II collagen, giving rise to a large (3/4-length) amino-terminal fragment and a smaller (1/4-length) carboxy-terminal fragment.
  • Specific collagenase isotypes have been implicated in the pathologic loss of cartilage. Billinghurst, R. et al., J. Clin.
  • Collagenase 3 (MMP-13) cleaves collagen Type II at a rate that is 5-10 times faster than collagenase 1.
  • MMP- 13 has been identified in the syonvium of humans with RA and OA, as well as in human OA cartilage. Fibrillar collagen can be damaged by helical cleavage, resulting in denaturation, or by telopeptide cleavage, leading to removal of cross-links.
  • neoepitopes will be located within the N-terminal 3/4 fragment sequence of the alphal(II) chain, which is known to contain epitopes to AH12L3 and CBl IB (recognized by antibody COL2- 3/4m).
  • Another aspect of this invention is the use of such monoclonal antibodies or fragments thereof that bind to neoepitopes on aggrecan or aggrecan fragments to target nanoparticles containing a combination of an anabolic promoting agent and a catabolic inhibitor.
  • the targeting antibodies or antibody fragments specifically bind to neoepitopes or cleavage sites of aggrecan or aggrecan fragments within cartilage, particularly such neoepitopes or cleavage sites generated by the individual or combined action of MMP-1, 3, 8 or 13, or other members of the MMP protein family or a member of the ADMATS protein family.
  • monoclonal antibodies that recognize different stractural epitopes or neoepitopes within aggrecan or aggrecan fragments are 8-A-4 or BC-3.
  • MAb 2-B-.6 has been used to detect the large number of aggrecan degradation products that result from either aggrecanase, MMP or other proteolytic activities at many sites along the core protein of aggrecan.
  • MAb 2-B-6 recognizes 4-sulphated unsaturated dissaccharides of chondroitin sulphate that are attached to these core protein fragments.
  • a related antibody, MAb 3-8-3 has also been used to identify different deglycosylated aggrecan metabolites containing 6-sulphated chondroiten sulfate oligosaccharides.
  • MAb BC-3 recognizes the N-terminal neoepitope sequence defined by the amino acid sequence, alanine-arginine, glycine (ARGxx%) generated after aggrecanase catabolism within the IGD domain of aggrecan.
  • the ADAMTS-4 gene (Genbank NM-005099) and ADAMTS-5 gene (Genbank 007038) encode a disintegrin and a metalloproteinase with thrombospondin motifs-4 and 5, which are members of the ADAMTS protein family.
  • Members of the family share several distinct protein modules, including a propeptide region, a metalloproteinase domain, a disintegrin-like domain, and a thrombospondin Type 1 (TS) motif.
  • Individual members of this family differ in the number of C- terminal TS motifs, and some have unique C-terminal domains.
  • the enzyme encoded by the ADAMTS-4 gene lacks a C-terminal TS motif.
  • the enzyme encoded by the ADAMTS-5 gene contains 2 C-terminal TS motifs and functions as aggrecanase to cleave aggrecan, a major proteoglycan of cartilage. Thus, both of these enzymes are responsible for the degradation of aggrecan and the generation of neoepitopes on aggrecan and aggrecan fragments (Tortorella, M., et al., J. Biol. Chem. 275(33):25791-25797 (2000); Tortorella, M. et al, J. Biol. Chem. 275(24): 18566-18573 (2000); Abbaszade, I. et al, J. Biol. Chem. 274(33):23443- 23450 (1999)).
  • an antibody for treatment of human cartilage in the setting of OA, an antibody is used that targets the early biochemical neoepitope marker of OA, termed 3-B-3(-).
  • the 3-B-3(-) epitope is an OA-related phenotypic change in the termini of the chondroitin sulfate (glycosaminoglycan) chains of aggrecan.
  • the term "antibody” is intended to include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules (i.e., molecules that contain an antigen binding site that specifically binds or immunoreacts with an antigen).
  • the term describes an immunoglobulin, whether it is naturally or synthetically produced.
  • the proteins comprising the antibody can be derived from natural sources, or synthetically produced either in part or in whole. Examples of antibodies include all immunoglobulin subtypes and Fab and F(ab') 2 , scFv, Fv, dAb, Fd fragments, as well as fragments disclosed in U.S. Patent 5,534,254.
  • ScFV refers to single-chain minimum binding domains of an immunoglobulin molecule.
  • antibody is also intended to refer to an antibody that functions in the extracellular space, within the plasma membrane of a cell, or in an intracellular region of a cell (e.g., the cytoplasm or nucleus) to modulate the expression or activity of one or more genes that regulate cartilage metabolism.
  • Preferred antibodies for use in the present invention include humanized, chimeric and human monoclonal antibodies.
  • fragment refers to any " sequence of amino acids that is part of any targeted polypeptide defined above, having common relevant elements of origin, stracture and mechanism and functional equivalence to the whole antigen for purposes of targeting within the present invention.
  • the calling out of an antibody in the compositions and methods described herein is also intended to include the use of fragments of such antibodies.
  • a preferred embodiment of the present invention employs humanized or human antibodies or fragments thereof that are covalently attached to the surface of the nanospheres or other particles in which the therapeutic chondroprotective agents of the present invention are encapsulated.
  • Antibodies or fragments thereof are preferred as the targeting molecules on the surface of particles in which therapeutic chondroprotective agents are encapsulated, because they must be sufficiently stable in vivo and exhibit a minimum potential of being removed from the surface of the particle by serum containing extracellular fluid proteins. It is envisioned that fully human monoclonal antibodies or humanized murine antibodies, which bind to any molecules of the cartilage extracellular matrix or to cells of the joint, will be most useful as the type of joint-targeting antibodies that direct the delivery of the therapeutic agent(s) to be administered to human patients because they will not generate an immune response upon administration.
  • a murine monoclonal antibody may be chimerized by genetically recombining the nucleotide sequence encoding the murine Fv region (i.e., containing the antigen binding sites) with the nucleotide sequence encoding a human constant domain region and an Fc region. Some murine residues may also be retained within the human variable-region framework domains to ensure proper target-site binding characteristics.
  • Humanized antibodies for use in targeting will be recognized to have the advantage of decreasing the immunoreactivity of the antibody or polypeptide in the host recipient, and may be useful for increasing the in vivo half- life and reducing the possibility of adverse immune reactions to the conjugated antibody on the surface of the nanoparticle or other encapsulating particle.
  • a preferred aspect of the targeted embodiment of the present invention will include at least one chondroprotective agent that is encapsulated or contained within a nanoparticle or other delivery vehicle to which a targeting antibody, antibody fragment or other targeting structure is attached.
  • the encapsulated or contained agent may be any one of anabolic chondroprotective agents or catabolic inhibitory agents disclosed herein having chemical or structural characteristics rendering it amenable to encapsulation or containment in a selected nanoparticle or other delivery vehicle.
  • agents that would otherwise be highly susceptible to metabolic degradation from systemic administration e.g., proteins and peptides
  • that cause harmful side effects if administered systemically without targeting are preferable for targeting.
  • each of the classes of anabolic chondroprotective agents disclosed herein below (including members of the transforming growth factor (TGF)- ⁇ superfamily, including TGF- ⁇ agonists and bone morphogenetic protein agonists, insulin-like growth factors and fibroblast growth factors) and certain of the classes of catabolic inhibitory agents disclosed herein below (Interleukin-1 receptor antagonists and TNF- ⁇ receptor antagonists) are proteins, and as such are well suited for delivery in targeted encapsulated form.
  • a most preferred targeted encapsulated composition of the present invention will include both an anabolic chondroprotective agent and a catabolic inhibitory chondroprotective agent encapsulated in the same targeted immunoparticles (or other targeted particles), preferably both agents being proteins.
  • each agent may be separately encapsulated, and an admixture of the two (or more than two) types of targeted particles may be administered, or less preferably the two or more targeted agents may be delivered separately, either concurrently or sequentially, to result in coincident presence of the agents within the joint.
  • anabolic chondroprotective agent or the catabolic inhibitory agent may be amenable to encapsulation, or one agent may not be associated with undesirable systemic side effects.
  • one agent may be delivered in encapsulated targeted form, while the other agent is delivered in ' non-targeted, non-encapsulated form, either together in the same dosage form as an admixture, or separately.
  • the size of a substance is a major factor determining whether it can permeate the wall of synovial capillaries and move from the systemic circulation into the joint.
  • the maximum diameter of particles that can move across the synovial capillary wall is generally regarded to be 50 nanometers.
  • some studies of the permeability of the synovial capillaries of the rat using lecitihin-coated polystyrene particles up to 240 nanometers in diameter can be transported across synovial capillary walls via transcytosis.
  • the present invention overcomes the limitation imposed by the synovial permeability barrier by preferably employing a class of encapsulation particles (e.g., nanoparticles (preferably nanospheres)), which are constrained in size distribution.
  • Sustained release dosage forms of the invention may comprise microparticles and/or nanoparticles having therapeutic agents dispersed therein, or may comprise the therapeutic agent in pure, preferably crystalline, solid form.
  • the therapeutic dosage forms of this aspect of the present invention may be of any configuration suitable for sustained release.
  • Preferred sustained release therapeutic dosage forms of the present invention will have the following size, biodegradation and biocompatibility characteristics.
  • the targeted delivery system of the present invention preferably utilizes nanoparticles that are limited in size from about 5 nanometers to about 750 nanometers in diameter, with about 10 to about 500 nanometers being more preferred, most preferably from about 20 to about 200 nanometers.
  • Preferred particles are biodegradable structures that biodegrade and release loaded drug at therapeutic levels over a period of time preferably between from about 1 to about 150 days, preferably from about 7 to about 60 days, with from about 14 to about 30 days being more preferred. It is understood by those in the art that drag release from nanoparticles and microparticles may occur by a combination of physical processes, which include, but are not limited to, diffusion and degradation and may be described by complex kinetic processes that are unique to each carrier formulation and combination of anabolic and anti-catabolic therapeutic agents. Preferred particles are biocompatible with targeted tissues of the joint and the local physiological environment into which the dosage form is administered, including yielding biocompatible biodegradation products.
  • Suitable compositions include biodegradable particles formulated from natural polymers, including hyaluronan, chitosan, collagen, gelatin and alginate. These natural polymers may be combined with other polymers to produce copolymer particles composed of, for example, chitosan and gelatin. Synthetic biodegradable poly(alpha-hydroxy esters) such as polylactic acid (PLLA), polyglycolic acid (PGA) and the copolymer PLGA have been used successfully for the production of microparticles that incorporate protein therapeutics, such as human growth hormone.
  • PLLA polylactic acid
  • PGA polyglycolic acid
  • the use of targeted, biodegradable polymeric nanospheres has the advantage of providing selected differential release profiles for the encapsulated therapeutic agents.
  • the optimal release kinetics may consist of a dual-release process, wherein each active agent demonstrates a different sustained release kinetic profile to provide the most optimal drug pharmacokinetics within the joint.
  • the optimal release kinetics from the nanoparticles or microparticles will vary for each individual drag, and will also be a function of the amount of drag loaded into particles during formulation, the size of the particles, and other physiochemical properties that are determined by the composition of the particles.
  • Quantitative release rates for each drag from the encapsulating particles resident in the joint will be adjusted to obtain the optimal therapeutic concentration in the synovial fluid and intra-articular space to achieve the desired therapeutic concentrations.
  • in vitro studies can be conducted to characterize the dual-release kinetics for the sustained release formulation in which each component (e.g., the anabolic drug and the catabolic inhibitor) demonstrate sustained release over a period of 7-30 days, by way of example.
  • Methods to quantitate the amount of each drug released into the synovial fluid are well known in the art, and may include measurements of radioactively labeled drug. Alternatively, it is possible to covalently attach fluorescent or other optical reporter molecules to prepare labeled drugs.
  • ELISAs or mass-specfrometry which are specific to each agent.
  • anabolic agent such as human IL-10, which is 160 amino acids in length and has a MW of approximately 18 Kda.
  • Independent control of the sustained release rates for each agent can be achieved by varying the stractural composition of the particles and/or by creating an admixture of two or more immunoparticles.
  • one set of particles is homogeneous with respect to the encapsulated anabolic agent, while a distinct set of particles is homogeneous with respect to the encapsulated catabolic inhibitor.
  • the two sets of admixed particles may vary in their respective sizes and polymeric composition, but will be characterized, if appropriate, by similar release rates for their active agents, or by release rates that are consistent, optimizing the local therapeutic effects of each of the encapsulated agents, respectively.
  • Liposomes are not preferred delivery vehicles for the targeted systemic delivery of chondroprotective agents in accordance with the present invention. Relative to nanospheres and other sustained release particle delivery systems, liposomes have a short half-life within the circulatory system. Liposome drag conjugates may be trapped in the liver and spleen, resulting in liposomal breakdown and release of the active agents. The agents are thus distributed systemically in the active state rather than being protected until localized within the joint. The release of agents from targeted liposomal delivery systems is not highly sustained and is much less localized than for targeted immunoparticle delivery systems. For these reasons, the use of particles (e.g., nanospheres) is highly preferred relative to the use of liposomes. None the less, for systemically delivered anabolic chondroprotective agents, or combinations of chondroprotective agents including an anabolic agent, for which targeting is highly desired, targeted liposomes may prove to be suitable and offer advantages relative to naked drag delivery.
  • Representative "coupling" methods for linking the targeting antibody to the sustained release nanoparticle through covalent or non-covalent bonds include chemical cross-linkers and heterobifunctional cross-linking compounds (i.e., "linkers") that react to form a bond between reactive groups (such as hydroxyl, amino, amido, or sulfhydryl groups) of the targeting antibody and other reactive groups (of a similar chemical nature) that are present on the surface of the nanoparticle or other targeting vehicle.
  • This bond formed between the targeting antibody and the particle or other delivery vehicle may include but is not limited to the following: a peptide bond, a disulfide bond, a thioester bond, an amide bond and a thioether bond
  • Direct conjugation of sustained release dosage forms to the targeting protein may disrupt recognition of the targeted molecule or cell by the modified targeting antibody.
  • Ligand sandwich attachment techniques are useful alternatives to achieve attachment of the sustained release dosage form to the targeting binding proteins (antibodies). These techniques may involve the formation of a primary peptide or protein shell using a protein that does not bind to the target cell population. Binding protein is then bound to the primary peptide or protein shell to provide the resultant particle with functional binding protein peptide.
  • An exemplary ligand sandwich approach involves covalent attachment of avidin or streptavidin to the particles through functional groups as described above with respect to the "direct" binding approach.
  • the binding protein is derivatized, preferably minimally, via functionalized biotin (e.g., through active ester, hydrazide, iodoacetal, maleimidyl or like functional groups).
  • Ligand i.e., binding peptide or protein/functionalized biotin attachment to the available biotin binding sites of the avidin/streptavidin primary prptein shell occurs through the use of a saturating amount of biotinylated protein/peptide.
  • EFFECT DELIVERY METHODS Other methods of targeting the chondroprotective agents and combinations thereof in the present invention, or in achieving preferential effectiveness of such agents or combinations at the joint relative to other sites of the body, are also within the scope of the present invention.
  • the two principle cell types of the synovium, macrophage synoviocytes (Type A) and fibroblast synoviocytes (Type B), and chondrocytes are known to express a variety of unique proteins that are present on the surfaces of these cells, and that could serve as epitopes for specific cellular targeting.
  • inflamed or diseased joint may preferentially increase local effect while minimizing unwanted systemic effects.
  • molecular targets within the joint may include components of the cartilage extracellular matrix, such as cartilage specific collagens, including collagen Type II, IX, X, XI, aggrecan and other small leucine-rich proteoglycans (e.g., decorin, biglycan, fibromodulin and lumican). Also included as targets are cartilage oligomeric matrix protein (COMP) and glycoprotein-39 (HC- gp39), also termed YKL-40.
  • a desirable target for use in this aspect of the invention would be a biochemical cartilage marker that may be either absent from normal adult cartilage, or present at very low levels, but that is present in certain stages of either RA or OA.
  • selection of chondroprotective agents may also preferentially increase local effect relative to other areas of the body.
  • an encapsulated agent or agents may be targeted to one or more stractures within the joint by coupling the encapsulated agent to a corresponding targeting antibody (or antibody fragment). Potentially such antibodies may also be coupled to the naked drugs themselves, using a linkage that is cleaved within the local environment of the joint to achieve targeting and delivery of the systemically admimstered drags to the joint.
  • the chondroprotective agents that are included in an anabolic-promoting and catabolic inhibitory composition of the present invention may be selected such that they preferentially act on sites within the joint, relative to the rest of the body, thereby preferentially exerting their effects on synoviocytes and/or chondrocytes and/or components of the extracellular matrix.
  • the specific nanosphere system and targeting antibody or fragment to be used in accordance with the present invention to deliver selected agents, and the precise loading or dosage of therapeutic agents to include in the targeted compositions, may be determined analytically in accordance with the invention.
  • the analytical method includes administering an antibody-labeled nanosphere (or other targeted particle) containing encapsulated therapeutic agents to a patient in need of such a diagnostic test, and subjecting the patient to imaging analysis to determine the location of the drug-containing nanospheres. The extent of deposition in the joint of a patient may then be determined by using imaging analysis.
  • Imaging analysis is well known in the medical art, and includes, without limitation, x-ray analysis, magnetic resonance imaging (MRI) or computed tomography (CT).
  • the joint- targeting antibodies may be labeled with a detectable agent that can be imaged in a patient.
  • the targeting antibody may be labeled with a contrast agent, such as barium, which can be used for x-ray analysis, or a magnetic contrast agent, such as a gadolinium chelate, which can be detected using MRI or CT.
