KR20040094413A - Compositions and methods for systemic inhibition of cartilage degradation - Google Patents

Abstract

Described herein are compositions and methods for inhibiting articular cartilage degradation. The composition of the present invention preferably comprises a plurality of cartilage protective agents, including one or more agents that enhance cartilage anabolic activity and one or more agents that inhibit cartilage catabolic activity. The compositions of the present invention may also include one or more pain and inflammation inhibitory agents. The compositions of the present invention may be administered systemically to treat patients at risk of cartilage degradation in several joints and may be suitably formulated in a carrier or delivery vehicle targeted to the joint. Alternatively, the compositions of the present invention may be injected or injected directly into the joint.

Description

COMPOSITIONS AND METHODS FOR SYSTEMIC INHIBITION OF CARTILAGE DEGRADATION

Diseases and disorders that cause cartilage destruction in joints raise public health concerns, which are particularly important from the demographic perspective of the aging population. Articular cartilage of the joints represents a complex system of many different molecules. Multiple mechanisms are involved in the articular cartilage breakdown in arthritis, such as rheumatoid arthritis (RA) and osteoarthritis (OA). OA, a non-inflammatory arthritis, is the most common form of joint disease and is most common after cardiovascular disease as a cause of premature retirement and physical disability. Some individuals have OA in a single or limited number of joints, which may result from traumatic injuries from accidents or surgery. Many other individuals suffer from OA in several joints due to prolonged exercise, occupational activity, or wear associated with aging. RA is the most common form of inflammatory arthritis, affecting 3% of women and 1% of men. Most RA patients have symptoms in several joints, particularly the joints of the hands, elbows, lower back and shoulders.

Superarticular cartilage destruction is characteristic of OA and incapable RA. Although various therapeutic approaches may provide symptomatic relief, no therapeutic regimen has been identified that has been shown to delay the progression of articular cartilage degradation. Progressive deterioration and loss of articular cartilage cause irreversible defects in joint motion. This change in cartilage is the final pathogenic event common to osteoarthritis (OA) and rheumatoid arthritis (RA).

The cartilage destruction process may be associated with or initiated by surgical treatment of the joint. Arthroscopy is a surgical procedure in which a camera with a remote light source and a video monitor is attached to these joints through a small incision in the flap and the skin covering the anatomical joints (eg knee, shoulder, etc.). Through similar lateral incisions, surgical instruments can be placed in the joints, the use of which can be guided by visualization using arthroscopy. As the arthroscopic skill level improves, the number of surgical procedures performed once by an "open" surgical technique can now be increased by using arthroscopy. Such treatments include, for example, partial meniscus resection and ligament reconstruction of the knee, shoulder acroplasty and circumcision palliative resection and elbow synovial resection. As surgical signs expand and small-diameter arthroscopy develops, waist and ankle arthroscopy have become commonplace.

Throughout each arthroscopy, physiological irrigation (e.g., normal saline or lactated Ringer's solution) is continuously introduced into the joint to provide cleaner intra-articular visualization by inflating the articular capsule and removing surgical debris. US Pat. No. 4,504,493 describes an equimolar solution of glycerol in non-conductive water and an optically clear irrigation solution for use in arthroscopy. Conventional physiological irrigation does not provide analgesic, anti-inflammatory or anticartilage degrading effects.

Reference to related application

This application claims priority to US Provisional Patent Application No. 60 / 353,552, filed February 1, 2002, which claims priority to US Provisional Patent Application No. 60 / 144,904, filed July 21, 1999. And a partial continuation of US Patent Application No. 10 / 031,546 (filed Jan. 18, 2002), which is the US national stage of the international patent application PCT / US00 / 19864 (filed Jul. 21, 2000) with the US as the designated country. Is a priority, and claims priority to US Provisional Patent Application No. 60 / 107,256, filed November 5, 1998, filed international patent application PCT / US99 / 26330 (November 5, 1999) And US Provisional Patent Application No. 60 / 105,026, filed Oct. 20, 1998, with an international patent application PCT / US99 / 24625, filed Oct. 20, 1999. US patent application Ser. No. 09 / 839,633, filed Apr. 20, 2001, filed partly.

The present invention relates to therapeutic compositions and methods for protecting articular cartilage.

The invention will be described in more detail by way of example with reference to the accompanying drawings in which:

1 is a schematic diagram of chondrocytes showing signaling information flow and molecular targets that induce the production of inflammatory mediators and the migration of cartilage metabolism. Of exogenous signals through various classes of cell surface receptors, including cytokine receptors such as the interleukin-1 (IL-1) receptor family and tumor necrosis factor (TNF) receptor family, TGF-b receptor super-family and integrin Integration is on a common intracellular signaling pathway comprising a major group of therapeutically targeted protein molecules of the drug included in the solution of the invention (MAP kinase, PKC, tyrosine kinase, SH2 protein, COX, PLA2 and NF-6B). It is shown to be focused on. Activation of these signaling pathways leads to chondrocyte expression and inflammation and / or cartilage degradation of numerous inducible gene products, including IL-1, TNF-a, IL-6, IL-8 and stromelysin (MMP-3). Or control chondrocyte expression of other mediators (nitric oxide (NO) and PGE2) that can lead to matrix molecule synthesis and chondrocyte proliferation.

2 is a schematic diagram of synovial cells showing signaling information flow and molecular targets leading to the generation of inflammatory mediators and to cartilage metabolic migration. Cytokine receptors such as the interleukin-1 (IL-1) receptor family and tumor necrosis factor (TNF) receptor family; G-protein coupled receptors including bradykinin, histamine and serotonin subtypes; And incorporation of exogenous signals through various classes of cell surface receptors, including integrins, is a major group of protein molecules that are therapeutic targets of drugs included in the solutions of the invention (MAP kinase, PKC, tyrosine kinase, SH2 protein, COX, It is shown to be concentrated on common intracellular signaling pathways including PLA2 and NF-6B). Activation of these signaling pathways can lead to inflammation and / or cartilage degradation of numerous inducible gene products, including IL-1, TNF-a, IL-6, IL-8 and stromelysin (MMP-3). Control synovial cell expression.

3 is common to both chondrocytes and synovial cells, including the major signaling protein responsible for "crosstalk" between the pro-inflammatory cytokine pathway and the GPCR activating receptor pathway leading to inflammation and / or cartilage degradation. Diagram of the signaling path.

4 is a diagram of a signaling pathway common to both chondrocytes and synovial cells, including the main signaling protein responsible for "crosstalk" between the pro-inflammatory cytokine pathway and the GPCR activating receptor pathway. Specific molecular action sites for some drugs in the preferred cartilage protective solution of the present invention are identified.

5 is a diagram of molecular targets present on chondrocytes or synovial cells that enhance the anabolic response of cartilage. Specific site of action for some drugs in the preferred cartilage protective solution of the invention is identified.

6 is a diagram of molecular targets present on chondrocytes or synovial cells that enhance the catabolic response of cartilage. Specific site of action for some drugs in the preferred cartilage protective solution of the invention is identified.

FIG. 7 is a graph showing prostaglandin E2 production in synovial cultures with G-protein regulatory agonists after priming overnight with interleukin-1 (IL-1, 10U / ml). The culture was stimulated for the time indicated by histamine (100 μM, white bar), or bradykinin (1 μM, black bar), and prostaglandin E2 released into the culture supernatant was determined as described in the next study 1. Values shown are mean ± standard deviation from representative experimental values, which were corrected for baseline prostaglandin E2 production by unstimulated cultures.

8 is a graph showing inhibition of prostaglandin E2 production in synovial cultures by ketoprofen. Cultures were primed overnight with IL-1 (10 U / ml) in the presence (indicated as "n") or in the absence of indicated ketoprofen (indicated as "r" or "s"). After 1 day, prostaglandin E2 was measured in culture supernatant treated with ketoprofen overnight, the remaining cultures were washed, incubated with ketoprofen at the indicated concentrations for 10 minutes, and then the indicated amount of ketopro Prostaglandin E2 production was measured in response to 3 minutes of subsequent challenge with histamine (100 μM, s) or bradykinin (1 μM, r) in the constant presence of pens. The data presented are each normalized to the maximal response obtained for each agonist, representing the mean ± standard deviation from three experiments performed on different cell lines.

FIG. 9 is a graph showing the effect of ketoprofen on IL-6 production by synovial cultures at 16 hours in the presence of the indicated concentrations of IL-1 with the addition of G-protein coupled receptor ligands. The culture was incubated with IL-1 for 16 hours at the indicated concentrations (0.3, 1.0 and 3.0 pg / ml) in the absence and presence of 0.75 μM ketoprofen in experimental growth medium using one of the following additional receptor ligands. Cultures were: 1) 1.0 μM isoproterenol (ISO) to activate the cAMP pathway, or 2) 100 μM histamine (HIS) to activate the IP3 / calcium pathway. The culture supernatant is collected and replaced with fresh medium aliquots containing the same agonist adduct at 8 hour intervals. After treatment, supernatant media corresponding to 8-16 hour interval treatments were collected and analyzed for IL-6.

Summary of the Invention

The present invention provides methods and compositions for reducing or preventing articular cartilage destruction in joints by administering a combination of two or more metabolic active cartilage protectors. Metabolic active agents include, but are not limited to, compounds that act to directly or indirectly modulate or change the biological, biochemical or biophysical state of a cell, including, but are not limited to, altering the potential of plasma membrane, Agents that change ligand binding or enzymatic activity of receptors, intracellular or extracellularly located enzymes, protein-protein interactions, RNA-protein interactions, or DNA-protein interactions are included. In one aspect of the present invention, there is provided a pharmaceutical composition of metabolic active cartilage protective agent based on a combination of two or more agents acting simultaneously on separate molecular targets.

Representative cartilage protective agents include, for example, (1) antagonists of receptors for interleukin-1 family proteins including, for example, IL-1β, 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-β receptor superfamily, including, for example, BMP-2, BMP-4 and BMP-7; (5) inhibitors of the MAP kinase family, including, for example, p38 MAP kinase; (7) inhibitors of matrix metalloproteinase (MMP) family proteins, including, for example, MMP-3 and MMP-9; (8) inhibitors of NF-kB family proteins, including, for example, p50 / p65 dimer complexes with IkB; (9) inhibitors of the nitric oxide synthase (NOS) family, including, for example, iNOS; (10) agonists and antagonists of integrin receptors, including, for example, agonists of α _V β ₃ integrins; (11) inhibitors of the protein kinase C (PKC) family; (12) inhibitors of the protein tyrosine kinase family, including, for example, the src subfamily; (13) modulators of protein tyrosine phosphatase; And (14) inhibitors of the protein src homology 2 (SH2) domain. Additional cartilage protective agents include other growth factors, examples of which are insulin-like growth factors (eg IGF-1) and fibroblast growth factors (eg bFGF).

In a preferred embodiment, the at least one agent is a cytokine or growth factor receptor agonist (also referred to herein as "anabolic agent") that directly provides anti-inflammatory activity and / or enhances cartilage anabolic processes, At least a second agent is a receptor antagonist or enzyme inhibitor that acts to inhibit cartilage catabolic processes and may inhibit pro-inflammatory processes (sometimes referred to herein as "cartilage catabolic inhibitors" or "catabolic inhibitors"). )to be. The term "cartilage protective agent" as used herein includes both anabolic agents and cartilage catabolic inhibitors.

In this aspect of the invention, at least the first cartilage protector is an anti-inflammatory / synergistic cytokine that functions to functionally inhibit the role of pro-inflammatory cytokines in certain joints, enhance cartilage matrix synthesis and inhibit matrix degradation. . These receptor agonists include, for example, specific anti-inflammatory and anabolic cytokines such as interleukin (IL) agonists (such as IL-4, IL-10 and IL-13), and transforming growth factors. specific members of the -β super-family (eg TGFβ and BMP-7), insulin-like growth factor (eg IGF-1) and fibroblast growth factor (eg bFGF). At least the second cartilage protector is a class of cartilage catabolism inhibitors (eg, IL-1 receptor antagonists, TNF-α receptor antagonists), including receptor antagonists or enzyme inhibitors that act to inhibit and reduce the activity or expression of pro-inflammatory molecular targets. , Cyclooxygenase-2 inhibitors, MAP kinase inhibitors, nitric oxide synthase (NOS) inhibitors, and nuclear factor kappa B (NFκB) inhibitors). Such second cartilage protectors include inhibitors of matrix metalloproteinases that inhibit cartilage catabolism; Cell adhesion molecules that inhibit cartilage catabolism (including integrin agonists and integrin antagonists); Intracellular signaling inhibitors that inhibit cartilage catabolism (including protein kinase C inhibitors and protein tyrosine kinase inhibitors); And inhibitors of the SH2 domain that inhibit cartilage catabolism.

Articular cartilage is a special extracellular matrix produced and maintained by metabolically active articular cartilage cells. The maintenance of a normal and healthy extracellular matrix reflects a dynamic balance of the rate of biosynthesis and incorporation of the matrix components and the rate at which these components degrade from cartilage into the synovial fluid and subsequently lose. Although the regulatory mechanisms underlying the matrix bio homeostasis are not widely recognized, it is clear that these mechanisms change in response to joint trauma in joint inflammatory diseases, so that the rate of matrix breakage exceeds the new rate of synthesis of matrix components. Matrix bio homeostasis is generally considered to represent a dynamic balance between the effects of catabolic cytokines and the effects of anabolic cytokines (including growth factors). The optimal combination of therapeutic agents useful for cartilage protection shifts the dynamic matrix equilibrium by promoting the rate of synthesis while also inhibiting the rate of breakage, optimizing the anabolic process and enhancing repair.

Catabolic cytokines such as IL-1β and TNF-α act at the specific receptors on chondrocytes to induce the production of MMPs that induce matrix degradation, whereas the degradation is anabolic cytokines, eg For example, it is inhibited by TGF-β, BMP-2 and IGF-1. Thus, therapeutic approaches based solely on inhibiting catabolic processes (eg, combinations of MMP inhibitors and IL-1 antagonists) are not optimal for cartilage repair, which induces component assembly and biosynthesis for matrix production. This is because anabolic preparations are required to promote or promote them. Secondly, the multiplicity of catabolic cytokines (IL-1, TNF, IL-17, IL-18, LIF), which contribute to cartilage matrix destruction, indicates that this will not be practical to block all of the catabolic cytokine activity. . Conversely, approaches that rely solely on the use of anabolic agents, such as IGF-1, BMP-2 or BMP-7, are also not optimal, which does not emphasize the counter-regulatory role of catabolic cytokines. Because. TGF-β, BMP-2 and IGF-1 also play a role in inducing specific chondrocytes that produce matrix components and are inhibited by IL-1b, TNF-α, IL-17 and LIF. Thus, it is believed that the optimal therapeutic combination for cartilage protection will consist of one or more anabolic agents and one cartilage catabolic inhibitor.

In one aspect of the invention, multiple cartilage protectants are administered by the systemic route to patients at risk of articular cartilage degradation. Many such formulations administered systemically include one or more agents that enhance cartilage anabolic activity and one or more agents that inhibit cartilage catabolic activity. Each agent is provided in an amount sufficient to provide a therapeutically effective combination when delivering a solution of the invention to a patient joint to inhibit cartilage catabolic processes and to enhance cartilage anabolic processes. In addition, one or more agents that act to inhibit pain and / or inflammation may be administered in conjunction with cartilage protectants. Systemic administration of multiple cartilage protectors may be desirable for patients suffering from cartilage degradation or suffering from degenerative diseases in several joints simultaneously.

In one aspect of the systemic delivery embodiments of the present invention, a therapeutic strategy to minimize adverse or unwanted systemic effects is to deliver the combination of agents in a carrier or delivery vehicle that targets a particular joint. In a preferred targeting embodiment, one or more anabolic cartilage protectors and / or one or more catabolic cartilage protectors, and preferably both anabolic cartilage protectors and catabolic cartilage protectors are delivered to the delivery vehicle, eg, nano. The film can be formed in a nanosphere. The targeting antibody or antibody fragment is coupled with these nanospheres. Such antibodies or antibody fragments are specific for targeting antigenic determinants localized within the joint. The method of treatment of the present invention comprises systemic administration of a composition of one or more cartilage protectants, targeted and encapsulated as described above, to patients at risk of cartilage degradation, preferably by intravascular, intramuscular, subcutaneous or inhalation administration.

In a further aspect of the present invention there is provided a composition for systemic administration comprising a plurality of cartilage protective agents, including one or more agents that enhance cartilage anabolic activity and one or more agents that inhibit cartilage catabolic activity. In addition, one or more agents that act to inhibit pain and / or inflammation may be included in the composition. All formulations are included in dosages sufficient to provide a cartilage protective therapeutic effect on the joint (s) when administered systemically. Also provided is a method of making a medicament comprising the composition for use in treating a patient at risk of cartilage degradation.

In order for such systemically administered compositions to target the joint, one or more anabolic cartilage protectors and / or one or more catabolic cartilage protectors, and preferably both anabolic cartilage protectors and catabolic cartilage protectors An envelope is formed in a Dar delivery vehicle, eg, a nanosphere, to which an antibody or antibody fragment specific for an antigenic determinant localized in the joint is coupled. Encapsulated cartilage protective agent (s) coupled with an antibody or antibody fragment (the antibody or antibody fragment targets an antigenic determinant localized in the joint) for use in treating a patient at risk of cartilage degradation. Also provided are methods of making the medicament.

In a different aspect of the invention, in a pharmaceutically effective carrier, one or preferably multiple metabolic active cartilage protectors are included with one or more agents for the inhibition of pain, inflammation, or more preferably assimilation Functional Agents and Catabolism Inhibitors A composition comprising a plurality of combinations of agents can be made for use in intraarticular delivery directly to a patient joint. Although systemic delivery of the cartilage protective composition of the present invention may be desirable for a disease or condition in which several joints are ill, local shearing of the composition of the present invention may be desirable in other cases. In such cases, treatment of a patient with a diseased cartilage degeneration disease or disease in only one joint or a limited number of joints, periprocedural administration associated with surgery or interventional treatment in a particular joint, or preferred Unfavorable side effects may be associated with systemic administration. In this local delivery aspect of the present invention, intraarticular injection (osteoarthritis or rheumatoid arthritis), including administration of the composition around the time of treatment (ie, preoperative and / or during and / or postoperative) during surgical treatment with arthroscopy. To treat cartilage degenerative diseases such as arthritis) or by injection.

This topical delivery aspect of the present invention provides a solution consisting of a low concentration of multiple agent mixtures instructed to locally inhibit modulators of pain, inflammation and cartilage degradation in a physiological electrolyte carrier solution. The present invention also provides a method of delivering irrigation solutions containing these agents directly to the surgical site at the perioperative time, which preemptively limits pain, inflammation and cartilage degradation at that site. Acts locally at the receptor and enzyme levels. Due to the method of the present invention that delivers locally at a time around the surgery, the desired treatment may be achieved with a lower dose of the agent than necessary when using other methods of delivery (ie, intravenous, intramuscular, subcutaneous and oral). Effect can be achieved.

Analgesic and / or anti-inflammatory and / or anti-cartilage degradants in such solutions include agents selected from several classes of receptor antagonists and agonists and enzyme activators and inhibitors, each class inhibiting pain and / or inflammation and / or It acts through different molecular mechanisms of action on cartilage degradation.

In addition to the anti-cartilage degrading agent (s), the compositions of the present invention may comprise analgesic and / or anti-inflammatory agents. Representative agents for inhibiting 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 neurokinin ₁ 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 route to arachidonic acid metabolites, including (a) phospholipase inhibitors, including PLA ₂ isotype inhibitors and PLC isotype 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 B ₄ receptor subtype antagonists and leukotriene D ₄ receptor subtype antagonists; (12) opioid agent receptor agonists including μ-opioid agents, d-opioid agents, and k-opioid agent receptor subtype agonists; (13) _purinoceptor antagonists including P _2X receptor antagonists and P _2Y receptor antagonists; And (14) calcium channel antagonists. Each of these agents acts as an anti-inflammatory agent and / or as an anti-invasive solution (ie, analgesic). The choice of agent from these classes of compounds depends on the particular use.

The present invention also provides a method for preparing a compound that is compounded in one aspect of the present invention as a dilute irrigation solution for use in continuous irrigation at the surgical site, typically at the joint site of the patient, during arthroscopic surgery. In this topical delivery embodiment of the invention, the method dissolves one or more anti-cartilage degradants and preferably one or more analgesic / anti-inflammatory agents, and in some applications anti-cartilage degradants in physiological electrolyte carrier fluid. Entraining, preferably at a concentration of about 100,000 nanomol or less, more preferably about 25,000 nanomol or less, most preferably about 10,000 nanomol or less.

The method of topical delivery aspect of the present invention provides a combination of multiple receptor antagonists and agonists and enzyme inhibitors and activators for wound sites during therapeutic or diagnostic treatment to inhibit cartilage degradation, pain and / or inflammation. Provides direct delivery to the surgical site. Because the active ingredients in solution are applied topically directly to surgical tissues in a continuous manner, even at extremely low doses compared to those required for therapeutic effects in the case of oral, intramuscular, subcutaneous or intravenous delivery of the same drug The drug can be used effectively. The term "topical" as used herein encompasses the application of certain drugs to and around wounds or other surgical sites, and excludes oral, subcutaneous, intravenous and intramuscular administration. As used herein, the term "continuous" does not stop except for uninterrupted administration, repeated administrations at frequent intervals, and brief pauses to allow for the introduction of other drugs, drugs or treatment equipment. Administration, which allows a substantially constant predetermined concentration to be maintained locally at the wound or surgical site.

According to this aspect of the present invention, there are three advantages of administering the formulation in low doses. Most importantly, there are no systemic side effects that often limit the usefulness of these agents. In addition, the agents selected for particular uses in the solutions of the present invention are highly specific for the mediators and mediators on which they operate. This specificity is also maintained by the low dose utilized. Finally, the cost of these active agents in a single treatment is low.

Advantages of topically administering a formulation via irrigation or other fluid application in accordance with this aspect of the invention are as follows: (1) Topical administration is known at the target site, regardless of diversity among patients in metabolism, blood flow, etc. Ensure a concentrated concentration; (2) because of the direct mode of delivery, the therapeutic concentration is obtained immediately, providing improved dosage control; (3) Direct topical administration of the active agent to the wound or surgical site may result in degradation of the agent through a systemic process that may occur when such agent is administered orally, intravenously, subcutaneously, or intramuscularly (e.g., And second-pass metabolism). This is particularly true of protein and peptide active agents that are rapidly metabolized. Thus, topical administration allows the use of agents or compounds that could not be used therapeutically. For example, some 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 antagonists; TGF-b receptor agonists; BMP-2 and BMP-7 receptor agonists; IL4, IL10 and IL-13 receptor agonists; And integrin receptor agonists and antagonists. Continuous local delivery to the wound or surgical site minimizes drug degradation or metabolism while continuously replacing a portion of the agent that may degrade, resulting in a topical therapeutic concentration sufficient to maintain receptor occupancy or enzyme saturation. It is maintained throughout the surgical procedure.

In accordance with this aspect of the present invention, by administering the solution locally throughout the surgical procedure at the time of the surgery, a proactive analgesic, anti-inflammatory and cartilage protective effect is achieved. The term “periodic timing” as used herein encompasses application during, before and during treatment, during and after treatment, and before, during and after treatment. In order to maximize the effect of preemptive anti-inflammatory, analgesic (for certain applications) and cartilage protection (for certain applications), it is most preferred to apply the solutions of the present invention before, during and after surgery. Prior to initiating a local surgical trauma, by occupying the target receptor or by inactivating or activating the targeting enzyme, the formulation of the solution of the invention modulates a specific pathway that preemptively inhibits targeted pathological processes. . If the inflammatory mediators and processes proactively inhibit them in accordance with the present invention prior to tissue damage, this is much more effective than if provided after the tissue damage was initiated.

Inhibiting one or more pain, inflammation, or cartilage mediators by administering the multiple agent solutions of the present invention has been found to significantly reduce inflammation and pain levels, which should theoretically provide a cartilage protection effect. The irrigation solution of the present invention comprises a drug combination, each solution acting on a plurality of receptors or enzymes. Thus, drug preparations are simultaneously effective against both pathological processes, including pain and inflammation, and loss of cartilage bio homeostasis. The combination of multiple receptor antagonists and inhibitory agonists of the present invention is considered synergistic in that disproportionately increased efficacy is provided relative to the efficacy of the individual agents. The synergism of some agents according to the invention is discussed by way of example in the following.

When used at the time of surgery, the solution of the present invention significantly reduces surgical site pain and inflammation and cartilage degradation compared to the currently used irrigation, thereby requiring a patient's postoperative analgesic (ie, opiate) requirements. The patient should be allowed to move the surgical site earlier in some cases. In the case of using the solution of the present invention, the surgeon and the operating room staff do not need to make special efforts compared to the conventional irrigation solution. In order to optimally protect cartilage according to this aspect of the invention, the solution of the invention is administered directly to the joint before, during and / or after surgical treatment.

In a further aspect of the present invention, there is provided a cartilage protective composition comprising an anabolic enhancer and a catabolic inhibitor. Such combinations include cartilage anabolic activity equivalent to or greater than cartilage catabolic activity; Maintenance of cartilage tissue to maintain or increase existing cartilage volume; Or at the same time increasing the synthesis of the cartilage matrix by the articular cartilage cells, the degradation of the cartilage matrix.

Detailed Description of the Preferred Embodiments

The present invention provides a composition and method for cartilage protection. In a first aspect, a method of topically administering to a particular joint a composition comprising at least a first agent acting to enhance cartilage anabolic activity and at least a second agent acting to inhibit cartilage catabolism Is provided. In a first aspect of this aspect of the invention, the composition is delivered locally by injecting into the joint a composition that may comprise a sustained release delivery vehicle. In a second aspect of this aspect of the invention, the composition comprises a liquid irrigation carrier and is delivered locally to the joint at a time around the surgery during surgery or interventional treatment.

In a second aspect of the invention, a method of systemically administering to a patient a composition comprising at least a first agent acting to enhance cartilage anabolic activity and a composition comprising at least a second agent acting to inhibit cartilage catabolism Is provided.

In a third aspect of the invention, a composition comprising at least a first agent acting to enhance cartilage anabolic activity and / or at least a second agent acting to inhibit cartilage catabolic activity (at least one of these agents A method of systemically administering a target) to a patient is provided. Before each of these embodiments is described in more detail, although not bound by theory, a rationale for cartilage protection according to the present invention is presented.

I. Theoretical basis for cartilage protection

Recent research progress in understanding the biochemical and molecular biology of inflammation and cartilage destruction has had a close impact on the role of numerous endogenous cytokines. Various pro-inflammatory mediators that have had a profound effect on the induction of cartilage loss in inflamed joints are the cytokines TNF-α, IL-1, IL-6 and IL-8. These numerous pro-inflammatory cytokine levels increase rapidly in the synovial fluid of rapidly damaged knee joints and remain at elevated levels in patients for more than four weeks (see Cameron, ML et al., “Synovial fluid cytokine concentrations”). as possible prognostic indicators in the ACL-deficient knee, " Knee Surg. Sports Traumatol. Arthroscopy 2: 38-44 (1994)). These cytokines are produced locally in the joints from several activated cell types, including synovial fibroblasts, synovial macrophages, and chondrocytes. These locally produced cytokines mediate pathophysiological events in acute and chronic inflammatory diseases, which are important autosecretory and lateral secretion mediators of cartilage catabolism. The action of these cytokines is characterized by the ability to elicit multiple effects on separate cellular targets and the ability to interact with other cytokines in a positive or negative synergistic manner. Of particular interest are IL-1 and TNF-α, which disrupt the cartilage matrix component by modulating the activity of endogenous proteins (eg matrix metalloproteinases (MMPs)) and tissue inhibitors of metalloproteinases (TIMPs). This is because the cartilage destruction effect is also initiated by disrupting the balance between and normal metabolic rotation. Cytokine control of cartilage bio homeostasis indicates that the balance between active mediators acting on chondrocytes is highly regulated, which determines whether matrix degradation or repair occurs.

Joint damage can often cause an inflammatory response in the joint space, including synovial tissue, and cause articular cartilage degradation. Significant shifts in synovial and cartilage metabolism of the human knee have been reported to occur after joint damage and arthroscopic surgery (Cameron, ML et al., Supra (1994) Cameron, ML et al., "The natural history of the anterior cruciate ligament-deficient knee: Changes in synovial fluid cytokine and keratan sulfate concentrations, "Am. J. Sports Med. 25: 751-754 (1997)). Specific pro-inflammatory cytokine levels in the knee joint synovial fluid increase sharply (up to 2-4 orders of magnitude) during the acute inflammatory phase observed after anterior cruciate ligament (ACL) rupture. Significant changes also occur in cartilage matrix molecule concentrations due to overproduction of matrix metalloproteinases (MMPs) such as collagenase and stromelysin-1, which are elevated in the synovial fluid of patients after acute trauma (see : Lohmander, LS 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)) . Temporarily, changes in cytokines and cartilage matrix markers (such as proteoglycans) in synovial fluid, which are associated with cartilage degeneration, are greatest during the acute injury cycle, but slowly prolonged (3 months to 1 year) and slower than baseline levels before injury. Remains larger.