  • a contrast agent such as barium, which can be used for x-ray analysis
  • a magnetic contrast agent such as a gadolinium chelate
  • Other labeling agents include, without limitation, radioisotopes, such as 99 Tc.
  • a biopsy can be obtained from the patient to determine the presence and concentration of the particles in the joint (e.g., synovial tissues).
  • the local delivery embodiment of the present invention was described in terms of suitable concentrations for the therapeutic agents when delivered locally, sufficient to provide a predetermined level of inhibitory or therapeutic effect at the local delivery site (e.g., the joint).
  • a greater concentration or dosage of the agents will need to be administered.
  • This systemic dosage and/or concenfration is that which is required to result, after any metabolic transformation processes, in the supply of sufficient active agent(s) at the desired site(s) of potential cartilage degradation necessary to achieve a desired level of local therapeutic effect.
  • suitable therapeutic and preferred levels for systemic dosages and/or concentrations are those which result in the supply of active agents at the local site (e.g., the joint) at a concentration level that is within the local delivery therapeutic and preferred concentration ranges, respectively, previously described.
  • a sufficient dosage or load of the agent is included in the composition to result in a local concentration at the joint or site of action over a predetermined sustained release period that achieves a desired level of therapeutic effect over the substantial duration of the desired sustained release period.
  • a sufficient dosage or load of the agent is included to result in a predetermined amount of the encapsulated agent that is taken up by the joint, accounting for any metabolic transformation of the agent that occurs before reaching the joint or in the local environment of the joint.
  • This predetermined amount of encapsulated agent reaching the joint will be determined in accordance with the disclosure contained herein such that as the nanosphere or other encapsulating delivery system degrades, agent is released at the local site of action to provide a local concentration that is within the therapeutic concentration range for that agent during a desired period of sustained release (e.g., over a period of 1 day to 4 weeks, more preferably between 1 day and 2 weeks).
  • a desired period of sustained release e.g., over a period of 1 day to 4 weeks, more preferably between 1 day and 2 weeks.
  • exemplary classes of chondroprotective agents and exemplary drugs within each class, that are suitable for use in the compositions of the present invention. While not wishing to be limited by theory, the justification behind the selection of the various classes of agents that is believed to render the agents operative is also set forth.
  • the interleukin IL-1 exists in two forms, IL-l ⁇ and IL-l ⁇ , which are polypeptides derived from separate gene products that share a similar spectrum of immunoregulatory and pro-inflammatory functions.
  • IL-1 is a 17 kD polypeptide that can both act upon and be produced by a number of cell types in the joint, including synovial fibroblasts and macrophages, chondrocytes, endothelial. cells and monocytes and macrophages.
  • synovial fibroblasts and macrophages include synovial fibroblasts and macrophages, chondrocytes, endothelial. cells and monocytes and macrophages.
  • IL-1 plays a pivotal role in joint inflammation and in the pathophysiological loss of articular cartilage that occurs in the injured joint.
  • IL-1 receptors IL-1 receptors
  • Type I IL-1 or Type II IL-1 receptors IL-1 receptors
  • IL-1 receptors are structurally distinct and belong to a separate superfamily characterized by the presence of immunoglobulin binding domains. These receptors bear close amino acid homology with other receptors containing immunoglobulin domains. Expression of the larger Type I IL-1 receptor is present on T cells and fibroblasts while the smaller Type II IL-1 receptor is present on B cells, monocytes, neutrophils, " and bone marrow cells.
  • Type II IL-1 receptors bind IL-l ⁇ with high affinity, but IL-l ⁇ binding does not initiate intracellular signal transduction as it does upon binding to the Type I IL-1 receptor.
  • the Type II receptor serves as a precursor for a soluble IL-1 binding factor that has been shown to be shed from cells and this soluble receptor acts as a physiological IL-l ⁇ antagonist.
  • a naturally occurring IL-1 binding protein has been described which corresponds to the soluble external portion of the Type II receptor.
  • IL-1 receptor antagonist sIL-IRA, IL-lRa, IL-lra
  • IL-lRa IL-1 receptor antagonist
  • IL-lRa competitively inhibits the binding of IL-l ⁇ and IL-l ⁇ to both Type I and II IL-1 receptors.
  • IL-lRa is a pure receptor antagonist since its binding to the receptor does not activate the cellular signal fransduction machinery of membrane associated IL-1 receptors.
  • IL-lRa Despite high affinity binding of this protein to the IL-IRs, a 10-100 fold molar excess is required to inhibit IL-1 biological responses of cells that express the Type I IL-1R.
  • Cells known to produce IL-lRa include monocytes, neutrophils, macrophages, synoviocytes and chondrocytes.
  • IL-lRa has been shown to inhibit PGE 2 synthesis, induction of pro-inflammatory cytokines and MMPs, and nitric oxide production. Secreted IL-lRa is released in vivo during experimentally induced inflammation. Importantly, IL-lRa is expressed in synovial tissue and is present in normal human synovial fluid.
  • IL-lRa In patients with knee injuries, levels of IL-lRa in the synovial fluid dramatically increase in the acute phase after injury, and subsequently decrease to below normal levels in sub-acute and chronic states. Thus, the IL-lRa has been shown to play a physiological role in responses of the joint to injury.
  • IL-1 is considered the dominant cartilage destructive cytokine that plays a pivotal role in joint destruction due to its ability to stimulate the production of degradative enzymes and pro-inflammatory cytokines by both chondrocytes and synoviocytes.
  • IL-l ⁇ is a potent inhibitor of proteoglycan and collagen synthesis by chondrocytes.
  • IL-l ⁇ -induced responses of synovial fibroblasts include increased production of PGE2, collagenase and other neutral proteases and the upregulation of pro-inflammatory cytokines, IL-6 and IL-8.
  • IL-1 which is present in the joint fluid of patients with arthritic diseases, stimulates chondrocytes to: 1) synthesize elevated amounts of enzymes such as stromelysin, fibroblast and neutrophil collagenase and plasminogen activator, and 2) inhibit synthesis of plasminogen activator inhibitor- 1 and TIMP.
  • IL-l ⁇ is a potent inhibitor of the synthesis of matrix constituents such as Type II collagen, the predominant form of collagen in articular cartilage, and proteoglycans.
  • the imbalance between the levels of inhibitors and proteases leads to an increase in the amount of active proteases. This increase, combined with a suppression of matrix biosynthesis, results in degradation of cartilage.
  • injection of IL-1 into rabbit knee joints causes depletion of proteoglycan from the articular cartilage.
  • IL-1 is one of the key cytokines involved in the pathogenesis of chronic synovitis and cartilage degradation
  • reducing its production or blocking its action represents an appropriate strategy for new treatments to reduce synovial inflammation and to provide a chondroprotective effect.
  • a variety of therapeutic approaches for antagonizing the interaction of the agonist, IL-1, with its natural membrane bound receptor can be utilized which include: 1) naturally occurring specific inhibitors of IL-1 activity that have been characterized to date, including IL- lRa and soluble IL-1 receptors; 2) anti-IL-1 Abs; and 3) small molecule antagonists which may be either peptidic or nonpetidic.
  • IL-1 receptor antagonist should block the propagation of the inflammatory response by IL-1 and thereby interrupt the disease process.
  • the therapeutic potential of a number of IL-1 receptor antagonists have been established in animal models of inflammation and arthritis (RA and OA). Patients suffering from RA have improved clinically following a subcutaneous injection of IL-lRa or an intra-articular injection of soluble Type I IL-1R.
  • IL-l ⁇ and IL-lRa depend on their respective local concentrations.
  • IL-l ⁇ levels were threefold higher than those of IL-lRa.
  • the spontaneous local production of IL- lRa is not sufficient to inhibit IL-l ⁇ effects because a larger (10 to 100-fold) molar excess of IL-lRa is required to inhibit IL-1 -induced biological responses in cells that express Type I IL-1R.
  • high doses of IL-lRa have been used in vivo to block IL-1 in human volunteers in patients with RA.
  • IL-lRa present locally in the synovium provides a negative signal, down-regulating at least part of the IL-1- mediated processes in synovitis, such as leukocyte accumulation in the inflamed tissue, PGE 2 production and collagenase production by synovial cells.
  • a chondroprotective effect of IL-lRa has been demonstrated using direct injection of IL-lRa into the joint in a canine ACL model and by employing a gene therapy approach based upon transfection of the IL-lRa gene into human synovial fibroblasts.
  • the present invention discloses local and systemic delivery of an IL-1 soluble receptor protein, which is comprised of an extracellular domain of a IL-1R, and which is capable of binding an IL-1 cytokine molecule in solution.
  • a soluble human IL-1 receptor (shuIL-lR) polypeptide comprising essentially the amino acid sequence 1-312, as disclosed within U.S. Patent No. 5,319,071 and U.S. Patent No. 5,726,148, is disclosed herein for use in combination with one or more drags chosen from either an anti-inflammatory class, anti-pain class, or chondroprotective class.
  • the local or systemic delivery of a fusion protein consisting of the sIL-lR binding domain polypeptide is proposed for use to promote chondroprotection, as disclosed in U.S. Patent 5,319,071.
  • the local or systemic delivery of an IL-1 receptor antagonist as disclosed within U.S. Patent 5,817,306 is disclosed for use in the present invention.
  • the shuIL-lR soluble receptor has been shown to bind IL-1 with nanomolar affinity.
  • Local delivery of a therapeutically effective concentration of an IL-1R soluble receptor may occur by direct injection of the joint or in an irrigation solution (e.g., during an arthroscopic surgical procedure) in combination with one or more chondroprotective drugs, anti-inflammatory drags, or anti-pain drags and is disclosed herein as a cartilage protective agent when applied locally to tissues of the joint in a variety of inflammatory or pathophysiological conditions.
  • a cartilage protective agent when applied locally to tissues of the joint in a variety of inflammatory or pathophysiological conditions.
  • such agents may be delivered systemically, such as in a targeted systemic delivery system.
  • Such treatment will preemptively inhibit IL-1 stimulation of production of collagenase- 1 and stromelysin- 1.
  • AF11567 is a truncated version of AF12198, lacking the 4 C- terminal residues and exhibiting slightly lower affinity for the Type I IL-1 receptor but possessing a similar plasma half-life of 2.3-2.6 hrs. Poor solubility and rapid metabolism appeared to limit the in vivo efficacy of AF 12198 when administered systemically via intravenous infusion. These limitations are in part overcome through direct, local delivery methods such as injection into the intra-articular joint space or by inclusion in the surgical irrigation fluid or other infusion, or through systemic delivery using targeted delivery vehicles, as described above. Examples of IL-1 receptor antagonist agents suitable for the present invention are listed below.
  • the optimal dose and/or concentration of each suitable agent is that which is therapeutically effective.
  • the preferred and most preferred concentrations of an irrigation solution containing the listed agent are provided. Such concentrations are expected to be therapeutically effective.
  • systemic compositions in accordance with the present invention will suitably include a dosage or load of the agent sufficient to result in a local concenfration at the joint or site of action within the listed therapeutic range.
  • a sufficient dosage or load of the agent is included in the composition to result in a local concentration at the joint or site of action within the listed therapeutic range over a predetermined sustained release period.
  • TNF- ⁇ a cytokine mainly produced by activated macrophages
  • TNF- ⁇ has many biological actions including transcriptional regulation of several genes that are mediated by specific TNF receptors, as well as immunoregulatory activities.
  • TNF-R1 and TNF-R2 two different receptors termed TNF-R1 and TNF-R2 were cloned and characterized and also found to be produced as soluble receptors.
  • TNFR-II transmembrane protein of 439 amino acids and has an apparent molecular weight of 75kDa.
  • the second receptor type termed TNFR-I (or Type B or 55 kDa) shows an apparent molecular weight of 55 kDa and encodes a transmembrane protein of 426 amino acids.
  • TNFRl contains an intracellular domain that can initiate signaling through the NF- ⁇ B pathway.
  • sTNFR Soluble TNF receptors
  • TNF BPI Soluble TNF receptors
  • TNF BPII sTNFRII
  • TNF- ⁇ plays a central role in the sequence of cellular and molecular events underlying the inflammatory response and cartilage destruction. Many of the effects of TNF- ⁇ overlap with the pro-inflammatory effects of IL-1. Among the pro- inflammatory actions of TNF- ⁇ is its stimulation of the release of other pro- inflammatory cytokines including IL-1, IL-6 and IL-8. TNF- ⁇ also induces the release of matrix metalloproteinases from neutrophils, fibroblasts and chondrocytes that degrade cartilage, in part through the stimulation of collagenase. Furthermore, TNF- ⁇ upregulates COX-2 in normal human articular chondrocytes and synovial fibroblasts, resulting in increased PGE2 production.
  • This cytokine along with IL-1, is considered to initiate and produce pathological effects on cartilage in the joint, including leukocyte infiltration, synovial hyperplasia, synovial cell activation, cartilage breakdown and inhibition of cartilage matrix synthesis.
  • increased levels of TNF- ⁇ are found in synovial fluid of joints and increased production of TNF- ⁇ by synovial cells occurs.
  • systemic delivery including in a targeted delivery system, or local delivery of a soluble TNF- ⁇ receptor in an irrigation solution, infusion, or injection will bind free TNF- ⁇ and function as an antagonist of TNF receptors in the surrounding tissue, thus, providing a cartilage protective effect.
  • the present invention describes the use of functional antagonists of TNF- ⁇ that act extracellularly to block interaction of the ligand with their cognate membrane receptors either by scavenging of available free ligand or by direct competitive interaction with the receptor itself, alone or in combination with other agents to provide a chondroprotective effect.
  • a variety of therapeutic approaches for antagonizing the interaction of the agonist, TNF- ⁇ , with its natural membrane bound receptor can be utilized which include: 1) the use of naturally occurring specific inhibitors of TNF- ⁇ activity that have been characterized to date, including soluble TNF- ⁇ receptors; 2) the use of anti- TNF- ⁇ antibodies and 3) the use of small molecule antagonists which may be either peptidic or nonpeptidic.
  • the present invention discloses the use of a chimeric soluble receptor (CSR) protein, in which the extracellular domain of a TNF receptor, which possesses binding activity for a TNF molecule, is covalently linked to a domain of an IgG molecule.
  • CSR chimeric soluble receptor
  • a chimeric polypeptide comprising the extracellular domain of the TNF receptor extracellular polypeptide coupled to the CH2 and CH3 regions of a mouse IgGl heavy chain polypeptide could be used, as disclosed in U.S. Patent No. 5,447,851.
  • the chimeric TNF soluble receptor also termed the "chimeric TNF inhibitor" in U.S. Patent No.
  • a second example is a chimeric fusion construct comprised of the ligand binding domain of the TNF receptor with portions of the Fc antibody (termed Fc fusion soluble receptors) that have been created for TNF- ⁇ receptors.
  • the present invention also discloses the use of a soluble TNF receptor: Fc fusion protein, or any modified forms, as disclosed in U.S. Patent No. 5,605,690.
  • the molecular form of the active soluble receptor fusion protein can be either monomeric or dimeric.
  • soluble receptors includes, but is not limited to: (1) soluble receptors which correspond to naturally (endogenous) produced amino acid sequences or soluble fragments thereof consisting of an extracellular domain of full- length membrane receptor, (2) recombinant soluble receptors which are truncated or partial sequences of the full length, naturally occurring receptor amino acid sequences which retain the ability to bind cognate ligand and retain biological activity and analogs thereof, and (3) chimeric soluble receptors which are recombinant soluble receptors comprised of truncated or partial sequences corresponding to a portion of the extracellular binding domain of the full length receptor amino acid sequences attached through oligomers (e.g., amino acids) to a sequence corresponding to a portion of an IgG polypeptide (e.g., IgG hinge and Fc domain) which retain biological activity and the ability to bind cognate ligand.
  • oligomers e.g., amino acids
  • Soluble, extracellular ligand-binding domains of cytokine receptors occur naturally in body fluids and are thought to be involved in the regulation of the biologic activities of cytokines.
  • the naturally occurring existence of soluble, truncated forms of a number of hematopoietic cytokine receptors has been reported (IL-1R, IL-4R, IL-6R, TNFR).
  • soluble TNFR is found at U541PC1 '
  • these cytokine binding proteins arise as a result of alternative splicing of the mRNA for the complete receptor sequence (membrane- bound form) or as a result of proteolytic cleavage and release of the membrane- bound form of the receptor.
  • these soluble truncated receptors appear to act as physiological antagonists of their complementary endogenous cytokines. This antagonism occurs because (1) scavenging of the free ligand through binding to its cognate soluble receptor reduces the effective free concentration available to the membrane-bound receptors and (2) actions of the cytokines are only produced subsequent to binding to cell surface receptors.
  • the TNF- ⁇ soluble receptor will function as a natural antagonist of the TNF- Rl and TNF-R2 by competing with these cell surface receptors for common pool of free ligand. Pharmacologically, the TNF soluble receptor will function as an antagonist through its ability to decrease free ligand bioavailability rather than by a mechanism of competitive inhibition (i.e., competing with an endogenous ligand for a common binding site on a membrane receptor). Addition of a therapeutically effective amount of the TNF soluble receptor to the joint should effectively neutralize the biological activity of the ligand. Experiments in which recombinant soluble receptors have been admimstered in vivo have demonstrated the capacity to inhibit inflammatory responses and act as antagonists.
  • agents suitable as chondroprotective agents for use in combination with other chondroprotective, anti-pain and/or anti-inflammatory agents to inhibit cartilage destruction include soluble TNFR, the human chimeric polypeptide (recombinant chimera) comprising the extracellular domain of the TNF- ⁇ receptor (p80) linked to the Fc portion of human IgGl, and the anti-TNF- ⁇ antibody.
  • the optimal dose and/or concentration of each suitable agent is that which is therapeutically effective.
  • the preferred and most preferred concentrations of an irrigation solution containing the listed agent are provided, such concentrations expected to be therapeutically effective.
  • compositions in accordance with the present invention will suitably include a dosage or load of the agent sufficient to result in a local concentration at the joint or site of action within the listed therapeutic range.
  • a sufficient dosage or load of the agent is included in the composition to result in a local concentration at the joint or site of action within the listed therapeutic range over a predetermined sustained release period.
  • cytokines are signaling glycoproteins that are important mediators of synovial inflammation and cartilage destruction.
  • IL-1 pro-inflammatory master cytokine
  • cytokine control of cartilage homeostasis is governed by the balance of catabolic and anabolic regulatory cytokines, and anabolic growth factors. If the balance between IL-l ⁇ and IL-lRa production is altered in the inflammatory state in favor of IL-l ⁇ , then it will contribute to the pathogenesis of chronic inflammatory conditions and cartilage destruction, such as is known to occur after knee joint surgery.
  • anti-inflammatory cytokines include the anti-inflammatory cytokines, IL-4, IL-10, and IL-13. These cytokines have been observed to greatly reduce articular cartilage destraction in vitro and in vivo via their effect on a range of pathways that reduce the impact of IL-1.
  • anti-inflammatory cytokines such as IL-4, IL-10, and IL-13, may be useful in reducing inflammation by: 1) reducing the production of pro-inflammatory cytokines, and 2) inducing the production of natural anti-inflammatory cytokines such as IL-lRa, as recently demonstrated in vivo for IL- 4.
  • IL-4 appears to attenuate the inflammatory process in the synovium of rheumatoid arthritis (RA) patients.
  • RA rheumatoid arthritis
  • IL-4 has been shown to inhibit the production of pro-inflammatory cytokines by pieces of synovium, to inhibit proliferation of synoviocytes and decrease bone resorption.
  • IL-4 may promote a direct chondroprotective effect through suppression of matrix metalloproteinase-3 (MMP-3) synthesis in human articular chondrocytes.
  • MMP-3 matrix metalloproteinase-3
  • a cell culture system employing human articular chondrocytes was used to evaluate the effect of IL-4 on IL-1 -induced production of MMP-3 and tissue inhibitor of metalloproteinase- 1 (TIMP-1).
  • IL-4 suppressed IL-1 -stimulated MMP-3 protein and enzyme activity.
  • IL-4 suppressed IL-1 -induced MMP-3 mRNA.
  • Induction of iNOS can be inhibited by IL-4, IL-10 and IL-13.
  • IL-4 may be characterized as a protective mediator of joint destruction seen in inflammatory joint diseases.
  • the effects of IL-4 on the balance of IL-1 regulatory cytokine levels have also been found to support a cartilage protective role.
  • IL-4 and IL-10 were found to suppress the production of inflammatory cytokines by freshly prepared rheumatoid synovial cells.
  • IL-4 and IL-10 synergistically inhibited the IL-1 and TNF- ⁇ stimulated production of IL-6 and IL-8, without effects on cell viability.
  • the addition of IL-4 to RA synovium cultures increased the production of IL-lRa and decreased that of IL-l ⁇ .
  • In vivo treatment with IL-4 has recently been reported to promote a reduction in rat experimental arthritis by acting differentially on the IL-l ⁇ /IL-IRa balance.
  • IL-13 another cytokine that shares many properties with IL-4, also induced IL-lRa in RA synovium. Therefore, the systemic or local delivery of an IL-4 and IL- 13 combination may provide a synergistic therapeutic value.
  • IL-10 has a number of properties that indicate that it is a good candidate to inhibit cartilage destruction. It inhibits both IL-1 and TNF- ⁇ release and stimulates TIMP-1 production while inhibiting MMP -2.
  • the production of IL-10 inside the RA synovium has recently been reported and anti-inflammatory effects of IL-10 have been characterized.
  • IL-10 suppressed IL-l ⁇ production in an ex vivo RA model using pieces of synovium, but to a lesser extent than IL-4.
  • a protective effect of IL-4 and IL-10 treatment on cartilage destraction has been found in animal models of arthritis employing non-local methods of delivery for the cytokines.
  • combination treatment of IL-4 and IL-10 produced substantial improvement.
  • combined treatment with IL-4 and IL- 10 also reduced cellular infiltrates in the synovial tissue and caused pronounced protection against cartilage destraction.
  • levels of mRNA for TNF- ⁇ and IL-1 were highly suppressed both in the synovial tissue and in the articular cartilage.
  • IL-1 receptor antagonist IL-1 receptor antagonist
  • endogenous IL-4 and IL-10 The role of endogenous IL-4 and IL-10 and the therapeutic effect of addition of these cytokines on joint inflammation and cartilage destraction in the early stages of the macrophage dependent murine streptococcal cell wall (SCW) arthritis model have also been investigated. It was demonstrated that endogenous IL-10 plays a role in the regulation of SCW arthritis. Addition of exogenous IL-10 further enlarged the suppressive effect of endogenous IL-10. An even more pronounced effect was found with the combination of IL-4 and IL-10. The combination resulted in a reduced swelling and an increase in chondrocyte proteoglycan synthesis.
  • SCW macrophage dependent murine streptococcal cell wall
  • Severe combined immunodeficient mice were used as a model to assess the effect of IL-4 or IL-10 injection on cartilage degradation and mononuclear cell (MNC) recruitment to human rheumatoid synovium in vivo.
  • Human rheumatoid synovium and cartilage from five rheumatoid arthritis patients were injected with recombinant human IL-4 (rhIL-4, 100 ng; rhIL-10, 100 ng), a combination of IL-4 and IL-10, or TNF-alpha (1000 U), or phosphate-buffered saline twice a week for 4 weeks.
  • Human IL-13 has been cloned and sequenced and has been found to share many of the properties of IL-4.
  • IL-1'3 is about 25% homologous to IL-4.
  • IL-13 decreases the production of pro-inflammatory cytokines, including IL-1 and TNF- ⁇ , by synovial fluid mononuclear cells.
  • IL-13 exhibits anti-inflammatory effects in vivo and thus has therapeutic potential in the treatment of cartilage destruction in the joint.
  • Compounds useful as IL-4, IL-10 and IL-13 agonists include naturally occurring human IL-4, IL-10 and IL-13, human recombinant IL-4 (rhIL-4), rhIL-10, and rhIL-13 as well as . partial sequences thereof, or peptide sequences which have been constructed using recombinant DNA techniques to recognize the IL-4, IL-10 and IL-13 receptors and are capable of activating these receptors on a cell surface.
  • interleukin agonist includes, but is not limited to: (1) peptide sequences which correspond to naturally (endogenous) produced amino acid sequences or fragments thereof, (2) recombinant interleukins which are truncated or partial sequences of the full length naturally occurring interleukin amino acid sequences which retain the ability to bind cognate receptor and retain biological activity and analogs thereof, and (3) chimeric interleukins which are recombinant polypeptides comprised of truncated or partial, sequences corresponding to a portion of the of the full length amino acid sequences attached through oligomers (e.g., amino acids) to a sequence corresponding to a portion of an IgG polypeptide (e.g., IgG hinge and Fc domain) which retain the ability to bind the cognate receptor and retain biological activity.
  • oligomers e.g., amino acids
  • interleukin agonists suitable for the present invention are listed below.
  • the optimal dose and/or concentration of each suitable agent is that which is therapeutically effective.
  • the preferred and most preferred concentrations of an irrigation solution containing the listed agent are provided, such concentrations expected to be therapeutically effective.
  • systemic compositions in accordance with the present invention will suitably include a dosage or load of the agent sufficient to result in a local concentration at the joint or site of action within the listed therapeutic range.
  • a sufficient dosage or load of the agent is included in the composition to result in a local concentration at the joint or site of action within the listed therapeutic range over a predetermined sustained release period.
  • TGF- ⁇ subfamily members are 25 kD pleiotropic, multifunctional proteins capable of influencing a variety of cellular functions and are known to be involved in tissue repair and remodeling. In many cases, it enhances the cell interaction with the extracellular matrix (ECM) and increases accumulation of ECM by stimulating production and secretion of ECM proteins and protease inhibitors. TGF- ⁇ also has been shown to have synergistic interactions with other cytokines, generally showing anti-inflammatory activities. Multiple isoforms of TGF- ⁇ have been identified which share close amino acid sequence homologies. TGF- ⁇ 1, TGF- ⁇ 2, and TGF- ⁇ 3 have been found in human tissue and are active in mammalian cells, although differing in binding affinity.
  • TGF- ⁇ 1 are potent modulators of chondrocyte proliferation, differentiation and extracellular matrix accumulation.
  • TGF- ⁇ 1 regulates metabolism of proteoglycans and stimulates collagen and glycpsaminoglycan synthesis by rabbit articular chondrocytes.
  • TGF- ⁇ 1 increases TIMP expression in human articular chondrocytes and down-regulates expression of IL-1 receptors in articular cartilage.
  • Bone morphogenetic proteins are multifunctional regulators of cell growth, differentiation and apoptosis that belong to the transforming growth factor (TGF)- ⁇ superfamily. More than a dozen members of the BMP protein family have been identified in mammals, which can be subclassified into several groups depending on their stractures.
  • BMP-2 and BMP -4 are highly similar to each other.
  • BMP-5, BMP-6, osteogenic protein (OP)-l (also called BMP-7), and OP-2/BMP-8 are structurally similar to each other.
  • Growth-differentiation factor (GDF)-5 also termed cartilage-derived morphogenetic protein-1
  • GDF-6 also cartilage-derived morphogenetic protein-2
  • GDF-7 form another related group.
  • GDF-5, GDF-6, and GDF-7 In contrast to BMP-2, BMP-4, BMP-6, and OP- 1 /BMP-7, which induce bone and cartilage formation in vivo, GDF-5, GDF-6, and GDF-7 more efficiently induce cartilage and tendon-like structures in vivo (Wolfinan et al., 1997).
  • TGF- ⁇ superfamily exert their effects via binding to two types of serine/threonine kinase receptors, both of which are essential for signal transduction (Massague, 1998).
  • the Type II receptors are constitutively active kinases, which fransphosphorylate Type I receptors upon ligand binding.
  • Type I receptors activate infracellular substrates such as Smad proteins and it is through this mechanism that specificity of infracellular signal transduction occurs.
  • Seven different Type I receptors have been isolated in mammals, which were originally termed activin receptor-like kinase (ALK)-1-ALK7.
  • BMP Type IA receptor (BMPR- IA or ALK-3) and BMP Type IB receptor (BMPR-IB or ALK-6) are structurally similar to each other and specifically bind BMPs together with Type II receptors.
  • ALK-2 has been shown to bind activin, but recent data revealed that it is a Type I receptor for certain BMPs (e.g., OP-l/BMP-7) (Macias-Silva et al., 1998).
  • ALK-1 is structurally highly similar to ALK-2, but its physiological ligand is still unknown.
  • ALK-5 and ALK-4 are Type I receptors for TGF- ⁇ (T ⁇ R-I) and activin (ActR-IB), respectively.
  • ALK-7 is structurally similar to ALK-4 and ALK-5, but its ligand has not been determined yet.
  • TGF- ⁇ and BMP agonists as well as synthetic or human recombinant (rh) agonists suitable for use in the cartilage-protective solution of the present invention may interact with any of the BMP receptors described above.
  • TGF- ⁇ and BMP agonists includes fragments, deletions, additions, amino acid substitutions, mutations, and modifications thereof that retain the biological characteristics of the naturally occurring human TGF- ⁇ and BMP agonist ligands.
  • the TGF- ⁇ or BMP agonists may be used alone or in synergistic combination with other members of the TGF- ⁇ superfamily as anabolic cartilage agents (chondrogenic or promoting cartilage matrix repair) or in combination with inhibitory agents that block cartilage catabolism.
  • Type I receptors function as downstream components of Type II receptors.
  • The, specificity of the intracellular signals by Type I receptors is determined by a specific region in the serine/threonine kinase domain, termed the L45 loop.
  • the structures of the L45 loop of BMPR-IA/ALK-3 and BMPR-IB/ALK-6 are identical to each other, and they may fransduce similar signals in cells.
  • the L45 loops of T ⁇ R-I/ALK-5, ActR-IB/ALK-4, and ALK-7 T ⁇ R-I groups
  • T ⁇ R-I groups are identical to each other, and they activate similar substrates (Chen et al., 1998).
  • ALK-1 and ALK-2 are most divergent from the other Type I receptors, but they activate substrates similar to that of the Type I receptors of the BMPR-I group (Armes et al., 1999).
  • Various proteins may transduce signals from the TGF- ⁇ and BMP serine/threonine kinase receptors.
  • the best-studied molecules are proteins of the Smad family. Eight different Smad proteins have been identified in mammals, and these proteins are classified into three subgroups, (i.e., receptor- regulated Smads (R-Smads), common partner Smads (Co-Smads), and inhibitory Smads).
  • R-Smads are directly activated by Type I receptors, from complexes with Co-Smads, and translocate into the nucleus.
  • the Smad heteromers bind to DNA directly and indirectly via other DNA-binding proteins and thus regulate the transcription of target genes.
  • Smadl, Smad5, and Smad ⁇ are activated by BMPs, whereas Smad2 and Smad3 are activated by TGF- ⁇ .
  • Smad2 in combination with Smad4 that functions as a Co-Smad is translocated to the nucleus where it activates the transcription of genes that mediate the biological effects of TGF.
  • Smad6 and Smad7 are structurally distantly related to the other Smads and act as inhibitory Smads.
  • BMPs induce new cartilage and bone formation in vitro and in vivo and regulate chondrocyte growth and differentiation. Furthermore, these proteins are also implicated in the cartilage repair process.
  • BMPs also promote and maintain the chondrogenic phenotype, which is indicated by their ability to stimulate proteoglycan synthesis in chick limb bud cells culture and fetal rat chondroblasts, as well as in rabbit and bovine articular chondrocytes. The importance of BMPs for cartilage and bone formation has been proven by transgenic approach in which specific BMP gene knockouts were studied.
  • osteogenic ' protein appears particularly important for cartilage homeostasis under normal and pathological conditions, such as during repair of cartilage.
  • OP-1 appears to be the only member of the BMP family, along with cartilage-derived morphogenetic proteins, which is expressed by adult articular chondrocytes (Chubinskaya, S., J. Histochemistiy and Cytochemistry 48:239-50 (2000)).
  • OP-1 was originally purified from bone matrix and shown to induce cartilage and bone formation.
  • the human OP-1 gene has been cloned and biologically active recombinant OP-1 homodimers have been produced.
  • Human recombinant OP-1 can stimulate synthesis of aggrecan and collagen Type II by human articular chondrocytes in vitro. It can also counteract the deleterious effects of IL-1 on the metabolism of these chondrocytes and block bovine cartilage damage mediated by fibronectin fragments. This effect was demonstrated by studying the effects of recombinant human OP-1 on the production of proteoglycan, prostaglandin E2, and IL-1 receptor antagonist by human articular chondrocytes cultured in the presence of interleukin- lbeta. Treatment of human articular chondrocytes with OP-1 was effective in overcoming the down-regulation of proteoglycan synthesis induced by low doses of IL-l ⁇ .
  • OP-1 stimulates the synthesis of hyaluronan and CD44, other molecules required for matrix assembly by human chondrocytes.
  • OP-1 (BMP-7) induces cartilage and bone formation when implanted at intra- and extraskeletal sites in vivo.
  • the influence of OP-1 on healing of full-thickness articular cartilage defects was investigated by drilling two adjacent holes through articular cartilage of rabbit knee joint.
  • OP-1 induced articular cartilage healing and regeneration of the joint surface that contained cells resembling mature joint chondrocytes.
  • cartilage in humans after surgical trauma could include systemic or local application of a member of the TGF- ⁇ superfamily, preferably either TGF ⁇ 2, BMP-7 (OP-1) or BMP-2, or an equivalent agonist which acts through the same receptors employed by these ligands.
  • the systemic or local delivery may occur in combination with a drug or drugs that are inhibitors of cartilage catabolic processes (e.g. such as MAP kinase inhibitors, MMP inhibitors or nitric oxide synthase inhibitors) and/or other agents for the inhibition of pain and inflammation.
  • TGF- ⁇ and BMP agonists include, but is not limited to: (1) peptide sequences which correspond to naturally (endogenous) produced amino acid sequences or fragments thereof, (2) recombinant TGF- ⁇ s and BMPs which are truncated or partial sequences of the full length naturally occurring TGF- ⁇ and BMP amino acid sequences which retain the ability to bind cognate their respective receptor and retain biological activity and analogs thereof, and (3) chimeric TGF- ⁇ s and BMPs which are recombinant polypeptides comprised of truncated or partial sequences corresponding to a portion of the of the full length amino acid sequences attached through oligomers (e.g., amino acids) ' to a sequence corresponding to a portion of an IgG polypeptide (e.g., IgG hinge and Fc domain) which retain the ability to bind the cognate receptor and retain biological activity.
  • oligomers e.g., amino acids
  • TGF- ⁇ and BMP agonists suitable for the present invention are listed below.
  • the optimal dose and/or concentration of each suitable agent is that which is therapeutically effective.
  • the preferred and most preferred concentrations of an irrigation solution containing the listed agent are provided, such concentrations expected to be therapeutically effective.
  • systemic compositions in accordance with the present invention will suitably include a dosage or load of the agent sufficient to result in a local concentration at the joint or site of action within the listed therapeutic range.
  • a sufficient dosage or load of the agent is included in the composition to result in a local concentration at the joint or site of action within the listed therapeutic range over a predetermined sustained release period.