Trauma due to arthroscopic surgery itself causes significant post-surgical inflammation, which reflects the additional inflammatory activation of cells in that joint, including upregulation of cyclooxygenase-2 and other pro-inflammatory cytokines. A significant number of patients with ACL rupture (60-90%) show knee changes in which radiographs indicate osteoarthritis (OA) 10-15 years after injury (see Cameron, ML et al., Supra (1994). )). Thus, the combined effect of early joint injury and surgical trauma can lead to a persistent inflammatory state and associated changes in cartilage matrix metabolism, which leads to subsequent degeneration of articular cartilage and the development of early osteoarthritis. It is believed to be the trigger. This health problem is significant, with an estimated 10.8 million total arthroscopic treatments performed in the United States in 1996, an increase of about 10% annually. Accordingly, it is desirable to provide a pharmaceutical method for preventing articular cartilage degradation in joints.

Although postoperative pain and inflammation have been recognized as clinically important issues, current pharmacological treatment regimens for arthroscopic surgery have focused only on acute postoperative analgesics. Existing surgical treatment embodiments do not focus on the chronic inflammatory state induced after surgery and the need to suppress cartilage destruction of the joints treated through the surgery. Thus, there is a need to develop effective and integrated drug therapies that focus on both acute and chronic aspects of pain and inflammation, as well as on pathological changes in cartilage metabolism of damaged and surgically treated joints.

According to this first aspect of this aspect of the invention, in a pharmaceutically effective carrier for intraarticular delivery, one or preferably a number of metabolic active cartilage protectors are provided as long as they are for inhibiting pain and / or inflammation as described above. Included with at least two agents, or preferably a combination of two or more metabolic active cartilage protectors, at least one of which promotes cartilage anabolic processes, at least one of which is an inhibitor of cartilage catabolic processes By directly administering a composition to a joint of a patient, a method of reducing or preventing articular cartilage destruction in such joint is provided. Metabolic active agents include, but are not limited to, any compound that acts to directly or indirectly modulate or change the biological, biochemical or biophysical state of a cell, including but not limited to altering the potential of the plasma membrane, Agents that alter ligand binding or enzymatic activity of sex receptors, intracellular or extracellularly located enzymes, protein-protein interactions, RNA-protein interactions, or DNA-protein interactions are included. For example, the agent may contain receptor agonists that initiate a signal transduction cascade, antagonists of receptors that inhibit signaling pathways, activators and inhibitors of intracellular or extracellular enzymes, and modulate binding of transcription factors to DNA. Agents may be included.

Representative cartilage protective agents include, for example, (1) antagonists of receptors for interleukin-1 family of proteins, including, for example, IL-1β, 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 TGF-β receptor super-family, 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 matrix metalloproteinase (MMP) family proteins, including, for example, MMP-3 and MMP-9; (8) inhibitors of NF-κB family proteins, including, for example, p50 / p65 dimer complexes with IkB; (9) inhibitors of the nitric oxide synthase (NOS) family, including, for example, iNOS; (10) agonists and antagonists of integrin receptors, including, for example, agonists of α _V β ₃ integrins; (11) inhibitors of the protein kinase C (PKC) family; (12) inhibitors of the protein tyrosine kinase family, including, for example, the src subfamily; (13) modulators of protein tyrosine phosphatase; And (14) inhibitors of the protein src homology 2 (SH2) domain. Other cartilage protective agents suitable for use in the present invention include other growth factors, examples of which are insulin-like growth factors (eg IGF-1) and fibroblast growth factors (eg bFGF).

A first aspect of the present invention provides a method of pharmacologically treating a damaged or surgically treated joint using a combination of locally delivered cartilage protectors to achieve maximum therapeutic benefit. A second aspect of the present invention provides a method for pharmacologically providing a therapeutic treatment by systemically administering a combination of cartilage protective agents. Limitations of existing therapeutic approaches based on the use of a single agent to block the multifactorial cartilage destruction process, whereby the shift between synthesis and degradation occurs to support the catabolic process by using a combination of cartilage protectors Overcome This aspect of the present invention is to inhibit the inflammatory process to the maximum and to maintain the cartilage bio homeostasis to achieve a cartilage protection effect in the joint, to inhibit the upregulated or excessive cartilage catabolic process and promote cartilage assimilation The combination approach of agents running simultaneously on separate molecular targets is uniquely utilized. Inhibiting biochemical mechanisms or single molecule targets known to induce cartilage destruction (catabolism), for example, inhibiting interleukin-1 (IL-1) binding to the IL-1 receptor, is optimal. This is not expected because, for example, the action of TNF-α mediated through its unique receptor shares pro-inflammatory and cartilage catabolic functions that overlap heavily with IL-1, which is cartilage in the joints. It is recognized as a major mediator of destruction. Similarly, utilizing a pharmaceutical agent that enhances only cartilage anabolic processes without inhibiting catabolic processes will also not optimally interfere with catabolic factors present in the damaged joint.

Specifically, one aspect of the present invention provides a pharmaceutical composition of metabolic active cartilage protective agents based on a combination of two or more agents acting simultaneously on separate molecular targets. In an exemplary embodiment, at least one agent is a cytokine or growth factor receptor agonist that directly provides anti-inflammatory activity and / or enhances cartilage anabolic processes, and at least the second agent is pro-inflammatory and / or cartilage catabolic. Receptor antagonists or enzyme inhibitors that act to inhibit the process. Representative drug combinations include one or more agents selected from the class of anti-inflammatory / synergogenic cytokines that function to inhibit the role of pro-inflammatory cytokines in certain joints, enhance cartilage matrix synthesis and inhibit matrix degradation. . These receptor agonists include specific anti-inflammatory and anabolic cytokines such as interleukin (IL) agonists (eg IL-4, IL-10 and IL-13), and transforming growth factor-β super-series (Eg, TGFβ and BMP-7), insulin-like growth factors (eg IGF-1) and specific members of fibroblast growth factor (eg bFGF), but are not limited thereto. At least a second agent is a class of receptor antagonists or enzyme inhibitors (e.g., IL-1 receptor antagonists, TNF-α receptor antagonists, cyclooxygenase-2 inhibitors) that act to inhibit and decrease the activity or expression of pro-inflammatory molecular targets. , MAP kinase inhibitors, nitric oxide synthase (NOS) inhibitors and nuclear factor kappa B (NFκB) inhibitors. Metabolic active agents include both functional agonists and antagonists of receptors localized on the cell surface, as well as inhibitors of membrane bound or extracellularly secreted enzymes such as stromelysin and collagenase. Many formulations also transduce signals from surface receptors, including inhibitors of enzymes NOS, COX-2 and cleavage promoter-activated protein kinases (MAPKs) and protein-DNA interactions, such as inhibitors of the transcription factor NFκB. It is directed towards novel agents that are intracellularly localized enzymes and transcription factors that incorporate and integrate. In this way, the prototype of cartilage can be preserved by promoting cytokine-driven anabolic processes while simultaneously inhibiting catabolic processes.

Compositions of preferred embodiments of the present invention constitute a novel therapeutic approach by combining multiple pharmacological agents and / or enzyme molecular targets that function at separate receptors. To date, pharmacological strategies have focused on the development of highly specific drugs that are selective for individual receptor subtypes and enzyme isotypes that mediate responses to individual signaling neurotransmitters and hormones. Moreover, despite the inactivation of a single receptor subtype or enzyme, activation of other receptor subtypes or enzymes and the resulting signal transduction can often trigger cascade effects. This explains the difficulty of using a single receptor-specific drug to block pathophysiological processes in which multiple signaling mediators (eg cytokines, growth factors or eicosonoids) play a role. Therefore, targeting only individual specific receptor subtypes or isotypes is expected to be ineffective.

Unlike standard approaches to pharmacological therapies, the therapeutic approach of the present invention inhibits the full range of events underlying the development of pathophysiological conditions, with drug combinations acting simultaneously on separate molecular targets highly. Based on rationale for being effective. In addition, instead of targeting only specific receptor subtypes, the compositions of the present invention include drugs that target common molecular mechanisms that operate on different cellular physiological processes associated with pain, inflammation, and cartilage development. (See Figure 1). In this way, cascading of additional receptors and enzymes in the invasion uptake, inflammation and cartilage degradation pathways is minimized. In these pathophysiological pathways, the compositions of the present invention inhibit the cascade effect of both "top" and "bottom".

An example of "top" inhibition is a cyclooxygenase antagonist in pain and inflammation settings. Cyclooxygenase enzymes (COX ₁ and COX ₂ ) catalyze the conversion of arachidonic acid to prostaglandin H, an intermediate in the biosynthesis of inflammatory and invasive water-soluble mediators, including prostaglandins, leukotrienes and thromboxanes. Cyclooxygenase inhibitors "top" block the formation of these inflammatory and nociceptive water-soluble mediators. This strategy precludes the need to block the interaction of the seven subtypes of the prostanoid receptors mentioned above with the prostanoid products of the COX biochemical pathway. A similar "top" inhibitor is aprotinin, a kallikrein inhibitor. The enzyme kallikrein, a serine protease, cleaves high molecular weight kininogen in plasma to produce bradykinin, an important mediator of pain and inflammation. By inhibiting kallikrein, aprotinin effectively inhibits the synthesis of bradykinin, providing effective "top" inhibition of these inflammatory mediators.

The compositions of the present invention may also use "bottom" inhibitors to control pathophysiological pathways. In synovial and chondrocyte preparations treated with various inflammatory cytokines (eg, IL-1β and TNFα), which have a close effect on progressive articular cartilage degradation, MAP kinase inhibitors have a cartilage protective effect. p38 MAP kinase is the transport point in the signaling pathway for many catabolic cytokines, and its inhibition prevents the upregulation of many cellular products that mediate cartilage degradation. Thus, MAP kinase inhibitors provide a significant benefit to the setting of joint inflammation by providing a "bottom" cartilage protective effect independent of the physiological combination of cytokine receptor agonists that initiate mobile cartilage bio homeostasis.

II. Local delivery of cartilage protective composition

Certain preferred embodiments of the solution of the present invention for use in cartilage protection and surgical treatment are preferably upregulated to achieve a cartilage protection effect in the joints by maximally inhibiting the inflammatory process and maintaining cartilage bio homeostasis. Combinations of agents that simultaneously inhibit excessive cartilage catabolic processes and enhance cartilage assimilation are performed on separate molecular targets.

The irrigation and injection solution of one aspect of the invention is a dilute solution of one or preferably one or more cartilage protectors and optionally one or more pain and / or inflammation inhibitors in a physiological carrier. Carriers as used herein include biocompatible solvents, suspensions, polymerizable and non-polymerizable gels, pastes and slaves, as well as sustained release delivery vehicles such as proteins, liposomes, carbohydrates, synthetic organic compounds or inorganic compounds. It is a liquid solution encompassing the composed fine particles, microspheres or nanoparticles. Preferably the carrier is an aqueous solution which may comprise a physiological electrolyte, such as normal saline or lactated Ringer's solution.

In each of the topically delivered surgical solutions of this aspect of the invention, the formulation is included in the liquid or fluid solution at a concentration lower than the concentration and dose required for the systemic administration of the drug to achieve the desired therapeutic effect. And locally delivered at low doses. "Liquid" or "fluid" as used herein encompasses pharmaceutically acceptable and biocompatible solvents, suspensions, polymerizable and non-polymerizable gels, pastes and slaves. Preferably the carrier is an aqueous solution which may comprise a physiological electrolyte, such as normal saline or lactated Ringer's solution. Equivalent treatment when delivering similarly administered agents via other drug routes of administration (ie, intravenous, subcutaneous, intramuscular or oral) because systemically administered drugs are metabolized first- and second-pass. It is impossible or impossible to obtain a scientific effect. The concentration of each agent is determined in part based on its receptor dissociation constant K _d or enzyme inhibition constant K _i . As used herein, the term dissociation constant refers to both the equilibrium dissociation constant for each of its agonist-receptor or antagonist-receptor interactions and its equilibrium inhibition constant for each activator-enzyme or inhibitor-enzyme interaction. Comprehensive Each agent is preferably included at a low concentration of 0.1 to 10,000 times K _d or K _i . Preferably, each agent is included at a low concentration of K _d or K _i 1.0 to 1,000 times, most preferably at a low concentration of about 100 times K _d or K _i . These concentrations are adjusted as necessary to account for dilution in the absence of metabolic transformation at the local delivery site. The exact formulation selected for use in the solutions of the present invention and the concentration of such formulations will vary depending upon the particular application, as described below.

The solution according to an aspect of the invention is a single or multiple cartilage protector (s), preferably a plurality of cartilage protectors (at least one of which is an anabolic cartilage protector, at least one of which is an inhibitor of cartilage catabolism) Or combinations of both cartilage protective agent (s) with pain and / or inflammation inhibitors at low concentrations. However, due to the synergistic effects of the multiple formulations described above and the desire to broadly inhibit cartilage destruction and to optionally block pain and inflammation, it is desirable to utilize multiple formulations.

Multiple drug combinations, either alone or with a post-operative sustained delivery agent (gi) by means of a controlled pump or by using a sustained release delivery vehicle, can be used for surgical perioperative treatment (ie, preoperative and And / or by intra-articular injection or infusion, including administration during and / or after surgery). 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. Thus, in some embodiments, the present invention provides a combination of agents to be delivered via injection or infusion, delivered alone or in combination with analgesics and / or anti-inflammatory agents. Rapid onset of action achieved by topical direct delivery of cartilage protectants (eg, perioperative administration) immediately after or at the point of injury has the potential to inhibit the initial process prior to triggering a subsequent response, thereby preventing local tissue damage. And subsequent cartilage loss can be restrained preemptively.

Advantages of the present invention include the following: 1) Combination drug therapy focuses on multifactorial causes of cartilage destruction during acute and / or chronic diseases; 2) a combination of cartilage protectors can be combined with anti-inflammatory and analgesics; 3) by topical delivery of the drug combination (if applicable), to achieve an immediate therapeutic concentration of cartilage protectant in the joint; 4) the use of irrigation solution at the time of treatment (if applicable) to maintain drug levels in the joints at therapeutically desirable ranges during arthroscopic surgical treatment; 5) topical delivery (for embodiments of the present invention) may reduce total drug dose and frequency of administration compared to systemic delivery; 6) topical site-directed delivery to the joint (for embodiments of the invention above) avoids systemic toxicity and reduces side effects; 7) Use of novel pharmaceutically active peptides and proteins (including cytokines and growth factors) that may be directly localized to the joint (in the embodiments of the present invention), which may not be therapeutically useful when limited to the systemic route of administration. Makes it possible.

From the molecular and cellular mechanisms of action defined for these cartilage protectors, these compounds are administered at perioperative time in irrigation solution (in combination with other cartilage protectors or in combination with other analgesics and anti-inflammatory agents described herein), or It is expected to exhibit cartilage protection when administered directly to the joint via infusion or injection. In particular, these agents are expected to be effective drugs when delivered by irrigation solution during arthroscopic surgical procedures. Each metabolic active cartilage protector is preferably combined with one or more other cartilage protectors, including small molecule drugs, peptides, proteins, recombinant chimeric proteins, antibodies, oligonucleotides or gene therapy vectors (viral and non-viral). Can be delivered to the joint space. Drugs, such as MAPK inhibitors, may exhibit their action on joint-containing structures that are associated with or due to pathological conditions of the joint and on all cells associated with the fluid space of the joint. These cells and structures include synovial cells, including both type A fibroblasts and type B macrophage cells; Cartilaginous components of the joint, such as chondrocytes 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 neurons; And combinations thereof, but is not limited thereto.

This aspect of the invention also provides formulations of the active therapeutic agent (s) which may be delivered in formulations useful for introducing and administering the drug into the joint, which may enhance the delivery, absorption, stability or pharmacokinetics of the cartilage protective agent (s). To provide. Such formulations may include, but are not limited to, microparticles, microspheres, nanospheres or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds or inorganic compounds. The present invention provides a combination of cartilage protectors, or one or preferably a combination of multiple cartilage protectors, as a plurality of pharmaceutically active substances in a homogeneous vehicle (eg single encapsulated microspheres) or individually Delivery is provided with one or more analgesic and / or anti-inflammatory agents that exist as separate mixtures of delivery vehicles (eg, groups of microspheres encapsulated with one or more agents). Examples of formulation molecules include antibodies, hydrophilic polymers, polyions (e.g. protamine, spermidine) that can target substances to specific cell types, gels, sustained release matrices (ie, sustained delivery vehicles, soluble and insoluble particles). , Polylysine), and peptide or synthetic ligands, as well as formulation elements not listed, are not limited thereto.

In one aspect, the invention provides two or more cartilage protective agents, or one or preferably, via a drug-containing infusion, irrigation solution, which is present in a therapeutically effective low dose and allows the drug to be delivered directly to the diseased tissue or joint. Provides for local delivery of a combination of multiple cartilage protectors alone or in combination with one or more analgesic and / or anti-inflammatory agents. Such drug-containing infusions or irrigation solutions may be used preoperatively and / or during and / or postoperatively in conjunction with surgical procedures, or may be administered at other times not related to the surgical procedure. In systemic drug delivery methods (eg, intramuscular, intravenous, subcutaneous), higher concentrations of drug (and higher total doses) must be administered to the patient to achieve the actual therapeutic concentration in the targeted joint. Systemic administration may be provided in high concentrations in tissues other than the targeted joint, which is undesirable and depending on the dose may cause adverse side effects. These systemic methods limit the duration of therapeutically effective concentrations by metabolic and rapid degradation of drugs. Since a combination of cartilage protective agents (with or without one or more analgesic and / or anti-inflammatory agents) is administered directly to the joint by infusion or irrigation, vascular perfusion is not required to deliver the drug to the targeting tissue. . This substantial advantage enables local delivery of lower therapeutically effective total doses for various cartilage protective drugs.

A. Topical Delivery Methods

The solution of the present invention is applied to a variety of surgical / interventional procedures, including surgical, diagnostic and therapeutic techniques. Combinations of cartilage protectors of the invention can be administered by injection or irrigation. In the case of injectable solutions, the amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending on the patient to be treated, the nature of the active agent in the solution, and the particular mode of administration. However, the specific dose level for all particular patients is determined by 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 release of the drug combination, and the specific progression of therapy. It should be recognized that this will depend on a variety of factors, including the severity of the disease.

Suitable dispersing or wetting agents and suspending agents can be used to formulate injectable preparations, for example, sterile injectable aqueous or oily suspensions, according to known techniques. Such sterile injectable preparations may also be sterile injectable solutions or suspensions in a parenterally acceptable non-toxic diluent or solvent, for example, present as a solution in propanediol. Acceptable vehicles and solvents that may be employed include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, all biocompatible oils may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

Injectable solutions of the present invention may be administered in conjunction with an arthroscopic surgical procedure or at a point in time determined by the attending physician to be preferred.

The irrigation solution of the present invention can be applied at a time around the surgery during arthroscopic surgery of anatomical joints. As used herein, the term “periodical period” encompasses application during, before and during treatment, during and after treatment, and before, during and after treatment. Preferably the solution of the invention is applied before and / or after treatment as well as during the treatment. Such treatment typically utilizes a physiological irrigation solution, such as normal saline or lactated Ringer's solution, applied to the surgical site by techniques well known to those skilled in the art. The method of the invention involves replacing the commonly applied irrigation solution with the analgesic / anti-inflammatory / cartilage protective irrigation solution of the invention. This irrigation solution is applied to the wound or surgical site prior to initiation of the treatment, preferably prior to tissue trauma, and continuously applied throughout the treatment period to preemptively block pain and inflammation, and cartilage degradation. As used herein, the term “irrigation” means flushing a wound or anatomical structure with a liquid stream. The term "application" refers to irrigation methods and other methods of topically introducing a solution of the invention, such as introducing a gelable variant of the solution of the invention into a surgical site, wherein the gelling solution is operated throughout the treatment. Remaining at the site). The term "continuously" as used throughout this application is intended to refer to the situation of frequent irrigation of the wound frequently and often enough to maintain a predetermined therapeutic topical concentration of the applied agent, and to the irrigation flow that may be necessary for surgical techniques. Intermittent interruptions are included.

The concentrations listed above for each agent in the solution of the present invention are the concentrations of the agent delivered locally to the site of surgery in the absence of metabolic transformation to achieve a predetermined level of effect at the site of surgery. The drug concentration in a given solution needs to be adjusted to account for the local dilution at delivery. Solution concentrations in this embodiment are not adjusted to account for the dilution rate or metabolic conversion by systemic distribution, as these situations can be avoided by local delivery as opposed to oral, intravenous, subcutaneous or intramuscular application. to be.

Arthroscopic techniques that may be used in the present invention include, but are not limited to, partial meniscus resection and ligament restoration of the knee, shoulder acroplasty and circumferential plate resection, elbow synovectomy, and waist and ankle arthroscopy. The irrigation solution of the present invention is continuously supplied to the joint during surgery at a flow rate sufficient to swell the articular sac, remove surgical debris and enable intra-articular visualization without occlusion.

Arthroscopic irrigation solutions suitable for inhibiting cartilage degradation and controlling pain and inflammation during this arthroscopic technique are provided in Examples 1-3 below.

In each of the solutions of the present invention intended for topical delivery, the formulation is included at a lower concentration and at a lower dose than the concentration and dose required for the systemic administration of the drug to achieve the desired therapeutic effect. Is passed to. Because systemically administered drugs are first- and second-pass metabolized and are often quickly removed from the systemic circulation, similarly administered agents via other drug administration routes (ie, intravenous, subcutaneous, intramuscular or oral). In the case of delivering, it is impossible to obtain an equivalent therapeutic effect.

The practice of the present invention should be distinguished from conventional intraarticular injection of opiates and / or local anesthetics upon completion of treatment of arthroscopy or “open” joints (eg knees, shoulders, etc.). This inventive solution is used for continuous infusion throughout surgical procedures to preempt pain and inflammation. In contrast, the high concentrations needed to achieve therapeutic efficacy with constant infusion of current local anesthetics can cause significant systemic toxicity.

Upon completion of the treatment of the present invention, as a substitute or supplement for opiates, the same cartilage protective agent (s) and / or pain and / or inflammation inhibitors as used in the irrigation solution are injected or otherwise applied to the surgical site at a higher concentration May be desired. In addition, it may be desirable to deliver a sufficient amount of the solution of the invention to the joint after surgical treatment in order for the round mass of the solution of the invention to remain in the synovial sac of the patient after the surgical treatment.

As previously recognized, a composition of the present invention, preferably comprising a plurality of cartilage protectors, including one or more catabolic inhibitors and one or more anabolic-enhancing agents, may be adapted for direct injection into anatomical joints. Preferably, the agent is selected; Each agent in an amount sufficient to provide a therapeutically effective combination when the solution of the invention is delivered locally to the patient's joint to inhibit cartilage catabolic processes and at the same time enhance the cartilage anabolic process. Included. Such compositions may be topically delivered to provide cartilage protection to patients suffering from chronic diseases such as osteoarthritis or rheumatoid arthritis, or acute diseases such as trauma or accidental injury from surgery. Can be. One composition suitable for topical injection is provided in Example 4 below.

III. Systemic Administration of Cartilage Protective Composition

Embodiments of the present invention have been described so far in terms of local delivery of cartilage protective compositions, eg, intraarticular injection. Topical administration is said to have several advantages over systemic administration (no systemic side effects). Although topical delivery of the compositions of the present invention is desirable in many cases, it may not be practical for some cartilage degenerative diseases. This is especially true in patients with chronic cartilage degenerative diseases, such as rheumatoid arthritis, polyarthritis osteoarthritis and other polyarthritis, which simultaneously present a risk of cartilage degradation at several sites. In such patients, injecting the cartilage protective composition into each affected area (eg, joints) or most of the affected areas may be painful, impractical, expensive or discourage treatment. The cartilage protective composition mentioned above for topical delivery may be adapted for administration via the systemic route, according to another aspect of the invention. Systemic delivery of the compositions of the present invention is suitable for treating, but not limited to, patients at risk of cartilage degradation at several sites. In addition to polyarticular osteoarthritis and rheumatoid arthritis, which are discussed further below, aspects of the present invention include neuropathic arthrosis, acute rheumatic fever, histopathopathy, systemic lupus erythematosus, infantile rheumatoid arthritis, psoriatic arthritis, ankylosing arthritis. It can be useful for treating spondylitis, and other non-inflammatory and inflammatory arterial pneumonia, including but not limited to spondyloarthropathy and crystalline arthrosis.

A. Arthritis Mechanism

This aspect of the invention can be better recognized by understanding the mechanisms involved in articular cartilage degradation in rheumatoid arthritis (e.g., rheumatoid arthritis (RA) and osteoarthritis (OA)). RA is the most common form of inflammatory arthritis, affecting 3% of women and 1% of men. The majority of patients have difficulty with multiple and usually symmetric joints, especially the joints of the hands, elbows, lower back and shoulders. OA is the most common form of joint disease and is the second most common cause of premature retirement and physical disability after cardiovascular disease. OA is usually polyarticular. Superarticular cartilage destruction is characteristic of OA and incapable RA.

Although various therapeutic approaches may provide symptomatic relief, therapies that have been demonstrated to delay the progression of articular cartilage degradation have not yet been identified. In OA, normal cartilage cell function may be inhibited or these cells may not constitutively have the ability to match the rate of repair to the rate of matrix degradation increase. Various cytokines and inflammatory mediators create imbalances in the synthesis of chondrocytes or increase cartilage matrix catabolism by upregulating various matrix-degrading enzymes, including matrix metalloproteinases (including collagenase). It turned out to be.

In order to optimally treat diseases related to cartilage degeneration, it is expected that a treatment regimen that stimulates anabolic processes and at the same time inhibits cartilage catabolism. Thus, the therapeutic approaches described above for inhibiting cartilage destruction in joint diseases, based on a combination of cartilage anabolic agents and cartilage catabolic inhibitors, are expected to be useful in the treatment of arthritis diseases such as OA and RA. As previously recognized, practical considerations indicate that the disease occurring concurrently in various parts of the body is treated by systemic administration of these therapeutic agents.

B. Combinations of Formulations

Accordingly, aspects of the invention provide compositions comprising a combination of cartilage protectors, and methods of systemic administration of such compositions. Agents targeting different receptor or molecular targets are utilized in a multifactorial approach as described above. Preferably, the therapeutic composition of the present invention comprises one or more cartilage protectors that enhance cartilage anabolic activity and one or more agents that inhibit cartilage catabolic activity. Such combinations are expected to optimize biocompatibility, and are expected to be preferred over conventional therapies that focus only on cartilage breakdown or more recent studies for drug development that focus only on cartilage synthesis. Anabolic enhancers and catabolic inhibitors suitable for topical administration have been described above and are expected to be useful in the systemic compositions of the present invention. It should be appreciated that the aspects and advantages of the compositions of the present invention mentioned above in connection with topical delivery may also be applied to the systemic aspects of the present invention to the extent applicable.

Thus, the cartilage protective composition of the present invention may suitably include one or more of the following anabolic enhancers: but not limited to: interleukin (IL) agonists (eg, IL-4, IL) -10, IL-13 agonists), TGF-β agonists (eg TGFβ1, TGFβ2, TGFβ3) and bone morphogenic protein agonists (eg BMP-2, BMP-4, BMP-6, BMP-7) Members of the transforming growth factor-b super-family, including insulin-like growth factors (eg IGF-1) and fibroblast growth factors (eg bFGF), and fragments retaining the biological characteristics of these natural agents, Deletions, adducts, amino acid substitutions, mutations and modifications.

Cartilage protective compositions of the invention may suitably include one or more of the following cartilage catabolic inhibitors, including but not limited to: IL-1 receptor antagonist, TNF-α receptor antagonist, cyclooxygenase -2 specific inhibitors, MAP kinase inhibitors, nitric oxide synthase inhibitors, nuclear factor κB inhibitors, matrix metalloproteinase inhibitors, cell adhesion molecules that inhibit cartilage catabolism (including integrin agonists and integrin antagonists), cartilage Intracellular signaling inhibitors that inhibit catabolism (including protein kinase C inhibitors and protein tyrosine kinase inhibitors), and inhibitors of the SH2 domain that inhibit cartilage catabolism.

As described with respect to the foregoing embodiments, one or more inhibitors of cartilage catabolic activity in the systemic anabolic agent / catabolic inhibitor combination are soluble receptors that inhibit cartilage catabolic, such as soluble IL-1 receptor or 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 factor useful for incorporation in the present invention include the functional TNF-α antagonists described in US Pat. No. 5,605,690 to Jacobs et al., While examples of soluble human IL-1 receptors useful in the present invention include US patents. And those described in US Pat. No. 6,159,460 (Thompson et al.), The entirety of which is incorporated herein by reference. Particularly prominent catabolic inhibitors useful in combination with anabolic agents for systemic delivery in accordance with this aspect of the invention include inhibitors of IL-1ra, TNFR1-IgG1 fusion proteins and matrix metalloproteinases.

As further described below, the cartilage protective composition of the present invention may comprise one or more pain and / or inflammation inhibitors or other therapeutic agents. Examples of cartilage protective compositions suitable for systemic delivery are provided in Examples 5-20 that follow.