  • a range of therapeutic concentrations for local delivery or local action in the surgical solution to the joint may be estimated from values of the dissociation constants (Kd) of each ligand for its cognate receptor. While these values will vary for particular cell types and tissues, the following example is given for BMP -4.
  • Binding experiments with I-BMP-4 revealed the presence of specific, high- affinity binding sites with an apparent dissociation constant of 110 pM and about 6000 receptors per cell. Therefore, at 11 nM BMP -4, binding of the ligand will be maximal and the available receptors will be fully occupied (saturated). The presence of functional receptors for BMP-4 on primary articular chondrocytes has been demonstrated.
  • TGF- ⁇ 1 0.05-500 0.5-100
  • TGF- ⁇ 2 0.05-500 0.5-100
  • Nonsteroidal anti-inflammatory drugs are widely used as anti-inflammatory agents, but have not been specifically developed or therapeutically employed as chondroprotective agents.
  • the direct molecular target for an NSAID drug is the first enzyme in the prostaglandin synthetic pathway, referred to either as prostaglandin endoperoxide synthase or fatty acid cyclooxygenase.
  • Two related forms of cyclooxygenase termed cyclooxygenase- 1 or Type 1 (COX-1) and cyclooxygenase-2 (COX-2) have been characterized.
  • isozymes are also known as Prostaglandin G/ H Synthase (PGHS)-l and PGHS-2. Both enzymes catalyze the rate-limiting step in the formation of prostanoids that is the conversion of arachidonic acid to prostaglandin H2.
  • COX-1 is present in platelets and endothelial cells and exhibits constitutive activity.
  • COX-2 has been identified in endothelial cells, macrophages, fibroblasts and other cells in the joint and its expression is induced by pro-inflammatory cytokines, such as IL-1 and TNF- ⁇ .
  • COX-2 expression is upregulated and it has been shown that large increases in activity of COX-2 occur concomitant with its upregulation, leading to increased synthesis of prostaglandins which are present in the synovial fluid of patients suffering from inflammatory arthropathies.
  • Cellular sources of prostaglandins (PGs) in the joint include activated chondrocytes, Type A and B synoviocytes and infiltrating macrophages.
  • PGs prostaglandins
  • Cellular functions important in cartilage metabolism modulated by PGs include gene expression, exfracellular matrix synthesis and proliferation. Because COX-2 is expressed in inflamed joint tissue or after exposure to mediators of inflammation (e.g., as a result of injury or surgical trauma), the use of a COX-2 inhibitor is expected to provide both anti-inflammatory and cartilage protective activity.
  • Cartilage destruction in inflammatory arthropathies can be triggered as a consequence of joint injury and as a result of arthroscopic surgical procedures.
  • Chondrocytes are the only cell type in articular cartilage and are known to participate in the breakdown of their own matrix through release of endogenous inflammatory mediators, including PGs.
  • PGs endogenous inflammatory mediators
  • Studies have shown that COX-2 gene expression, protein synthesis, and PG release in normal human articular chondrocytes is rapidly induced by cytokines, including IL-1, TNF- ⁇ and IL-6.
  • cytokines including IL-1, TNF- ⁇ and IL-6.
  • Levels of mRNA are detected as early as 2 hours after cytokine induction, reach high levels at 6 hours and show a remarkably long duration of expression for at least 72 hours.
  • NSAIDs are commonly used in the treatment of patients with osteoarthritis or rheumatoid arthritis, but their effects on articular cartilage metabolism in the context of these arthritic diseases remains a subject of debate. For instance, the clinical treatment of osteoarthritis and rheumatoid arthritis with NSAIDs is successful in reducing inflammation. However, it is thought that some NSAIDs which are not selective for COX-2, primarily salicylates and indomethacin, accelerate osteoarthritic cartilage destraction by impairing proteoglycan synthesis by chondrocytes, whereas other NSAIDS are thought to have a somewhat chondroprotective effect by stimulating cartilage repair.
  • a criteria for defining selectivity is the ratio of the. COX-1 /COX-2 inhibitory constants (or COX-2/COX-1) obtained for a given biochemical or cellular assay system.
  • the selectivity ratio accounts for different absolute ICso values for inhibition of enzymatic activity that are obtained between microsomal and cellular assay systems (e.g., platelet and macrophage, cell lines stably expressing recombinant human COX isozymes).
  • COX-2 mimics the inhibitory effects triggered by chondroprotective (inhibitory) cytokines, such as IL-4, which down-regulate infracell ⁇ lar COX-2 synthesis.
  • cytokines such as IL-4
  • Comparison of the selectivity of more than 45 NSAIDs and selective COX-2 inhibitors showed the following rank-ordered relative selectivity for COX-2 vs.
  • COX-2 inhibitors such as celecoxib and rotecoxib
  • these compounds are expected to exhibit chondroprotective action when applied perioperatively in an irrigation solution or in an injection directly to a joint.
  • COX-2 inhibitors are expected to be effective drags delivered in an irrigation solution during an arthroscopic surgical procedure or by direct injection into a joint prior to, during or after a surgical procedure or other injury to the joint. Examples of COX-2 inhibitors suitable for the present invention are listed below. For all modes of local delivery (i.e., injection, infusion and irrigation) the optimal dose and/or concentration of each suitable agent is that which is therapeutically effective.
  • compositions in accordance with the present invention will suitably include a dosage or load of the agent sufficient to result in a local concenfration at the joint or site of action within the listed therapeutic range.
  • a sufficient dosage or load of the agent is included in the composition to result in a local concentration at the joint or site of action within the listed therapeutic range over a predetermined sustained release period.
  • SC-58451 0.3-30,000 30-3,000 celecoxib (SC-58125) 0.3-30,000 30-3,000 meloxicam 0.5-50,000 50-5,000 nimesulide 0.5-50,000 50-5,000 diclofenac 0.3-30,000 30-3,000
  • the mitogen-activated protein (MAP) kinases are a group of protein serine/threonine kinases that are activated in response to a variety of extracellular stimuli and function in transducing signals from the cell surface to the nucleus.
  • the MAP kinase cascade is one of the major infracellular signaling pathways that transmit signals from growth factors,, hormones and inflammatory cytokines to intermediate early genes. In combination with other signaling pathways, these 2US4IFCT
  • MAPKs mitogen-activated protein-kinases
  • p38 a member of the MAPK family (p38) mediates the major biochemical ' signal fransduction pathways from the potent pro-inflammatory cytokines, IL-1 and TNF- ⁇ , leading to induction of cyclooxygenase-2 (COX-2) in stimulated macrophages, through cis-acting factors involved in the transcriptional regulation of the COX-2 gene.
  • COX-2 cyclooxygenase-2
  • the members of the MAP kinase class of agents are composed of at least three families that are known to differ in sequence, size of the activation loop, activation by extracellular stimuli and participation in distinct signal transduction pathways.
  • Prominent members among this family of MAP kinases include the extracellular signal-regulated kinases (ERKs), ERK1 and ERK2 (p44MAPK and p42MAPK, respectively); stress-activated protein kinase 1 (SAPK1) family which is also referred to as the JNK or jun N-terminal kinase family; and the p38 MAP kinase family which is also known as stress-activated kinase 2/3 (SAPK-2/3).
  • ERKs extracellular signal-regulated kinases
  • p44MAPK and p42MAPK p44MAPK and p42MAPK
  • SAPK1 stress-activated protein kinase 1
  • JNK or jun N-terminal kinase family
  • the p38 kinases are activated by stresses, most notably pro-inflammatory cytokines. Within the p38 family, there are at least four distinct homologs (isotypes or isoenzymes) which standard nomenclature ' refers to either as SAPK2a, SAPK2b, SAPK2d, . SAPK3, or p38 ⁇ , ⁇ , ⁇ (SAPK4) and ⁇ , respectively.
  • the inhibitors of MAP kinases useful for the practice of this invention may interact with any one or combination of the above MAP kinases.
  • the inhibitory constants characterized through assays of purified in vitro enzymes and in cellular assays may vary over a wide range of concentrations and demonstrate utility in this application.
  • Activation of p38 MAP kinase is mediated by dual phosphorylation of threonine and tyrosine residues. Both TNF- ⁇ and IL-1 treatment of cells has been shown to rapidly (within 5 min) increase phosphorylation and activate p38 MAP kinase.
  • the kinase selectivity of SB203580 inhibitory action for p38 was demonstrated by its failure or at best weak inhibition of at least 15 other protein kinases in vitro, including members of the PKC, PKA, src and receptor tyrosine kinase families (Lee, J., Pharmacol. Ther. 82:389-397 (1999)).
  • pre-incubation with SB 203580 has been shown to block the IL-1 and TNF- ⁇ induced phosphorylation of the . enyzme and subsequent IL-8 production. This supports the preemptive effect of delivering the inhibitors during the surgical procedure.
  • p38 mitogen-activated protein kinase (MAPK) in biochemical inflammatory responses resulting in destraction of cartilage has been studied using SB203580, which specifically inhibits the enzyme.
  • Actions of IL-1 that are selectively controlled by p38 MAPK are the regulation of prostaglandin H synthase-2 (COX-2), metalloproteinases, and IL-6 (Ridley, S. et al., J. Immunol. 158:3165-73 (1997)).
  • SB203580 significantly inhibited IL-1 -stimulated IL-6 (30 to 50% at 1 ⁇ M) but not .
  • SB203580 strongly inhibited IL-1 -stimulated prostaglandin production by fibroblasts and human endothelial cells. This was associated with the inhibition of the induction of COX-2 protein and mRNA.
  • PGE 2 contributes to increased expression of matrix metalloproteinases that are important mediators of cartilage degradation. Both synovial fibroblasts and chondrocytes express the COX-2 gene at high levels upon activation by cytokines and exfracelluar stimuli.
  • the MAPK inhibitor provides chondroprotective activity due to its inhibitory activity on MAP kinases expressed in these and other cell types.
  • MAPK inhibitors are expected to be effective as cartilage protective agents when applied systemically or locally to tissues of the joint in • a variety of inflammatory or pathophysiological conditions.
  • SB 203580 has been characterized in several pharmacological models in vivo and demonstrated to have activity under long term, oral dosing. SB203580 was found to inhibit the stimulation of collagenase- 1 and stromelysin- 1 production by IL-1 without affecting synthesis of TIMP-1. Furthermore, SB203580 prevented an increase in IL-1 -stimulated collagenase- 1 and stromelysin- 1 mRNA.
  • p38 MAP kinase is involved in TNF-induced cytokine expression, and drugs which function as inhibitors of p38 MAP kinase activity block the production of pro- inflammatory cytokines (Beyaert, R. et al., EMBO J. 15:1914-23 (1996)).
  • TNF- ⁇ treatment of cells activated the p38 MAPK pathway as shown by increased phosphorylation of p38 MAPK itself and activation of its subsfrate proteins.
  • Prefreatment of cells with SB203580 completely blocked TNF- ⁇ induced activation of MAPK activating protein kinase-2 and hsp27 phosphorylation.
  • SB203580 also completely inhibited TNF- ⁇ induced synthesis of IL-6 and expression of a reporter gene that was driven by a minimal promoter containing two NF- ⁇ B elements.
  • inhibitors such as SB203580 and FR133605, on p38 MAPK interfere selectively with TNF- ⁇ - and IL-1 -induced activation of various proteins linked to the cartilage degradation.
  • MAP kinase inhibitors may provide a chondroprotective effect.
  • SB 2035.80 has been evaluated in several animal models of cytokine inhibition and inflammatory disease. It was demonstrated to be a potent inhibitor of inflammatory cytokine production in vivo in both mice and rats with IC 50 values of 15 to 25 mg/kg. SB 203580 possessed therapeutic activity in collagen-induced arthritis in DBA/LACJ mice with a dose of 50 mg/kg resulting in significant inhibition of paw inflammation. Antiarthritic activity was also observed in adjuvant- induced arthritis in the Lewis rat when SB203580 was administered p.o. at 30 and 60 mg/kg/day. Additional evidence was obtained for beneficial effects on bone resorption with an IC 50 of 0.6 ⁇ M.
  • p38 MAPK plays an important role in the regulation of responses to IL-1 and TNF- ⁇ and that it is involved in the regulation of mRNA levels of some inflammatory-responsive genes, such as COX-2.
  • Inhibitors of p38 block the production of pro-inflammatory cytokines and inhibit the production of MMPs, and have been demonstrated to inhibit collagen breakdown in cartilage explants.
  • MAPK inhibitor to block the actions of key pro-inflammatory cytokines, such as IL-1 and TNF- ⁇ , will have beneficial effects on many cell types in the joint, including synovial fibroblasts, macrophages and chondrocytes, thus inhibiting subsequent pathological effects such as infiltration of inflammatory cells into the joint, synovial hyperplasia, synovial cell activation, and cartilage breakdown.
  • a MAPK inhibitor should block the propagation of the inflammatory response by the aforementioned cytokines, and thereby interrupt the disease process.
  • MAPK inhibitors suitable for the present invention are listed below.
  • the optimal dose and/or concentration of each suitable agent is that which is therapeutically effective.
  • the preferred and most preferred concentrations of an irrigation solution contaimng the listed agent are provided, such concentrations expected to be therapeutically effective for local delivery.
  • systemic compositions in accordance with the present invention will suitably include a dosage or load of the agent sufficient to result in a local concentration at the joint or site of action within the listed therapeutic range.
  • a sufficient dosage or load of the agent is included in the composition to result in a local concentration at the joint or site of action within the listed therapeutic range over a predetermined sustained release period.
  • Matrix metalloproteinases are a family of at least 15 zinc endopeptidases that function exfracellularly and play a key role in pathological degradation of tissue. Current nomenclature and alternative names for members of the MMP are provided in Table 7. Most MMPs are highly regulated and most are not constitutively expressed in normal tissues. However, pro-inflammatory cytokines, such as IL-1 and TNF- ⁇ , initiate transcription and expression. An imbalance created by upregulation and activation of tissue-degrading MMPs is a primary causative factor in the cartilage breakdown process during chronic inflammatory diseases and sustained synovial inflammatory responses subsequent to joint injury.
  • Cartilage matrix metabolism has been studied in patients with either a meniscal injury or anterior cruciate ligament rupture in the knee. It was shown that concentrations of stromelysin- 1 (MMP-3), collagenase, tissue inhibitor of metalloproteinases (TIMP- 1), and proteoglycan fragments increased in human knee synovial fluid after traumatic knee injury. Temporally, these values increased immediately over reference levels and remained significantly elevated (10-fold increase) over a period of one year. These changes likely drive the increase in the concenfration of proteoglycan fragments that are observed in synovial fluid after knee ligament injury.
  • MMP-3 stromelysin- 1
  • TMP-1 tissue inhibitor of metalloproteinases
  • Fibroblast X Fibroblast X
  • Gelatin Gelatin
  • aggrecan
  • MMP-3 Stromelysin- 1 EC3.4.24.17 Collagens (III, IV, V, IX); Gelatin; Transin aggrecan; versican and hyaluronidase-treated veriscan; perlecan; decorin; proteoglycan link protein; large tenascin-C; fibronectin; laminin; entactin; osteonection; elastin; casein; a t - ACHYM; aiitithrombin-III; ⁇ 2 M; ovostain; Substance P; MBP; GST- TNF/TNF peptide; IL-l ⁇ ; serum amyloid A; IGF-BP3; fibrinogen and cross-linked fibrin; plasminogen; MMP- r'superactivation", MMP-2/TIMP-2 complex; MMP-7; MMP-8; MMP- 9; MMP-13 _
  • MMP-7 Matrilysin EC3.4.24.23 Collagen IV and X; Gelatin; PUMP aggrecan; decorin; proteoglycan link protein; fribronectin and laminin; insoluble fibronectin fibrils; enactin; large and small tenascin-C; osteonectin; ⁇ 4 integrin; elastin; casein; transferrin; MBP; a t -AT; GST-TNF/TNF peptide; plasminogen; MMP-1; MMP-2; MMP-9; MMP-9/TIMP-1
  • MMP-8 Neutrophil EC3.4.24.34 Collagens (I, II, III, V, VII and X); Collagenase Gelatin; aggrecan; a t -AT; ai - Collagenase I ACHYM; a 2 -antiplasmin; fibronectin
  • Metalloelastase elastin casein; ⁇ —AT; fibronectin; vifronectin; laminin; enactin; proteoglycan monomer; GST-TNF;
  • MMP-13 Collagenase-3 EC3.4.24 Collagens (I, II and III, IV, IX, X and XIV); Gelatin, ⁇ l-ACHYM and plasminogen activator inhibitor 2; aggrecan; perlecan; large tenascin-C, fribronectin; osteonectin;MMP-9
  • MMP-14 MT-MMP-1 EC3.4.24 Collagen (I, II and III); Gelatin, casean, K-elastin, fribronectin, laminin, vitrbriectin and proteoglycans; large tenascin-C, enactin; ⁇ AT, ⁇ 2 M; GST-TNF; MMP-2; MMP-13 MMP-15 MT-MMP-2 Fibronectin, large tenascin-C, entactin, laminin, aggrecan, perlecan; GST-TNF; MMP-2
  • the MMP family of enzymes has been shown to be secreted from human chondrocytes and by cells of the synovium, such as synovial fibroblasts. Furthermore, using in situ hybridization, it was shown that human synovium synthesizes both stromelysin- 1 and collagenase. Stromelysin- 1 (MMP-3) is capable of degrading all of the components of the cartilage matrix. There is evidence that chondrocytes contribute to cartilage degradation by the release of the matrix- degrading enzyme, collagenase-3.
  • MMPs Upon activation by pro-inflammatory cytokines, MMPs are secreted from cells in a latent form, are activated exfracellularly, and are inhibited by tissue inhibitors of metalloproteinases (TlMPs).
  • TlMPs tissue inhibitors of metalloproteinases
  • the MMPs are regulated by cytokines, such as interleukin-1 (IL-1), and growth factors that act on chondrocytes and synoviocytes to enhance their protease production.
  • cytokines such as interleukin-1 (IL-1)
  • Other pro-inflammatory cytokines such as IL-6, IL-8 and TNF- ⁇ , also upregulate the production of matrix-degrading enzymes. This leads to cartilage destruction, which is usually assessed as the loss of sulfated glycosaminoglycans (GAGs) and the cleavage of collagen.
  • GAGs sulfated glycosaminoglycans
  • IL-1 which is present in the joint fluid of patients with arthritic diseases, stimulates chondrocytes to synthesize elevated amounts of enzymes such as stromelysin, fibroblast and ne trophil collagenase, and plasminogen activator.
  • enzymes such as stromelysin, fibroblast and ne trophil collagenase, and plasminogen activator.
  • IL-1 inhibits synthesis of plasminogen activator inhibitor- 1 and TIMP, and also inhibits synthesis of matrix constituents such as collagen.
  • the imbalance between the levels of inhibitors and enzymes leads to an increase in the amount of active proteases and, combined with a suppression of matrix biosynthesis, results in cartilage degradation.
  • collagenase inhibitors can inhibit either the IL-1 or IL-8 stimulated invasion of articular cartilage by rheumatoid synovial fibroblasts (RSF).
  • the collagenase inhibitors 1,10-o- phenanthroline and phosphoramidon, substantially inhibited the concentration-dependent penetration of cartilage by RSF cells at concentrations of 10-150 ⁇ M.
  • the selective effect of cytokines on the secretion of proteinases demonstrates that synovial fibroblast-like cell-mediated articular degradation is a highly regulated process.
  • the action of the inhibitors in the limited in vitro system suggests that therapeutic intervention using systemic or local delivery of synthetic MMP inhibitors with appropriate pharmokinetics will be effective as chondroprotective agents.
  • MMP inhibitors suitable for the present invention include U- 24522 ((R,S)-N-[2-[2-(hydoxylamino)-2-oxoethyl]-4-methyl-l-oxopentyl]-L-leucyl- L-phenylalaniamide); BB2516; (N 2 -[35[Hydroxy-4-(N-hydroxyamino)-2R-isobutyl]- L-leucine-NXmethylamide, also known as marimastat) peptides such as MMP Inhibitor I and MMP-3 Inhibitor, and larger proteins such as TIMP-1 or fragments thereof, and are listed in the Table below: For all modes of local delivery (i.e., injection, infusion and irrigation) the optimal dose and/or concentration of each suitable agent is that which is therapeutically effective.
  • compositions in accordance with the present invention will suitably include a dosage or load of the agent sufficient to result in a local concentration at the joint or site of action within the listed therapeutic range.
  • a sufficient dosage or load of the agent is included in the composition to result in a local concentration at the joint or site of action within the listed therapeutic range over a predetermined sustained release period.
  • MMPs Matrix Metalloproteinases
  • NFKB pro-inflammatory and cartilage-destructive cellular pathways are regulated by exfracellular and intracellular signaling mechanisms that are targets for novel therapeutic local and systemic drag delivery.
  • NFKB activity is mediated by a family of transcription factor subunits that bind to DNA either in the form of homodimers or heterodimers.
  • I ⁇ B inhibitory subunits
  • NFKB was found to be involved in IL-1 induced expression and was capable of increasing pro-inflammatory COX-2 protein expression in RA synovial fibroblasts.
  • NFKB as a key molecular target is based upon its role as a common downstream signaling element regulating gene expression of several critical inflammatory mediators linked to joint inflammation and cartilage-destructive pathways.
  • the response of many genes (COX-2, collagenase, IL-6, IL-8) are governed by promoters which contain both NFKB promoter elements.
  • Activation of NFKB mediates the induction of many proteins central to the inflammatory process, such as cytokines, cell-adhesion molecules, metalloproteinases and other proteins that participate in the production of prostaglandins and leukotrienes (COX-2) in synoviocytes.
  • this transcription factor represents a physiologically significant target in therapies directed to the injury responses of human synovial fibroblasts, human articular chondrocytes, as well as other cells in the joint.
  • rheumatoid synovial fibroblasts results in the coordinate upregulation of 85-kD phospholipase A2 (PLA2) and inducible cyclooxygenase (COX- 2). Together, these two enzymes promote, the subsequent biosynthesis of PGE 2 , a primary inflammatory mediator in the joint.
  • Oligonucleotide decoys and antisense were used to demonstrate the participation of the (NFKB), in the regulation of the prostanoid-metabolizing enzymes. Antagonizing NFKB mRNA using anti-sense oligonucleotide resulted in decreased binding to the COX gene promoter.
  • the specificity of the molecular target was shown through use of an analog, aldisine, and the protein kinase C inhibitor, RO 32-0432, which were inactive.
  • Direct action of hymenialdisine on IL-1 -induced NFKB activation was demonstrated by a significant reduction (approximately 80%) in NFKB binding to the classical KB consensus motif and inhibition of stimulated p65 migration from the cytosol of treated cells.
  • hymenialdisine-treated RSF did not transcribe the mRNAs for either COX-2 or PLA2 in response to IL-1. Consequently, reduced protein levels for these enzymes and reductions in the ability to produce PGE2 were observed. Furthermore, IL-1 -stimulated interleukin-8 (IL-8) production, which is known to be an NF ⁇ B-regulated event, was also inhibited by hymenialdisine, whereas IL-1 -induced production of vascular endothelial growth factor, a non-NF ⁇ B-regulated gene, was not affected by exposure to hymenialdisine.
  • IL-1 -stimulated interleukin-8 (IL-8) production which is known to be an NF ⁇ B-regulated event, was also inhibited by hymenialdisine, whereas IL-1 -induced production of vascular endothelial growth factor, a non-NF ⁇ B-regulated gene, was not affected by exposure to hymenialdisine.
  • hymenialdisine inhibits IL-1 -stimulated synovial fibroblast formation of PGE2 through its inhibitory effect on NFKB activation.
  • This provides a basis to define its use as a novel inhibitor to block the role of NFKB in joint inflammation and cartilage destruction.
  • NFKB inhibitors suitable for the present invention are listed below.
  • the optimal dose and/or concentration of each suitable agent is that which is therapeutically effective.
  • the preferred and most preferred concentrations of an irrigation solution containing the listed agent are provided, such concentrations expected to be therapeutically effective when delivered locally.
  • systemic compositions in accordance with the present invention will suitably include a dosage or load of the agent sufficient to result in a local concentration at the joint or site of action within the listed therapeutic range.
  • a sufficient dosage or load of the agent is included in the composition to result in a local concentration at the joint or site of action within the listed therapeutic range over a predetermined sustained release period.
  • Nitric oxide is a widespread intracellular and intercellular mediator involved in the pathophysiological mechanisms of some connective tissue diseases. NO is formed from L-arginine by a family of enzymes, the NO synthases, which are localized intracellularly. Three isoforms of NO synthase have been cloned and sequenced. Endothelial cell NO synthase (ecNOS) and brain NO synthase (bNOS) are constitutively active. A distinct isoform of NO synthase, inducible NOS (iNOS), is found in many cell types, including chondrocytes.
  • ecNOS Endothelial cell NO synthase
  • bNOS brain NO synthase
  • iNOS inducible NOS
  • IL-1 is a very potent stimulator of chondrocyte NO synthesis and that IL-1 acts through its ability to upregulate the level of the iNOS.
  • chondrocytes are the major source of NO and chondrocytic iNOS induced by pro-inflammatory cytokines is considered to mediate many effects of IL-1 in inflammatory arthropathies.
  • chondrocyte inducible NO synthase Drugs that specifically inhibit chondrocyte inducible NO synthase may have a therapeutic role in the prevention of chondrodestraction that occurs due to joint injury (e.g., surgical procedures involving the joint).
  • iNOS chondrocyte inducible NO synthase
  • S-methyl isothiourea and S-(aminoethyl) isothiourea were 2-4 times more potent than N G - monomethyl-L-arginine, 5-10 times more potent than aminoguanidine and over 300 times more potent than N ⁇ -nitro-L-arginine and N ⁇ -nitro-L-arginine methyl ester.
  • These isothiourea compounds provide a potent and relatively specific class of inhibitors of iNOS in cartilage and thus are suitable for systemic or local delivery in accordance with aspects of the invention (Jang, D., Eur. J. Pharmacol. 312:341- 347(1996)).
  • the cartilage protective therapeutic potential of NO synthase inhibitors as also been assessed using in vitro systems such as isolated chondrocytes to define effects on the cartilage matrix.
  • Inhibition of endogenous NO production by N G - monomethyl-L-arginine (L-NMMA), an established NO synthase inhibitor leads to the suppression of gelatinase, collagenase, and stromelysin production by IL- l ⁇ -stimulated chondrocytes.
  • Inhibition of NO production also partially reduces the increase in the lactate production that occurs from the exposure of chondrocytes to IL-l ⁇ .
  • SMT S- methylisothiourea
  • AETU S-aminoethylisothiourea
  • L-NMMA N-nitro- L-arginine methyl ester
  • the above NOS inhibitors inhibited NO production by cartilage cells treated with IL-l ⁇ and had marked effects on restoring proteoglycan synthesis in chondrocytes. Therefore, if NO production can be blocked using a therapeutically effective concentration and dose, then IL-l ⁇ inhibition of proteoglycan synthesis will be prevented.
  • NO synthase is expressed in cartilage obtained from the joint of patients with arthritic disease.
  • increased levels of nitrite have been observed in the synovial fluid and it has been shown that a significant source of NO production in these patients is derived from articular cartilage.
  • sustained systemic delivery of L-NIL, a potent inhibitor of iNOS reduces the progression of experimental OA in dogs (induced by sectioning of the ACL) and causes a substantial decrease in IL-l ⁇ , PGE 2) NO and MMP production.
  • NO synthase inhibitors suitable for the present invention are listed below.
  • the optimal dose and/or concentration of each suitable agent is that which is therapeutically effective.
  • the preferred dose and/or concentration of each suitable agent is that which is therapeutically effective.
  • the preferred dose and/or concentration of each suitable agent is that which is therapeutically effective.
  • compositions in accordance with the present invention will suitably include a dosage or load of the agent sufficient to result in a local concentration at the joint or site of action within the listed therapeutic range.
  • a sufficient dosage or load of the agent is included in the composition to result in a local concentration at the joint or site of action within the listed therapeutic range over a predetermined sustained release period.
  • the preferred NO synthase inhibitors for inclusion in the solutions of the invention is 1400 W ((N-3- (aminomethyl)benzyl)acetamidine), a selective, slow, tight binding inhibitor of iNOS, diphenyleneiodinium and 1,3-PBIT.
  • Integrins are heterodimer receptors located on the plasma membrane that contain ⁇ and ⁇ subunits that bind ligands which are extracellular matrix (ECM) components or may be other large proteins, such as collagen, laminin, vitronectin, osteopontin (OPN) and fibronectin (FN). Degradation of the cartilage matrix is regulated by chondrocytes through mechanisms that depend upon the interaction of these cells with the ECM. Chondrocyte gene expression is, in part, controlled through cellular contacts involving the interaction of integrins with components of ECM in the environment surrounding the chondrocyte. Hence, integrins on chondrocytes are involved in confrol of cartilage homeostasis, and this family of receptors represents a class of therapeutic targets for preventing cartilage degradation.
  • ECM extracellular matrix
  • OPN osteopontin
  • FN fibronectin
  • Human chondrocytes express an array of integrin receptors composed of distinct ⁇ and ⁇ subunits, including ⁇ 5 ⁇ , ⁇ V ⁇ 3 and lesser quantities of others. Of particular importance is the ⁇ V ⁇ 3 integrin, which is known to bind OPN.
  • the ⁇ V ⁇ 3 complex-specific function blocking monoclonal antibody (mAb) LM609 acts as an agonist in a manner that is similar to the ligand, OPN. It attenuates the production of a number of proinflammatory and cartilage destructive mediators, such as IL-1, NO and PGE2.
  • the agonistic mAb LM609 is thought to be suitable for use in the present invention.
  • Anti-chemotactic agents prevent the chemotaxis of inflammatory cells.
  • Representative examples of anti-chemotactic targets at which these agents would act include, but are not limited to, F-Met-Leu-Phe receptors, IL-8 receptors, MCP-1 receptors, and MIP-1-I/RANTES receptors. Drags within this class of agents are early in the development stage, but it is theorized that they may be suitable for use in the present invention. 12. Intracellular Signaling Inhibitors
  • PKC Protein Kinase C
  • PKC Protein kinase C plays a crucial role in cell-surface signal transduction for a number of physiological processes. PKC isozymes can be activated as downstream targets resulting from initial activation of either G-protein coupled receptors (e.g., serotonin, bradykinin, etc.) or pro-inflammatory cytokine receptors. Both of these receptor classes play important roles in mediating cartilage destraction.
  • PKC exists as a large family consisting of at least 8 subspecies (isozymes). These isozymes differ substantially in structure and mechanism for linking receptor activation to changes in the proliferative response of specific cells. Expression of specific isozymes is found in a wide variety of cell types, including: synoviocytes, chondrocytes, neutrophils, myeloid cells, and smooth muscle cells. Inhibitors of PKC are therefore likely to effect signaling pathways in several cell types unless the inhibitor shows isozyme specificity. Thus, inhibitors of PKC can be predicted to be effective in blocking the synoviocyte and chondrocyte activation and may also have an anti-inflammatory effect in blocking diverophil activation and subsequent attachment.
  • G-6203 (also known as Go 6976) is a new, potent PKC inhibitor with high selectivity for certain PKC isotypes with IC 50 values in the 2-10 ⁇ M range. Concentrations of these and another drag, GF 109203X, also known as Go 6850 or bisindoylmaleimide I (available from Warner-Lambert), that are believed to be suitable for local delivery use in the present invention are set forth below.
  • systemic compositions in accordance with the present invention will suitably include a dosage or load of the agent sufficient to result in a local concentration at the joint or site of action within the listed therapeutic range.
  • a sufficient dosage or load of the agent is included in the composition to result in a local concentration at the joint or site of action within the listed therapeutic range over a predetermined sustained release period.
  • Protein Kinase C Inhibitors calphostin C 0.5-50,000 100-5,000
  • RTK tyrosine-kinase
  • tyrphostin compounds have potential as specific inhibitors of tyrosine kinase activity (IC 50 s in vitro in the 0.5-1.0 ⁇ M range), since they have little effect on other protein kinases and other signal transduction systems.
  • IC 50 s in vitro in the 0.5-1.0 ⁇ M range IC 50 s in vitro in the 0.5-1.0 ⁇ M range
  • suitable concentrations for these agents as used in the present invention are set forth below.
  • staurosporine has been reported to demonstrate potent inhibitory effects against several protein tyrosine kinases of the src subfamily and a suitable concentration for this agent as used in the present invention for local delivery also is set forth below.
  • compositions in accordance with the present invention will suitably include a dosage or load of the agent sufficient to result in a local concentration at the joint or site of action within the listed therapeutic range.
  • a sufficient dosage or load of the agent is included in the composition to result in a local concentration at the joint or site of action within the listed therapeutic range over a predetermined sustained release period.
  • Protein Kinase Inhibitors lavendustin A 10-100,000 100-10,000 tyrphostin 10-100,000 100-10,000
  • protein tyrosine phosphatases containing src-homology2 SH2 domains are known and nomenclature refers to them as SH-PTP1 and SH-PTP2.
  • SH-PTP1 is also known as PTP1C, HCP or SHP.
  • SH-PTP2 is also known as PTP1D or PTP2C.
  • SH-PTP1 is expressed at high levels in hematopoietic cells of all lineages and all stages of differentiation, and the SH-PTP1 gene has been identified as responsible for the motheaten (me) mouse phenotype and this provides a basis for predicting the effects of inhibitors that would block its interaction with its cellular substrates.
  • PTPase activity modulates agonist induced activity by reversing the effects of tyrosine kinases activated in the initial phases of cell stimulation.
  • Agents that could stimulate PTPase activity could have potential therapeutic applications as anti- inflammatory mediators.
  • PTPases have also been shown to modulate the activity of certain RTKs. They appear to counter-balance the effect of activated receptor kinases and thus may represent important drag targets.
  • SH2 domains originally identified in the src subfamily of protein tyrosine kinases (PTKs), are noncatalytic protein sequences and consist of about 100 amino acids conserved among a variety of signal transducing proteins (Cohen, et al., 1995). SH2 domains function as phosphotyrosine-binding modules and thereby mediate critical protein-protein associations in signal transduction pathways within cells (Pawson, Nature, 573-580, 1995). In particular, the role of SH2 domains has been clearly defined as critical for receptor tyrosine kinase (RTK) mediated signaling such as in the case of the platelet-derived growth factor (PDGF) receptor.
  • RTK receptor tyrosine kinase
  • Phosphotyrosine-containing sites on autophosphorylated RTKs serve as binding sites for SH2-proteins and thereby mediate the activation of biochemical signaling pathways (see FIGURE 2) (Carpenter, G., FASEB J. 6:3283-3289 (1992); Sierke, S.et al, J. Biochem. 32:10102-10108 (1993)).
  • the SH2 domains are responsible for coupling the activated growth-factor receptors to cellular responses that include alterations in gene expression, and ultimately cellular proliferation.