C. Whole body delivery

Aspects of the present invention involve systemic delivery of cartilage protectors, and potentially anti-inflammatory and / or analgesics or other therapeutic agents, to provide a therapeutic effect on several joint sites. As used herein, the terms "systemic delivery" and "systemic administration" include one or a number intended for oral, intramuscular, subcutaneous, intravenous, inhalation, sublingual, intranasal, topical, transdermal, intranasal, and therapeutic actions. Other routes of administration include, but are not limited to, the formulations that are effectively dispersed in dog sites. Preferred systemic delivery routes for the compositions of the present invention are intravenous, intramuscular, subcutaneous and inhaled administration. It will be appreciated that the exact systemic route of administration for the selected agent utilized in the particular compositions of the present invention will be determined in part taking into account the susceptibility of the agent to the metabolic transformation pathways associated with the given route of administration. For example, the peptide agonist may be most suitably administered by a route other than oral administration.

The compositions of the present invention can be administered systemically periodically at intervals determined to maintain the desired level of therapeutic effect. For example, the composition may be administered by subcutaneous injection at intervals of two to four weeks. Dosage regimens will be determined by the attending physician taking into account various factors that may affect the formulation combination action. These factors include the cartilage area to be treated, the size of the joint (if necessary), the amount of cartilage tissue to be treated, the cartilage damage site, the condition of the cartilage damaged during treatment, the patient's age, sex and weight, and other clinical factors. will be. Dosages for each individual agent may vary as a function of other anabolic and catabolic inhibitors included in the compositions of the present invention, as well as depending on the presence and type of all drug delivery vehicles (e.g., sustained release delivery vehicles). Will vary. In addition, the dosage may be adjusted taking into account variables of frequency of administration and pharmacokinetic behavior of the delivered agent (s). The treatment progress of an individual can be monitored through various methods known in the art, including clinical assessment, radiographic and magnetic resonance imaging, computed tomography, biochemical markers, and arthroscopic evaluation.

D. Delivery Vehicles and Targeting

Methods of combining compositions for various systemic routes of administration are known and can be adapted for use in the compositions of the present invention. It is suitable to combine cartilage protectors and, if included, inflammation and pain inhibitors or other therapeutic agents in a physiological carrier or delivery vehicle as appropriate for a given systemic route of administration, for example as described above. In one aspect of the invention, systemic delivery of these formulation combinations, or all of the ingredient (s) thereof, may be incorporated into or combined with drug delivery vehicles, eg, sustained release delivery vehicles and / or depots.

As used herein, the term “delivery vehicle” includes all structures containing, coupled to or accompanying a therapeutic agent, such as nanospheres and other nanoparticles, microspheres and other particulates, micelles and liposomes. This includes the vehicle formed of a protein, lipid, carbohydrate, synthetic organic compound or inorganic compound. Preferred delivery vehicles for the targeted systemic compositions of the present invention, further described below, are "particles", which include nanospheres and other nanoparticles, microspheres and other particulates, micelles, and other delivery vehicles, provided that Less preferred liposomes as described are excluded). The term “delivery system” refers to a delivery vehicle and one or more therapeutic agents contained or coupled thereto.

The term "sustained release system" means a delivery system for prolonging, enhancing or regulating the delivery, duration of action or utilization of all incorporated agents. Examples of sustained release systems include drug-containing microparticles, microspheres, nanoparticles, proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds and injectable hydrogels, for example, US Patent Application Serial No. 09 / 861,182 (Jun Li et al. ) (Which is incorporated herein by reference), including, but not limited to. Sustained release systems suitable for other medicaments are known and are adapted according to the invention to deliver cartilage protectants at relatively consistent therapeutic levels, thereby providing a longer duration of action compared to the systemic delivery system of the formulation and reducing side effects. You can.

As used herein, the term “depot” refers to a drug delivery system delivered to a site of intended action or delivered to a remote site from an intended site of action that provides a reservoir of therapeutic agent for sustained release.

Individual formulations may be combined in a homogeneous mixture, or they may be administration mixtures of separate particulates, microspheres, or the like, or may be administered continuously and separately.

In order to minimize adverse or undesirable systemic effects, a therapeutic strategy in one aspect of the invention involves delivering a combination of agents in a body part (s) containing cartilage, particularly in delivery vehicles that preferentially target joints. will be. As used herein, a "targeted delivery vehicle" can be used to systemically deliver a particular drug; A delivery vehicle that is adapted to reach the joint or desired local action site (ie, greater than the amount of drug when using an unadapted delivery vehicle or in the absence of the delivery vehicle to reach the joint or desired local action site) The drug is preferentially localized in the joint because the targeted delivery vehicle is preferentially associated with the molecules, cells, or anatomical structures of these joints). Likewise, a "target delivery system" is the targeted delivery vehicle containing one or more therapeutic agents. "Targeting drug" refers to a therapeutic agent that is directly linked or coupled with a targeting construct. As used herein, systemically administered drugs that exhibit a "priority effect" at the joint or desired local action site exhibit greater pharmacological activity at the joint or desired local action site than at most other sites in the body. .

1. Theoretical Basis for Targeting

In OA, normal cartilage cell function may be inhibited or these cells may not constitutively have the ability to match the rate of repair to the rate of matrix degradation increase. Various cytokines and inflammatory mediators create imbalances in the synthesis of chondrocytes or increase cartilage matrix catabolism by upregulating various matrix-degrading enzymes, including matrix metalloproteinases (including collagenase). It turned out to be. Thus, the loss of the circular preservation of cartilage extracellular matrix (CEM) is a consequence of the dynamic imbalance between the catabolic activity leading to degradation and the synthetic activity of the cartilage anabolic process.

Systemic administration of cytokines, growth factors and other bioactive molecules often causes serious side effects. For example, pathological effects have been correlated with systemic administration of TGF-1 and other factors. In addition, systemic delivery of unprotected (exposed) polypeptides, often desirable for arthrosis arthrosis as previously recognized, is a problem associated with the stability of protein preparations due to the rapid degradation and inactivation of these therapeutic proteins in circulation. Often limited.

The breakdown of articular cartilage in OA and RA is expected to be related to the synthesis and release of catabolic factors under the microenvironment of the joint. Previous studies demonstrate that pro-inflammatory cytokines (such as IL-1) and inducible genes (such as NO synthase, COX-2, MMPs) are highly expressed in the synovial membrane from patients with inflammatory arthritis. It became. Similarly, these mediators and genes are frequently expressed in OA-infected chondrocytes. In both cases, this is a special microenvironment of the joint, which defines a pathophysiological environment that critically affects articular cartilage conditions. For this reason, it is desirable to target therapeutic molecules to preferentially act locally on the intended target in the joint space. This aspect of the invention provides a mechanism by which systemically administered anabolic cartilage protectors and / or systemically administered catabolic cartilage protectors, and preferably both, are targeted to the joint for cartilage protection of the joint. .

Thus, the preferred route of delivery is to target these protein factors to the site of action in the joint. For clinical use, there is a need for a safe method of delivering these agents into a patient's joints in a continuous and local manner. Also desired is a biodegradable drug delivery vehicle that provides unique targeting of drug delivery vehicles to the joint while protecting and stabilizing systemically administered anabolic and / or catabolic factors while leaving the joint. .

Cartilage anabolic growth factors are potent mediators secreted by the body locally in the joints in fine amounts to induce local biological responses in articular cartilage. Under normal physiological conditions, the appropriate anabolic growth factor, the synovial membrane in cartilage cells and other joint structures in cartilage at a concentration sufficient to provide a signal necessary to maintain cartilage in a healthy and stable state by affecting cartilage matrix metabolism. Produced by the cell.

2. Antibody Targeted Delivery Vehicles

Preferred targeting cartilage protective compositions of the invention comprise one or more anabolic-enhancing cartilage protectors and / or one or more catabolic inhibitors, and preferably an anabolic-enhancing cartilage protector, contained within a targeted delivery vehicle. Both catabolic inhibitors. Such targeted delivery vehicles preferably include particles encapsulated in at least one of the cartilage protectors, and preferably both, and most preferably nanoparticles. The particles target the joint by targeting an antibody or antibody fragment coupled to the particle, which antibody or fragment is specific for antigenic determinants localized in the joint (ie, compared to most other sites in the body). Preferentially expressed in the joint, preferably highly expressed in such joint, more preferably limited to expression for this joint). Antibody-targeting particles, also referred to herein as "targeting immunoparticles," and the cartilage protective agent (s) encapsulated therein are systemically administered. A portion of the targeting composition is absorbed by the joint. The remaining composition is excreted and / or metabolized. Within such joints, the targeting antibody or fragment is bound to the targeting antigen. Over time, the particles are degraded within the joint, such that the therapeutic concentration of cartilage protector (s) is delivered locally within the joint in a sustained release manner over a predetermined time period, such as cells that are intended to be controlled (eg, primary cells of the joint (On both synovial cells and chondrocytes). The therapeutic agent may diffuse or be released into the synovial fluid and subsequently bind with the cell surface of the articular structure, may be absorbed or introduced into the cells of the articular structure, or may act directly on cytokines and / or proteases that may be present in the synovial fluid.

The targeting composition of this aspect of the invention can be targeted to the joints of a patient according to the invention without knowledge of the particular molecular pathology underlying the joint disease. Targeting antibodies allow the encapsulating agent to be preferentially localized in the joint, and more preferably localized to or bound to components of the articular cartilage.

Accordingly, this aspect of the invention is directed to administering a pharmaceutical formulation comprising a targeted drug delivery vehicle, preferably containing both cartilage anabolic and anti-catabolic agents, to one or several joints and Methods of treating patients suffering from related inflammatory, non-inflammatory or other joint diseases are provided. Such patients may suffer from OA, RA, or other joint diseases, such as non-inflammatory and inflammatory arthritis, including neuropathic arthrosis, acute rheumatic fever, histopathopathy, systemic lupus erythematosus, infant rheumatism. Arthritis, psoriatic arthritis, ankylosing spondylitis, and other spinal arthritis and crystalline arthrosis. The targeted therapeutic methods and compositions of the present invention are particularly well suited to patients with osteoarthritis. By systemic delivery of a combination of agents in a carrier targeting the joint, the disease can be treated with minimal adverse or unwanted systemic effects.

A. Identification and Characterization of Targets in Joints

The present invention provides methods and compositions for targeting drugs to joints and specifies preferred targets in the joints, including antigenic determinants associated with molecules, cells and tissues of articular cartilage and other joint tissues. Examples of such targets include collagen, including collagen type II and minor collagen types V, VI, IX, X and XI; Proteoglycans, including large cohesive proteoglycans, aggrecans, decorin, bigglycans, fibromodulin and lumicans; Cartilage oligomeric matrix protein, glycoprotein-39; Proteoglycan chondroitin-sulfate and glycosaminoglycans; Macrophage synovial cells and fibroblast synovial cells; And chondrocytes.

In a preferred embodiment, the targeting immunoparticle is associated with, irreversibly reacts to, or bound in a reversible manner with certain components of the articular cartilage (also known as ultrasonable joints) in the joint. Other molecular targets in the joint are components of the articular cartilage extracellular matrix, such as cartilage specific collagen, aggrecan, and other small leucine- including collagen types II, V, VI, IX, X and XI. Abundant proteoglycans (including decorin, bigglycan, fibromodulin and lumican). Proteoglycans are high molecular weight complexes of proteins and polysaccharides, which are found throughout the structural tissues of the vertebrate (eg cartilage) as well as on the cell surface. Glycosaminoglycans (GAGs), the polysaccharide units of proteoglycans, are polymers of acidic disaccharides containing derivatives of amino sugar glucosamine or galactosamine and are useful targets. Cartilage oligomeric matrix protein (COMP) and glycoprotein-39 (HC-gp39) (also called YKL-40) are similarly useful targets.

Articular cartilage contains a variety of genetically distinct types of collagen useful in the present invention as molecular targets to which immunoparticles (including corresponding antibodies) can be attached, allowing the encapsulated therapeutic agent to be delivered to the antibody binding site. Type II collagen, the primary collagen of articular cartilage, accounts for 90-95% of the total collagen content of articular or vitreous cartilage, which forms a cross-linked fibrillar structure recognized by electron microscopy. Type II collagen is also a unique and specific marker of chondrocytes (Hollander, et al., J. Clin. Invest 93 Exp. Cell Res. 240: 58 (1998)). The major extracellular modification of collagen molecules, which occurs after fibrillar formation, is the generation of covalent interfiber cross-links. Antibodies that bind to epitopes specific for type II collagen have been reported (Kafienah, W. et al., Tissue Engineering 8 : 817-826 (2002); Kolettas, E, et al., Rheumatology 40 : 1146-) 1156 (2001)). Type II collagen and its associated epitopes in articular cartilage represent a preferred target for the present invention.

For example, monoclonal antibodies against type II collagen, isotype IgG ₁ (designated clone 6B3) (Linsenmayer, TF et al., Biochem. Biophys. Res. Commun 92 (2): 440-6 (1980) recognizes both α1 (II) and α3 (XI) chains with the same primary structure. In western blotting, these mAbs react with TC ^A fragments 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 shows no cross reaction with type I or type III collagen. Immunblotting of the cyanogen bromide (CNBr) peptide of collagen II shows that the mAb reacts with the CB11 fragment, which is an immunogenic epitope site along the original type II molecule.

In another example, using human cartilage specific CNBr-cleaved collagen II as an immunogen, a monoclonal antibody (isotype IgG ₁ ) against type II collagen (Miller, EJ, Biochemistry 11: 4903-4909 ( 1972; Glant, TT, et al., Histochemistry 82: 149-158 (1985a); British Journal of Haematology 90: 757-766 (1995) . The mAb, commercially available from Chemicon International (Temecula, Calif.), Reacts with both pepsin-solubilized and CNBr-cleaved human and bovine type II collagen. No cross-reactivity is observed using collagen types I, III, V and IX. In a preferred embodiment of the invention, the antibody against type II collagen is bound to the target epitope with a dissociation constant in the range of 0.1-10 nM.

Quantitatively minor collagen of articular cartilage also affects the structure of the matrix, which serves as a useful target for the present invention. For example, type IX collagen, which is short, non-fibrous collagen, which is also considered proteoglycan because it contains glycosaminoglycan chains, covalently binds to type II collagen fibrils and binds the fibrils together. May help to connect or the fibrils may be bound to other matrix molecules. Type XI collagen, which is a minor fibrillar collagen, may be involved in controlling the diameter of type II fibrils. Other collagen, including type V and type VI, may form part of the matrix. These minor collagens of articular cartilage may serve as targets for antibody-directed immunoparticle binding with appropriate antibodies.

In another aspect of the invention, a separate type of proteoglycan contained in articular cartilage is useful in the present invention as a molecular target for binding an immunoparticle to enable delivery of a therapeutic agent to an antibody binding site. In articular cartilage, the proteoglycans make up the second largest solid phase portion, which accounts for 5-10% of the wet weight. The proteoglycans of the cartilage matrix consist mainly of large cohesive (50-85%) proteoglycans and large non-aggregating (10-40%) proteoglycans. There are also separate small proteoglycans.

Cartilage proteoglycans that have the most substantial effect on the material properties of tissues are high molecular weight large monomers (molecular weights 1 to 4 × 10 ⁶ ). Structurally, large proteoglycans consist of an extended protein core with several distinct regions: the N-terminal region with two globular domains (G1 and G2), domains rich in keratan sulfate; Longer domains enriched in chondroitin sulfate, which may contain some interspersed keratan sulfate and neutral oligosaccharide chains; And C-terminal globular domain G3. Aggregates are formed by the binding of many proteoglycan monomers to the hyaluronate chain in the G1 globular domain. Each proteoglycan-hyaluronate bond is stabilized by a separate globular linker protein (molecular weight 41,000 to 48,000). The large size (200-400 nm) of the chondroitin sulfate-rich region and the amount of chain present in the chondroitin-sulfate proteoglycan aggregates make it a preferred target for the targeted immunoparticles of this aspect of the invention.

Additional targets for antibodies or fragments thereof coupled with immunoparticles of the invention are provided by HC gp-39. Within the joint, fragments of with appropriate antigenic properties are also sufficient to target the drug delivery vehicle.

Antibodies or fragments thereof can be used to target immunoparticles to associate with, irreversibly react, or bind in a reversible fashion (most common) to specific structures of the joint synovial membrane. Special target cells of the joint, which are preferred targets, include the two major cell types of the synovial membrane: macrophage synovial cells (type A) and fibroblast synovial cells (type B).

An additional target for an antibody or fragment thereof coupled with an immunoparticle of the invention is chondrocytes. These cells are known to express a variety of proteins that are present on these surfaces and can serve as epitopes for cellular targeting.

In another embodiment of the invention, chondroitin-sulfate proteoglycans associated with articular cartilage represent a preferred target for a targeted drug delivery system. Monoclonal antibodies useful in the present invention that bind to epitopes specific for chondroitin-sulfate proteoglycans have been reported (Morgan Jr., A. et al., Hybridoma 1: 27-36 (1981); Schrappe, M. et al., Cancer Res 52: 3838-3844 (1992); Schrappe, M. et al., Cancer Res. 51: 4986-93 (1991)). One such example is a mouse-anti-human chondroitin-sulfate proteoglycan monoclonal antibody, designated clone 9.2.27 (IgG _2a isotype). This 9.2.27 antibody recognizes 250 kDa mature chondroitin sulfate proteoglycan core glycoproteins as well as precursor polypeptides of 210, 220 and 240 kDa. The mouse anti-human agrecan monoclonal antibody, clone 2A2.1, is also suitable for the present invention, which is commercially available from United States Biological; Swampscott, Mass. The antibody does not react with the chondroitin sulfate chain region. Transmission electron microscopy indicates that it is bound within the N-terminal portion of the chondroitin sulfate-attachment region.

B. Targeting Neoepitopes Associated with Cartilage Degeneration

Although not present in normal adult cartilage or may be present at extremely low levels, biomolecular components of cartilage that are increased or more highly expressed at certain stages of RA or OA may be provided as targets for the targeted drug delivery system of the present invention. . Preferred targets associated with cartilage degenerative diseases are neoepitopes that appear on the articular cartilage of patients diagnosed with OA, RA or other degenerative joint diseases, such as neoepitopes of agrecan or other cartilage proteoglycans. And, specifically, neoepitopes immunolocalized to the articular cartilage superficial layer of the patient.

In one aspect of the invention, the targeting antibody or antibody fragment is a neoepitope or cleavage site of type II collagen or type II collagen fragment, in particular matrix metalloproteinase (MMP) -1, 3, 8 or 13, or MMP protein Specifically binds to said neoepitope or cleavage site caused by the individual or combined action of other members of the family, or members of the disintegrin and metalloproteinase (ADAMTS) protein family members with thrombospondine motifs. ADMATS is further described in WO 00/04917, EP 0 823 478 and US 5,811,535 and in Tang, B. et al., FEBS Lett. 445 (2-3): 223-225 (1999). It is.

Antibodies directed to specific epitopes defined by the region of specification of type II collagen structure are useful in the targeting compositions of the invention. These structural regions are important in diseased cartilage, in part, by the breakdown of type II collagen that occurs in OA and RA. When Type II collagen is broken down, fragments of collagen protein are released that are released from the cartilage matrix, which is visible in the synovial fluid and moves to the circulatory system to be removed through urine. For maximum utility, the composition of the present invention targets epitopes that remain physically associated with the cartilage matrix rather than remain on the released fragments.

Each of the collagenase MMP-1 (EC 3.4.24.7), MMP-8 (EC 3.4.24.34), and MMP-13 (EC 3.4.24 -. ) Is a triple-the-forming type II collagen-helical fibrillar Because of its ability to cleave, large (3 / 4-length) amino-terminal fragments and smaller (1 / 4-length) carboxy-terminal fragments are produced (Kafienah, W. et al., Biochem. J. 331: 727-732 (1998). All of these are initially cleaved at specific Gly-Leu / Ile binding to produce characteristic 3/4 and 1/4 fragments. Specific collagenase isotypes have had a close effect on the pathological loss of cartilage (Billinghurst, R. et al., J. Clin. Invest. 99 : 1534-1545 (1997)). For example, in an animal model of OA, the focal regions of collagenase 1 and collagenase 3 proteins were localized in the extracellular matrix of the OA lesion site in the knee joint, which is associated with 3 / 4-1 / 4 collagen cleavage and Happen at the same time. Collagenase 3 protein is also abundant throughout the central tibial cartilage in diseased joints (Huebner, J. et al., Arthritis & Rheumatism 41 : 877-890 (1998)).

Collagenase 1 (MMP-1) was detected in synovial, synovial and cartilage samples from people with RA and those with OA. Collagenase 3 (MMP-13) cleaves collagen type II at 5-10 times faster than collagenase 1. Importantly, these have been identified in human OA cartilage as well as in the synovial membrane of people with RA and OA.

Fibrillar collagen may be damaged by denaturation due to helical cleavage or by removal of cross-linkages due to telopeptide cleavage. Two studies have shown that epitopes hide within the spiral region of type II collagen exposed to collagen unwind due to collagenase cleavage, and polyclonal antiserum specific for collagen neoepitopes caused by collagenase It has been demonstrated that there is active collagenase in cartilage with. These findings indicate that collagenase 1, collagenase 3, or both, are involved in cartilage degradation associated with various arterial pids. Thus, specific degradation of type II collagen from collagenase cleavage creates neoepitopes for antibody targeting useful for the specific targeting aspects of the invention. Such neoepitopes will be localized in the N-terminal 3/4 fragment sequence of the alpha (II) chain, which is known to contain epitopes for AH12L3 and CB11B (recognized by antibody COL2-3 / 4m).

In articular pidgin, increased catabolism of the aggrecan, the cartilage proteoglycan, is one of the major pathological processes leading to articular cartilage degradation. Accordingly, loss of sulfated glycosaminoglycans, an intrinsic (inherent) component of the aggrecan molecule, endangers both the functional and structural circular preservation of the cartilage matrix. Over time, the process causes cartilage breakdown. In situ degradation of agrecans is a proteolytic process involving cleavage at specific peptide bonds localized in the core protein. The enzymatic activity most widely found to affect this process is the result of the specific action of metalloproteinases. In vitro agrecan addition degradation reactions by matrix metalloproteinases (MMPs) have been extensively studied. However, it is now widely recognized that the major proteinase responsible for in situ aggrecan degradation in articular cartilage is agrecanase, the two recently identified isoforms of which are the thrombospondin motifs. Is a member of the disintegrin and metalloproteinase (ADAMTS) gene family. There is a monoclonal antibody technique for identifying novel neoepitopes on agrecan or agrecan degradation products. In addition, another aspect of the present invention provides such monoclonal binding to neoepitopes on agrecans or agrecan fragments for targeting nanoparticles containing a combination of anabolic enhancing agents and catabolic inhibitors. The use of an antibody or fragment thereof.

A temporal bone study has been established that agrecanase is primarily responsible for the loss and catabolism of agrecans from articular cartilage in the early stages of joint disease with arthritis. Although continuous, the process appears to occur much earlier than collagen catabolism. If collagen catabolism occurs at a later stage of the disease process, there is evidence that a small percentage of agrecan remaining in cartilage is mediated and degraded by MMP.

It was possible to develop and use monoclonal antibody technology to identify catabolic neoepitopes on proteolytic degradation products to identify specific cleavage sites characteristic and unique to different classes of matrix degrading enzymes. In these studies, catabolic neoepitopes (specific N- or C-terminus of proteolytic cleavage products) produced by the action of agrecanase (or MMPs) on the interglobular (IGD) domain of aggrecan Several monoclonal antibodies were identified that specifically identified new epitopes generated as amino acid sequences. These antibodies were used to monitor proteolysis of agrecan and linker proteins. In addition, those skilled in the art will recognize that antibodies that recognize neoepitopes on degradation products of matrix proteins generated during cartilage catabolism can be used to target immunoparticles to joints without departing from the scope of the present invention.

In another aspect of the invention, the targeting antibody or antibody fragment is a neoepitope or cleavage site of agrecan or agrecan fragment in cartilage, in particular MMP-1, 3, 8 or 13, or other member of the MMP protein family, Or specifically binds to said neoepitope or cleavage site generated by the individual or combined action of an ADAMTS protein family member. Examples of monoclonal antibodies that recognize different structural epitopes or neoepitopes in agrecans or agrecan fragments are 8-A-4 or BC-3. MAb 2-B-6 was used to detect a significant number of agrecan degradation products resulting from agrecanase, MMP or other proteolytic activity at many sites along the core protein of agrecan. MAb 2-B-6 recognizes 4-sulfated unsaturated dissaccharides of chondroitin sulfate attached to these core protein fragments. The relevant antibody MAb 3-8-3 was also used to identify different deglycosylated aggrecan metabolites containing 6-sulfated chondroitin sulfate oligosaccharides. MAb BC-3 recognizes the N-terminal neoepitope sequence defined by the amino acid sequence, alanine-arginine, glycine (ARGxx ...), generated after agrecanase catabolism in the IGD domain of Agrecan. .

The ADAMTS-4 gene (genebank NM-005099) and the ADAMTS-5 gene (genebank 007038) encode disintegrin and metalloproteinases with thrombospondin motifs 4 and 5, members of the ADAMTS family of proteins. . This family member shares several distinct protein modules, including pro-peptide regions, metalloproteinase domains, disintegrin-like domains, and thrombospondin type 1 (TS) motifs. 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 the C-terminal TS motif. The enzyme encoded by the ADAMTS-5 gene contains two C-terminal TS motifs and acts as agrecanase, which cleaves agrecan, the main proteoglycan of cartilage. Thus, both of these enzymes are involved in the degradation of aggrecan and the production 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)).

In another embodiment of the invention for treating human cartilage in the setting of OA, an antibody is used that targets the initial biochemical neoepitope marker of OA, designated 3-B-3 (-). This 3-B-3 (-) epitope is an OA-related phenotypic change at the end of the chondroitin sulfate (glycosaminoglycan) chain of agrecan.

C. Targeting Antibody Features

The term “antibody” as used herein includes immunoglobulin molecules and immunologically active portions of the immunoglobulin molecules (ie, molecules containing antigen binding sites that specifically bind or immunoreact with an antigen). The term refers to immunoglobulins, whether natural or synthetically produced. Proteins, including antibodies, may be derived from natural sources or may be produced partially or fully synthetically. Examples of antibodies include all immunoglobulin subtypes and Fab and F (ab ') ₂ , scFv, Fv, dAb, Fd fragments, as well as fragments described in US Pat. No. 5,534,254. ScFV refers to the single chain minimum binding domain of an immunoglobulin molecule. The term “antibody” also refers to an antibody that acts in an intracellular region (eg, cytoplasm or nucleus), extracellular space, or plasma membrane of a cell to modulate the expression or activity of one or more genes that regulate cartilage metabolism. Refers to. Preferred antibodies for use in the present invention include human adaptation, chimeric and human monoclonal antibodies.

The term “fragment” as used in the context of an antibody is an amino acid sequence which is part of all the targeting polypeptides defined above, which have common relevant elements in origin, structure and mechanism and which are functionally equivalent to the complete antigen for the purpose of targeting within the present invention. Refers to. Use of antibodies in the compositions and methods described herein also includes the use of fragments of such antibodies.

Preferred embodiments of the present invention utilize human adaptations or human antibodies or fragments thereof wherein the therapeutic cartilage protectant of the present invention is covalently attached to the surface of the encapsulated nanoparticles or other particles. Antibodies or fragments thereof are preferred as targeting molecules on particle surfaces encapsulated with therapeutic cartilage protectors, which must be sufficiently stable in vivo and have minimal potential for removal from the particle surface by serum containing extracellular fluid proteins. Because it is. Fully human monoclonal antibodies or human adapted murine antibodies that bind to all molecules or articular cells of the cartilage extracellular matrix are most useful as joint-targeting antibody types that direct delivery of the therapeutic agent (s) to be administered to a human patient. It is estimated that these antibodies will not elicit an immune response upon administration.

For example, murine monoclonal antibodies can be chimerized by genetically recombining nucleotide sequences encoding murine Fv regions (ie, containing antigen binding sites) with nucleotide sequences encoding human constant domain regions and Fc regions. . Some murine residues may be maintained within the human variable-region construct domain to ensure proper-target-site binding characteristics. Human adapted antibodies for use in targeting will be appreciated to have the advantage of lowering the immune reactivity of the antibody or polypeptide in the host recipient, which is useful for increasing half-life in vivo and conjugation on the surface of nanoparticles or other encapsulated particles. It may be useful to reduce the likelihood of an adverse immune response to a given antibody.

It is quite advantageous to use antibodies or their homologues with full human characteristics to treat human patients. Similar methods may be used as described in US Pat. Nos. 6,075,181, 6,235,883 and 6,492,160, and EP 1 167 537 A1, the entirety of which is incorporated herein by reference. The method has previously been used to generate a variety of complete human antibodies against human IL-8 and epidermal growth factor receptors. In the most preferred embodiment of the invention, the antibody or antibody fragment binds the target epitope with a dissociation constant in the range of 0.1 to 10 nanomolar.

Although complete human or human adaptation antibodies are preferred for use in the present invention to treat human patients, the methods and compositions of the present invention may also be used in veterinary medicine to treat other mammals (eg, horses and dogs) that are susceptible to joint degeneration. It should be recognized that it is also useful for the application.

D. Cartilage Protectors for Targeted Delivery

Preferred aspects of the targeting aspect of the invention will include one or more cartilage protectors encapsulated or contained within nanoparticles or other delivery vehicles to which the targeting antibody, antibody fragment or other targeting construct is attached. The formulation encapsulated or contained may be any of the anabolic cartilage protectors or catabolic inhibitors described herein having chemical or structural characteristics that make them suitable for encapsulation or inclusion in selected nanoparticles or other delivery vehicles. have. In addition, agents that are highly sensitive to metabolic degradation from systemic administration (eg proteins and peptides), or agents that cause adverse side effects when administered systemically without targeting, are preferred for targeting. For example, a class of anabolic cartilage protectors (transforming growth factor (TGF) -β super-family, insulin-like growth factor and fibroblast growth, including TGF-β agonists and bone morphogenic protein agonists, as described below) As each agent in the factor) and the particular agent in the catabolic inhibitor classes described below (interleukin-1 receptor antagonists and TNF-α receptor antagonists) are proteins, they are very useful for delivering in targeted encapsulated form. Suitable.

Most preferred targeting encapsulant compositions of the invention will include both anabolic cartilage protectors and catabolic cartilage protectants encapsulated within the same targeting immunoparticle (or other targeting particles), preferably both agents are proteins. . Alternatively, each agent may be encapsulated separately and a mixture of two (or more than two) types of targeting particles may be administered, or less preferably two or more targeting agents may be continuously or sequentially separated. Delivery so that the agent is present simultaneously in the joint.

There are some cases where only anabolic cartilage protectors or catabolic inhibitors may be suitable for encapsulation or one agent may not be associated with undesirable systemic side effects. In such cases, one agent may be delivered in encapsulated targeting form, while the other agent is delivered in non-targeted, non-encapsulated form, which are delivered together or separately in the same dosage form as a mixture.

While it may not be desirable to administer both anabolic agents and catabolic inhibitors, provided all of the benefits provided by the combinations mentioned above, a single cartilage protector encapsulated in an immunoparticle targeting an antigen localized in the joint (Promotes assimilation or catabolic).

E. Film Formation of Formulations

The size of a particular material is an important factor in determining whether it can penetrate the synovial capillary wall and move from the systemic circulation into the joint. The maximum diameter of particles that can cross the synovial capillary wall is generally considered to be 50 nanometers. However, several studies on the synovial capillary penetrating ability of rats using lecithin-coated polystyrene particles up to 240 nanometers in diameter have shown that they can be transported across the synovial capillary wall through transcytosis. . The present invention preferably overcomes the limitations imposed by the synovial permeability barrier by using a class of encapsulating particles, such as nanoparticles (preferably nanospheres), which are limited in size distribution.

Sustained release dosage forms of the invention may comprise particulates and / or nanoparticles in which the therapeutic agent is dispersed, or may comprise the therapeutic agent in pure, preferably crystalline solid form. This aspect of the therapeutic dosage form may be any steric configuration suitable for sustained release. Preferred sustained release therapeutic dosage forms of the invention will have the following size, biodegradability and biocompatibility characteristics.

Targeted delivery systems of the present invention provide for nanoparticles that are limited in size to about 5 nanometers to about 750 nanometers in diameter, more preferably about 10 to about 500 nanometers, and most preferably about 20 to about 200 nanometers in diameter. It is desirable to utilize. Useful, but less preferred, when proven to bring sufficient penetration in a disease state is from about 1 to about 100 micrometers in diameter, more preferably from about 1 to about 25 micrometers, most preferably from about 1 to about 10 Particulates in the size range that are micrometers.

Preferred particles biodegrade the drug loaded at therapeutic levels over a period of time, preferably from about 1 to about 150 days, preferably from about 7 to about 60 days, more preferably from about 14 to about 30 days. Biodegradable structures that release and release. One skilled in the art can combine drug release from nanoparticles and particulates by combining physical processes, including but not limited to diffusion and degradation, which combinations of anabolic and anti-catabolic therapeutic agents and respective carrier formulations. It should be recognized that this can be described as a unique complex dynamics process.

Preferred particles are biocompatible with the targeting tissue of the local physiological environment and joints in which the dosage form is administered (including producing biocompatible biodegradable products). Suitable compositions include biodegradable particles formulated from natural polymers including hyaluronan, chitosan, collagen, gelatin and alginate. These natural polymers can be combined with other polymers to produce copolymer molecules composed of, for example, chitosan and gelatin. Synthetic biodegradable poly (alpha-hydroxy esters), such as polylactic acid (PLLA), polyglycolic acid (PGA) and copolymer PLGA, produce microparticles incorporating protein therapeutics such as human growth hormone. Used successfully. Another example of a biodegradable polymer that may be suitable for use in preparing the targeting particles of the present invention is an amphiphilic ABA triblock copolymer such as poly (ethylene oxide): poly (3-hydroxybutyrate): poly (Ethylene oxide).

In selecting polymeric nanoparticle systems for use with selected cartilage protectors of the present invention, it may be important to ensure the proper bioactivity of the encapsulated drug.

The use of targeted biodegradable polymeric nanospheres has the advantage of providing selected differential release profiles for encapsulated therapeutics. For some drug combinations, the optimal release kinetics may consist of a dual-release process, where each active agent has been shown to exhibit a different sustained release kinetics profile to provide the most optimal pharmacokinetics in the joint. Those skilled in the art will, based on the present specification, the optimal release kinetics from the nanoparticles or particulates will vary for each individual drug, which also depends on the amount of drug loaded on the particles, particle size, and particle composition during formulation. It will be appreciated that it will be a function of other physicochemical properties determined by. The desired therapeutic concentration will be achieved by adjusting the quantitative release rate for each drug from the encapsulated particles remaining in the joint to obtain optimal therapeutic concentrations in the synovial fluid and intraarticular space. In vitro studies can be performed to confirm double-release kinetics for sustained release formulations in which each component (eg, anabolic drug and catabolic drug) exhibits sustained release over 7-30 days as an example. . Methods of quantifying the amount of each drug released into the synovial fluid are well known in the art and may include methods of measuring radiolabeled drugs. Alternatively, it is possible to covalently attach fluorescent or other optical reporter molecules to prepare labeled drugs. Those skilled in the art will recognize that there are many indirect quantification methods specific to each agent, such as ELISAs or mass spectroscopy.

For practical reasons, it is difficult to achieve similar release kinetics for two drugs of varying sizes, as is the case for many assimilation and catabolic combinations. For example, in one preferred embodiment of the invention, catabolic inhibitors, such as p38 MAP kinase inhibitors, which are characterized by a molecular weight of 200 to 500 (eg SB203580, MW = 377), may be 160 in length. Delivery with an anabolic agent, eg, human IL-10, of dog amino acid and MW of approximately 18 Kda is desired. Independent control of the sustained release rate for each agent can be achieved by varying the structural composition of the particle in question and / or creating a mixture of two or more immunoparticles. In this mixture, one set of particles is homogeneous for the encapsulated anabolic agent, while a separate set of particles is homogeneous for the encapsulated catabolic inhibitor. These two sets of mixed particles may vary in their respective size and polymeric composition, but in some cases may be characterized by a similar release rate for their active agent or by a consistent release rate. Thus, the topical therapeutic effects of each encapsulant will each be optimized.

Liposomes are not preferred delivery vehicles for targeted systemic delivery of cartilage protectors according to the present invention. Compared to nanospheres and other sustained release particle delivery systems, liposomes have a short half-life in the circulation. Liposomal drug conjugates can be trapped in the liver and spleen, resulting in liposome breakage and release of the active agent. Thus, the agent is not protected until localized in the joint, but rather distributed throughout the body in an active state. Agent release from the targeted liposome delivery system is not highly persistent and much less localized than for the targeted immunoparticle delivery system. For these reasons, it is highly desirable to use particles (eg nanospheres) rather than liposomes. Nevertheless, for systemically delivered anabolic cartilage protectors, or combinations of cartilage protectors including anabolic agents, where targeting is highly desired, targeted liposomes provide a suitable and advantageous over exposed drug delivery. Can be proved.

F. Antibody Coupling for Encapsulating Agents

Representative “coupling” methods for linking a targeting antibody to sustained release nanoparticles via covalent or non-covalent bonds include reactive groups of the targeting antibody (eg, hydroxyl, amino, amido or sulfhydryl groups), and Chemical cross-linkers and hetero-bifunctional cross-linking compounds (ie, "linkers") that react to form bonds between other reactive groups (of similar chemical nature) present on the nanoparticles or other targeting vehicle surfaces Included. Such bonds formed between the targeting antibody and the particles or other delivery vehicles may include, but are not limited to: peptide bonds, disulfide bonds, thioester bonds, amide bonds and thioether bonds.

Direct conjugation of sustained release dosage forms to the targeting protein (antibody) can disrupt the recognition of the targeting molecule or cell by the modified targeting antibody. Ligandsandwich attachment techniques are useful alternatives for attaching sustained release dosage forms to targeting binding proteins (antibodies). These techniques may include forming a primary peptide or protein shell using a protein that does not bind to the target cell population. The binding protein is then bound to the primary peptide or protein shell, thereby providing a particle with the functional binding protein / peptide produced thereby. Examples of ligand sandwich approaches include a method of covalently attaching avidin or streptavidin to a particle via a functional group as mentioned above for the "direct" binding approach. Such binding proteins are derivatized via functionalized biotin (eg, via active esters, hydrazides, iodoacetals, maleimidyl or other functional groups), preferably to a minimum. Ligand (ie, binding peptide or protein / functionalized biotin) attachment to the available biotin binding site of the avidin / streptavidin primary protein shell is achieved by using a saturated amount of biotinylated protein / peptide.

3. Alternative targeting and delivery of preferential effects

Other methods of targeting cartilage protectants and combinations thereof in the present invention, or other methods by which such agents or combinations preferentially effect joints over other parts of the body, are also within the scope of the present invention. For example, the two major cell types of the synovial membrane, macrophage synovial cells (type A) and fibroblastic synovial cells (type B), and chondrocytes are present on the surface of these cells and for specific cellular targeting. It is known to express a variety of unique proteins that can serve as epitopes. Undesired systemic effects of selecting agents for proteins that are preferentially expressed in the joint or that are inflamed or preferentially expressed in a diseased joint, and preferably a combination of anabolic-enhancing agents and catabolic inhibitors Local effects can be increased preferentially while minimizing

As discussed above, molecular targets in joints include components of the cartilage extracellular matrix, such as cartilage specific collagen (including collagen type II, IX, X, XI), aggrecan and other small leucine-rich Proteoglycans such as decorin, bigglycan, fibromodulin, and lumican. Also included as targets are cartilage oligomeric matrix proteins (COMP) and glycoprotein-39 (HC-gp39) (also termed YKL-40). Preferred targets for use in this aspect of the invention may be biochemical cartilage markers present at certain stages of RA or OA, although they may be absent or at extremely low levels in normal adult cartilage. Likewise, the selection of cartilage protectors specific for receptors that are upregulated in a diseased or inflamed state, and preferably a combination of anabolic and catabolic agents, may be topical compared to other parts of the body. The effect may be increased preferentially.

As noted above, by coupling the encapsulated agent (s) with the corresponding targeting antibody (or antibody fragment), such encapsulated agent (s) can be targeted to one or more structures in the joint. Potentially, the antibody may be coupled to the exposed drug itself using a linkage cleaved within the local environment of the joint to achieve targeting and delivery of the drug systemically administered to the joint. Similarly, cartilage protectors included in the assimilation-promoting and catabolic inhibitory compositions of the present invention are characterized in that synovial and / or chondrocytes and / or It may be chosen to exert their effect on extracellular matrix components preferentially.

E. Methods of Selecting Delivery Vehicles and Dosages

The exact loading or dosage of the particular nanosphere system and targeting antibody or fragment to be used in accordance with the present invention to deliver the selected agent and the therapeutic agent to be included in the targeting composition can be determined analytically in accordance with the present invention. This assay involves administering antibody-labeled nanospheres (or other targeting particles) containing the encapsulated therapeutic agent to a patient in need of such a diagnostic test and performing imaging analysis on the patient to determine the location of drug-containing nanospheres. It includes determining. Imaging analysis can then be used to determine the degree of deposition in the patient's joints. Imaging analysis is well known in the medical arts, including but not limited to x-ray analysis, magnetic resonance imaging (MRI) or computed tomography (CT). In a preferred embodiment, the joint-targeted antibody can be labeled with a detectable agent that can be imaged in the patient. For example, the targeting antibody can be labeled with a contrast agent (eg barium) that can be used for x-ray analysis, or a magnetic contrast agent (eg gadolinium chelate) that can be detected using MRI or CT. Other labeling agents include, but are not limited to, radioisotopes, such as ⁹⁹ Tc. In addition to imaging analysis, biopsies can be obtained from a patient to determine the presence and concentration of particles in the joint (eg, synovial tissue).

Topical delivery embodiments of the invention are mentioned in terms of suitable concentrations for therapeutic agents, when delivered locally, sufficient to provide a predetermined level of inhibition or therapeutic effect at the local delivery site (eg, joint). If systemically delivered, larger concentrations or dosages of formulation will be needed. Such systemic dosages and / or concentrations are those required to provide sufficient active agent (s) to the target site of potential cartilage degradation necessary to achieve the desired level of topical therapeutic effect after all metabolic transformation processes. In particular, therapeutic and preferred levels suitable for systemic dosages and / or concentrations are those which provide the active agent to a local site (eg joint) at a concentration level within the previously described topical delivery therapeutic and preferred concentration ranges.

In the case of a targeted sustained release delivery system, a dosage of the agent sufficient to provide a local concentration at the joint or site of action during a predetermined sustained release period to achieve a desired level of therapeutic effect over a substantial period of the desired sustained release period, or The loading is included in the composition of the present invention. Thus, formulation doses or loadings sufficient to provide a predetermined amount of encapsulated preparation absorbed by the joint are included, taking into account all the metabolic transformation of the formulation that occurs prior to reaching the joint or local environment of the joint. Since such predetermined amount of encapsulating agent reaching the joint will be determined in accordance with the present disclosure, as the nanospheres or other encapsulating delivery systems degrade, the desired sustained release period (eg, 1 to 4 weeks, More preferably from 1 to 2 weeks) to provide a local concentration within the therapeutic concentration range for the agent.

IV. Formulation for inhibiting cartilage degradation

The following describes examples of classes of cartilage protectors suitable for use in the compositions of the present invention and examples of drugs in each class. While not wishing to be bound by any theory, a clarification behind the selection of the various classes of formulations that are believed to render the formulations operable is also presented.

1. Interleukin-1 (IL-1) Receptor Antagonists

Interleukin IL-1 exists in two forms, IL-1α and IL-1β, which are polypeptides derived from separate gene products that share a similar spectrum of immunomodulatory and pro-inflammatory functions. IL-1 is a 17 kD polypeptide that can be produced and acted on by numerous cell types in the joint, including synovial fibroblasts and macrophages, chondrocytes, endothelial cells and monocytes and macrophages. There is substantial evidence that it plays a decisive role in inflammation of the joints and plays a decisive role in the pathophysiological loss of articular cartilage that occurs in damaged joints.

The action of both cartilage destructive cytokine forms is mediated by two IL-1 receptors (IL-1R), either type I IL-1 or type II IL-1 receptors. IL-1 receptors are structurally separate and they belong to a separate super-family characterized by the presence of an immunoglobulin binding domain. These receptors have close amino acid homology with other receptors containing immunoglobulin domains. While expression of larger type I IL-1 receptors is present on T cells and fibroblasts, smaller type II IL-1 receptors are present on B cells, monocytes, neutrophils and myeloid cells.

The type II IL-1 receptor binds with high affinity with IL-1β but does not initiate intracellular signal transduction as IL-1b binding is done upon binding to the type I IL-1 receptor. In contrast, type II receptors serve as precursors to soluble IL-1 binding factors found to be separated from cells, and these soluble receptors act as physiological IL-1β antagonists. Natural IL-1 binding proteins have been reported that correspond to the soluble outer portion of the type II receptor.

Cloning and sequencing of naturally secreted soluble ligands that bind to IL-1 receptor, also referred to as IL-1 receptor antagonist (sIL-1RA, IL-1Ra, IL-1ra), encodes a 22 kD protein Turned out to be. IL-1Ra competitively inhibits binding of IL-1α and IL-1β to both type I IL-1 receptors and type II IL-1 receptors. IL-1Ra is a pure receptor antagonist because its binding to the receptor does not activate the cellular signal transduction moving parts of the membrane-associated IL-1 receptor. Despite the high affinity binding of these proteins to IL-1Rs, a 10-100 fold molar excess is required to inhibit the IL-1 biological response of cells expressing type I IL-1R. Cells known to produce IL-1Ra include monocytes, neutrophils, macrophages, synovial cells, and chondrocytes. IL-1Ra has been shown to inhibit PGE ₂ synthesis, induction of pro-inflammatory cytokines and MMPs, and nitric oxide production. Secreted IL-1Ra is released in vivo during experimentally induced inflammation. Importantly, IL-1Ra is expressed in synovial tissue and is present in normal human synovial fluid. In patients with injured knees, the level of IL-1Ra in synovial fluid increases rapidly in the acute phase after injury, and subsequently decreases below normal levels in subacute and chronic conditions. It has been shown to play a physiological role.

IL-1 is considered a predominant cartilage destructive cytokine that plays a decisive role in joint destruction due to its ability to stimulate the production of destructive enzymes and pro-inflammatory cytokines by both chondrocytes and synovial cells. Moreover, IL-1b is a potent inhibitor of proteoglycan and collagen synthesis by chondrocytes. At the cellular level, IL-1β-induced responses of synovial fibroblasts include increased production of PGE2, collagenase and other neutral proteases, and upregulation of the pro-inflammatory cytokines IL-6 and IL-8 .

IL-1 present in the articular fluid of patients with arthritis stimulates chondrocytes to 1) synthesize an increased amount of enzymes such as stromelysine, fibroblasts and neutrophil collagenase and plasminogen activator; 2) inhibits the synthesis of plasminogen activator inhibitor-1 and TIMP. In addition, IL-1β is a potent inhibitor of the synthesis of matrix components, such as type II collagen, a prodominant form in articular cartilage, and proteoglycans. An imbalance between inhibitor levels and protease levels leads to an increase in the amount of active protease. This increase results in cartilage degradation in combination with matrix biosynthesis inhibition. In animal studies, injection of IL-1 into rabbit knee joints results in depletion of proteoglycans from articular cartilage.

Since IL-1 is one of the major cytokines involved in the pathogenesis of chronic synovitis and cartilage degradation, reducing its production or blocking its action is a suitable strategy for new therapies that reduce synovial inflammation and provide cartilage protection. Indicates. 1) the use of naturally specific inhibitors of IL-1 activity so far identified, including IL-1Ra and soluble IL-1 receptors; 2) use of anti-IL-1 Abs; 3) Various therapeutic approaches can be utilized to antagonize the interaction of agonist IL-1 with its natural membrane bound receptor, including the use of small molecule antagonists, which may be peptidic or non-peptidic.

The ability to block the action of these major cytokines exerts effects on many cell types in the joints (eg, synovial fibroblasts and chondrocytes) such that subsequent pathological effects, such as infiltration of inflammatory cells into the joints, It will inhibit synovial hyperplasia, synovial cell activation as well as inhibiting cartilage breakage and cartilage matrix synthesis. IL-1 receptor antagonists should interfere with the disease process by blocking the proliferation of inflammatory responses by IL-1. The therapeutic potential of numerous IL-1 receptor antagonists has been established in animal models of inflammation and arthritis (RA and OA). RA patients were clinically improved after subcutaneous injection of IL-1Ra or intraarticular injection of soluble Type I IL-1R.

The effects of IL-1β and IL-1Ra depend on their respective local concentrations. In RA synovial fragment supernatant, IL-1β levels were about three times higher than IL-1Ra levels. Thus, spontaneous local production of IL-1Ra is not sufficient to inhibit the IL-1β effect, which is greater (10 to 100) to inhibit IL-1-induced biological responses in cells expressing type I IL-1R. 2) molar excess of IL-1Ra is required. Therefore, high doses of IL-1Ra have been used to block IL-1 in human volunteers in RA in vivo. Locally present in the synovial membrane, IL-1Ra down-regulates at least some of the IL-1-mediated processes in synoviitis, such as leukocyte accumulation, PGE ₂ production, and collagenase production by synovial cells in inflamed tissues. To provide a negative signal. Demonstrating the cartilage protection effect of IL-1Ra by using a gene therapy approach based on direct injection of IL-1Ra into the joints and transfecting the IL-1Ra gene into human synovial fibroblasts in a canine ACL model It was.

The present invention describes local and systemic delivery of IL-1 soluble receptor proteins composed of the extracellular domain of IL-1R and capable of binding to IL-1 cytokine molecules in solution. By way of example, in particular, soluble human IL-1 receptor (shuIL-1R) polypeptides essentially comprising amino acid sequences 1-312 as described in US Pat. Nos. 5,319,071 and 5,726,148 are anti-inflammatory class, analgesic class or cartilage protecting class. It is described herein for use in combination with one or more drugs selected from. On the other hand, local or systemic delivery of fusion proteins consisting of sIL-1R binding domain polypeptides is proposed to be used to enhance cartilage protection, as described in US Pat. No. 5,319,071. In addition, local or systemic delivery of IL-1 receptor antagonists as described in US Pat. No. 5,817,306 is described for use in the present invention. The shuIL-1R 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, such as shuIL-1R, may be achieved in irrigation solution (eg, arthroscopy in combination with one or more cartilage protective drugs, anti-inflammatory drugs, or analgesic drugs). Or by direct injection into the joint, which is described herein as a cartilage protectant when applied topically to joint tissue of various inflammatory or pathophysiological diseases. Alternatively, the agent can be systemically delivered, for example, in a targeted systemic delivery system. This therapy will proactively inhibit IL-1 stimulation of collagenase-1 and stromelysin-1 production. Using completely different methods based on gene delivery for the local production of type 1 soluble receptors for IL-1 and / or a, the presence of soluble receptors for these cytokines is rabbet during the acute inflammatory phase of antigen-induced arthritis It has been found to provide protection for the knee joint.

IL-1 receptor antagonist peptides (11-15 amino acids) that specifically bind to human type I IL-1 receptors with high affinity are suitable for use in the present invention as cartilage protectors. These small peptides offer many advantages over larger recombinant IL-1 soluble receptors or recombinant IL-1ra, which are easy to synthesize, inexpensive to synthesize, and capable of penetrating biological barriers. Two of the most potent peptides based on in vitro efficacy are Ac-FEWTPGWYQJYALPL-NH ₂ (AF12198, IC ₅₀ = 0.5-2 nM) and Ac-FEWTPGWYQJY-NH ₂ (AF11567). AF11567 is a truncated variant of AF12198, lacking a 4 C-terminal residue and exhibiting a slightly lower affinity for the type I IL-1 receptor, but having a similar plasma half-life of 2.3 to 2.6 hours. When administered systemically via intravenous infusion, poor solubility and rapid metabolism are believed to limit the in vivo efficacy of AF12198. These limitations may be partially due to direct local delivery methods, such as injection into intraarticular joint spaces, or inclusion in surgical irrigation or other infusions, or through systemic delivery using a targeted delivery vehicle as described above. To be overcome. Examples of IL-1 receptor antagonist formulations suitable for the present invention are listed below. For all modes of local delivery (ie injection, infusion and irrigation) and systemic delivery (including the use of targeted delivery systems), the optimal dose and / or concentration of each suitable agent is therapeutically effective. As an example, for each of the listed formulations, the preferred and most preferred concentrations of irrigation solutions containing the listed formulations are provided. Such concentrations are expected to be therapeutically effective. Similarly, the systemic composition according to the present invention will suitably comprise a dosage or loading of the agent sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges. In the case of targeted sustained release delivery systems, formulation dosages or loadings sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges over a predetermined sustained release period are included in the compositions of the present invention.

Therapeutic and Preferred Concentrations of Interleukin-1 Receptor Antagonists compound Local delivery therapeutic concentration (nM) Most desirable local delivery concentration (nM) rshuIL-1R 0.2-2000 200 rhIL-1ra 0.2-2000 200 Anti-IL1-antibody 0.2-2000 200 AF12198 0.2-2000 200 AF11567 0.2-2000 200

2. Tumor Necrosis Factor (TNF) Receptor Antagonist

TNF-α, a cytokine produced primarily by activated macrophages, has immunomodulatory activity as well as numerous biological actions, including transcriptional regulation of several genes mediated by specific TNF receptors. Cloning and sequencing of two different receptors, originally named and, revealed that it is produced as a soluble receptor.

Receptors of this family are single transmembrane proteins that show significant homology in their extracellular domains, but show little sequence homology in their relatively short intracellular domains. The action of TNF is the result of this factor binding to cell surface receptors present on almost all cell types studied. Two receptors were identified and cloned. One receptor type, designated TNFR-II (or Type A or 75 kDa), encodes a transmembrane protein of 439 amino acids and has an apparent molecular weight of 75 kDa. The second receptor type, designated TNFR-I (or Type B or 55 kDa), has an apparent molecular weight of 55 kDa and encodes a transmembrane protein of 426 amino acids. TNFR1 contains an intracellular domain capable of initiating signaling through the NF-κB pathway.

Both of these TNF receptors exhibit high affinity for TNFa binding. Soluble TNF receptor (sTNFR) was isolated and demonstrated to be caused as a result of the divergence of the extracellular domain of membrane bound receptor. Two types of sTNFRs have been identified, named sTNFR1 (TNF BPI) and sTNFRII (TNF BPII). Both of these soluble receptor forms have been shown to exhibit truncated forms of the two types of TNFR mentioned above.

TNF-α plays a pivotal role in the sequence of cellular and molecular events that underlie inflammatory responses and cartilage destruction. Many of the TNF-a effects overlap with the pro-inflammatory effects of IL-1. One pro-inflammatory action of TNF-α is stimulation of the release of other pro-inflammatory cytokines, including IL-1, IL-6 and IL-8. TNF-α also induces release of matrix metalloproteinases from chondrocytes, neutrophils and fibroblasts that degrade cartilage, in part through stimulation of collagenase. In addition, TNF-α upregulates COX-2 in normal human articular chondrocytes and synovial fibroblasts to increase PEG2 production.

Such cytokines, along with IL-1, are considered to initiate and induce pathological effects on cartilage within joints, including leukocyte infiltration, synovial hyperplasia, synovial cell activation, cartilage breakdown and inhibition of cartilage matrix synthesis. In particular, during synovial inflammation, increased levels of TNF-α are found in the synovial fluid of joints, and increased production of TNF-α by synovial cells occurs. Thus, systemic delivery, including delivery in targeted delivery systems, or local delivery of soluble TNF-α receptors in irrigation solutions, infusions or injections will bind free TNF-α, which acts as an antagonist of TNF receptors in surrounding tissues. Will act to provide cartilage protection.

The present invention, either alone or in combination with other agents that provide a cartilage protective effect, captures the available free ligands or direct competitive interactions with the receptors themselves, thereby exchanging the interactions of the ligands with their homologous membrane receptors. It describes the use of functional antagonists of TNF-α that acts to block cell sites. 1) the use of naturally specific inhibitors of TNF-α activity so far identified as soluble, including soluble TNF-α receptors; 2) use of anti-TNF-α antibodies; 3) Various therapeutic approaches may be utilized to antagonize the interaction of agonist TNF-α with its natural membrane bound receptor, including the use of small molecule antagonists, which may be peptidic or non-peptidic.

The present invention describes the use of chimeric soluble receptor (CSR) proteins in which the extracellular domain of the TNF receptor, which retains binding activity for the TNF molecule, is covalently linked to the domain of the IgG molecule. In particular, and as a first example, a chimeric polypeptide (recombinant chimera) comprising the extracellular domain of a TNF receptor extracellular polypeptide coupled with the CH2 and CH3 regions of a mouse IgG1 heavy chain polypeptide is used as described in US Pat. No. 5,447,851. Could. Chimeric TNF soluble receptors (also termed "chimeric TNF inhibitors" in US Pat. No. 5,447,851) have been shown to bind with high affinity with TNF-α and have been proven to be highly active as inhibitors of TNF-α biological activity. . Also a second example is a chimeric fusion construct consisting of the ligand binding domain of the TNF receptor with the Fc antibody portion (called Fc fusion soluble receptor) created for TNF-α. The invention also describes the use of soluble TNF receptor: Fc fusion proteins, or any modified form, as described in US Pat. No. 5,605,690. The molecular form of the active soluble receptor fusion protein may be monomeric or dimeric. Existing studies establish that the soluble TNF receptor: Fc fusion protein possesses high binding affinity for TNF-α.

With regard to defining soluble receptors as pharmacological antagonists, the term soluble receptors includes, but is not limited to: (1) soluble receptors corresponding to naturally occurring (endogenous) generated amino acid sequences, or of full length; Soluble fragments thereof consisting of the extracellular domain of a membrane receptor; (2) recombinant soluble receptors, and their analogs, which are part or truncated sequences of full-length native receptor amino acid sequences that possess the ability to bind homologous ligands and retain biological activity; And (3) complete attachment of an oligomer (eg, amino acid) to a sequence corresponding to a portion of the IgG polypeptide (eg, IgG hinge and Fc domain) that possesses biological activity and has the ability to bind homologous ligands. A chimeric soluble receptor, which is a recombinant soluble receptor consisting of a truncated sequence or a portion corresponding to a portion of an extracellular binding domain of a receptor amino acid sequence of length.