  • inhibitors that will selectively block the effects of activation of specific RTKs (excluding IGFR and FGFR) expressed on the surface of synoviocytes are predicted to be effective in blocking cartilage degradation after arthroscopy procedures.
  • cytosolic proteins that contain SH2 domains and function in infracellular signaling.
  • the distribution of SH2 domains is not restricted to a particular protein family, but found in several classes of proteins, protein kinases, lipid kinases, protein phosphatases, phospholipases, Ras-controlling proteins and some transcription factors.
  • Many of the SH2-containing proteins have known enzymatic activities while others (Grb2 and Crk) function as "linkers” and "adapters” between cell surface receptors and "downstream” effector molecules (Marengere, L., et al., Nature 369:502-505 (1994).
  • proteins containing SH2 domains with enzymatic activities that are activated in signal fransduction include, but are not limited to, the src subfamily of protein tyrosine kinases (src (pp60 c"src ), abl, lck, fyn, fgr and others), phospholipaseC ⁇ (PLC ⁇ ), phosphatidylinositol 3-kinase (PI-3-kinase), p21-ras GTPase activating protein (GAP) and SH2 containing protein tyrosine phosphatases (SH-PTPases) (Songyang, et al., Cell 72: 767-778 (1993).
  • T-cell activation via the antigen specific T-cell receptor (TCR) initiates a signal transduction cascade leading to lymphokine secretion and T-cell proliferation.
  • TCR antigen specific T-cell receptor
  • compositions of the present invention may also include other therapeutic agents.
  • one or more anti-inflammatory or analgesic agents also referred to herein as anti-pain agents
  • anti-inflammatory and/or analgesic agents are further described in detail below.
  • compositions of the present invention may include one or more disease modifying anti-rheumatic drugs (DMARDs), such as methotrexate, sulfasalazine, gold compounds such as oral gold, gold sodium thiornalate and aurothioglucose, azathioprine, cyclosporine, antimalarials, steroids, colchicines, cyclophosphanmide, hydroxychloroquine sulfate, leflunomide, minocycline and penicillamine.
  • DMARDs disease modifying anti-rheumatic drugs
  • methotrexate such as methotrexate, sulfasalazine, gold compounds such as oral gold, gold sodium thiornalate and aurothioglucose, azathioprine, cyclosporine, antimalarials, steroids, colchicines, cyclophosphanmide, hydroxychloroquine sulfate, leflunomide
  • Therapeutic agents aimed at treating postoperative pain while avoiding detrimental side effects are not easily developed because the molecular targets for these agents are distributed widely throughout the body and mediate diverse physiological actions.
  • systems for the delivery of inhibitors of pain, inflammation, and cartilage degradation at effective dosages while minimizing adverse systemic side effects have not been developed.
  • systemic i.e., intravenous, oral, subcutaneous or intramuscular
  • opiates in therapeutic doses frequently is associated with significant adverse side effects, including severe respiratory depression, changes in mood, mental clouding, profoimd . nausea and vomiting.
  • 5-HT applied to a human blister base has been demonstrated to cause pain that can be inhibited by 5-HT3 receptor antagonists.
  • bradykinin receptor antagonists Similarly, peripherally-applied bradykinin produces pain that can be blocked by bradykinin receptor antagonists. Sicuteri et al., 1965; Whalley et al., 1987; Dray, A., et al., "Bradykinin and Inflammatory Pain", Trends Neurosci. 16: 99-104 (1993). Peripherally-applied histamine produces vasodilation, itching and pain that can be inhibited by histamine receptor antagonists.
  • 5-HT is located in platelets and in central neurons, histamine is found in mast cells, and bradykinin is produced from a larger precursor molecule during tissue trauma, pH changes and temperature changes. Because 5-HT can be released in large amounts from platelets at sites. of tissue injury, producing plasma levels 20-fold greater than resting levels (Ashton, J.H., et al., "Serotonin as a Mediator of Cyclic Flow Variations in Stenosed Canine Coronary Arteries," Circulation 73:572-578 (1986)), it is possible that endogenous 5-HT plays a role in producing postoperative pain, hyperalgesia and inflammation.
  • Cyclooxygenase inhibitors are commonly used in non-surgical and postoperative settings to block the production of prostaglandins, thereby reducing prostaglandin-mediated pain and inflammation.
  • ibuprofen analgesic- Antipyretics and Anti-Inflammatojy Agents; Drugs Employed in the Treatment of Gout, in Goodman, L.S., et al., ed., The Pharmacological Basis of Therapeutics, MacMillan Publishing Company, New York, pp. 674-715 (1985).
  • Cyclooxygenase inhibitors are associated with some adverse systemic side effects when applied systemically. For example, indomethacin or ketorolac have well recognized gastrointestinal and renal adverse side effects.
  • 5-HT 5-HT
  • histamine histamine
  • bradykinin and prostaglandins cause pain and inflammation.
  • the various receptors through which these agents mediate their effects on peripheral tissues have been known and/or debated for the past two decades. Most studies have been performed in rats or other animal models. However, there are differences in pharmacology and receptor sequences between human and animal species.
  • Amitriptyline in addition to blocking the uptake of 5-HT and norepinephrine, is a potent 5-HT receptor antagonist. Therefore, the lack of efficacy in reducing postoperative pain in the previously mentioned studies would appear to conflict with the proposal of a role for endogenous 5-HT in acute pain. There are a number of reasons for the lack of acute pain relief found with amitriptyline in these two studies. (1) The first study (Levine et al., 1986) used amitriptyline preoperatively for one week up until the night prior to surgery whereas the second study (Kerrick et al.,
  • histaminei (Hi) receptor antagonists have been a few studies demonstrating the ability of extremely high . concenfrations (1% - 3% solutions -- i.e., 10 - 30 mg per milliliter) of histaminei (Hi) receptor antagonists to act as local anesthetics for surgical procedures.
  • This anesthetic effect is not believed to be mediated via Hi receptors but, rather, due to a non-specific interaction with neuronal membrane sodium channels (similar to the action of lidocaine).
  • side effects e.g., sedation
  • local administration of histamine receptor antagonists currently is not used in the perioperative setting.
  • the molecular switches responsible for cell signaling have been traditionally divided into major discrete signaling pathways, each comprising a distinct set of protein families that act as transducers for a particular set of extracellular stimuli and mediating distinct cell responses.
  • One such pathway transduces signals . from neurotransmitters and hormones through G-protein coupled receptors (GPCRs) to produce contractile responses that include the production of inflammatory mediators, such as PGE2.
  • GPCRs G-protein coupled receptors
  • the GPCRs couple to infracellular targets through activation of trimeric G proteins (see FIGURE 2). Examples of signaling molecules involved in activation of synoviocytes and chondrocytes through the GPCR pathway are histamine, bradykinin, serotonin and ATP.
  • a second major pathway transduces signals from pro-inflammatory cytokines, such as IL-1, through a kinase cascade and NF- ⁇ B protein into regulation of gene expression and the production of catabolic cytokines and other catabolic factors, including NO.
  • GPCRs composed of seven-helix transmembrane regions, or ligand-gated ion channels. "Downstream" signals from both kinds of receptors converge on controlling the concentration of cytoplasmic Ca 2+ (see FIGURE 3).
  • GPCR fransmembrane receptor activates a specific class of trimeric G proteins, including G q , G ⁇ or many others.
  • G q subunits activate phospholipase C ⁇ , resulting in activation of protein kinase C (PKC) and an increase in the levels of cytoplasmic calcium (FIGURE 3).
  • AA arachidonic acid
  • PKC activation also results in activation of MAP kinase leading to activation of NF- B and, in cells and tissues which have been primed by exposure to pro-inflammatory cytokines, modulates increased gene expression of proteins involved in cartilage catabolism.
  • Pro-inflammatory cytokine signaling such as mediated by both IL-1 and
  • TNF- ⁇ through their distinct cognate receptors also converges on regulation of cell gene expression.
  • the signal transduction pathways utilized by these distinct receptors employ distinct kinases that are proximal to the receptors but the signaling pathways subsequently converge at the level of MAP kinases (FIGURE 3 and 4).
  • Signal fransduction depends upon phosphorylation of residues in a cascade of kinases, including "downstream" enzymes such as p38 MAP kinase.
  • Activation of the IL-1 -receptor and TNF-receptor also leads to stimulation of MAP kinase, common steps shared by the Gq coupled GPCRs (see FIGURE 3).
  • Suitable classes of anti-inflammation and/or anti-pain agents for use in the compositions and methods of the present invention include: (1) serotonin receptor antagonists; (2) serotonin receptor agonists; (3) histamine receptor antagonists;
  • bradykinin receptor antagonists (5) kallikrein inhibitors; (6) tachykinin receptor antagonists, including neurokinin ! and neurokinin 2 receptor subtype antagonists; (7) calcitonin gene-related peptide (CGRP) receptor antagonists; (8) interleukin receptor antagonists; (9) inhibitors of enzymes active in the synthetic pathway for arachidonic acid metabolites, including (a) phospholipase inhibitors, including PLA 2 isoform inhibitors and PLC isoform inhibitors (b) cyclooxygenase inhibitors, and (c) lipooxygenase inhibitors; (10) prostanoid receptor antagonists including eicosanoid EP-1 and EP-4 receptor subtype antagonists and thromboxane receptor subtype antagonists; (11) leukotriene receptor antagonists including leukotriene B4 receptor subtype antagonists and leukotriene D4 receptor subtype antagonists; (12) opioid receptor agonists, including ⁇ -opioid,
  • compositions in accordance with the present invention will suitably include a dosage or load of the agent sufficient to result in a local concentration at the joint or site of action within the listed therapeutic range.
  • a sufficient dosage or load of the agent is included in the composition to result in a local concentration at the joint or site of action within the listed therapeutic range over a predetermined sustained release period. While not wishing to be limited by theory, the justification behind the selection of the various classes of agents which is believed to render the agents operative is also set forth.
  • Each agent is preferably included at a low concentration of 0.1 to 10,000 times Kd or Kj, except for cyclooxygenase inhibitors, which may be required at larger concenfrations depending on the particular inhibitor selected.
  • each agent is included at a concentration of 1.0 to 1,000 times K ⁇ or K j and most preferably at approximately 100 times K ⁇ j or Kj. These concenfrations are adjusted as needed to account for dilution in the absence of metabolic transformation at the local delivery site.
  • the exact agents selected for use in the solution, and the concentration of the agents varies in accordance with the particular application, as described below.
  • Serotonin Receptor Antagonists Serotonin (5-HT) is thought to produce pain by stimulating serotonm.2 (5-HT 2 ) and/or serotonm ⁇ (5-HT3) receptors on nociceptive neurons in the periphery. Most researchers agree that 5-HT3 receptors on peripheral nociceptors mediate the immediate pain sensation produced by 5-HT (Richardson et al., 1985). In addition to inhibiting 5-HT-induced pain, 5-HT 3 receptor antagonists, by inhibiting nociceptor activation, also may inhibit neurogenic inflammation. Barnes P.J., et al.,
  • 5-.HT2 receptor is responsible for nociceptor activation by 5-HT. Grubb, B.D., et al., "A Study of 5-HT-Receptors Associated with Afferent Nerves Located in Normal and Inflamed Rat Ankle Joints", Agents Actions 25: 216-18 (1988). Therefore, activation of 5-HT 2 receptors also may play a role in peripheral pain and neurogenic inflammation.
  • One goal of the solution of the present invention is to block pain and a multitude of inflammatory processes.
  • 5-HT2 an ⁇ ' 5-HT3 receptor antagonists are both suitably used, either individually or together, in the solution of the present invention, as shall be described subsequently.
  • Amitriptyline (ElavilTM) is a suitable 5-HT2 receptor antagonist for use in the present invention.
  • Amitriptyline has been used clinically for numerous years as an anti-depressant, and is found to have beneficial effects in certain chronic pain patients.
  • Metoclopramide (ReglanTM) is used clinically as an anti-emetic drag, but displays moderate affinity for the 5-HT 3 receptor and can inhibit the actions of 5-HT at this receptor, possibly inhibiting the pain due to 5-HT release from platelets. Thus, it also is suitable for use in the present invention.
  • 5-HT 2 receptor antagonists include imipramine, trazodone, desipramine and ketanserin. Ketanserin has been used clinically for its anti- hypertensive effects. Hedner, T., et al., "Effects of a New Serotonin Antagonist, Ketanserin, in Experimental and Clinical Hypertension", Am J of Hypertension 317s- 23s (Jul. 1988).
  • Other suitable 5-HT 3 receptor antagonists include cisapride and ondansefron.
  • Suitable serotonin ⁇ receptor antagonists include yohimbine, N-[-methoxy-3-(4-methyl-l-piperanzinyl)phenyl]-2'-methyl-4'-(5-methyl-l, 2, 4- oxadiazol-3-yl)[l, l-biphenyl]-4-carboxamide ("GR127935") and methiothepin.
  • Therapeutic and preferred concentrations for local delivery use of these drugs in the solution of an aspect the present invention are set forth in Table 14.
  • systemic compositions in accordance with the present invention will suitably include a dosage or load of the agent sufficient to result in a local concentration at the joint or site of action within the listed therapeutic range.
  • a sufficient dosage or load of the agent is included in the composition to result in a local concentration at the joint or site of action within the listed therapeutic range over a predetermined sustained release period.
  • Agent s Local Delivery Local Delivery Therapeutic Preferred Class of Agent Concentrations Concentrations (Nanomolar) (Nanomolar)
  • Tropisefron 0.01 - 100 0.05 - 50 Metoclopramide 10 - 10,000 200 - 2,000 Cisapride 0.1 - 1,000 . 20 - 200 Ondansefron 0.1 - 1,000 20 - 200
  • SerotoniniB (Human IDR) Antagonists Isamoltare 0.1 - 1,000 50 - 500
  • Serotonin Receptor Agonists 5-HTiA s 5-HTIB and 5-HTJD receptors are known to inhibit adenylate cyclase activity.
  • these serotonin ⁇ , serotonini ⁇ and serotoninir j ) receptor agonists in the solution should inhibit neurons mediating pain and inflammation.
  • the same action is expected from serotonin ⁇ E and serotoninip receptor agonists because these receptors also inhibit adenylate cyclase.
  • Buspirone is a suitable IA receptor agonist for use in the present invention.
  • Sumatriptan is a suitable 1 A, IB, ID and IF receptor agonist.
  • a suitable IB and ID receptor agonist is dihydroergotamine.
  • a suitable IE receptor agonist is ergonovine.
  • Therapeutic and preferred concenfrations for these receptor agonists when delivered locally are provided in Table 15.
  • systemic compositions in accordance with the present invention will suitably include a dosage or load of the agent sufficient to result in a local concentration at the joint or site of action within the listed therapeutic range.
  • a sufficient dosage or load of the agent is included in the composition to result in a local concenfration at the joint or site of action within the listed therapeutic range over a predetermined sustained release period.
  • Histamine receptors generally are divided into histamine ⁇ (H ⁇ ) and histamine2 (H 2 ) subtypes.
  • the classic inflammatory response to the peripheral administration of histamine is mediated via the Hi receptor. Douglas, 1985. Therefore, the solution of the present invention preferably includes a histamine H ⁇ receptor antagonist.
  • Promethazine PhenerganTM
  • this drug also has been shown to possess local anesthetic effects but the concenfrations necessary for this effect are several orders higher than that necessary to block H j receptors, thus, the effects are believed to occur by different mechanisms.
  • the histamine receptor antagonist concentration in the solution is sufficient to inhibit Hi receptors involved in nociceptor activation, but not to achieve a "local anesthetic" effect, thereby eliminating the concern regarding systemic side effects.
  • H j receptor antagonists include terfenadine, diphenhydramine, amitriptyline, mepyramine and tripolidine. Because amitriptyline is also effective as a serotonin 2 receptor antagonist, it has a dual function as used in the present invention.
  • Suitable therapeutic and preferred concenfrations for each of these H j receptor antagonists for local delivery are set forth in Table 16.
  • systemic compositions in accordance with the present invention will suitably include a dosage or load of the agent sufficient to result in a local concenfration at the joint or site of action within the listed therapeutic range.
  • a sufficient dosage or load of the agent is included in the composition to result in a local concentration at the joint or site of action within the listed therapeutic range over a predetermined sustained release period.
  • Tripolidine 0.01 - 100 5 - 20
  • Bradykinin receptors generally are divided into bradykinin! (B ⁇ ) and bradykinin2 (B ) subtypes. Studies have shown that acute peripheral pain and inflammation produced by bradykinin are mediated . by the B2 subtype whereas bradykinin-induced pain in the setting of chronic inflammation is mediated via the Bj subtype.
  • bradykinin receptor antagonists are not used clinically. Some of these drugs are peptides, and thus they cannot be taken orally, because they would be digested. Antagonists to B 2 receptors block bradykinin-induced acute pain and inflammation. Dray et al., 1993. Bj receptor antagonists inhibit pain in chronic inflammatory conditions. Perkins et al., 1993; Dray et al., 1993. Therefore, depending on the application, the solution of the present invention preferably includes either or both bradykinin B ⁇ and B 2 receptor antagonists. For example, arthroscopy is performed for both acute and chronic conditions, and thus an irrigation solution for arthroscopy could include both B ⁇ and B2 receptor antagonists.
  • bradykinin receptor antagonists for use in the present invention include the following bradykinin ⁇ receptor antagonists: the [des-Arg 10 ] derivative of
  • bradykinin2 receptor antagonists include: [D-Phe 7 ]-BK;
  • compositions in accordance with the present invention will suitably include a dosage or load of the agent sufficient to result in a local concentration at the joint or site of action within the listed therapeutic range.
  • a sufficient dosage or load of the agent is included in the composition to result in a local concentration at the joint or site of action within the listed therapeutic range over a predetermined sustained release period.