Soluble extracellular ligand-binding domains of cytokine receptors occur naturally in body fluids and are believed to be involved in the regulation of the biological activity of cytokines. Many of the soluble truncated forms of hematopoietic cytokine receptors that exist in their natural state have been reported (IL-1R, IL-4R, IL-6R, TNFR). For example, soluble TNFR is found at a concentration of about 1 to 2 ng / ml in serum and urine of healthy subjects. Lacking signal transduction function, these cytokine binding proteins result from alternative splicing of mRNA to the complete receptor sequence (membrane bound form), or as a result of proteolytic cleavage and release of the receptor in membrane bound form. Is generated as. Although the in vivo function of these soluble cleaved receptors has not been fully established, it appears to act as the physiological antagonist of their complementary endogenous cytokines. (1) the capture of a free ligand through its binding to homologous soluble receptors reduces the effective free concentration available for membrane-bound receptors, and (2) the action of cytokines is associated with the binding of cell surface receptors. Such antagonisms occur because they occur only in a subsequent action.

TNF-α soluble receptors will function as natural antagonists of TNF-R1 and TNF-R2 by competing with these cell surface receptors for a common pool of free ligands. Pharmacologically, the TNF soluble receptor will function as an antagonist through its ability to reduce free ligand bioavailability, not by competitive inhibitory mechanisms (ie, competing with endogenous ligands for a common binding site on the membrane receptor). . Adding a therapeutically effective amount of TNF soluble receptor to the joint should effectively neutralize the biological activity of the ligand. In vivo administration of recombinant soluble receptors demonstrated the ability to inhibit inflammatory responses and act as antagonists.

In the present invention, agents suitable as cartilage protectors for use in combination with other cartilage protectors, analgesics and / or anti-inflammatory agents to inhibit cartilage destruction include soluble TNFR, cells of TNF-α receptor (p80) linked to the Fc portion of human IgG1. Human chimeric polypeptides (recombinant chimeras) comprising an extradomain, and anti-TNF-α antibodies. For all modes of topical delivery (ie, injection, infusion and irrigation), the optimal dose and / or concentration of each suitable agent is therapeutically effective. As an example, for each of the listed formulations, a preferred and most preferred concentration of irrigation solution containing the listed formulations is provided, which concentration is expected to be therapeutically effective. Similarly, the systemic composition according to the present invention will suitably comprise a dosage or loading of the agent sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges. In the case of targeted sustained release delivery systems, formulation dosages or loadings sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges over a predetermined sustained release period are included in the compositions of the present invention.

Therapeutic and preferred concentrations of TNF-receptor antagonist compound Local delivery therapeutic concentration (nM) Most desirable local delivery concentration (nM) STNFR 0.1-2000 200 Chimera rhTNFR: Fc 0.1-2000 200 Anti-TNF-α antibody 0.2-2000 200

3. Interleukin Receptor Agonists

Some cytokines are signaling glycoproteins that are important mediators of synovial inflammation and cartilage destruction. Recent analyzes of cartilage destruction mechanisms indicate that absolute levels of pro-inflammatory master cytokine IL-1 are important in determining cartilage loss, as well as cytokine control of cartilage bio homeostasis, which regulates catabolic and anabolic regulatory cytokines. It suggests that it depends on the balance between growth factors. If the balance between IL-1β production and IL-1Ra production changes in the inflammatory state for IL-1β, this will contribute to the pathogenesis of chronic inflammatory disease and cartilage destruction, as is known to occur after knee joint surgery. Potential therapeutic agents that inhibit the production of pro-inflammatory cytokines at inflammatory sites in the joints include anti-inflammatory cytokines IL-4, IL-10 and IL-13. These cytokines have been observed to significantly reduce articular cartilage destruction in vitro and in vivo through their effects on a range of pathways that reduce the effects of IL-1. Thus, anti-inflammatory cytokines such as IL-4, IL-10 or IL-13 1) reduce the production of pro-inflammatory cytokines, and 2) natural anti-inflammatory cytokines, as recently demonstrated in vivo for IL-4. By inducing the production of (eg, IL-1Ra), it may be useful to reduce inflammation.

IL-4 is believed to attenuate the inflammatory process in the synovial membrane of rheumatoid arthritis (RA) patients. In rheumatic synovial membranes, IL-4 has been found to inhibit the production of pro-inflammatory cytokines, inhibit the proliferation of synovial cells and reduce bone absorption as part of the synovial membrane. IL-4 can enhance the direct cartilage protective effect through inhibition of matrix metalloproteinase-3 (MMP-3) synthesis in human articular cartilage cells. Using a Cell Culture System Using Human Articular Cartilage Cells to Assess the Effect of IL-4 on Tissue Inhibitors of Metalloproteinase-1 (TIMP-1) and IL-1-Induced Production of MMP-3 It was. It has been found that IL-4 inhibits IL-1-stimulated MMP-3 protein and enzyme activity. In addition, IL-4 inhibited IL-1-induced MMP-3 mRNA. Induction of iNOS can be inhibited by IL-4, IL-10 and IL-13. Thus, IL-4 can be identified as a protective mediator of joint destruction observed in inflammatory joint disease.

In addition, the effect of IL-4 on the balance between IL-1 regulatory cytokine levels has also been found to support the cartilage protective role. IL-4 and IL-10 have been shown to inhibit the production of inflammatory cytokines by freshly prepared rheumatic synovial cells. Although each interleukin is effective alone, the combination of IL-4 and IL-10 synergistically stimulates IL-1 and TNF-α stimulated production of IL-6 and IL-8 without affecting cell viability. Suppressed. Addition of IL-4 to RA synovial culture increased production of IL-1Ra and decreased production of IL-1β. In vivo treatment with IL-4 has recently been reported to enhance the degradation of experimental rat arthritis by differentially acting on the IL-1β / IL-1Ra balance. Another cytokine, IL-13, which shares many properties with IL-4, also induced IL-1Ra in the RA synovial membrane. Thus, systemic or local delivery of an IL-4 and IL-13 combination may provide synergistic therapeutic value.

IL-10 has a number of properties that indicate that it is a good candidate for inhibiting cartilage destruction. It inhibits both IL-1 release and TNF-α release and stimulates TIMP-1 production, while inhibiting MMP-2. The production of IL-10 in the RA synovial membrane has recently been reported and the anti-inflammatory effect of IL-10 has been confirmed. IL-10 inhibited IL-1b production in an ex vivo RA model using synovial fragments, but to a lesser extent than IL-4.

Using a non-local delivery method to cytokines, the protective effect of IL-4 and IL-10 treatment on cartilage destruction has been demonstrated in arthritis animal models. In a collagen-induced arthritis rat model, the combination treatment of IL-4 and IL-10 led to significant improvement. In addition to suppressing the visual signs of inflammation, the combination treatment of IL-4 and IL-10 lowered cellular invasion in synovial tissue and resulted in significant protection against cartilage destruction. Moreover, mRNA levels for TNF-α and IL-1 were highly inhibited in both synovial tissue and articular cartilage. In contrast, the levels of IL-1 receptor antagonist (IL-1Ra) mRNA remained elevated, indicating that the protective mechanism is accompanied by upregulation of the IL-1Ra / IL-1 balance, TNF-a and IL-1. It suggests that it may be related to production suppression. These data are consistent with the predominant role of IL-10 in the endogenous inhibition of inflammatory responses and articular cartilage destruction, and the combination treatment of IL-4 and IL-10 is considered to have potential therapeutic value.

The therapeutic effects of endogenous IL-4 and IL-10 addition on cartilage destruction and joint inflammation and the role of these cytokines in the early stages of the macrophage dependent murine streptococcal cell wall (SCW) arthritis model were also investigated. Endogenous IL-10 has been shown to play a role in the regulation of SCW arthritis. The addition of exogenous IL-10 further expanded the inhibitory effect of endogenous IL-10. Even when a combination of IL-4 and IL-10 was used, even better effects were found. This combination reduced swelling and increased chondrocyte proteoglycan synthesis. Treatment with a combination of IL-4 and IL-10 not only substantially reduced the level of TNF-α as occurred for IL-10 alone treatment, but also strongly reduced the level of IL-1β in the synovial membrane and potentially It also has the added benefit of clinical benefit. Overall, these data are consistent with the role of IL-4 and IL-10 as cartilage protectors delivered systemically or locally to the joint to prevent cartilage destruction, a combination containing IL-4 and IL-10. It is indicated that this agent can provide greater potential therapeutic value than when used alone.

Severe combined immunodeficiency (SCID) mice were used as models to assess the effect of IL-4 or IL-10 injection on mobilizing mononuclear cells (MNC) and cartilage degradation into human rheumatic synovial membranes in vivo. Recombinant human IL-4 (rhIL-4, 100ng; rh IL-10, 100ng), or TNF-alpha, which is a combination of IL-4 and IL-10 in human rheumatoid synovial and cartilage from five patients with rheumatoid arthritis 1000 U), phosphate buffered saline was injected twice weekly for 4 weeks. The combination of IL-4 and IL-10 inhibited cartilage degradation and invasion by human synovial tissue, demonstrating the cartilage protective properties of these interleukin agonists.

Cloning and sequencing of human IL-13 revealed that it shares many of the properties of IL-4. IL-13 exhibits about 25% homology with IL-4. Like IL-4, IL-13 reduces the production of pro-inflammatory cytokines (including IL-1 and TNF-α) by synovial mononuclear cells. IL-13 has an anti-inflammatory effect in vivo and has therapeutic potential in treating cartilage destruction in joints.

Compounds useful as IL-4, IL-10 and IL-13 agonists include native human IL-4, IL-10 and IL-10, human recombinant IL-4 (rh IL-4), rhIL-10 and rhIL-. 13 as well as their partial sequences, or peptide sequences constructed using recombinant DNA technology to recognize IL-4, IL-10 and IL-13 receptors and capable of activating these receptors on specific cell surfaces. This includes, inter alia, multiple specifics consisting of an anti-Fc receptor moiety and an anti-IL-4, anti-IL-10 and anti-IL-13 receptor moiety (at least one part is constructed using recombinant DNA technology). Molecules are included. With regard to defining an interleukin agonist as a pharmacological agonist, the term interleukin agonist includes but is not limited to: (1) peptide sequences corresponding to naturally occurring (endogenous) generated amino acid sequences, or Fragments thereof; (2) a recombinant interleukin and its analogs that are truncated or partial sequences of full-length native interleukin amino acid sequences that possess the ability to bind homologous receptors and retain biological activity; And (3) complete attachment of oligomers (e.g., amino acids) to sequences corresponding to portions of the IgG polypeptide (e.g., IgG hinge and Fc domain) that possess the ability to bind homologous receptors and retain biological activity. A chimeric interleukin, which is a recombinant polypeptide consisting of a truncated or partial sequence corresponding to a portion of an amino acid sequence of length.

Examples of suitable interleukin agonists for the present invention are listed below. For all modes of topical delivery (ie injection, infusion and irrigation), the optimal dose and / or concentration of each suitable agent is therapeutically effective. As an example, for each of the listed formulations, a preferred and most preferred concentration of irrigation solution containing the listed formulations is provided, which concentration is expected to be therapeutically effective. Similarly, the systemic composition according to the present invention will suitably comprise a dosage or loading of the agent sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges. In the case of targeted sustained release delivery systems, formulation dosages or loadings sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges over a predetermined sustained release period are included in the compositions of the present invention.

Therapeutic and Preferred Concentrations of Interleukin Agonists compound Topical delivery therapeutic concentration (nanomol) Preferred Local Delivery Concentration (Nanomol) rh IL-4 0.5-500 5-500 rh IL-10 0.5-500 5-500 rh IL-13 0.5-500 5-500

4. Transforming Growth Factor-β Super-Series Agonists

Transforming growth factor-β (TGF-β) super-family members are 25 kD polyphase genetically functional proteins that can affect various cellular functions, which are known to be involved in tissue repair and remodeling. In many cases, this enhances the accumulation of ECM by enhancing cellular interaction with the extracellular matrix (ECM) and stimulating the production and secretion of ECM proteins and protease inhibitors. TGF-β has also been shown to exhibit synergistic interactions with other cytokines, which generally exhibit anti-inflammatory activity. Multiple isotypes of TGF-β have been identified that share intimate amino acid sequence homology. TGF-β1, TGF-β2, and TGF-β3 have been found in human tissues, and although they have different binding affinity, they are active in mammalian cells.

Members of the TGF-β super-family are potent modulators of chondrocyte proliferation, differentiation and extracellular matrix accumulation. In cartilage organ cultures, TGF-β1 regulates the metabolism of proteoglycans and stimulates collagen and glycosaminoglycan synthesis by rabbit articular chondrocytes. In addition, TGF-β1 increases TIMP expression in human articular cartilage cells and downregulates expression of the IL-1 receptor in articular cartilage.

Bone morphogenic proteins (BMPs) are multifunctional regulators of cell growth, differentiation and apoptosis, belonging to the transforming growth factor (TGF) -β super-family. More than one dozen members of the BMP family of proteins have been identified in mammals, which can be classified into several groups depending on their structure. BMP-2 and BMP-4 are highly similar to each other. BMP-5, BMP-6, osteogenic protein (OP) -1 (also referred to as BMP-7), and OP-2 / BMP-8 are structurally similar to each other. Growth-differentiation factor (GDF) -5 (also referred to as cartilage-derived morphogenic protein-1), GDF-6 (also referred to as cartilage-derived morphogenic protein-2) and GDF-7 Form a group. 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 are in vivo Induce cartilage and tendon-like structures more efficiently (Wolfman et al., 1997).

TGF-β super-family members exert their effects through binding to two types of serine / threonine receptors, both of which are essential for signal transduction (Massague, 1998). Type II receptors are constitutively active kinases that cause phosphate transfer reactions of type I receptors upon ligand binding. Type I receptors activate intracellular substrates, such as Smad proteins, through the mechanism by which the specificity of intracellular signal transduction occurs. Seven different type I receptors were isolated in mammals, initially named active receptor-like kinases (ALK) -1-ALK7. BMP type IA receptors (BMPR-IA or ALK-3) and BMP type IB receptors (BMPR-IB or ALK-6) are structurally similar to each other and specifically bind BMPs with type II receptors. ALK-2 has been shown to bind actibin, but recent data have shown that it is a type I receptor for certain BMPs (eg OP-1 / BMP-7) (Macias-Silva et al., 1998). ALK-1 is structurally highly similar to ALK-2, but its physiological ligands are still unknown. ALK-5 and ALK-4 are type I receptors for TGF-β (TβR-I) and type I receptors for actin (ActR-IB), respectively. ALK-7 is structurally similar to ALK-4 and ALK-5, but its ligand has not yet been determined.

Natural TGF-β and BMP agonists as well as synthetic or human recombination (rh) agonists suitable for use in the cartilage-protecting solution of the present invention can interact with all of the BMP receptors mentioned above. As used herein, the term “TGF-β and BMP agonist” includes fragments, deletions, adducts, amino acid substitutions, mutations thereof that retain the biological characteristics of natural human TGF-β and BMP agonist ligands. Water and modifications. TGF-β or BMP agonists may be used alone or as an anabolic cartilage agent (cartilage or promote cartilage matrix repair) and with other members of the TGF-β super-family. It can be used in combination synergistically or in combination with inhibitory agents that block cartilage catabolism.

Type I receptors act as bottom components of type II receptors. The specificity of intracellular signals by type I receptors is determined by specific regions within the serine / threonine kinase domain (named L45 loops). Thus, the L45 loop structures of BMPR-IA / ALK-3 and BMPR-IB / ALK-6 (BMPR-I group) are identical to each other and they can transduce similar signals in cells. Similarly, the L45 loops of TβR-I / ALK-5, ActR-IB / ALK-4, and ALK-7 (TβR-I group) are identical to one another and they activate similar substrates (see Chen et al. , 1998). The L45 loops of ALK-1 and ALK-2 (ALK-1 group) are the most deviating from other type I receptors, but they activate a substrate similar to that of the type I receptors of the BMPR-I group (Arms et al., 1999).

Various proteins can transduce signals from TGF-β and BMP serine / threonine kinase receptors. Of these, the best studied molecules are the Smad family of proteins. Eight different Smad proteins have been identified in mammals, which are classified into three subgroups: receptor-regulated Smads (R-Smads), common partners Smads (Co-Smads), and inhibitory Smads. R-Smads are directly activated by type I receptors and complexed into the nucleus from complexes with Co- Smads. Smad heterologs bind DNA directly and indirectly through other DNA-binding proteins to regulate transcription of target genes. Smad1, Smad5 and Smad8 are activated by BMPs, while Smad2 and Smad3 are activated by TGF-β. For example, Smad2 is translocated into the nucleus in combination with Smad4, which acts as Co-Smad, where it activates the transcription of genes that mediate the biological effects of TGF. Smad6 and Smad7 are structurally related to other Smads and act as inhibitory Smads. BMPs have been shown to induce new cartilage and bone formation in vitro and in vivo and to regulate chondrocyte growth and differentiation. In addition, these proteins may have a close effect on the process of cartilage repair. Various studies have shown that BMPs also promote and maintain chondrogenic phenotypes, which stimulate proteoglycan synthesis in rabbit and bovine articular chondrocytes as well as in chick limb bud cell cultures and fetal rat chondrocytes. It is indicated by their ability to let them. The importance of BMPs for cartilage and bone formation was demonstrated by a transgenic approach that studied specific BMP gene knockouts.

Osteogenic proteins (OP-1 or BMP-7), other members of the BMP family, are believed to be particularly important for cartilage bio homeostasis during normal and pathological conditions, such as cartilage repair. OP-1, along with cartilage-derived morphogenic proteins, is believed to be the only member of the BMP family expressed by adult articular chondrocytes (Chubinskaya, S., J. Histochemistry and Cytochemistry 48: 239-50 ( 2000)). OP-1 was first purified from the bone matrix and was found to induce cartilage and bone formation. The human OP-1 gene was cloned and produced a biologically active recombinant OP-1 homodimer. Human recombinant OP-1 can stimulate the synthesis of agrecan and collagen type II by human articular chondrocytes in vitro. It may also counteract the deleterious effects of IL-1 on the metabolism of these chondrocytes and may block cartilage damage in cattle mediated by fibronectin fragments. This effect was demonstrated by studying the effect of recombinant human OP-1 on the production of proteoglycans, prostaglandin E2, and IL-1 receptor antagonists by human articular chondrocytes cultured in the presence of interleukin-1beta. Treatment of human articular cartilage cells with OP-1 was effective to overcome the down regulation of proteoglycan synthesis induced by low doses of IL-1β. In addition, studies have shown that OP-1 stimulates the synthesis of hyaluronan and CD44, another molecule required for matrix assembly by human chondrocytes. These studies on the regulation and expression of OP-1 in human adult cartilage suggest a role for OP-1 in cartilage protection and repair, and OP-1 promotes cartilage assimilation and repair of human articular cartilage. It indicates that it can be used as a therapeutic agent.

OP-1 (BMP-7) induces cartilage and bone formation when implanted in intraskeletal and extraskeletal sites in vivo. The influence of OP-1 on the healing of penetrating articular cartilage defects was investigated by drilling two adjacent holes in the articular cartilage of the rabbit knee joint. OP-1 induced regeneration of articular surface and healing of articular cartilage containing cells resembling mature articular cartilage cells.

These data indicate that one preferred embodiment of a solution useful in the practice of the present invention for maintaining biological bio homeostasis of articular cartilage and preventing cartilage degradation in humans after surgical trauma is a member of the TGF-β super-family, preferably It suggests that it could include systemic or topical application of TGFβ2, BMP-7 (OP-1) or BMP-2, or equivalent agonists acting through the same receptors used by these ligands. Such systemic or local delivery occurs in combination with inhibitors of cartilage catabolic processes (eg, MAP kinase inhibitors, MMP inhibitors or nitric oxide synthase inhibitors) and / or drug (s), which are other agents for inhibiting pain and inflammation. Can be.

With regard to defining TGF-β and BMP agonists as pharmacological agonists, the terms TGF-β and BMP agonists include, but are not limited to: (1) naturally occurring (endogenous) amino acids Peptide sequences corresponding to the sequences or fragments thereof; (2) recombinant TGF-β and BMP, which are truncated or partial sequences of full-length native TGF-β and BMP amino acid sequences that possess the ability to bind homologous these respective receptors and retain biological activity, and their Homologues; And (3) complete attachment of oligomers (e.g., amino acids) to sequences corresponding to portions of the IgG polypeptide (e.g., IgG hinge and Fc domain) that possess the ability to bind homologous receptors and retain biological activity. Chimeric TGF-β and BMP, which are recombinant polypeptides composed of truncated or partial sequences corresponding to a portion of an amino acid sequence of length.

Examples of TGF-β and BMP agonists suitable for the present invention are listed below. For all modes of topical delivery (ie, injection, infusion and irrigation), the optimal dose and / or concentration of each suitable agent is therapeutically effective. As an example, for each of the listed formulations, a preferred and most preferred concentration of irrigation solution containing the listed formulations is provided, which concentration is expected to be therapeutically effective. Similarly, systemic compositions according to the present invention will suitably include a dosage or loading of the formulation sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges. In the case of targeted sustained release delivery systems, formulation dosages or loadings sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges over a predetermined sustained release period are included in the compositions of the present invention.

The therapeutic concentration range for local delivery or local action in surgical solutions to the joint can be estimated from the dissociation constant (Kd) value of each ligand for its homologous receptor. While these values will vary for specific cell types and tissues, the following examples for BMP-4 are presented. Binding experiments with ¹²⁵ I-BMP-4 revealed specific and high affinity binding sites with an apparent dissociation constant of 110 pM and about 6000 receptors per cell. Thus, at 11 nM BMP-4, binding of the ligand will be maximal and fully occupied (saturated) with available receptors. It has been demonstrated that functional receptors for BMP-4 are present on primary articular cartilage cells.

Therapeutic and Preferred Concentrations of TGF-β and BMP-Receptor Agonists compound Topical delivery therapeutic concentration (nanomol) Most desirable local delivery concentration (nanomol) TGF-β1 0.05-500 0.5-100 TGF-β2 0.05-500 0.5-100 BMP-2 0.1-2000 1-200 BMP-4 0.1-2000 1-200 BMP-7 (OP-1) 0.1-2000 1-200

5. Cyclooxygenase-2 (COX-2) Inhibitors

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used as anti-inflammatory agents, but have not been specifically developed or therapeutically used as cartilage protective agents. The direct molecular target for NSAID drugs is the first enzyme in the prostaglandin synthesis pathway, referred to as prostaglandin endoperoxide synthase or fatty acid cyclooxygenase. The properties of two related forms of cyclonase, cyclooxygenase-1 or type 1 (COX-1) and cyclooxygenase-2 (COX-2), were identified. These isozymes are also known as prostaglandin G / H synthase (PGHS) -1 and PGHS-2. Both enzymes catalyze the rate-limiting step in the formation of prostanoids that convert arachidonic acid to prostaglandin H2. COX-1 is present in platelets and endothelial cells and exhibits constitutive activity. In contrast, COX-2 has been identified in endothelial cells, macrophages, fibroblasts and other cells in the joints, the expression of which is induced by pro-inflammatory cytokines such as IL-1 and TNF-α.

In inflamed joints, COX-2 expression is upregulated and, at the same time as its upregulation, a significant increase in the activity of COX-2 occurs, leading to an increase in the synthesis of prostaglandins present in the synovial fluid of patients suffering from inflammatory arthrosis. Cellular sources of prostaglandins (PGs) in these joints include activated chondrocytes, type A and B synovial cells, and invasive macrophages. Cellular functions important for cartilage metabolism mediated by PGs include gene expression, extracellular matrix synthesis and proliferation. Since COX-2 is expressed in inflamed joint tissues or after exposure to inflammatory mediators (eg, as a result of injury or surgical trauma), the use of inhibitors of COX-2 prevents anti-inflammatory activity and cartilage protection. It is expected to provide both activity.

Cartilage destruction in inflammatory arthrosis can be triggered as a result of joint damage and as a result of surgical treatment with arthroscopy. Cartilage cells are the only cell type in articular cartilage and are known to participate in breaking their matrix through the release of endogenous inflammatory mediators, including PGs. Studies have shown that PG release, COX-2 gene expression and protein synthesis in normal human articular chondrocytes are rapidly induced by cytokines including IL-1, TNF-α and IL-6. Levels of mRNA are detected within a short period of time, about 2 hours after cytokine induction, reach high levels at 6 hours, and show a significantly longer expression period of 72 hours or more. Similarly, cell culture studies on IL-1α and TNF-α activation of human synovial cells showed a significant increase in COX-2 expression and prostaglandin E2 (PGE2) production. Treatment with various NSAIDS, such as ketoprofen, eliminates this induced PEG2 response. In a chondrocyte culture system, the specific COX-2 inhibitor compound NS-398 prevents increased cytokine-induced PEG2 production, while COX-1 levels remain stable (Morisset, S., 1998, J. Rheumatol. 25: 1146-53). Thus, it can be inferred that blocking PG production by activated chondrocytes, which is associated with expression of COX-2, can provide a cartilage protective effect.

NSAIDs are commonly used in the treatment of patients with osteoarthritis or rheumatoid arthritis, but their effects on articular cartilage metabolism in relation to these arthritis diseases are still a subject of debate. For example, clinical treatment of osteoarthritis and rheumatoid arthritis using NSAIDs has been successful in reducing inflammation. However, some NSAIDs, predominantly salicylate and indomethacin, which are not selective for COX-2, are thought to promote osteoarthritic cartilage destruction by damaging proteoglycan synthesis by chondrocytes, while other NSAIDS are involved in cartilage repair. It is thought to have some degree of cartilage protection effect by stimulation. Most studies have shown that NSAIDs have little or no effect on cartilage. At present, the use of this class of drugs for the treatment of traumatic joint injuries and cartilage destruction and synoviitis after surgical trauma requires the establishment of unique characteristics of each NSAID for the pathophysiological mechanisms responsible for cartilage destruction. will be.

Because the two COX isoenzymes are pharmacologically distinct, isoenzyme-specific (selective) cyclooxygenase inhibitors useful for anti-inflammatory therapy have been developed, and several of these same COX-2 inhibitors have been tested in joint inflammation models . However, the in vitro effects of COX-2 inhibitors on the synaptic production of IL-1, IL-6 and IL-8, and prostanoids, as well as the synthesis and degradation of cartilage proteoglycans, have shown that certain NSAIDs may be interleukin and eicoso Their in vivo effects on the cartilage and synovial production of the nod can vary considerably, indicating that the combined effects of these parameters can influence the results of COX-2 inhibitors on cartilage prototyping preservation. For example, some NSAIDS can promote joint damage in osteoarthritis by enhancing the production of pro-inflammatory cytokines or by inhibiting cartilage proteoglycan synthesis. However, despite possible clinical effect deviations between specific inhibitors, inhibition of C typically results in a reduction in synovitis and an expected reduced risk of cartilage damage.

Various biochemical and cellular and animal assays have been developed to assess the relative selectivity of inhibitors for COX-1 and COX-2 isotypes. In general, the criterion for defining selectivity is the ratio (or COX-2 / COX-1) of the COX-1 / COX-2 inhibition constant obtained for a given biochemical or cellular assay system. This selectivity ratio takes into account the different IC ₅₀ absolute values for inhibition of enzymatic activity obtained between microsomal assay systems and cellular assay systems (eg, platelet and macrophage cell lines stably expressing recombinant human COX isoenzymes). do. In addition, inhibition of COX-2 simulates the inhibitory effects triggered by cartilage protective (suppressive) cytokines such as IL-4, which downregulate intracellular COX-2 synthesis. Comparison of selectivity of more than 45 NSAIDs and selective COX-2 inhibitors ( Can. J. Physiol. Pharmacol 75 : 1088-95 (1997)) is a relative selection of the next rank-order for COX-2 vs. COX-1. Degrees are shown: DuP 697> SC-58451 = celecoxib> nimesulide = meloxycamp = pyroxicam = NS-398 = RS-57067>SC-57666>SC-58125>florulide> etodolak >L-745,337> DFU-T-614 (IC ₅₀ values range from 7 nM to 17 μM).

From animal studies as well as from the molecular and cellular mechanisms of action defined for selective COX-2 inhibitors, such as celecoxib and rotecoxib, these compounds have a perioperative time directly to the joint in irrigation solution or injection It is expected to show a cartilage protective effect when applied to. In particular, the COX-2 inhibitor is expected to be an effective drug delivered in irrigation solution during arthroscopically-assisted surgical treatment, or by direct injection into the joint before, during, or after surgical treatment or other damage to the joint. .

Examples of COX-2 inhibitors suitable for the present invention are listed below. For all modes of topical delivery (ie, injection, infusion and irrigation), the optimal dose and / or concentration of each suitable agent is therapeutically effective. As an example, for each of the listed formulations, a preferred and most preferred concentration of irrigation solution containing the listed formulations is provided, which concentration is expected to be therapeutically effective. Similarly, the systemic composition according to the present invention will suitably comprise a dosage or loading of the agent sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges. In the case of targeted sustained release delivery systems, formulation dosages or loadings sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges over a predetermined sustained release period are included in the compositions of the present invention.