  • peptide bradykinin is an important mediator of pain and inflammation, as noted previously.
  • -Bradykinin is produced as a cleavage product by the action of kallikrein on high molecular weight kininogens in plasma. Therefore kallikrein inhibitors are believed to be therapeutic in inhibiting bradykinin production and resultant pain and inflammation.
  • a suitable kallikrein inhibitor for use in the present invention is aprotinin.
  • Suitable concenfrations for use in the solutions of the present invention when delivered locally are set forth below in Table 18.
  • systemic compositions in accordance with the present invention will suitably include a dosage or load of the agent sufficient to result in a local concentration at the joint or site of action within the listed therapeutic range.
  • a sufficient dosage or load of the agent is included in the composition to result in a local concenfration at the joint or site of action within the listed therapeutic range over a predetermined sustained release period.
  • Tachykinins are a family of structurally related peptides that include substance P, neurokinin A (NKA) and neurokinin B (NKB). Neurons are the major source of TKs in the periphery. An important general effect of TKs is neuronal stimulation, but other effects include endothelium-dependent vasodilation, plasma protein exfravasation, mast cell recraitment and degranulation and stimulation of inflammatory cells. Maggi, C.A., Gen. Pharmacol, 22: 1-24 (1991). Due to the above combination of physiological actions mediated by activation of TK receptors, targeting of TK receptors is a reasonable approach for the promotion of analgesia and the treatment of neurogenic inflammation. 6a. Neurokinin ⁇ Receptor Subtype Antagonists
  • Substance P activates the neurokinin receptor subtype referred to as NK ⁇ .
  • Substance P is an undecapeptide that is present in sensory nerve terminals.
  • Substance P is known to have multiple actions that produce inflammation and pain in the periphery after C-fiber activation, including vasodilation, plasma extravasation and degranulation of mast cells.
  • a suitable Substance P antagonist is ([D-Pro 9 [spiro-gamma-lactam]Leu 10 ,Trp 11 ]physalaemin-(l-l l)) ("GR 82334").
  • Suitable antagonists for use in the present invention which act on the NK ⁇ receptor are: l-imino-2-(2-methoxy-phenyl)-ethyl)-7,7-diphenyl-4- perhy ⁇ roisoindolone(3aR,7aR) ("RP 67580"); and 2S,3S-cis-3-(2- methoxybenzylamino)-2-benzhydrylquinuclidine (“CP 96,345").
  • Suitable concentrations for these agents when delivered locally are set forth in Table 19.
  • systemic compositions in accordance with the present invention will suitably include a dosage or load of the agent sufficient to result in a local concentration at the joint or site of action within the listed therapeutic range.
  • a sufficient dosage or load of the agent is included in the composition to result in a local concentration at the joint or site of action within the listed therapeutic range over a predetermined sustained release period.
  • Neurokinin A is a peptide which is colocalized in sensory neurons with substance P and which also promotes inflammation and pain. Neurokinin A activates the specific neurokinin receptor referred to as NK 2 . Edmonds-Alt, S., et al., "A
  • NK 2 Neurokinin A Receptor
  • suitable NK 2 antagonists include: ((S)-N- methyl-N-[4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]- benzamide ("(+)-SR 48968"); Met-Asp-Trp-Phe-Dap-Leu ("MEN 10,627”); and cyc(Gln-Trp-Phe-Gly-Leu-Met) ("L 659,877").
  • compositions in accordance with the present invention will suitably include a dosage or load of the agent sufficient to result in a local concenfration at the joint or site of action within the listed therapeutic range.
  • a sufficient dosage or load of the agent is included in the composition to result in a local concentration at the joint or site of action within the listed therapeutic range over a predetermined sustained release period.
  • Calcitonin gene-related peptide is a peptide which is also colocalized in sensory neurons with substance P, and which acts as a vasodilator and potentiates the actions of substance P. Brain, S., et al., Br. J. Pharmacol. 99: 202 (1985).
  • An example of a suitable CGRP receptor antagonist is I-CGRP-(8-37), a truncated version of CGRP. This polypeptide inhibits the activation of CGRP receptors. Suitable concenfrations for this agent when delivered locally are provided in Table 21.
  • compositions in accordance with the present invention will suitably include a dosage or load of the agent sufficient to result in a local concentration at the joint or site of action within the listed therapeutic range.
  • a sufficient dosage or load of the agent is included in the composition to result in a local concenfration at the joint or site of action within the listed therapeutic range over a predetermined sustained release period.
  • CGRP Receptor Antagonist I-CGRP-(8-37) 1-1,000 10-500 8.
  • Interleukin Receptor Antagonist I-CGRP-(8-37) 1-1,000 10-500 8.
  • Interleukins are a family of peptides, classified as cytokines, produced by leukocytes and other cells in response to inflammatory mediators. Interleukins (IL) may be potent hyperalgesic agents peripherally. Ferriera, S.H., et al., Nature 334:
  • IL-l ⁇ receptor antagonist Lys-D-Pro-Thr, which is a truncated version of IL-l ⁇ . This tripeptide inhibits the activation of IL-l ⁇ receptors. Suitable concenfrations for this agent for local delivery are provided in
  • compositions in accordance with the present invention will suitably include a dosage or load of the agent sufficient to result in a local concentration at the joint or site of action within the listed therapeutic range.
  • a sufficient dosage or load of the agent is included in the composition to result in a local concentration at the joint or site of action within the listed therapeutic range over a predetermined sustained release period.
  • PLA 2 isoform inhibitor manoalide. Inhibition of the phospholipase C ⁇ (PLC ⁇ ) isoform also will result in decreased production of prostanoids and leukotrienes, and, therefore, will result in decreased pain and inflammation.
  • PLC ⁇ isoform inhibitor is 1 -[6-((l 7 ⁇ -3-methoxyestra-l ,3,5(10)-trien- 17-yl)amino)hexyl]- lH- ⁇ yrrole-2,5- dione.
  • Suitable concenfrations for this agent when delivered locally are included in Table 23.
  • systemic compositions in accordance with the present invention will suitably include a dosage or load of the agent sufficient to result in a local concenfration at the joint or site of action within the listed therapeutic range.
  • a sufficient dosage or load of the agent is included in the composition to result in a local concenfration at the joint or site of action within the listed therapeutic range over a predetermined sustained release period.
  • Nonsteroidal anti-inflammatory drags are widely used as anti-inflammatory, anti-pyretic, anti-thrombotic and analgesic agents.
  • Lewis, R.A. Prostaglandins and Leukotrienes, In: Textbook of Rheumatology, 3d ed. (Kelley W.N., et al., eds.), p. 258 (1989).
  • the molecular targets for these drags are Type I and Type II cyclooxygenases (COX-1 and COX-2).
  • the COX-2 enzyme has been identified in endothelial cells, macrophages, and fibroblasts. This enzyme is induced by IL-1 and TNF- ⁇ , and its expression is upregulated at sites of inflammation. Constitutive activity of COX-1 and induced activity of COX-2 both lead to synthesis of prostaglandins that contribute to pain and inflammation.
  • NSAIDs currently on the market are generally nonselective inhibitors of both isoforms of COX, but may show greater selectively for COX-1 over COX-2, although this ratio varies for the different compounds.
  • Use of COX-1 and 2 inhibitors to block formation of prostaglandins represents a better therapeutic strategy than attempting to block interactions of the natural ligands with the seven described subtypes of prostanoid receptors.
  • Reported antagonists of the eicosanoid receptors EP-1, EP-2, EP-3) are quite rare and only specific, high affinity antagonists of the thromboxane A2 receptor have been reported. Wallace, J. et al. Trends in Pharm. Sci, 15:405-406 (1994).
  • compositions in accordance with the present invention will suitably include a dosage or load of the agent sufficient to result in a local concenfration at the joint or site of action within the listed therapeutic range.
  • a sufficient dosage or load of the agent is included in the composition to result in a local concenfration at the joint or site of action within the listed therapeutic range over a predetermined sustained release period.
  • ketorolac 100 - 10,000 500 - 5,000 indomethacin 1,000 - 500,000 10,000 - 200,000
  • lipooxygenase inhibits the production of leukotrienes, such as leukotriene B 4 , which is known to be an important mediator of inflammation and pain.
  • leukotriene B 4 which is known to be an important mediator of inflammation and pain.
  • Lewis, R.A. Prostaglandins and Leukotrienes, In: Textbook of Rheumatology, 3d ed. (Kelley W.N., et al., eds.), p. 258 (1989).
  • An example of a 5-lipooxygenase antagonist is 2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-
  • compositions in accordance with the present invention will suitably include a dosage or load of the agent sufficient to result in a local concentration at the joint or site of action within the listed therapeutic range.
  • a sufficient dosage or load of the agent is included in the composition to result in a local concentration at the joint or site of action within the listed therapeutic range over a predetermined sustained release period.
  • Prostanoid Receptor Antagonists Specific prostanoids produced as metabolites of arachidonic acid mediate their inflammatory effects through activation of prostanoid receptors. Examples of classes of specific prostanoid antagonists are the eicosanoid EP-1 and EP-4 receptor subtype antagonists and the thromboxane receptor subtype antagonists.
  • a suitable prostaglandin E 2 receptor antagonist is 8-chlorodibenz[b,f][l,4]oxazepine-10(l lH)- carboxylic acid, 2-acetylhydrazide ("SC 19220").
  • a suitable thromboxane receptor subtype antagonist is [15-[l ⁇ , 2 ⁇ (5Z), 3 ⁇ , 4 ⁇ ]-7-[3-[2-(phenylamino)-carbonyl] hydrazino] methyl]-7-oxobicyclo-[2,2,l]-hept-2-yl]-5-heptanoic acid ("SQ 29548").
  • Suitable concentrations for these agents when delivered locally are set forth in Table 26.
  • systemic compositions in accordance with the present invention will suitably include a dosage or load of the agent sufficient to result in a local concentration at the joint or site of action within the listed therapeutic range.
  • a sufficient dosage or load of the agent is included in the composition to result in a local concenfration at the joint or site of action within the listed therapeutic range over a predetermined sustained release period.
  • the leukotrienes (LTB 4 , LTC 4 , and LTD 4 ) are products of the
  • LTB 4 receptor is found in certain immune cells including eosinophils and neutrophils. LTB 4 binding to these receptors results in chemotaxis and lysosomal enzyme release thereby contributing to the process of inflammation.
  • the signal fransduction process associated with activation of the LTB 4 receptor involves G-protein-mediated stimulation of phosphotidylinositol (PI) metabolism and elevation of intracellular calcium (see FIGURE 2).
  • a suitable leukotriene B 4 receptor antagonist is SC (+)-(S)-7- (3-(2-(cyclopropylmethyl)-3-methoxy-4-[(methylamino)- carbonyl]phenoxy(propoxy)-3 ,4-dihydro- 8-propyl-2H- 1 -b enzopyran-2-propanoic acid ("SC 53228").
  • SC 53228 a suitable leukotriene B 4 receptor antagonist
  • compositions in accordance with the present invention will suitably include a dosage or load of the agent sufficient to result in a local concenfration at the joint or site of action within the ' listed therapeutic range.
  • a sufficient dosage or load of the agent is included in the composition to result in a local concentration at the joint or site of action within the listed therapeutic range over a predetermined sustained release period.
  • Opioid Receptor Agonists Activation of opioid receptors results in anti-nociceptive effects and, therefore, agonists to these receptors are desirable.
  • Opioid receptors include the ⁇ -, ⁇ - and ⁇ -opioid receptor subtypes. The ⁇ -receptors are located on sensory neuron terminals in the periphery and activation of these receptors inhibits sensory neuron activity. Basbaum, A.I., et al., "Opiate analgesia: How Central is a Peripheral Target?", N. Engl. J. Med. 325:1168 (1991).
  • ⁇ - and K-receptors are located on sympathetic efferent terminals and inhibit the release of prostaglandins, thereby inhibiting pain and inflammation. Taiwo, Y.O., et al., "Kappa- and Delta-Opioids Block Sympathetically Dependent Hyperalgesia", J. Neurosci. 11:928 (1991).
  • The. opioid receptor subtypes are members of the G-protein coupled receptor superfamily. Therefore, all opioid receptor agonists interact and initiate signaling through their cognate G-protein coupled receptor. Examples of suitable ⁇ -opipid receptor agonists are fentanyl and Try-D-Ala-Gly-[N-MePhe]-NH(CH 2 )-OH (“DAMGO").
  • Suitable ⁇ -opioid receptor agonist is [D-Pen 2 ,D-Pen 5 ]enkephalin ("DPDPE").
  • An example of a suitable i -opioid receptor agonist is (trans)-3,4- dichloro-N-methyl-N-[2-(l-pyrrolidnyl)cyclohexyl]-benzene acetamide ("U50,488").
  • Suitable concenfrations for the local delivery of each of these agents are set forth in Table 28.
  • systemic compositions in accordance with the present invention will suitably include a dosage or load of the agent sufficient to result in a local concentration at the joint or site of action within the listed therapeutic range.
  • a sufficient dosage or load of the agent is included in the composition to result in a local concenfration at the joint or site of action within the listed therapeutic range over a predetermined sustained release period.
  • Agonist DAMGO 0.1-100 0.5-20 sufentanyl .0.01-50 1-20 fentanyl 0.1-500 10-200 PL 017 0.05-50 0.25-10 ⁇ -Opioid Agonist: DPDPE 0.1-500 1.0-100 ⁇ -Opioid Agonist: U50,488 0.1-500 1.0-100
  • Extracellular ATP acts as a signaling molecule through interactions with P 2 purinoceptors.
  • P 2x purinoceptors which are ligand-gated ion channels possessing intrinsic ion channels permeable to Na + , K , and Ca 2+ .
  • P 2x receptors described in sensory neurons are important for primary afferent neurotransmission and nociception.
  • ATP is known to depolarize sensory neurons and plays a role in nociceptor activation since ATP released from damaged cells stimulates P 2 ⁇ receptors leading to depolarization of nociceptive nerve-fiber terminals.
  • the P2X3 receptor has a highly restricted distribution (Chen, CC, et al.,
  • ATP and UTP did not alter cartilage matrix synthesis as measured by rate of incorporation of [35S]sulfate into glycosaminoglycan by cartilage explants or primary chondrocytes.
  • Matrix degradation measured by release of glycosaminoglycan from cartilage explants, was also unaltered by either agonist.
  • the presence of a functional P2Y purine receptor on the surface of primary articular chondrocytes enable concentrations of extracellular purines, such as ATP, to activate chondrocyte metabolism.
  • P2Y2 receptor agonists ATP and UTP stimulated a small release of PGE2 that was synergistically enhanced after prefreatment with human IL-l ⁇ .
  • PGE2 release in response to coaddition of ATP and UTP after IL-1 prefreatment was mimicked by phorbol myristate acetate.
  • the function of the P2Y2 receptor is to increase IL-1 -mediated PGE2 release, thereby promoting pain and inflammation within the joint.
  • the use of P2Y antagonists in the present invention should prevent activation of inflammatory mediator production by both synoviocytes and chondrocytes.
  • Suitable antagonists of P 2 ⁇ /ATP purinoceptors for use in the present invention include, by way of example, suramin and pyridoxylphosphate-6- azophenyl-2,4-disulfonic acid ("PPADS"). Suitable concentrations for the local delivery of these agents are provided in Table 29.
  • systemic compositions in accordance with the present invention will suitably include a dosage or load of the agent sufficient to result in a local concenfration at the joint or site of action within the listed therapeutic range.
  • a sufficient dosage or load of the agent is included in the composition to result in a local concenfration at the joint or site of action within the listed therapeutic range over a predetermined sustained release period.
  • Calcium channel antagonists are a distinct group of drugs that interfere with the fransmembrane flux of calcium ions required for activation of cellular responses mediating neuroinflammation. Calcium entry into synoviocytes and chondrocytes is a key event mediating activation of responses in these cells. Furthermore, the role of bradykinin, histamine, serotonin (SHT 2 ) and neurokinin receptors (NK ⁇ and NK2) in mediating the neuroinflammation signal transduction pathway includes increases in infracellular calcium, thus leading to activation of calcium channels on the plasma membrane.
  • calcium channel antagonists such as nifedipine
  • nifedipine can reduce the release of arachidonic acid, prostaglandins, and leukotrienes that are evoked by various stimuli.
  • calcium channel antagonists and either tachykinin, histamine or bradykinin antagonists exhibit synergistic effects in inhibiting neuroinflammation.
  • NK ⁇ neurokinin !
  • NK2 neurokinin 2
  • G-protein coupled superfamily signal transduction pathway includes increases in infracellular calcium, thus leading to activation of calcium channels on the plasma membrane.
  • activation of bradykinin 2 (BK 2 ) receptors is coupled to increases in intracellular calcium in synoviocytes and chondrocytes.
  • BK 2 bradykinin 2
  • calcium channel antagonists interfere with a common mechanism involving elevation of infracellular calcium, part of which enters through L-type channels. This is the basis for synergistic interaction between calcium channel antagonists and antagonists to neurokinin, histamine, P Y and bradykinin 2 receptors.
  • Suitable calcium channel antagonists for the practice of the present invention include nisoldipine, nifedipine, nimodipine, lacidipine, isradipine and amlodipine.
  • Suitable concenfrations for the local delivery of these agents are set forth in Table 30.
  • systemic compositions in accordance with the present invention will suitably include a dosage or load of the agent sufficient to result in a local concenfration at the joint or site of action within the listed therapeutic range.
  • a sufficient dosage or load of the agent is included in the composition to result in a local concenfration at the joint or site of action within the listed therapeutic range over a predetermined sustained release period.
  • composition is suitable for use in anatomic joint irrigation during arthroscopic procedures.
  • Each drag is solubilized in a carrier fluid containing physiologic elecfrolytes, such as normal saline or lactated Ringer's solution, as are the remaining solutions described in subsequent examples.
  • physiologic elecfrolytes such as normal saline or lactated Ringer's solution
  • TGF- ⁇ Agonist TGF- ⁇ 2 200
  • composition is an alternate formulation suitable for use in anatomic joint irrigation during arthroscopic procedures.
  • TGF- ⁇ Agonist TGF- ⁇ 2 100
  • composition is suitable for injection into an anatomic joint.
  • Each drag is solubilized in a carrier fluid containing physiologic electrolytes, such as normal saline or lactated Ringer's solution.
  • a dosage of 20 ml of the solution is suitable for administration to a patient.
  • the following chondroprotective composition is suitable for systemic delivery, such as by intramuscular or subcutaneous administration.
  • Each drug is included in the composition at a concentration that will result in the following concentration at the site of intended action, and is solubilized in a physiologic carrier fluid or delivery system.
  • TGF- ⁇ Agonist TGF- ⁇ 2 200
  • the following chondroprotective composition is suitable for systemic delivery, such as by intramuscular or subcutaneous administration.
  • Each drug is included in the composition at a concenfration sufficient to result in the following concentration at the site of intended action, and is solubilized in a physiologic carrier fluid or delivery system.
  • the following chondroprotective composition is suitable for systemic delivery, such as by intramuscular or subcutaneous adminisfration.
  • Each drag is included in the composition at a concentration sufficient to result in the following concentration at the site of intended action, and is solubilized in a physiologic carrier fluid or delivery system.
  • TGF- ⁇ Agonist TGF- ⁇ 2 100
  • the following chondroprotective composition is suitable for systemic delivery, such as by intramuscular or subcutaneous adminisfration.
  • Each drag is included in the composition at a concenfration sufficient to result in the following concentration at the site of intended action, and is solubilized in a physiologic carrier fluid or delivery system.
  • TGF- ⁇ Agonist TGF- ⁇ 2 200
  • the following chondroprotective composition is suitable for systemic delivery, such as by intramuscular or subcutaneous adminisfration.
  • Each drag is included in the composition at a concentration sufficient to result in the following concentration at the site of intended action, and is solubilized in a physiologic carrier fluid or delivery system.
  • Antagonist (KineretTM,
  • TGF- ⁇ Agonist TGF- ⁇ 2 200
  • the following chondroprotective composition is suitable for systemic delivery, such as by inframuscular or subcutaneous administration.
  • Each drag is included in the composition at a concentration sufficient to result in the following concenfration at the site of intended action, and is solubilized in a physiologic carrier fluid or delivery system.
  • the following chondroprotective composition is suitable for systemic delivery, such as by intramuscular or subcutaneous administration.
  • Each drag is admimstered at the noted dosage, and is solubilized in a physiologic carrier fluid or delivery system.
  • chondroprotective composition is suitable for systemic delivery, such as by inframuscular or subcutaneous adminisfration. Each drag is administered at the noted dosage, and is solubilized in a physiologic carrier fluid or delivery system.
  • the following chondroprotective composition is suitable for systemic delivery, such as by intravenous, inframuscular, subcutaneous or inhalation adminisfration.
  • the drags are encapsulated within a DL-lactide/glycolide copolymer (PLGA) nanosphere, to which is coupled an anti-human Type II collagen monoclonal antibody.
  • PLGA DL-lactide/glycolide copolymer
  • This antibody targets epitopes on human Type II collagen in articular cartilage.
  • Each drug is included in the composition at a concentration sufficient to result in the following average concenfration at the site of intended action upon degradation of the nanosphere and release of the agents over a period of sustained release.
  • the following chondroprotective composition is suitable for systemic delivery, such as by infravenous, inframuscular, subcutaneous or inhalation administration.
  • the drags are encapsulated within a biodegradable PLA/PLGA copolymeric nanosphere, to which is coupled an anti-human aggrecan monoclonal antibody.
  • This antibody targets neoeptitopes on human aggrecan in articular cartilage.
  • Each drug is included in the composition at a concentration sufficient to result in the following concentration at the site of intended action upon degradation of the nanosphere and release of the agents over a desired period of sustained release.
  • Class of Agent Concentration at Site of Action (Nanomolar)
  • the following chondroprotective composition is suitable for systemic delivery, such as by infravenous, intramuscular, subcutaneous or inhalation administration.
  • the drags are encapsulated within a chitosan/gelatin nanosphere, to which is coupled an anti-human Type II collagen monoclonal antibody. This antibody targets neoepitopes on human Type II collagen.
  • Each drug is included in the composition at a concenfration sufficient to result in the following concenfration at the site of intended action upon degradation of the nanosphere and release of the agents over a desired period of sustained release.
  • the following chondroprotective composition is suitable for systemic delivery, such as by intravenous, inframuscular, subcutaneous or inhalation administration.
  • the drags are encapsulated within an albumin nanosphere, to which is coupled an anti-human Type II collagen monoclonal antibody. This antibody targets neoeptitopes on human Type II collagen.
  • Each drag is included in the composition at a concentration sufficient to result in the following concenfration at the site of intended action upon degradation of the nanosphere and release of the agents over a desired period of sustained release.
  • the following chondroprotective composition is suitable for systemic delivery, such as by intravenous, intramuscular, subcutaneous or inhalation administration.
  • the drags are encapsulated within a poly(lactide-co- glycolide)/poly(ethyleneglycol) copolymer nanosphere, to which is coupled an anti- human Type II collagen monoclonal antibody.
  • This antibody targets neoeptitopes on human Type II collagen in articular cartilage.
  • Each drag is included in the composition at a concenfration sufficient to result in the following concenfration at the site of intended action upon degradation of the nanosphere and release of the agents over a desired period of sustained release.
  • the following chondroprotective composition is suitable for systemic delivery, such as by infravenous, inframuscular, subcutaneous or inhalation administration.
  • the BMP receptor agonist, BMP-7 is encapsulated within a poly(lactide-co-glycolide) (PLGA) nanosphere to which is coupled an anti-human Type II collagen monoclonal antibody.
  • the IGF Receptor agonist, IGF-1 is separately encapsulated within a chondroiten-6-sulfate/gelatin nanosphere to which is also coupled an anti-human aggrecan monoclonal antibody.
  • the antibody used for targeting each type of nanosphere binds to neoeptitopes on human Type II collagen and neoepitopes on aggrecan in articular cartilage.
  • Each drag is included in the composition at a concentration sufficient to result in. the following average concenfration at the site of intended action upon degradation of the nanosphere and release of the agents over a desired period of sustained release.
  • the following chondroprotective composition is suitable for systemic delivery, such as by infravenous, inframuscular, subcutaneous or inhalation administration.
  • the drags are encapsulated within an albumin nanosphere, to which is coupled an anti-human F(ab') 2 fragment that binds to Type II collagen monoclonal antibody.
  • This F(ab') 2 antibody targets neoeptitopes on human Type II collagen.
  • Each drag is included in the composition at a concenfration sufficient to result in the following concentration at the site of intended action upon degradation of the nanosphere and release of the agents over a desired period of sustained release.
  • the following chondroprotective composition is suitable for systemic delivery, such as by intravenous, inframuscular, subcutaneous or inhalation adminisfration.
  • the drugs are encapsulated within an albumin nanosphere, to which is coupled an anti-human single-chain minimum binding domain of an immunoglobulin molecule (scFV) that binds to Type II collagen monoclonal antibody.
  • scFV immunoglobulin molecule
  • This scFV antibody targets neoeptitopes on human Type II collagen.
  • Each drag is included in the composition at a concentration sufficient to result in the following concentration at the site of intended action upon degradation of the nanosphere and release of the agents over a desired period of sustained release.
  • STUDY 1 Synergistic stimulation of a rapid PGE2 burst upon exposure to IL-1 and GPCR agonists.
  • Fibroblast-like synoviocytes exhibit characteristics of inflammatory cells and seem to be crucial regulators of joint inflammation and cartilage degradation.
  • a synoviocyte cell culture model system was used to characterize the synergistic interactions between IL-1 and non-cytokine inflammatory mediators which are important in modulating the destruction of joint tissue, including damage that occurs as a consequence of tissue injury during arthroscopic surgery.
  • GPCR G-protein coupled receptor
  • Synovial tissue was obtained from osteoarthritis patients undergoing joint replacement surgery through the Clinical Research Center, MacNeal Hospital, and transported to the laboratory in Dulbecco's Modified Eagle's Medium (DMEM) containing penicillin (100 units/ml), streptomycin (100 ⁇ g/ml), and fungizone (0.25 ⁇ g/ml).
  • DMEM Dulbecco's Modified Eagle's Medium
  • penicillin 100 units/ml
  • streptomycin 100 ⁇ g/ml
  • fungizone 0.25 ⁇ g/ml
  • the synovium was dissected and minced with scissors, and plated as explants in culture medium composed of DMEM containing L-glutamine (2 mM), heat inactivated fetal bovine serum (10% v/v), plus antibiotics.
  • LB composition in mM NaCl, 154; KCl, 2.6; KH 2 PO , 2.15; K 2 HPO 4 , 0.85; MgCl 2 , 5; CaCl 2 , 2; D-glucose, 10; HEPES, 10; pH 7.4, BSA, 0.1% w/v
  • LB composition in mM NaCl, 154; KCl, 2.6; KH 2 PO , 2.15; K 2 HPO 4 , 0.85; MgCl 2 , 5; CaCl 2 , 2; D-glucose, 10; HEPES, 10; pH 7.4, BSA, 0.1% w/v
  • Pharmacological inhibitors typically were added during the 10 min preincubation interval, and agonists plus the specified inhibitors were present during the 3 min challenge interval. 3. Measurement of Prostaglandin E2. Following indicated freatment protocols, aliquots of culture supernatant (1 ml) were collected and rapidly frozen in liquid nitrogen. Samples were stored at -80° until processing. Aliquots of culture supernatant were analyzed by competitive binding radioimmunoassay as specified by the manufacturer (Sigma Chemical Co.), using an antibody with equivalent reactivity toward prostaglandins E2 and El. For quantitation, a standard curve was prepared with each assay using fixed concentrations of [ 3 H]prostaglandin E2, and increasing concenfrations of authentic competing prostaglandin E2.
  • IL-6 was measured by sandwich ELISA with alkaline phosphatase detection as described by the manufacturer (Pharmingen) and quantitated " using standard curves prepared with the respective pure recombinant human cytokines. Experimental determinations were performed on duplicate cultures.
  • Synoviocyte cell lines were routinely evaluated for competence to proliferate in response to IL-1, measured as incorporation of [ 3 H]thymidine (Kimball & Fisher,
  • Cell culture media were obtained from Sigma or Gibco/BRL. Fetal bovine serum was from Atlanta Biologicals Inc. (Norcross, GA).
  • Drugs Recombinant human interleukin-1 was obtained from Genzyme (Cambridge, MA). Ketoprofen was provided by Omeros Medical Systems, Inc. (Seattle, WA). Amitriptyline, forskolin, 5-hydroxytryptamine, isoproterenol, Bradykinin, histamine, and prostaglandin E2 were from Sigma.
  • Radiochemicals [ 3 H]Prostaglandin E2, was obtained from American Radiolabeled Chemicals, Inc. (St. Louis, MO). All other reagents were obtained in the highest purity available from standard commercial suppliers.
  • the bradykinin-stimulated PGE2 response continues to increase (2-fold) over the same time period.
  • naive synoviocytes show no detectable PGE2 production in response to stimulation with either GPCR agonist alone. Under conditions of IL-1 priming, histamine and bradykinin both synergistically potentiated PGE2 release.
  • IL-1 increases expression of cPLA2 (Hulkower et al., 1994). These two proteins act together to provide free arachidonic acid substrate for COX-2.
  • cPLA2 is the only known PLA2 that exhibits functional properties indicative of receptor regulation and is likely to be involved in eicosonoid production and infracellular signaling: Since cPLA2 is activated by increasing calcium concentrations for full activity and bradykinin B2 and histamine HI receptor activation is coupled to mobilization of intracellular calcium, this is likely the predominant factor regulating the rapid agonist-stimulated burst in PGE2 production. Finally, the very rapid and transient increase in cytoplasmic calcium triggered by B2 or HI receptor activation is similar to the kinetics known for cPLA2 activation, arachidonic acid release, and the observed PGE2 burst.
  • ketoprofen a cyclooxygenase inhibitor
  • IL-1 a cyclooxygenase inhibitor
  • FIGURE 8 The actions of ketoprofen, a cyclooxygenase inhibitor, to attenuate PGE2 formation were determined by co-incubation with IL-1 during prolonged exposure (16 hr); and by brief pre-incubation prior to a subsequent GPCR agonist challenge interval, as shown in FIGURE 8.
  • ketoprofen on the induction of cytokine production by IL-1 and GPCR agonists was examined.
  • the protocol also, tested the effects of IL-1 concentration dependence on the IL-6 steady state induction.
  • Synoviocyte cultures were exposed to indicated concentrations of IL-1 and GPCR agonists. Culture supernatants were collected and replaced with fresh media aliquots containing the same agonist additions at 8-hr intervals. PGE 2 , IL-6, and IL-8 in the supernatants were assayed as described.
  • FIGURE 9 Data for IL-6 production are shown in FIGURE 9, which shows IL-6 production at 16 hr (corresponding to treatment interval from 8-16 hr) in the presence of indicated concentrations of IL-1 plus added ligand.
  • Addition of histamine or isoproterenol does not enhance IL-6 production compared to IL-1 alone.
  • ketoprofen causes a partial ( ⁇ 50%) inhibition of IL-1 -elicited IL-6 production.
  • ketoprofen inhibited IL-6 production in the histamine or isoproterenol/IL-1 co-stimulated samples.
  • the synoviocyte cell culture model system was used to characterize the synergistic interactions between IL-1 and non-cytokine inflammatory mediators that are important in modulating the destraction of joint tissue, including damage that occurs as a consequence of tissue injury during arthroscopic surgery.
  • IL-1 induces large increases in PGE 2 , IL-6, and IL-8 in cultured synoviocytes, whereas quiescent cultures do not produce detectable quantities of these mediators
  • the induction of PGE 2 occurs most rapidly and results in release of PGE 2 to the culture supernatant at 4 hr, followed by IL-8 at 6 hr, and IL-6 at longer intervals
  • all three mediators remain elevated in the culture supernatant following 24 hr IL-1 exposure.
  • the GPCR agonists do not enhance IL-1 induction of IL-6 or IL-8 and also do not increase IL-6 and IL-8 release following priming with IL-1.
  • IL-1 induction of IL-6 and IL-8 appears to be reinforced by the concomitant induction of PGE 2 since ketoprofen reduces the production of these cytokines in response to IL-1. This result indicates that ketoprofen could provide a therapeutic chondroprotective effect when delivered to the joint during surgical procedures. Taken together, these results demonstrate interactions between specific
  • G-coupled receptor signaling pathways and the activation of synoviocytes by pro- inflammatory stimulation with IL-1 A similar mechanism is expected to be operative in chondrocytes.
  • These interactions provide a means of integrating and modulating pro-inflammatory responses of synoviocytes and chondrocytes depending on inputs from other autocoid or neurofransmitter receptor systems within the joint.
  • These findings underscore the rationale and potential clinical benefit of therapeutic interventions which target inhibition of G-protein coupled receptors that mediate signaling through calcium mobilization, phosphoinositide hydrolysis and PKC activation and are coupled to increases in production of PGE 2 in arthroscopic surgery.
  • These receptors on synoviocytes and chondrocytes include histamine Hi, bradykinin, Substance P, 5HT2, and the purinergic P2Y receptors.

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Abstract

L'invention concerne des méthodes et des compositions d'inhibition de la dégradation du cartilage articulaire. Les compositions comprennent, de préférence, de multiples agents chondroprotecteurs, comprenant au moins un agent favorisant l'activité anabolique du cartilage et au moins un agent d'inhibition du catabolisme du cartilage. Les compositions peuvent également comprendre un ou plusieurs agents d'inhibition de la douleur ou de l'inflammation. Les compositions peuvent être administrées de façon systémique, par exemple pour traiter les patients à risque pouvant présenter une dégradation du cartilage au niveau de multiples articulations, et peuvent être formulées de manière appropriée dans un excipient ou véhicule d'administration ciblant les articulations. En variante, les compositions peuvent être injectées ou administrées par perfusion directement dans l'articulation.
PCT/US2003/003175 2002-02-01 2003-01-31 Compositions et methodes d'inhibition systemique de la degradation du cartilage WO2003063799A2 (fr)

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JP2003563495A JP2005519917A (ja) 2002-02-01 2003-01-31 軟骨分解の全身阻害のための組成物および方法
AU2003212898A AU2003212898B2 (en) 2002-02-01 2003-01-31 Compositions and methods for systemic inhibition of cartilage degradation
MXPA04007124A MXPA04007124A (es) 2002-02-01 2003-01-31 Composiciones y metodos para la inhibicion sistemica de la degradacion del cartilago.
EP03708941A EP1496835A4 (fr) 2002-02-01 2003-01-31 Compositions et methodes d'inhibition systemique de la degradation du cartilage
KR10-2004-7011603A KR20040094413A (ko) 2002-02-01 2003-01-31 연골 분해를 전신 억제하기 위한 조성물 및 방법
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CA2474645A1 (fr) 2003-08-07
CA2474645C (fr) 2011-08-09
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JP2005519917A (ja) 2005-07-07
CN1697647A (zh) 2005-11-16

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