Therapeutic and Preferred Concentrations of Cyclooxygenase-2 Inhibitors compound Topical Delivery Therapeutic Preferred Concentration (nM) Most desirable local delivery concentration (nM) Lopecoxib (MK 966) 0.3-30,000 30-3,000 SC-58451 0.3-30,000 30-3,000 Celecoxib (SC-58125) 0.3-30,000 30-3,000 Melcock Seacamp 0.5-50,000 50-5,000 Nimesulide 0.5-50,000 50-5,000 Diclofenac 0.3-30,000 30-3,000 NS-398 0.3-30,000 30-3,000 L-745,337 0.2-100,000 20-10,000 RS57067 0.2-100,000 20-10,000 SC-57666 0.2-100,000 20-10,000 Floslide 0.2-100,000 20-10,000

6. MAP Kinase Inhibitors

Cleavage factor-activated protein (MAP) kinases are a group of protein serine / threonine kinases that are activated in response to various extracellular stimuli and functions in transducing signals from the cell surface to the nucleus. The MAP kinase cascade is one of the major intracellular signaling pathways that deliver signals from growth factors, hormones and inflammatory cytokines to intermediate early genes. In combination with other signaling pathways, these activated proliferation factor-activated protein-kinases (MAPKs) differentially change the phosphorylation status and activity of transcription factors and ultimately are cellular to cell proliferation, differentiation and environmental stress. Regulate the reaction. For example, a member of the MAPK family (p38) mediates major biochemical signal transduction pathways from the potent pro-inflammatory cytokines IL-1 and TNF-α, leading to the cyclooxygenase-2 (COX-2) gene. Through the cis-acting factor associated with the regulation of transcription, COX-2 induction is induced in stimulated macrophages.

Members of the MAP kinase family of agents consist of three or more families known to differ in sequence, activation loop size, activation by extracellular stimuli, and participation in separate signal transduction pathways. Outstanding members of this family of MAP kinases include extracellular signal-regulated kinases (ERKs), ERK1 and ERK2 (p44MAPK and p42MAPK, respectively); Stress-activated protein kinase 1 (SAPK1) family, also referred to as JNK or jun N-terminal kinase family; And the p38 MAP kinase family, also known as stress-activated kinase 2/3 (SAPK-2 / 3). p38 kinase is the best pro-inflammatory cytokine that is activated by stress. Within the p38 family there are four or more distinct homologues (isotypes or isoenzymes) whose standard names are referred to as SAPK2a, SAPK2b, SAPK2d, SAPK3, or p38 α, β, δ (SAPK4) and γ, respectively. MAP kinase inhibitors useful in the practice of the present invention may interact with any one or combination of these MAP kinases. For specific MAP kinase inhibitors, the inhibition constants identified through purified in vitro enzyme assays and in cellular assays can vary over a wide range of concentrations, which may prove useful herein. Activation of p38 MAP kinase is mediated by the double phosphorylation of threonine and tyrosine residues. Treatment of cells with both a and was found to rapidly increase phosphorylation (within 5 minutes) and activate p38 MAP kinase.

Previous studies have shown that small molecule inhibitors can specifically inhibit p38 MAP kinase (Lee, J. et al., Nature 372: 739-746 (1994)), and have anti-inflammatory effects at biochemical levels and in various animal models. It has been shown to cause. The following references (Cuenda, A. et al., FEBS Lett. 364: 229 (1995)) include compound SB203580 [4- (4-fluorophenyl) -2- (4-methylsulfinylphenyl) -5- (4-pyridyl) imidazole] inhibited p38 in vitro (IC ₅₀ = 0.6 μM), inhibited MAPK activating protein kinase-2, and in vivo responded to IL-1 and cellular stress in vivo. It has been reported to prevent phosphorylation of protein (hsp) 27. Kinase selectivity of SB203580 inhibitory action against p38 has failed or has been demonstrated to exhibit the weakest inhibition of at least 15 other protein kinases in vitro, including the PKC, PKA, src and receptor tyrosine kinase families (Lee, J., Pharmacol. Ther 82: 389-397 (1999)) In cellular studies, pre-incubation with SB 203580 blocked IL-1 and TNF-α induced phosphorylation and subsequent IL-8 production of the enzyme. Turned out to be. This supports the preemptive effect of delivering these inhibitors during surgical procedures.

The role of p38 cleavage factor-activated protein kinase (MAPK) in biochemical inflammatory responses leading to cartilage destruction was studied using SB203580, which specifically inhibits the enzyme. The action of IL-1, optionally controlled by p38 MAPK, is to modulate prostaglandin H synthase-2 (COX-2), metalloproteinases, and IL-6 (see Ridley, S. et al. , J. Immunol. 158: 3165-73 (1997)) In human fibroblasts and vascular endothelial cells, SB203580 is a further known IL-1-activated protein kinase pathway (p42 / p44 MAPK, p54 MAPK / c-Jun N -Terminal kinase) inhibited IL-1-induced phosphorylation of hsp 27 (indicator of p38 MAPK activity) in fibroblasts (IC ₅₀ = 0.5 μM). In addition, SB203580 significantly inhibited IL-1-stimulated IL-6 production (30-50% at 1 μM) but did not inhibit IL-8 production from human fibroblasts and endothelial cells.

Importantly, SB203580 strongly inhibited IL-1-stimulated prostaglandin production by fibroblasts and human endothelial cells. This is associated with the inhibition of induction of COX-2 protein and mRNA. PGE ₂ results in increased expression of matrix metalloproteinases, an important mediator of cartilage degradation. Both synovial fibroblasts and chondrocytes express high levels of COX-2 gene upon activation by cytokines and extracellular stimuli. MAPK inhibitors provide cartilage protective activity due to their inhibitory action 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 joint tissues of various inflammatory or pathophysiological diseases. SB 203580 has been characterized in several in vivo pharmacological models, which has been shown to show activity under prolonged oral administration. SB203580 has been shown to inhibit stimulation of collagenase-1 and stromelysin-1 production by IL-1 without affecting the synthesis of TIMP-1. In addition, SB203580 prevented the increase of IL-1-stimulated collagenase-1 and stromelysin-1 mRNA. In the cartilage disruption model, short-term IL-1-stimulated proteoglycan uptake and proteoglycan synthesis inhibition were not affected by SB203580, while longer-term collagen disruption was prevented. In addition, SB203580 has been shown to be effective in inhibiting IL-1-induced nitric oxide production in bovine articular cartilage explants and chondrocytes (Badger, 1998). These in vitro observations provide the basis for the cartilage protective activity of MAP kinase inhibitors administered systemically or directly and locally to these tissues in the joint.

p38 MAP kinase is involved in TNF-induced cytokine expression and drugs acting as p38 MAP kinase activity inhibitors block pro-inflammatory cytokine production (see Beyaert, R. et al., EMBO J. 15 : 1914-). 23 (1996)). Treatment of the cells with TNF-α activated the p38 MAPK pathway, as indicated by increased phosphorylation of p38 MAPK itself and activation of its substrate protein. Pretreatment of cells with SB203580 completely blocked TNF-α induced activation and hsp 27 phosphorylation of MAPK activating protein kinase-2. Under the same conditions, SB203580 also completely inhibited TNF-α-induced synthesis of IL-6 and expression of reporter genes driven by a minimal promoter containing two NF-6B elements. Thus, these studies and related studies on other p38 inhibitors suggest that the action of inhibitors, such as SB203580 and FR133605, on p38 MAPKs may inhibit TNF-α- and IL-1-induced activation of various proteins associated with cartilage degradation. Suggest that they interfere selectively. Thus, selective inhibition of the MAP kinase signaling pathway of these major pro-inflammatory cytokines by kinase bottom inhibition of the receptors indicates that MAP kinase inhibitors can provide a cartilage protective effect.

SB 203580 was evaluated in several animal models of cytokine inhibition and inflammatory disease. This has proven to be a potent inhibitor of inflammatory cytokine production in vivo in both mice and rats, with IC ₅₀ values of 15-25 mg / kg. SB 203580 showed therapeutic activity in collagen-induced arthritis of DBA / LACJ mice when using a dose of 50 mg / kg resulting in significant inhibition of foot inflammation. Antiarthritis activity was also observed in adjuvant-induced arthritis in Lewis rats when orally administered SB203580 at 30 and 60 mg / kg per day. Additional evidence of the beneficial effect on bone uptake was obtained (IC ₅₀ : 0.6 μM).

In summary, various biochemical, cellular and animal studies have shown that p38 MAPK plays an important role in regulating the response to IL-1 and TNF-a, which is responsible for the mRNA levels of several inflammatory-reactive genes, such as COX-2. Indicates that it is related to regulation. p38 inhibitors have been shown to block pro-inflammatory cytokine production, inhibit MMPs production, which inhibits collagen breakdown in cartilage explants.

The use of MAPK inhibitors to block the action of major pro-inflammatory cytokines such as IL-1 and TNF-a has been shown to affect numerous cell types in the joints, including synovial fibroblasts, macrophages and chondrocytes. As it will have a beneficial effect, it will inhibit subsequent pathological effects such as inflammatory cell infiltration into the joint, synovial hyperplasia, synovial cell activation and cartilage disruption. Thus, MAPK inhibitors must block disease processes by blocking the proliferation of inflammatory responses by the aforementioned cytokines.

Examples of MAPK inhibitors suitable for the present invention are listed below. For all modes of topical delivery (ie, injection, infusion and irrigation), the optimal dose and / or concentration of each suitable agent is therapeutically effective. As one example, for each of the listed formulations, a preferred and most preferred concentration of irrigation solution containing the listed formulations is provided, which concentration is expected to be therapeutically effective. Similarly, the systemic composition according to the present invention will suitably comprise a dosage or loading of the agent sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges. In the case of targeted sustained release delivery systems, formulation dosages or loadings sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges over a predetermined sustained release period are included in the compositions of the present invention.

Therapeutic and Preferred Concentrations of MAP Kinase Inhibitors compound Topical delivery therapeutic concentration (nanomol) Local delivery preferred concentration (nanomol) SB 203580 0.5-50,000 50-5,000 SB 203580 Iodine 0.5-50,000 50-5,000 SB 202 190 0.2-20,000 20-2,000 SB 242235 0.2-10,000 20-1,000 SB 220025 0.2-10,000 20-1,000 RWJ 67657 0.3-30,000 30-3,000 RWJ 68354 0.9-90,000 90-9,000 FR 133605 1-100,000 10-10,000 L-167307 0.5-50,000 50-5,000 PD 98059 0.1-10,000 10-1,000 PD 169316 1-100,000 10-10,000

7. Inhibitors of Matrix Metalloproteinases

Articular cartilage destruction is not only a common feature for joint diseases such as osteoarthritis and rheumatoid arthritis, but also occurs after damage to the joint. Pathophysiologically, structural breakdowns of proteoglycans and collagen have been observed which damage the biomechanical properties of cartilage. Maintenance of a normal healthy extracellular matrix reflects the balance between the rate of biosynthesis and incorporation of matrix components and the rate of their degradation and subsequent loss from cartilage into the synovial fluid. Various proteases have the potential to cleave cartilage, which is involved in the degradation process, the most prominent being matrix metalloproteinases.

Matrix metalloproteinases (MMPs), or matrixins, are a specific family of at least 15 zinc endopeptidase that act extracellularly and play an important role in the pathological degradation of tissues. Current and alternate names for MMP members 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-α initiate transcription and expression. Imbalances created by upregulation and activation of tissue-degradable MMPs are the primary triggers in the process of cartilage disruption during chronic inflammatory diseases and the sustained synovial inflammatory response after joint injury. Cartilage matrix metabolism was studied in patients with anterior cruciate ligament rupture or meniscus injury in the knee. As a result, concentrations of stromelysin-1 (MMP-3), collagenase, tissue inhibitors of metalloproteinases (TIMP-1), and proteoglycan fragments have been shown to increase in human knee synovial fluid after traumatic knee injury. Temporarily, these values increased just above the baseline level and remained significantly higher (10-fold increase) over the one-year period. These changes are expected to drive increased concentrations of proteoglycan fragments observed in synovial fluid after knee ligament injury.

Matrix metalloproteinase MMP Alternative name EC number temperament MMP-1 Collagenase fibroblast collagenase interstitial collagenase EC3.4.24.7 Collagen (I, II, II, VII, and X); gelatin; Agrecan; Hyaluronidase-treated versican; Proteoglycan linking protein; Large tenasin-C; α ₁ - antitrypsin / α ₁ - proteinase inhibitor (α ₁ -AT); α ₁ antichymotrypsin (α ₁ -ACHYM); α ₂ M; Rat α ₁ M; Pregnancy region protein; Rat α ₁ I ₃ (α ₁ -inhibitor 3); Ovostatin; Entaxin; MBP; GST-TNF / TNF peptides; L-selection; IL-1β; Serum amyloid A; IGF-BP5; IGF-BP3; MMP-2; MMP-13 MMP-2 72-kDa Gelatinase Gelatinase A Type IV Collagenase Neutrophil Gelatinase EC3.4.24.24 Collagen (I, IV, V, VI, X, XI, and XIV); gelatin; Elastin; Fibronectin; Laminin-1, laminin-5; Gelatin-3; Agrecan; Decorin; Hyaluronidase-treated versican; Proteoglycan linking protein; Osteonectin; MBP; GST-TNF / TNF peptides; IL-1β; Ab _1-40 ; Ab _10-20 ; APP ₆₉₅ ; α ₁ -AT; Prolysyl oxidase fusion protein; IGF-BP5; IGF-BP3; FGF R1; MMP-1; MMP-9; MMP-13 MMP-3 Trommelin-1Transcin EC3.4.24.17 Collagen (III, IV, V, IX); gelatin; Agrecan; Versican and hyaluronidase-treated versican; Perecan; Decorin; Proteoglycan linking protein; Large tenasin-C; Fibronectin; Laminin; Entaxin; Osteonecthione; Elastin; casein; α ₁ ACHYM; Antithrombin-III; α ₂ M; Obostein; Substance P; MBP; GST-TNF / TNF peptides; IL-1β; Serum amyloid A; IGF-BP3; Fibrinogen and crosslinked fibrin; Plasminogen; MMP-1 "superactivation", MMP-2 / TIMP-2 complex; MMP-7; MMP-8; MMP-9; MMP-13

MMP-7 Matlysine PUMP EC3.4.24.23 Collagen IV and X; gelatin; Agrecan; Decorin; Proteoglycan linking protein; Fibronectin and laminin; Insoluble fibronectin fibrils; Enactin; Large and small tenasin-C; Osteonectin; β4 integrins; Elastin; casein; Transferrin; MBP; α ₁ AT; GST-TNF / TNF peptides; Plasminogen; MMP-1; MMP-2; MMP-9; MMP-9 / TIMP-1 MMP-8 Neutrophil Collagenase Collagenase EC3.4.24.34 Collagen (I, II, III, V, VII and X); gelatin; Agrecan; α ₁ AT; α ₁ -ACHYM; α ₂ antiplasmin; Fibronectin MMP-9 92 kDa Gelatinase Gelatinase B EC3.4.24.35 Collagen (IV, V, VII, X and XIV); gelatin; Elastin; Galectin-3; Agrecan; Hyaluronidase-treated versican; Proteoglycan linking protein; Fibronectin; Entaxin; Osteonectin; α ₁ AT; MBP; GST-TNF / TNF peptides; IL-1β; Ab _1-40; Plasminogen MMP-10 Stromelysin-2 EC3.4.24.22 Collagen (III, IV and V); gelatin; casein; Agrecan; Elastin; Proteoglycan linking protein; MMP-1; MMP-8 MMP-11 Stromelysin-3 EC3.4.24 Human enzyme: α ₁ AT; α ₂ M; Casein, laminin, fibronectin, gelatin, collagen IV and carboxymethylated transferrin MMP-12 Macrophage metaloelastase EC3.4.24 Collagen IV; gelatin; Elastin and κ-elastin; casein; α ₁ AT; Fibronectin; Vitronectin; Laminin; Enactin; Proteoglycan monomers; GST-TNF; MBP; Fibronectin; Fibrin; Plasminogen MMP-13 Collagenase-3 EC3.4.24 Collagen (I, II and III, IV, IX, X and XIV); Gelatin, α1-ACHYM and plasminogen activator inhibitor 2; Agrecan; Perecan; Large tenasin-C, fibronectin; Osteonectin; MMP-9 MMP-14 MT-MMP-1 EC3.4.24 Collagen (I, II and III); Gelatin, casein, κ-elastin, fibronectin, laminin, vitronectin and proteoglycans; Large tenasin-C, enactin; α ₁ AT, α ₂ M; GST-TNF; MMP-2; MMP-13 MMP-15 MT-MMP-2 Fibronectin, large tenasin-C, entaxin, laminin, agrecan, perrecan; GST-TNF; MMP-2

These MMP family enzymes have been found to be secreted from human chondrocytes and secreted by synovial cells, for example, synovial fibroblasts. In addition, the use of in situ hybridization has shown that the human synovial membrane synthesizes both stromelysin-1 and collagenase. Tromelilysin-1 (MMP-3) is able to degrade all components of the cartilage matrix. There is evidence that chondrocytes contribute to cartilage degradation by release of collagenase-3, a matrix-degrading enzyme. Upon activation by pro-inflammatory cytokines, MMPs are secreted from cells in a latent form, activated extracellularly and inhibited by tissue inhibitors of metalloproteinases (TIMPs). The balance between MMPs activity and TIMPs activity is believed to be important for maintaining the original cartilage matrix. Under pathological diseases such as osteoarthritis and rheumatoid arthritis, several studies have shown elevated amounts of MMPs, which results in an imbalance between MMPs and TIMPs, which is considered to be responsible for the observed cartilage destruction.

MMPs are regulated by cytokines such as interleukin-1 (IL-1), and growth factors acting on chondrocytes and synovial cells to enhance their protease production. Other pro-inflammatory cytokines such as IL-6, IL-8 and TNF-a also upregulate the production of matrix-degrading enzymes. This leads to cartilage destruction, which is commonly assessed as loss of sulfated glycosaminoglycans (GAGs) and cleavage of collagen. IL-1 present in the articular fluid of arthritis patients stimulates cartilage cells to synthesize synergistic amounts of enzymes such as stromelysine, fibroblast and neutrophil collagenase, and plasminogen activator. In addition, IL-1 inhibits the synthesis of plasminogen activator inhibitor-1 and TIMP, and also inhibits the synthesis of matrix components such as collagen. Due to the imbalance between the inhibitor level and the enzyme level, the amount of active protease increases and, in combination with the inhibition of matrix biosynthesis, leads to cartilage degradation.

Using cartilage slices as an in vitro model has revealed that collagenase inhibitors can inhibit IL-1 or IL-8-stimulated invasion of articular cartilage by rheumatoid synovial fibroblasts (RSF) . Collagenase inhibitors 1,10-o-phenanthroline and phosphoramidone significantly inhibited the concentration-dependent penetration of cartilage by RSF cells at concentrations of 10-150 mM. The selective effect of cytokines on proteinase secretion demonstrates that synovial fibroblast-like cell-mediated articular degradation is a highly regulated process. Thus, the ability to inhibit protease activity and the ability of matrix degradation associated locally within the joint is expected to inhibit the cartilage destruction process. Inhibitor action in a limited in vitro system suggests that therapeutic intervention using systemic or local delivery of synthetic MMP inhibitors with appropriate pharmacokinetics would be effective as cartilage protectors.

Examples of MMP inhibitors suitable for the present invention include U-24522 ((R, S) -N- [2- [2- (hydroxylamino) -2-oxoethyl] -4-methyl-1-oxopentyl] -L -Louisyl-L-phenylalaniniamide); BB2516; (N ^2- [35 [hydroxy-4- (N-hydroxyamino) -2R-isobutyl] -L-leucine-N ¹ -methylamide; also known as marimastat] peptide, for example , MMP inhibitor I and MMP-3 inhibitors, and larger proteins, such as TIMP-1 or fragments thereof, are listed in the following table: All of topical delivery (ie, injection, infusion and irrigation) In such a manner, the optimal dose and / or concentration of each suitable agent is therapeutically effective, for example, for each of the listed agents, a preferred and most preferred concentration of the irrigation solution containing the listed agents is provided. Such concentrations are expected to be therapeutically effective.Similarly, systemic compositions according to the invention are suitably formulated with a dosage of an agent sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges, or Load In the case of targeted sustained release delivery systems, formulation dosages or loadings sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges over a predetermined sustained release period are included in the compositions of the present invention. .

Therapeutic and preferred concentrations of matrix metalloproteinase (MMPs) inhibitors compound Topical delivery therapeutic concentration (nanomol) Most desirable local delivery concentration (nanomol) 1.BB2516 0.2-2000 2-200 2. GM1489 0.2-400 2-100 3.GM6001 0.4-800 2-200 4.U-24522 0.2-2,000 20-200 Minocycline 30-500,000 300-3,000 MMP inhibitor I4-Abz-Gly-Pro-D-Ala-NHOH 0.3-3,000 3-600 MMP-3 Inhibitor Ac-Arg-Cys-Gly-Val-Pro-Asp-NH ₂ 0.5-5,000 5-500 rhuman TIMP1 0.5-5,000 5-500 rhuman TIMP2 0.3-3,000 3-600

8. Inhibitors of nuclear factor kappa B (NFκB)

Pro-inflammatory and cartilage-destructive cellular pathways are regulated by extracellular and intracellular signaling mechanisms targeted for therapeutic local and systemic drug delivery. The complete molecular signaling mechanisms utilized by pro-inflammatory cytokine interleukin-1 (IL-1) to activate the transcription factor nuclear factor kappa B (NFκB) are difficult to define. Nevertheless, the major molecule involved in intracellular signaling at the gene transcription level is pro-inflammatory transcription factor (NFκB). NFκB activity is mediated by a family of transcription factor subunits that bind to homodimers or heterodimeric forms of DNA. These subunits are typically present in the cytoplasm of cells in inactive form due to the binding of inhibitory subunits called IκB. Activation of the IL-1 receptor and other extracellular signals leads to the degradation of IκB, which simultaneously releases NFκB from the inhibitor and then translocates it to the nucleus. NFκB has been shown to be involved in IL-1 induced expression, which can increase pro-inflammatory COX-2 protein expression in RA synovial fibroblasts.

Identifying NFκB as a major molecular target is based on its role as a common bottom signaling element that regulates gene expression of several crucial inflammatory mediators associated with joint inflammation and cartilage-destructive pathways. The response of many genes (COX-2, collagenase, IL-6, IL-8) is governed by a promoter containing both NFκB promoter elements. Activation of NFκB leads to the induction of many proteins critical to the inflammatory process, such as cytokines, cell-adherent molecules, metalloproteinases, and other proteins involved in the production of prostaglandins and leukotrienes (COX-2) in synovial cells. Mediate. Thus, these transcription factors represent physiologically important targets in therapies directed at damaging responses of human synovial fibroblasts, human articular chondrocytes, as well as other cells in the joint.

Specifically, when human rheumatic synovial fibroblasts (RSF) are exposed to interleukin 1 beta (IL-1 beta), 85-kD phospholipase A2 (PLA2) and inducible cyclooxygenase (COX-2) ) Can be adjusted up equally. Together these two enzymes enhance the subsequent biosynthesis of PGE ₂ , the primary inflammatory mediator in the joint. Oligonucleotide handouts and antisenses were used to demonstrate the involvement of NFκB in the regulation of prostanoid-metabolic enzymes. Antagonizing NFκB mRNA with antisense oligonucleotides reduced binding to the COX gene promoter.

The marine natural product hymenadisycin has recently been identified as an inhibitor of NFkB activation and exposure of IL-1-stimulated RSF-inhibited PEG2 production in a concentration-dependent manner (IC _{50 =} 1 μM). Specificity of this molecular target was shown by the use of the homologue aldicin and the inactive protein kinase C inhibitor RO 32-0432. The direct action of hymenadisycin on IL-1 induced NFκB activation is evidenced by a significant reduction (approximately 80%) of NFκB binding to traditional κB consensus motifs and the inhibition of stimulated p65 strains from treated cell cytosol. It became. As expected for NFκB transcriptional regulator inhibitors, hymenadisycin-treated RSFs did not transcrib mRNAs for COX-2 or PLA2 in response to IL-1. As a result, reduced protein levels and decreased PGE2 production capacity for these enzymes were observed. In addition, IL-1-stimulated interleukin-8 (IL-8) production, which is known to be an NFκB-regulated event, is also inhibited by hymenadicin, while vascularity is a non-NFκB-regulated gene. IL-1-induced production of endothelial growth factor was not affected by exposure to hymenadisycin. Thus, hymenadisycin inhibits IL-1-stimulated synovial fibroblast formation of PGE2 through its inhibitory effect on NFκB activation. This provides the basis for defining its use as a novel inhibitor that blocks the role of NFκB in joint inflammation and cartilage destruction.

Examples of NFκB inhibitors suitable for the present invention are listed below. For all modes of topical delivery (ie, injection, infusion and irrigation), the optimal dose and / or concentration of each suitable agent is therapeutically effective. As an example, for each of the listed formulations, a preferred and most preferred concentration of irrigation solution containing the listed formulations is provided, which concentration is expected to be therapeutically effective. Similarly, the systemic composition according to the present invention will suitably comprise a dosage or loading of the agent sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges. In the case of targeted sustained release delivery systems, formulation dosages or loadings sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges over a predetermined sustained release period are included in the compositions of the present invention.

Therapeutic and preferred concentrations of inhibitors of NFκB compound Topical delivery therapeutic concentration (nanomol) Most desirable local delivery concentration (nanomol) Caffeic acid phenylethyl ester (CAPE) 1-100,000 50-20,000 DM-CAPE 0.5-50,000 50-5,000 SN-50 Peptide 0.1-100,000 100-20,000 Hymenicidine 1-100,000 100-10,000 Pyrrolidonedithiocarbamate 1-50,000 50-10,000

9. Nitric Oxide Synthase Inhibitor

Nitric oxide (NO) is a wide range of intracellular and extracellular mediators involved in the pathophysiological mechanisms of some connective tissue diseases. NO is formed from L-arginine by a family of NO synthase enzymes localized intracellularly. Three isotypes of NO synthase were cloned and sequenced. Endothelial cell NO synthase (ecNOS) and brain NO synthase (bNOS) are constitutively active. Separate isotypes of NO synthase, i.e. inducible NOS (iNOS), are found in many cell types, including chondrocytes. It does not exist under basal conditions but is upregulated in response to pro-inflammatory mediators such as IL-1β and TNF-α. Recent studies show that IL-1 is a very potent stimulator of chondrocyte NO synthesis and that IL-1 acts through its ability to upregulate iNOS levels. In joints, chondrocytes are a major source of NO and chondrocytes induced by pro-inflammatory cytokines are considered to mediate many effects of IL-1 in inflammatory arthrosis.

Drugs that specifically inhibit cartilage cell-induced NO synthase (iNOS) can play a therapeutic role in preventing cartilage destruction caused by joint damage (eg, surgical intervention involving the joint). Evidence supporting this beneficial therapeutic effect has been evaluated in various iNOS inhibitors for their ability to inhibit inducible NO synthase activity in cartilage explants and cultured chondrocytes from osteoarthritis patients. Based on a significant number of studies. A specific class of compounds named S-substituted isothioureas has been identified as potent inhibitors of NO biosynthesis in cartilage. S-methyl isothiourea and S- (aminoethyl) isothiourea are 2-4 times more potent than N ^G -monomethyl-L-arginine, 5-10 times more potent than aminoguanidine, and N ^w 300 times more potent than -nitro-L-arginine and N ^w -nitro-L-arginine methyl esters. These isothiourea compounds provide a relatively specific class of iNOS inhibitors that are potent in cartilage and are therefore suitable for systemic or local delivery in accordance with aspects of the present invention (Jang, D., Eur. J. Pharmacol . 312: 341-). 347 (1996)).

In vitro systems such as isolated chondrocytes to define the effect on the cartilage matrix were also used to assess the cartilage protective therapeutic potential of NO synthase inhibitors. Inhibition of endogenous NO production by N ^G -monomethyl L-arginine (L-NMMA), an established NO synthase inhibitor, inhibits gelatinase, collagenase and stromelysin by IL-1β-stimulated chondrocytes. Causes production inhibition. Inhibition of NO production also partially lowers the increased lactate production resulting from exposure of chondrocytes to IL-1β. Treatment of cartilage fragments with L-NMMA partially reverses the IL-1β inhibitory effect of glycosaminoglycan synthesis, inhibits IL-1β-stimulated MMP activity, and inhibits IL-1 receptor antagonist (IL-1ra) Production is increased. NO can also modulate proteoglycan synthesis by indirectly lowering TGF-β1 production by chondrocytes exposed to IL-1β. This prevents an increase in autosecretory-stimulated TGF-β, thus reducing the anabolic effect of the cytokines on chondrocytes.

New aminoguanidine, S-methylisothiourea (SMT), S-aminoethylisothiourea (AETU), L-NMMA and N-nitro on the inhibitory effect of recombinant human IL-1 response on proteoglycan synthesis and NO production A study comparing the efficacy of -L-arginine methyl ester (L-NAME) NOS inhibitors was conducted. Different culture systems have been shown to respond in a concentration dependent manner to IL-1β challenge involving significant increase in NO production and significant inhibition of proteoglycan synthesis. The NOS inhibitors (1 to 1000 μM) inhibited NO production by chondrocytes treated with IL-1β and showed significant effects in restoring proteoglycan synthesis in chondrocytes. Thus, if NO production can be blocked using therapeutically effective concentrations and doses, IL-1β inhibition of proteoglycan synthesis will be prevented.

NO synthase is expressed in cartilage obtained from the joints of patients with arthritis disease. In patients with rheumatoid arthritis or osteoarthritis, increased levels of nitrite have been observed in the synovial fluid, and a substantial source of NO production in these patients has been shown to be derived from articular cartilage. In addition, sustained systemic delivery of L-NIL, a potent inhibitor of iNOS, slows the progression of experimental OA in dogs (induced by sectioning of ACL) and prevents the substantial degradation of IL-1β, PGE _2, NO and MMP production. Cause. These findings suggest that NO is a potent regulator of IL-1β effect, which contributes to the pathophysiology of joint disease.

Thus, these in vitro and in vivo results indicate that specific inhibitors of NO synthase are potential new drugs for the clinical treatment of synovial inflammation, and anti-inflammatory, cartilage protection to treat surgically treated joints or other damaged joints. And in combination with one or more drugs selected from the analgesic class, can provide a cartilage protective effect when administered systemically or topically.

Examples of NO synthase inhibitors suitable for the present invention are listed below. For all modes of topical delivery (ie, injection, infusion and irrigation), the optimal dose and / or concentration of each suitable agent is therapeutically effective. As an example, for each of the listed formulations, a preferred and most preferred concentration of irrigation solution containing the listed formulations is provided, which concentration is expected to be therapeutically effective. Similarly, the systemic composition according to the present invention will suitably comprise a dosage or loading of the agent sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges. In the case of targeted sustained release delivery systems, formulation dosages or loadings sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges over a predetermined sustained release period are included in the compositions of the present invention. In one embodiment, the preferred NO synthase inhibitor to be encapsulated in the solution of the invention is 1400 W ((N-3- (aminomethyl) benzyl) acetamidine), diphenylene, which is a selective, slow and tight binding inhibitor of iNOS. Orium and 1,3-PBIT.

Therapeutic and Preferred Concentrations of Nitric Oxide Synthase Inhibitors compound Local delivery therapeutic concentration (μM) Most Preferred Local Delivery Concentration (μM) N ^G -monomethyl-L-arginine 50-50,000 3,000 1400 W 0.1-1,000 1-20 Diphenyleneiodinium 0.1-1,000 1-100 S-methyl isothiourea 1-1,000 10-100 S- (aminoethyl) isothiourea 1-1,000 10-100 LN ^6- (1-iminoethyl) lysine 1-1,000 10-100 1,3-PBITU 0.5-500 5-100 2-ethyl-2-thioshudorea 2-20,000 20-2,000

10. Cell adhesion molecule

10a. Integrin agonists and antagonists

Integrins contain α and β subunits that bind to ligands that may be extracellular matrix (ECM) components or other large proteins such as collagen, laminin, vitronectin, osteopontin (OPN) and fibronectin (FN). Heterodimeric receptors localized on the containing plasma membrane. Degradation of the cartilage matrix is regulated by cartilage cells through a mechanism that depends on the interaction of these cells with the ECM. Chondrocyte gene expression is controlled in part through cellular contact associated with the interaction between integrin and ECM components in the environment surrounding the chondrocyte. Thus, integrins on chondrocytes are involved in controlling cartilage bio homeostasis, and this class of receptors represents a class of therapeutic targets for preventing cartilage degradation.

Human chondrocytes express an array of integrin receptors (including α ₁ β ₁ , α ₅ β ₁ , αVβ ₃ and less, etc.) composed of distinct α and β subunits. Of particular importance is the αVβ ₃ integrin, which is known to bind OPN. The αVβ ₃ complex-specific function of blocking monoclonal antibody (mAb) LM609 acts as an agonist in a manner similar to the ligand OPN. This attenuates the production of numerous pro-inflammatory and cartilage destructive mediators such as IL-1, NO and PGE2. Thus, agonistic mAb LM609 is considered suitable for use in the present invention.

In addition, two peptide-mimetics, MK-383 (Merck) and RO 4483 (Hoffmann-LaRoche), were studied in the Phase II clinic. Because they are both small molecules, they have a short half-life and high potency. However, they are believed to have less specificity and interact with other closely related integrins. These peptide- mimetics are also suitable for use in the present invention.

Therapeutic and Preferred Concentrations of Integrins Formulation Category Topical delivery therapeutic concentration (㎍ / ml ) Local delivery preferred concentration (µg / ml) Integreen αVβ3 mAb LM 609 0.05-5,000 5-500 Echistatin 0.1-10,000 100-1,000

11.Anti -Chemical Agents

Anti-chemotactic agents prevent chemotaxis of inflammatory cells. Representative examples of anti-chemotactic targets against which these agents can act include, but are not limited to, the F-Met-Leu-Phe receptor, IL-8 receptor, MCP-1 receptor, and MIP-1-I / RANTES receptor Do not. Drugs belonging to this class of formulations are early in the development phase, but it is theorized that they may be suitable for use in the present invention.

12. Intracellular Signaling Inhibitors

12a. Protein kinase inhibitors

i. Protein Kinase C (PKC) Inhibitors

Protein kinase C (PKC) plays a crucial role in cell-surface signal transduction for many physiological processes. PKC isoenzymes can be activated as the bottom target resulting from the initial activation of G-protein coupled receptors (eg serotonin, bradykinin, etc.) or pro-inflammatory cytokine receptors. Both of these receptor classes play an important role in mediating cartilage destruction.

Molecular cloning analysis revealed that PKC exists as a large family of eight or more subspecies (isozymes). These isoenzymes are substantially different in the mechanism and structure of linked receptor activation that alters the proliferative response of specific cells. Expression of specific isoenzymes is found in a wide range of cell types, including synovial cells, chondrocytes, neutrophils, myeloid cells, and smooth muscle cells. Thus, inhibitors of PKC appear to affect signaling pathways in some cell types unless these inhibitors show isoenzyme specificity. Thus, inhibitors of PKC can be expected to be effective in blocking synovial and chondrocyte activation, which may have an anti-inflammatory effect in blocking neutrophil activation and subsequent adhesion. Several inhibitors have been reported and initial reports indicate IC _{50 50} μM for calfostin C inhibitory activity. G-6203 (also known as Go 6976) is a novel and potent PKC inhibitor with high selectivity for certain PKC isotypes with IC ₅₀ values ranging from 2-10 μM. The concentrations of these drugs and GF 109203X (also known as Go 6850) or non-sindoylmaleimide I (Source: Warner-Lambert), which are believed to be suitable for topical delivery use in the present invention, are shown below. do. Similarly, the systemic composition according to the present invention will suitably comprise a dosage or loading of the agent sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges. In the case of targeted sustained release delivery systems, formulation dosages or loadings sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges over a predetermined sustained release period are included in the compositions of the present invention.

Therapeutic and Preferred Concentrations of Inhibitory Agents for Cartilage Destruction Formulation Class Topical delivery therapeutic concentration (nanomol) Local delivery preferred concentration (nanomol) Protein Kinase C Inhibitors: Calfostine C 0.5-50,000 100-5,000 GF 10903X 0.1-10,000 1-1,000 G-6203 (Go 6976) 0.1-10,000 1-1,000

ii. Protein Tyrosine Kinase Inhibitors

Although tremendous diversity exists among the numerous members of the receptor tyrosine-kinase (RTK) family, the signaling mechanisms used by these receptors share many common features. Biochemical and molecular genetic studies have shown that binding of ligands to the extracellular domain of RTK rapidly activates the intracellular domain specific tyrosine kinase catalytic activity (see FIG. 5). This increased activity results in tyrosine-specific phosphorylation of numerous intracellular substrates containing consensus sequence motifs. As a result, this leads to the activation of numerous "lower" signaling molecules and to cascades of intracellular pathways that regulate phospholipid metabolism, arachidonate metabolism, protein phosphorylation (including mechanisms other than protein kinases), calcium recruitment and transcriptional activation. Induced (see FIG. 2). Growth factor-dependent tyrosine kinase activity of the RTK cytoplasmic domain is the primary mechanism for the generation of intracellular signals leading to cellular proliferation. Thus, inhibitors have the potential to block this signaling, thereby preventing synovial cells and chondrocyte activation.

Several tyrphostin compounds in this regard all have the potential as specific inhibitors of tyrosine kinase activity (in vitro, IC ₅₀ s ranges from 0.5-1.0 μM), which has been linked to other protein kinases. This is because it has little effect on other signal transduction systems. To date, only a few of the many tyrphostin compounds are commercially available and suitable concentrations for these formulations as used in the present invention are given below. It has also been reported that staurosporin exhibits a potent inhibitory effect on several protein tyrosine kinases of the src subfamily, and the concentrations suitable for such formulations as used in the present invention for topical delivery are given below. Similarly, the systemic composition according to the present invention will suitably comprise a dosage or loading of the agent sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges. In the case of targeted sustained release delivery systems, formulation dosages or loadings sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges over a predetermined sustained release period are included in the compositions of the present invention.

Therapeutic and Preferred Concentrations of Inhibitory Agents Formulation Category Topical delivery therapeutic concentration (nanomol) Local delivery preferred concentration (nanomol) Protein kinase inhibitors Lavender stin a 10-100,000 100-10,000 Tyrfostin AG1295 10-100,000 100-10,000 Tyrfostin AG1295 10-100,000 100-10,000 Staurosporin 1-100,000 10-10,000 PD 158780 0.1-10,000 10-500 PD 174265 0.1-10,000 10-500

12b. Modulators of Intracellular Protein Tyrosine Phosphatase

Non-membrane protein tyrosine phosphatase (PTPases) containing src-homologous ₂ SH2 domains are known and are named SH-PTP1 and SH-PTP2. SH-PTP1 is also known as PTP1C, HCP or SHP. SH-PTP2 is also known as PTP1D or PTP2C. Similarly, SH-PTP1 is expressed at high levels in hematopoietic cells of all lineages and all differentiation stages, and the SH-PTP1 gene has been shown to be responsible for the mouse mouse phenotype. It provides a basis for estimating the effects of inhibitors that can block the interaction of Stimulating neutrophils with chemotactic peptides is known to result in the activation of tyrosine kinases that mediate neutrophil responses (Cui, et al., J. Immunol. (1994)), and PTPase activity is an early stage of cell stimulation. Agonist induced activity is modulated by reversing the effects of tyrosine kinases activated in the phase. Agents capable of stimulating PTPase activity may have potential therapeutic uses as anti-inflammatory mediators.

These same PTPases have also been found to modulate specific RTKs activity. These are believed to balance the activating receptor kinase effect and may therefore represent important drug targets. In vitro experiments have shown that infusion of PTPase blocks insulin stimulated phosphorylation of tyrosyl residues on endogenous proteins. Thus, activators of PTPase activity may act to reverse the activation of RTK-receptor action in relapsing stenosis, which is believed to be useful in the solution of the present invention. In addition, receptor-linked PTPases also act as extracellular ligands, similar to cell adhesion molecules. Although the functional consequences of specific ligand binding to extracellular domains have not yet been defined, it is reasonable to assume that binding will act to modulate phosphatase activity in cells (Fashena et al., Current Biology , 5 ; 1367-1369 (1995). This action can block attachment mediated by other cell surface adhesion molecules (NCAMs) and can provide an anti-inflammatory effect. However, no drug has yet been developed for these applications.

12c. Inhibitors of the SH2 domain (src homology ₂ domain)

The SH2 domain, first identified in the src subfamily of protein tyrosine kinases (PTKs), is a non-catalytic protein sequence, consisting of about 100 amino acids conserved in various signal transducing proteins (Cohen, et al. , 1995). The SH2 domain acts as a phosphotyrosine-binding module, thereby mediating protein-protein associations critical in signal transduction pathways in cells (Pawson, Nature , 573-580, 1995). In particular, the role of the SH2 domain has been clearly defined as crucial for receptor tyrosine kinase (RTK) mediated signaling as in the case of platelet-induced growth factor (PDGF) receptors. Phosphotyrosine-containing sites on autophosphorylation RTKs serve as binding sites for SH2-protein, thereby mediating the activation of biochemical signaling pathways (see FIG. 2) (Carpenter, G., FASEB J. 6 : 3283-3289). (1992); Sierke, S. et al., J. Biochem. 32: 10102-10108 (1993)). The SH2 domain is responsible for the coupling of activating growth-factor receptors to cellular responses, including changes in gene expression, and ultimately changes in cellular proliferation. Thus, inhibitors that would selectively block the activating effect of specific RTKs (except IGFR and FGFR) expressed on the surface of synovial cells are expected to be effective in blocking cartilage destruction after arthroscopic treatment.

More than 20 cytosolic proteins containing SH2 domains and acting on intracellular signaling were identified. The distribution of the SH2 domain is not limited to specific protein families, but is found in several classes of proteins, protein kinases, lipid kinases, protein phosphatase, phospholipases, Ras-controlled proteins and some transcription factors. While many SH2-containing proteins have known enzymatic activity, other (Grb2 and Crk) proteins act as "connectors" and "adapters" between cell surface receptors and "bottom" effector molecules (Marengere, L., et al., Nature 369 : 502-505 (1994)). Examples of SH2 domain containing proteins with enzymatic activity activated in signal transduction include src subtype protein tyrosine kinases (src (pp60 ^c-src ), abl, lck, fyn, fgr, etc.), phospholipase C (PLC) ), Phosphatidylinositol 3-kinase (PI-3-kinase), p21-ras GTPase activating protein (GAP), and SH2 containing protein tyrosine phosphatase (SH-PTPases), including but not limited to Songyang, et al. , Cell 72 : 767-778 (1993)). Blocks specific SH2 protein binding due to the central role these SH2-proteins play in delivering signals from the activating cell surface receptors into a cascade of additional molecular interactions, which ultimately define cellular responses. Inhibitors (such as c-src) are desired as agents that are potentially therapeutically applied to cartilage protection.

In addition, the regulation of many immune / inflammatory responses is mediated through receptors that signal through non-receptor tyrosine kinases containing the SH2 domain. T-cell activation via antigen specific T-cell receptors (TCR) initiates a signal transduction cascade, leading to lymphokine secretion and T-cell proliferation. One of the first biochemical reactions that occur after TCR activation is increased tyrosine kinase activity. In particular, neutrophil activation is partially controlled through the response of cell surface immunoglobulin G receptors. Activation of these receptors mediates the activation of unidentified tyrosine kinases known to carry the SH2 domain. Additional evidence suggests that several src-family kinases (lck, blk, fyn) participate in signal transduction pathways derived from cytokines and integrin receptors, and thus may act to integrate stimuli received from several independent receptor constructs. Instruct Thus, inhibitors of specific SH2 domains have the potential to block many neutrophil functions and serve as anti-inflammatory mediators.

At present, efforts to develop drugs targeting the SH2 domain are undertaken at the biochemical in vitro and cellular levels. If these efforts are successful, it is theorized that the resulting drug would be useful in the practice of the present invention.

V. Additives

In addition to the cartilage protective agent (s) mentioned above, the topically and systemically delivered compositions of the present invention may comprise other therapeutic agents. For example, one or more anti-inflammatory or analgesics may be included. Suitable examples of anti-inflammatory and / or analgesics are further described in detail below. As a further example, the compositions of the present invention may be used in combination with one or more disease-controlling anti-rheumatic drugs (DMARDs) such as methotrexate, sulfasalazine, gold compounds such as oral gold, gold sodium thioralate and Aurothioglucose, azathioprine, cyclosporin, antimalarial agents, steroids, colchicine, cyclophosphamide, hydroxychloroquinine sulfate, leflunomide, minocycline and penicillamine. Anti-inflammatory, analgesic and / or DMARD agents may be included in the compositions of the present invention or they may be administered simultaneously or sequentially, separately.

Many aspects of the present invention regarding the advantages of topical administration, targeted systemic administration, and the use of multiple agents in connection with cartilage protective agents, addressed above, also apply to the administration of other agents. Relieving patient pain and pain after surgery is a particularly focused area of clinical medicine, especially as the number of outpatient surgeries performed annually increases. The most widely used systemic preparations, cyclooxygenase inhibitors (eg ibuprofen) and opioids (eg morphine, fentanyl), have serious side effects including gastrointestinal irritation / bleeding and respiratory depression. The high incidence of nausea and vomiting associated with opioids is particularly problematic in the postoperative period. Therapies aimed at treating postoperative pain while avoiding adverse side effects are not readily developed because the molecular targets for these agents are widely distributed throughout the body and mediate various physiological actions. Despite the significant clinical need to inhibit cartilage degradation as well as pain and inflammation, no method has been developed to deliver effective doses of inhibitors of pain, inflammation and cartilage degradation while minimizing adverse systemic side effects. As one example, methods of administering therapeutic doses of opiates systemically (ie, intravenously, orally, subcutaneously or intramuscularly) often have significant adverse side effects, including severe respiratory depression, mood changes, mental turbidity, severe nausea and vomiting. Is associated with

Previous studies have demonstrated the ability of endogenous agents, such as serotonin (5-hydroxytrytamine; often referred to as "5-HT"), bradykinin and histamine, to cause pain and inflammation (see Sicuteri, F., et al., Serotonin-Bradykinin Potentiation in the Pain Receptors in Man, Life Sci. 4: 309-316 (1965); Rosenthal, SR, Histamine as the Chemical Mediator for Cutaneous Pain, J. Invest. Dermat .: 98-105 (1977); Richardson, BP, et al., Identification of Serotonin M Receptor Subtypes and their Specific Blockade by a New Class of Drugs, Nature 316 :, 126-131 (1985); Whalley, ET, et al., The Effect of Kinin Agonists and Antagonists, Naunyn-Schmiedeb Arch. Pharmacol. 36: 652-57 (1987); Lang, E., et al., "Chemo-Sensitivity of Fine Afferents from Rat Skin In Vitro" J. Neurophysiol .: 887-901 (1990)).

For example, 5-HT applied to human bleb substrates (epidermal skin) has been shown to cause pain that can be inhibited by 5-HT ₃ receptor antagonists (Richardson et al., (1985)). . Similarly, peripherally applied bradykinin causes pain that can be blocked by the bradykinin receptor antagonist ( Trends Neurosci. 16 ) .Histamine, which is applied peripherally, is vasodilatation that can be inhibited by histamine receptor antagonists, Causes pruritus and pain (see 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 causes vasodilation, pruritus and pain that can be inhibited by histamine receptor antagonists (Rosenthal, 1977; Douglas, WW, "Histamine and 5 Hydroxytryptamine (Serotonin) and their Antagonists", in Goodman, LS, et al., Ed., The Pharmacological Basis of Therapeutics, MacMillan Publishing Company, New York, pp. 605-638 (1985); Rumore, MM, et al., Analgesic Effects of Antihistaminics, Life Sci 36, pp. 403-416 (1985)). The combination of these three agonists (5-HT, -bradykinin, and histamine) resulted in a synergistic pain-inducing effect, resulting in long-lasting and focused pain signals (see Sicuteri et al. ., 1965; Richardson et al., 1985; Kessler, W., et al., "Excitation of Cutaneous Afferent Nerve Endings In Vitro by a Combination of Inflammatory Mediators and Conditioning Effect of Substance P," Exp. Brain Res. 91: 467-476 (1992).

In the body, 5-HT is localized in platelets and central neurons, histamine is found in mast cells, and bradykinin is produced from larger precursor molecules during tissue trauma, pH changes, and temperature changes. Since 5-HT is released in large quantities from platelets at the site of tissue damage, it can produce plasma levels that are about 20 times greater than resting levels (see Ashton, JH, et al., "Serotonin as a Mediator of Cyclic Flow Variations in Stenosed Canine Coronary Arteries, "Circulation 73: 572-578 (1986)), and endogenous 5-HT may play a role in inducing postoperative pain, hyperalgesia and inflammation. In fact, activating platelets has been shown to induce excitability of peripheral invasive receptors in vitro (Ringkamp, M., et al., "Activated Human Platelets in Plasma Excite Nociceptors in Rat Skin, In Vitro," Neurosci Lett. 170: 103-106 (1994)). Similarly, histamine and bradykinin are also released into tissue during trauma (Kimura, E., et al., "Changes in Bradykinin Level in Coronary Sinus Blood After the Experimental Occlusion of a Coronary Artery," Am Heart J. 85: 635-647 (1973); Douglas, 1985; Dray et al. (1993).

Prostaglandins are also known to cause pain and inflammation. Cyclooxygenase inhibitors (such as ibuprofen) are commonly used in non-surgical and postoperative settings to reduce prostaglandin-mediated pain and inflammation by blocking the production of prostaglandins (see Flower, RJ, et al., Analgesic-Antipyretics and Anti-Inflammatory Agents; Drugs Employed in the Treatment of Gout, in Goodman, LS, et al., Ed., The Pharmacological Basis of Therapeutics, MacMillan Publishing Company, New York, pp. 674-715 (1985) ). Cyclooxygenase inhibitors are associated with several adverse systemic side effects when administered systemically. For example, indomethacin or ketorolac exhibits widely recognized gastrointestinal and kidney side effects.

As discussed, 5-HT, histamine, bradykinin and prostaglandins cause pain and inflammation. Various receptors for which these agents mediate these effects on peripheral tissues are known and / or have been reviewed for the last two decades. Most studies have been conducted in rats or other animal models. However, there are differences in pharmacology and receptor sequences between human and animal species.

In addition, antagonists of these mediators are not currently used to treat postoperative pain. Drug classes, designated 5-HT and norepinephrine uptake antagonists (including amitriptyline), have been used orally with moderate levels of success for chronic pain diseases. However, the mechanism of chronic pain state versus acute pain state is thought to be quite different. In fact, two studies in acute pain settings using amitriptyline at the perioperative period showed no pain-relieving effect of amitriptyline (Levine, JD et al., "Desipramine Enhances Opiate Postoperative Analgesia , Pain 27: 45-49 (1986); Kerrick, JM et al., "Low-Dose Amitriptyline as an Adjunct to Opioids for Postoperative Orthopedic Pain: a Placebo-Controlled Trial Period," Pain 52: 325-30 (1993) In both studies, the drug is given orally, a second study found that oral amitriptyline actually lowers the overall well-being of the patient after surgery, which affects a number of amine receptors in the brain. May be due to drug affinity.

Amitriptyline is a potent 5-HT receptor antagonist in addition to blocking the absorption of 5-HT and norepinephrine. Thus, the lack of postoperative pain-lowering efficacy in the aforementioned studies appears to contradict the suggestion 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 at least one week until the night before surgery, while the second study (Kerrick et al., 1993) showed amitripps Tilin was used only after surgery. Thus, the levels of amitriptyline present in surgical site tissue during the actual tissue injury phase and the time at which 5-HT is released are not known. (2) Amitriptyline is known to be widely metabolized by the liver. For oral administration, the concentration of amitriptyline in the surgical site tissue may not be high enough for a long time sufficient to inhibit the activity of 5-HT released after surgery in the second study. (3) Because studies have demonstrated a large number of inflammatory mediators and demonstrate synergy between these inflammatory mediators, blocking only one agent (5-HT) may inhibit the inflammatory response to tissue damage. It cannot be suppressed enough.

There are several studies showing the ability of an extremely high concentration (1% to 3% solution-ie 10 to 30 mg / ml) of histamine ₁ (H ₁ ) receptor antagonist to act as a local anesthetic for surgical treatment. This anesthetic effect is not believed to be mediated through the H ₁ receptor, but rather may be due to non-specific interactions (similar to the action of lidocaine) with neuronal membrane sodium channels. If side effects (eg, sedation) are associated with high “anesthetic” concentrations of these histamine receptor antagonists, topical administration of histamine receptor antagonists is currently not available for setting the time around surgery.

A. Synergistic Interactions Induced from Therapeutic Combinations of Analgesic and / or Anti-inflammatory and Cartilage Protectors

Given the complexity of the disease process associated with loss of cartilage bio homeostasis and inflammation following arthroscopic treatment and the multiplicity of molecular targets involved, blockade or inhibition of single molecule targets is adequate for preventing cartilage degradation and osteoarthritis development. Will not provide. Indeed, numerous animal studies targeting different individual molecular receptors and / or enzymes have not proven effective in animal models or have shown no efficacy in clinical trials to date. Thus, therapeutic combinations of drugs that act on separate molecular targets and delivered locally or systemically appear to be desirable for clinical effectiveness in therapeutic approaches for cartilage protection. As described below, the rationale for this synergistic molecular targeting therapy is the basic biochemistry that integrates stimuli exposed to synovial cells and chondrocytes during arthroscopic treatment and delivers these synovial cells and chondrocytes in the synovial membrane and cartilage. It is a result of recent research progress in understanding the mechanism.

Molecular switches responsible for cell signaling have traditionally been divided into separate major signaling pathways, each containing a separate set of protein families that act as transducers for a particular set of extracellular stimuli, and separate cellular responses Mediate. One such pathway transduces signals from neurotransmitters and hormones through G-protein coupled receptors (GPCRs) to trigger contractile responses involving the production of inflammatory mediators such as PGE2. GPCRs couple to intracellular targets through activation of trimeric G proteins (see FIG. 2). Examples of signaling molecules involved in the activation of synovial cells and chondrocytes through the GPCR pathway are histamine, bradykinin, serotonin and ATP. The second major pathway is to transduce signals from pro-inflammatory cytokines such as IL-1 via the kinase cascade and the NF-6B protein, regulating gene expression and catabolic cytokines and other catabolic factors (NO). Inclusive).

Signals transmitted from neurotransmitters and hormones stimulate two classes of receptors: GPCRs consisting of seven-helix transmembrane regions, or ligand-gated ion channels. The "bottom" signal from these two types of receptors is focused on controlling cytoplasmic Ca ²⁺ concentrations (see Figure 3). Each GPCR transmembrane receptor activates a specific class of trimeric G proteins, including G _q , G _i , and the like. G _q subunits activate phospholipase Cγ, activate protein kinase C (PKC) and increase cytosolic calcium levels (FIG. 3). Eventually, due to intracellular calcium elevation, cPLA2 is activated and arachidonic acid (AA) is produced. This AA serves as a substrate for COX in both synovial cells and chondrocytes, causing PGE2 production. PKC activation also activates MAP kinase, leading to activation of NF-B and, in cells and tissues primed by exposure to pro-inflammatory cytokines, regulates increased gene expression of proteins involved in cartilage catabolism. .

Pro-inflammatory cytokine signaling, as mediated by both IL-1 and TNF-α through distinct homologous receptors, is also focused on regulating cellular gene expression. Although the signal transduction pathways utilized by these separate receptors use the separate kinases closest to these receptors, these signaling pathways are continuously focused on the level of MAP kinase (FIGS. 3 and 4). Signal transduction depends on the phosphorylation of residues in the cascade of kinases, including "bottom" enzymes such as p38 MAP kinase. Activation of the IL-1-receptor and TNF-receptor also leads to MAP kinase stimulation, a common step shared by Gq coupled GPCRs (see FIG. 3). It is now recognized that ligand-independent “confusion” can induce a synergistic cellular response by treating kinase pathways in response to co-stimulation of specific GPCRs and cytokines such as IL-1 (see FIG. 3). ). Thus, a combination of selective inhibitors that block common signaling pathway processing (as shown in FIGS. 1 and 2) resulting in increased gene expression of pro-inflammatory cytokines, iNOS, COX-2, and MMPs was used with arthroscopy. It will act synergistically to prevent inflammation and cartilage degradation after surgical intervention.

B. Anti-inflammatory and Analgesics

Suitable classes of anti-inflammatory and / or analgesics for use in the compositions and methods of the present invention include: (1) serotonin receptor antagonists; (2) serotonin receptor agonists; (3) histamine receptor antagonists; (4) bradykinin receptor antagonists; (5) kallikrein inhibitors; (6) tachykinin receptor antagonists (including neurokinin ₁ 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 route to arachidonic acid metabolites, including (a) phospholipase inhibitors, including PLA ₂ isotype inhibitors and PLC isotype 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 B ₄ receptor subtype antagonists and leukotriene D ₄ receptor subtype antagonists; (12) opioid agent receptor agonists including μ-opioid agents, δ-opioid agents, and κ-opioid agent receptor subtype agonists; (13) _Purinoceptor agonists and antagonists, including P _2X receptor antagonists and P _2Y receptor antagonists; And (14) calcium channel antagonists.

In the following, suitable concentrations for use in the solutions of the invention to be delivered locally, as well as suitable drugs belonging to each of the aforementioned anti-inflammatory / analgesic classes, are described. Similarly, the systemic composition according to the present invention will suitably comprise a dosage or loading of the agent sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges. In the case of targeted sustained release delivery systems, formulation dosages or loadings sufficient to bring a local concentration to a joint or site of action within the listed therapeutic ranges over a predetermined sustained release period are included in the compositions of the present invention. While not wishing to be bound by any theory, a clarification behind the selection of the various classes of formulations that are believed to render the formulations operable is also presented.

Each agent is preferably included at low concentrations of 0.1 to 10,000 times K _d or K _i , except for cyclooxygenase inhibitors, which may be required at higher concentrations depending on the particular inhibitor selected. Preferably, each agent is included at a concentration of 1.0 to 1,000 times K _d or K _i , most preferably at a concentration of approximately 100 times K _d or K _i . These concentrations are adjusted as needed to account for the dilution without metabolic transformation at the local delivery site. The exact formulation selected for use in the solutions of the present invention and the concentration of such formulations will vary depending upon the particular application, as described below.

1. Serotonin Receptor Antagonists

Serotonin (5-HT) is thought to cause pain by stimulating serotonin ₂ (5-HT ₂ ) and / or serotonin ₃ (5-HT ₃ ) receptors on peripherally invading neurons. Most researchers agree that 5-HT ₃ receptors on peripheral invasive receptors mediate the immediate pain caused by 5-HT (Richardson et al., 1985). In addition to inhibiting the induced pain, and ₃ receptor antagonists by inhibiting the infringement of receptor activation, it is also possible to inhibit neurogenic inflammation (See: Barnes PJ, et al,. "Modulation of Neurogenic Inflammation: Novel Approaches to Inflammatory Disease", Trends in Pharmacological Sciences 11: 185-189 (1990)). However, studies in rat ankle joints claim that 5-HT ₂ receptors are responsible for 5-HT activation by 5-HT (Grubb, BD, 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)). Thus, activation of the 5-HT ₂ receptor may play a role in peripheral pain and neurological inflammation.

One object of the solution of the present invention is to block pain and many inflammatory processes. Thus, both 5-HT ₂ and 5-HT ₃ receptor antagonists are suitably used individually or together in the solution of the invention, as described below. Imitriptyline (Elavil ^™ ) is a suitable 5-HT ₂ receptor antagonist for use in the present invention. Imitriptyline has been used clinically as an antidepressant for many years, and it has been found to have a beneficial effect on certain chronic pain patients. Metoclopramide (Reglan ^™ ) is used clinically as an antiemetic agent, but it exhibits moderate affinity for the 5-HT ₃ receptor and can inhibit the action of 5-HT at this receptor, thus reducing It is possible to suppress pain due to -HT release. Therefore, it is suitable for use in the present invention.

Other suitable 5-HT ₂ receptor antagonists include imipramine, trazodone, desipramine and ketanserine. Ketanserine has been used clinically for its antihypertensive effects (see Hedner, T., et al., "Effects of a New Serotonin Antagonist, Ketanserin, in Experimental and Clinical Hypertension", Am J of Hypertension 317s-). 23 s (Jul. 1988)). Other suitable 5-HT ₃ receptor antagonists include cisapride and ondansetron. Suitable serotonin _1B receptor antagonists include yohimbine, N-[-methoxy-3- (4-methyl-1-piperazinyl) phenyl] -2'-methyl-4 '-(5-methyl-1, 2, 4- Oxadiazol-3-yl) [1, 1-biphenyl] -4-carboxamide (“GR127935”) and methiotepine. Therapeutic concentrations and preferred concentrations for the topical delivery use of these drugs in solutions of certain aspects of the invention are shown in Table 14. Similarly, the systemic composition according to the present invention will suitably comprise a dosage or loading of the agent sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges. In the case of targeted sustained release delivery systems, formulation dosages or loadings sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges over a predetermined sustained release period are included in the compositions of the present invention.

Therapeutic and preferred concentrations of pain and inflammation inhibitory agents Formulation Category Topical delivery therapeutic concentration (nanomol) Local delivery preferred concentration (nanomol) Serotonin ₂ receptor antagonist Amitriptyline 0.1-1,000 50-500 MDL-11,939 0.1-1,000 50-500 AMI-193 0.1-2,000 50-500 Desipramine 0.1-1,000 50-500 Ketanserin 0.1-1,000 50-500 Serotonin ₃ receptor antagonist Trophysetron 0.01-100 0.05-50 Metoclopramide 10-10,000 200-2,000 Sisafried 0.1-1,000 20-200 Ondansetron 0.1-1,000 20-200 Serotonin _1B (human 1D _β ) antagonist Isa Maltare 0.1-1,000 50-500 GR 127935 0.1-1,000 10-500 Methiotepine 0.1-500 1-100 SB 216641 0.2-2,000 2-200

2. Serotonin Receptor Agonists

5-HT _1A , 5-HT _1B and 5-HT _1D receptors are known to inhibit adenylate cyclase activity. Therefore, the inclusion of these serotonin _1A , serotonin _1B and serotonin _1D receptor agonists in the solution of the present invention at low doses should inhibit neuronal mediated pain and inflammation. The same action is expected from serotonin _1E and serotonin _1F receptor agonists because these receptors also inhibit adenylate cyclase.

Buspyrone is a 1A receptor agonist suitable for use in the present invention. Sumatriptan is a suitable 1A, 1B, 1D and 1F receptor agonist. Suitable 1B and 1D receptor agonists are dihydroergotamine. Suitable 1E receptor agonists are ergonobins. Therapeutic and preferred concentrations of these receptor agonists when delivered locally are shown in Table 15. Similarly, the systemic composition according to the present invention will suitably comprise a dosage or loading of the agent sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges. In the case of targeted sustained release delivery systems, formulation dosages or loadings sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges over a predetermined sustained release period are included in the compositions of the present invention.

Therapeutic and preferred concentrations of pain and / or inflammation inhibition Formulation Category Topical delivery therapeutic concentration (nanomol) Local delivery preferred concentration (nanomol) Serotonin _1A agonists Buspyron 1-1,000 10-200 Sumatriptan 1-1,000 10-200 Serotonin _1B agonists Dehydroergotamine 0.1-1,000 10-100 Sumatriptan 1-1,000 10-200 Nara Triptan 1-1,000 10-200 Lisa Triptan 1-1,000 10-200 Zolmitriptan 1-1,000 10-200 L-694,247 1-1,000 10-200 Serotonin _1D agonists Dehydroergotamine 0.1-1,000 10-100 Sumatriptan 1-1,000 10-200 Nara Triptan 1-1,000 10-200 Lisa Triptan 1-1,000 10-200 Zolmitriptan 1-1,000 10-200 L-694,247 1-1,000 10-200 Serotonin _1E agonists Ergonobin 10-2,000 100-1,000 Serotonin _1F agonists Sumatriptan 1-1,000 10-200

3. Histamine Receptor Antagonists

Histamine receptors are generally divided into histamine ₁ (H ₁ ) and histamine ₂ (H ₂ ) subtypes. Traditional inflammatory responses to peripheral administration of histamine are mediated through the H ₁ receptor (Douglas, 1985). Thus, the solution of the present invention preferably comprises a histamine H ₁ receptor antagonist. Promethazine (Phenergan ^™ ) is a commonly used antiemetic agent that strongly blocks the H ₁ receptor, which is suitable for use in the present invention. Interestingly, the drug has also been found to have a local anesthetic effect, but since the concentration required for this effect is several orders of magnitude higher than the concentration required to block the H ₁ receptor, the effect is believed to be caused by a different mechanism. The histamine receptor antagonist concentration in the solution of the present invention is sufficient to inhibit the H ₁ receptor involved in nociceptor activation, but not enough to achieve the "local anesthetic" effect, thus removing concerns about systemic side effects.

Other suitable H ₁ receptor antagonists include terpenadine, diphenhydramine, amitriptyline, mepyramine and tripolydine. Since amitriptyline is also effective as a serotonin ₂ receptor antagonist, it has a dual function as used in the present invention. Suitable therapeutic concentrations and preferred concentrations for each of these H ₁ receptor antagonists for local delivery are shown in Table 16. Similarly, the systemic composition according to the present invention will suitably comprise a dosage or loading of the agent sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges. In the case of targeted sustained release delivery systems, formulation dosages or loadings sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges over a predetermined sustained release period are included in the compositions of the present invention.

Therapeutic and preferred concentrations of pain and / or anti-inflammatory agents Formulation Category Topical delivery therapeutic concentration (nanomol) Local delivery preferred concentration (nanomol) Histamine ₁ receptor antagonist Promethazine 0.1-1,000 50-200 Diphenhydramine 0.1-1,000 50-200 Amitriptyline 0.1-1,000 50-500 Terpenadine 0.1-1,000 50-500 Mepyramine (pyrilimine) 0.1-1,000 5-200 Tripolidine 0.01-100 5-20

4. Bradykinin Receptor Antagonists

Bradykinin receptors are generally divided into bradykinin ₁ (B ₁ ) and bradykinin ₂ (B ₂ ) subtypes. While acute peripheral pain and inflammation caused by bradykinin are mediated by subtype ₂ , studies have shown that bradykinin-induced pain in the setting of chronic inflammation is mediated through subtype ₁ (see Perkins). , MN, et al., "Antinociceptive Activity of the Bradykinin B1 and B2 Receptor Antagonists, des-Arg9, [Leu8] -BK and HOE 140, in Two Models of Persistent Hyperalgesia in the Rat", Pain 53: 191-97 (1993 Dray, A., et al., "Bradykinin and Inflammatory Pain", Trends Neurosci. 16: 99-104 (1993); each of which is incorporated herein by reference).

Currently, bradykinin receptor antagonists are not used clinically. Since some of these drugs are peptides, they cannot be taken orally because they can degrade. Antagonists against the B ₂ receptor block bradykinin-induced acute pain and inflammation (Dray et al., 1993). B ₁ receptor antagonists inhibit pain in chronic inflammatory diseases (Perkins et al., 1993; Dray et al., 1993). Thus, depending on the field of application, the solution of the present invention preferably comprises either or both of bradykinin B ₁ and B ₂ receptor antagonists. For example, because arthroscopy is performed for both acute and chronic diseases, the irrigation solution for arthroscopy may include both B ₁ and B ₂ receptor antagonists.

Suitable bradykinin receptor antagonists for use in the present invention include the following bradykinin ₁ receptor antagonists: [des-Arg ¹⁰ of D-Arg- (Hyp ³ -Thi ⁵ -D-Tic ⁷ -Oic ⁸ ) -BK ] Derivatives (“[des-Arg ¹⁰ ] derivatives of HOE 140”, Hoechst Pharmaceuticals); And [Leu ⁸ ] des-Arg ⁹ -BK. Suitable bradykinin ₂ receptor antagonists include [D-Phe ⁷ ] -BK; D-Arg- (Hyp ³ -Thi ^5,8- D-Phe ⁷ ) -BK ("NPC 349"); D-Arg- (Hyp ³ -D-Phe ⁷ ) -BK (“NPC 567”); And D-Arg- (Hyp ³ -Thi ⁵ -D-Tic ⁷ -Oic ⁸ ) -BK ("HOE 140"). These compounds are described in more detail in the earlier incorporated references (Perkins et al. 1993 and Dray et al. 1993). Therapeutic and preferred concentrations suitable for topical delivery are shown in Table 17. Similarly, the systemic composition according to the present invention will suitably comprise a dosage or loading of the agent sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges. In the case of targeted sustained release delivery systems, formulation dosages or loadings sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges over a predetermined sustained release period are included in the compositions of the present invention.

Therapeutic and preferred concentrations of pain and / or anti-inflammatory agents Formulation Category Topical delivery therapeutic concentration (nanomol) Local delivery preferred concentration (nanomol) Bradykinin ₁ Receptor Antagonist Leu ⁸ des-Arg ⁹ -BK 1-1,000 50-500 [Des-Arg ¹⁰ ] derivatives of HOE 140 1-1,000 50-500 [leu ⁹ ] [des-Arg ¹⁰ ] Caliden 0.1-500 10-200 Bradykinin ₂ Receptor Antagonist [D-Phe ⁷ ] -BK 100-10,000 200-5,000 NPC 349 1-1,000 50-500 NPC 567 1-1,000 50-500 HOE 140 1-1,000 50-500

5. Kallikrein Inhibitors

Peptide bradykinin, as mentioned above, is an important mediator of pain and inflammation. Bradykinin is produced as a cleavage product by the action of kallikrein on high molecular weight kininogen in plasma. Thus, kallikrein inhibitors are considered therapeutic in inhibiting bradykinin production and the pain and inflammation caused thereby. Suitable kallikrein inhibitors for use in the present invention are aprotinin. Suitable concentrations for use in the solution of the invention when delivered locally are shown in Table 18 below. Similarly, the systemic composition according to the present invention will suitably comprise a dosage or loading of the agent sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges. In the case of targeted sustained release delivery systems, formulation dosages or loadings sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges over a predetermined sustained release period are included in the compositions of the present invention.

Therapeutic and preferred concentrations of pain and / or anti-inflammatory agents Formulation Category Topical delivery therapeutic concentration (nanomol) Local delivery preferred concentration (nanomol) Kallikrein Inhibitors: Aprotinin 0.1-1,000 50-500

6. Takkinin Receptor Antagonists

Tachykinin (TKs) is a family of structurally related peptides that includes substance P, neurokinin A (NKA), and neurokinin B (NKB). Neurons are the major source of TKs in the periphery. Important common effects of TKs are neurogenic stimulation, but other effects include endothelial-dependent vasodilation, plasma protein extravasation, mast cell recruitment, and degranulation and stimulation of inflammatory cells (see Maggi, CA, Gen. Pharmacol). ., 22: 1-24 (1991)). Because of the combination of physiological actions mediated by the activation of TK receptors, targeting TK receptors is a reasonable approach to pain relief and neurological inflammation treatment.

6a. Neurokinin ₁ receptor subtype antagonist

Substance P activates neurokinin receptor subtypes referred to as NK ₁ . Substance P is an undecapeptide present at the sensory nerve terminus. Substance P is known to have multiple effects causing peripheral pain and inflammation after C-fiber activation, including vasodilation, plasma extravasation and degranulation of mast cells (Levine, JD, et al., "Peptides and the Primary Afferent Nociceptor", J. Neurosci. 13: 2273 (1993)). Suitable substance P antagonist is ([D-Pro ⁹ [spiro-gamma-lactam] Leu ¹⁰ , Trp ¹¹ ] pisalamin- (1-11)) (“GR 82334”). Other antagonists suitable for use in the present invention which act on the NK ₁ receptor are 1-imino-2- (2-methoxy-phenyl) -ethyl) -7,7-diphenyl-4-perhydroisoindoleone (3aR, 7aR) ("RP 67580"); And 2S, 3S-cis-3- (2-methoxybenzylamino) -2-benzhydrylquinuclidin (“CP 96,345”). Appropriate concentrations of these agents when delivered locally are shown in Table 19 below. Similarly, the systemic composition according to the present invention will suitably comprise a dosage or loading of the agent sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges. In the case of targeted sustained release delivery systems, formulation dosages or loadings sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges over a predetermined sustained release period are included in the compositions of the present invention.

Therapeutic and preferred concentrations of pain and / or anti-inflammatory agents Formulation Category Topical delivery therapeutic concentration (nanomol) Local delivery preferred concentration (nanomol) Neurokinin ₁ receptor subtype antagonist GR 82334 1-1,000 10-500 CP 96,345 1-10,000 100-1,000 RP 67580 0.1-1,000 100-1,000

6b. Neurokinin ₂ receptor subtype antagonist

Neurokinin A, along with substance P, is a peptide localized in sensory neurons and also promotes inflammation and pain. Neurokinin A activates a specific neurokinin receptor, referred to as ₂ (Edmonds-Alt, S., et al., "A Potent and Selective Non-Peptide Antagonist of the Neurokinin A (NK2) Receptor" , Life Sci. 50: PL101 (1992). Examples of 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") The concentrations of these agents suitable for topical delivery are shown in Table 20. Similarly, systemic compositions according to the invention are suitably adapted to bringing local concentrations to joints or sites of action within the listed therapeutic ranges. Sufficient agent dosage or loading will be included In the case of targeted sustained release delivery systems, a dosage or loading of agent sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges over a predetermined sustained release period. It is included in the composition of the present invention.

Therapeutic and preferred concentrations of pain and / or anti-inflammatory agents Formulation Category Topical delivery therapeutic concentration (nanomol) Local delivery preferred concentration (nanomol) Neurokinin ₂ Receptor Subtype Antagonists: MEN 10,627 1-1,000 10-1,000 L 659,877 10-10,000 100-10,000 (±) -SR 48968 10-10,000 100-10,000

7. CGRP Receptor Antagonists

Calcitonin gene-related peptide (CGRP) is a peptide that is localized to sensory neurons with substance P, acts as an vasodilator and enhances the action of substance P (see Brain, S., et al.,Br. J. Pharmacol. 99:202 (1985)). An example of a suitable receptor antagonist is is a truncated modification of CGRP. Such polypeptides inhibit the activation of CGRP receptors. Suitable concentrations for these formulations when delivered locally are shown in Table 21. Similarly, the systemic composition according to the present invention will suitably comprise a dosage or loading of the agent sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges. In the case of targeted sustained release delivery systems, formulation dosages or loadings sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges over a predetermined sustained release period are included in the compositions of the present invention.

Therapeutic and preferred concentrations of pain and / or anti-inflammatory agents Formulation Category Topical delivery therapeutic concentration (nanomol) Local delivery preferred concentration (nanomol) CGRP Receptor Antagonists I-CGRP- (8-37) 1-1,000 10-500

8. Interleukin Receptor Antagonists

Interleukins are a class of peptides classified as cytokines, produced by leukocytes and other cells in response to inflammatory mediators. Interleukin (IL) can be a peripherally potent nociceptive hypersensitivity agent (Feriri, SH, et al., Nature 334 : 698 (1988)). An example of a suitable IL-1b receptor antagonist is Lys-D-Pro-Thr, which is a truncated variant of IL-1b. Such tripeptides inhibit the activation of the IL-1b receptor. The concentrations of the agents suitable for topical delivery are shown in Table 22. Similarly, systemic compositions according to the present invention will suitably include a dosage or loading of the formulation sufficient to bring a local concentration at the joint or site of action within the therapeutic ranges listed. In the case of targeted sustained release delivery systems, formulation dosages or loadings sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges over a predetermined sustained release period are included in the compositions of the present invention.

Therapeutic and preferred concentrations of pain and / or anti-inflammatory agents Formulation Category Topical delivery therapeutic concentration (nanomol) Local delivery preferred concentration (nanomol) Interleukin receptor antagonists Lys-D-Pro-Thr 1-1,000 10-500

9. Inhibitors of Enzymes Active in Synthetic Pathways to Arachidonic Acid Metabolites

9a. Phospholipase inhibitors

The production of arachidonic acid by phospholipase A ₂ (PLA ₂ ) enzymes (cPLA ₂ , iPLA ₂ , sPLA ₂ ) and phospholipase C (PLC) produces a number of inflammatory mediators known as eicosanoids. A reaction cascade occurs. Since there are numerous steps that can be inhibited throughout this pathway, it is possible to lower the production of these inflammatory mediators. Examples of inhibition at these various steps are given below.

Inhibiting the enzyme PLA ₂ isoform inhibits the release of arachidonic acid from the cell membrane, thus inhibiting the production of prostaglandins and leukotrienes, thus reducing inflammation and pain. Glaser, KB, Their Pharmacological Potential ", Adv. Pharmacol. 32:31 (1995). One example of a suitable PLA ₂ isotype inhibitor is amanolide. Inhibition of the phospholipase C _g (PLC _g ) isoform will also reduce the production of prostanoids and leukotrienes, thus reducing pain and inflammation. One example of a PLC _g isotype inhibitor is 1- [6-((17-3-methoxyestra-1,3,5 (10) -trien-17-yl) amino) hexyl] -1H-pyrrole-2, 5-dione. Suitable concentrations for these formulations when delivered locally are shown in Table 23. Similarly, the systemic composition according to the present invention will suitably comprise a dosage or loading of the agent sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges. In the case of targeted sustained release delivery systems, formulation dosages or loadings sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges over a predetermined sustained release period are included in the compositions of the present invention.

Therapeutic and preferred concentrations of pain and / or anti-inflammatory agents Formulation Category Topical delivery therapeutic concentration (nanomol) Local delivery preferred concentration (nanomol) Phospholipase Inhibitors: Manoalid 100-100,000 500-10,000 Aristoloki acid 40-400,000 400-40,000 ACA 10-100,000 100-10,000 HELSS 6-6,000 60-6,000

9b. Cyclooxygenase inhibitors

Non-steroidal anti-inflammatory drugs are widely used as anti-inflammatory, antipyretic, antithrombotic and analgesics (Lewis, RA, Prostaglandins and Leukotrienes, In: Textbook of Rheumatology, 3d ed. (Kelley WN, et al., Eds .), p. 258 (1989)). Molecular targets for these drugs are type I and type II cyclooxygenases (COX-1 and COX-2). These enzymes are also known as prostaglandin H synthase (PGHS) -1 (constitutive) and -2 (inducible) and catalyze the conversion of arachidonic acid to prostaglandin H, an intermediate in prostaglandin and thromboxane biosynthesis. . These COX-2 enzymes have been identified in endothelial cells, macrophages and fibroblasts. This enzyme is induced by IL-1 and TNF-a and its expression is upregulated at the site of inflammation. Both constitutive and inducible activity of prostaglandin synthesis contribute to pain and inflammation.

Many NSAIDs currently on the market (Diclofenac, Naproxen, Indomethacin, Ibuprofen, etc.) are generally non-selective inhibitors for both isoforms of COX, but have higher selectivity for COX-1 over COX-2. These ratios vary for different compounds. The use of COX-1 and 2 inhibitors to block the formation of prostaglandins is a better therapeutic strategy than attempting to block the interaction of natural ligands with the seven subtypes of prostanoid receptors mentioned above. to be. Rarely reported as antagonists of eicosanoid receptors (EP-1, EP-2, EP-3), only high affinity specific antagonists of thromboxane A2 receptors have been reported (Wallace, J. et al. Trends in Pharm. Sci. , 15 : 405-406 (1994)).

Representative therapeutic and preferred concentrations of cyclooxygenase inhibitors for topical delivery in the solutions of the present invention are shown in Table 24. Similarly, the systemic composition according to the present invention will suitably comprise a dosage or loading of the agent sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges. In the case of targeted sustained release delivery systems, formulation dosages or loadings sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges over a predetermined sustained release period are included in the compositions of the present invention.

Therapeutic and preferred concentrations of pain and / or anti-inflammatory agents Formulation Category Topical delivery therapeutic concentration (nanomol) Local delivery preferred concentration (nanomol) Cyclooxygenase Inhibitors: Ketorolac 100-10,000 500-5,000 Indomethacin 1,000-500,000 10,000-200,000

9c. Lipooxygenase inhibitors

Inhibition of the enzyme lipooxygenase inhibits the production of leukotrienes, such as leukotriene ₄ , which are known as important mediators of inflammation and pain (Lewis, RA, Prostaglandins and Leukotrienes, In: Textbook of Rheumatology, 3d ed. (Kelley WN, et al., eds.), p. 258 (1989)). One example of a 5-lipooxygenase antagonist is 2,3,5-trimethyl-6- (12-hydroxy-5,10-dodecadiinyl) -1,4-benzoquinone ("AA 861"), Suitable local delivery concentrations are listed in Table 25. Similarly, the systemic composition according to the present invention will suitably comprise a dosage or loading of the agent sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges. In the case of targeted sustained release delivery systems, formulation dosages or loadings sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges over a predetermined sustained release period are included in the compositions of the present invention.

Therapeutic and preferred concentrations of pain and / or anti-inflammatory agents Formulation Category Topical delivery therapeutic concentration (nanomol) Local delivery preferred concentration (nanomol) Lipooxygenase Inhibitors: AA 861 100-10,000 500-5,000 Caffeic acid 500-50,000 2,000-20,000

10. Prostanoid Receptor Antagonists

Specific prostanoids produced as metabolites of arachidonic acid mediate their inflammatory effects through activation of prostanoid receptors. Examples of specific prostanoid antagonist classes are eicosanoids EP-1 and EP-4 receptor subtype antagonists and thromboxane receptor subtype antagonists. Suitable prostaglandin E ₂ receptor antagonists are 8-chlorodibenz [b, f] [1,4] oxazepine-10 (11H) -carboxylic acid, 2-acetylhydrazide ("SC 19220"). Suitable thromboxane receptor subtype antagonists [15- [1, 2 (5Z), 3, 4] -7- [3- [2- (phenylamino) -carbonyl] hydrazino] methyl] -7-oxo Bicyclo- [2,2,1] -hept-2-yl] -5-heptanoic acid ("SQ 29548"). Suitable concentrations for these formulations when delivered locally are shown in Table 26. Similarly, the systemic composition according to the present invention will suitably comprise a dosage or loading of the agent sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges. In the case of targeted sustained release delivery systems, formulation dosages or loadings sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges over a predetermined sustained release period are included in the compositions of the present invention.

Therapeutic and preferred concentrations of pain and / or anti-inflammatory agents Formulation Category Topical delivery therapeutic concentration (nanomol) Local delivery preferred concentration (nanomol) Eicosanoid EP-1 Antagonists: SC 19220 100-10,000 500-5,000

11. Leukotriene Receptor Antagonists

Leukotriene (LTB ₄ , LTC ₄ , and LTD ₄ ) is a 5-lipooxygenase pathway product of arachidonic acid metabolism, which is enzymatically generated and has important biological properties. Leukotriene has a close influence on many pathological diseases, including inflammation. Many pharmaceutical companies now seek to develop specific antagonists for potential therapeutic interventions in these pathologies (Halushka, PV, et al., Annu. Rev. Pharmacol. Toxicol. 29: 213-239 (1989) Ford-Hutchinson, A., Crit. Rev. Immunol. 10: 1-12 (1990)). LTB ₄ receptors are found in certain immune cells, including eosinophils and neutrophils. LTB ₄ binding to these receptors leads to chemotaxis and lysosomal enzyme release leading to inflammatory processes. Signal transduction processes associated with the activation of ₄ receptors include G-protein-mediated stimulation of phosphatidylinositol metabolism and intracellular calcium elevation (see FIG. 2).

One example of 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-benzopyran-2-propanoic acid ("SC 53228") Other suitable leukotriene B ₄ receptor antagonists include [3-[-2 (7-chloro-2-quinolinyl) ethenyl] phenyl] [[3- (dimethylamino-3-oxopropyl) thio] methyl] thiopropanoic acid ("MK 0571") and drug LY 66,071 and ICI 20 , 3319. MK 0571 also acts as an LTD ₄ receptor subtype antagonist The concentrations of such agents suitable for carrying out the topical delivery methods of the invention are shown in Table 27. Similarly, for systemic use according to the invention The composition will suitably comprise a dosage or loading of the agent sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges. In the case of a system, formulation dosages or loadings sufficient to bring a local concentration to a joint or site of action within the listed therapeutic ranges over a predetermined sustained release period are included in the compositions of the present invention.

Therapeutic and preferred concentrations of pain and / or anti-inflammatory agents Formulation Category Topical delivery therapeutic concentration (nanomol) Local delivery preferred concentration (nanomol) Leukotriene B ₄ Antagonist: SC 53228 100-10,000 500-5,000

12. Opiate Receptor Agonists

Activation of opioid receptors results in anti-invasive effects, so agonists for these receptors are desired. Opiate formulation receptors include μ-, δ- and κ- opioid receptor receptor subtypes. μ-receptors are localized on the peripheral sensory neuronal ends, and activation of these receptors inhibits sensory neuronal activity (Babaum, AI, et al., "Opiate analgesia: How Central is a Peripheral Target?", N Engl. J. Med. 325: 1168 (1991)). δ- and κ-receptors localize on the sympathetic nervous system distal ends and inhibit pain and inflammation by inhibiting the release of prostaglandins (see Taiwo, YO, et al., "Kappa- and Delta-Opioids Block Sympathetically Dependent"). Hyperalgesia ", J. Neurosci. 11: 928 (1991)). The opioid agent receptor subtype is a member of the G-protein coupled receptor super-family. Thus, all opioid receptor receptor agonists interact and initiate signaling through their homologous G-protein coupled receptors. Examples of suitable μ-opioid receptor agonists are fentanyl and Try-D-Ala-Gly- [N-MePhe] -NH (CH ₂ ) -OH (“DAMGO”). An example of a suitable δ-opioid receptor receptor agonist is [D-Pen ² , D-Pen ⁵ ] enkephalin (“DPDPE”). Examples of suitable κ- opioid receptor agonists include (trans) -3,4-dichloro-N-methyl-N- [2- (1-pyrrolidinyl) cyclohexyl] -benzene acetamide ("U50,488 ")to be. Concentrations suitable for topical delivery of each of these agents are shown in Table 28. Similarly, the systemic composition according to the present invention will suitably comprise a dosage or loading of the agent sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges. In the case of targeted sustained release delivery systems, formulation dosages or loadings sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges over a predetermined sustained release period are included in the compositions of the present invention.

Therapeutic and preferred concentrations of pain and / or anti-inflammatory agents Formulation Category Topical delivery therapeutic concentration (nanomol) Local delivery preferred concentration (nanomol) μ-opioid preparation agonists: DAMGO 0.1-100 0.5-20 Sufentanil 0.10-50 1-20 Fentanyl 0.1-500 10-200 PL 017 0.05-50 0.25-10 δ-opioid formulation agonists: DPDPE 0.1-500 1.01-00 κ-opioid preparation agonists: U50,488 0.1-500 1.0-100

13. Purinoceptor antagonists

Extracellular ATP acts as a signaling molecule through interaction with P ₂ purinoceptors. One major class of purinoceptors is the P _2x purinoceptor, which is a ligand-gated ion channel that has an inherent ion channel that can penetrate against Na ⁺ , K ⁺ , and Ca ²⁺ . P _2x receptors described in sensory neurons are important for primary afferent neurotransmission and uptake. ATP is known to depolarize sensory neurons, which play a role in invasive receptor activation, whereby ATP released from damaged cells stimulates P _2X receptors, leading to depolarization of nociceptive nerve-fiber ends. P2X ₃ receptors exhibit a highly limited distribution (Chen, CC, et al., Nature, Vol. 377, pp. 428-431 (1995)), which is selective in sensory C-fiber nerves that progress into the spinal cord. This is because many of these C-fibers are known to carry receptors for painful stimuli. Thus, due to the highly limited localization of expression for P2X ₃ receptor subunits, these subtypes are excellent targets for analgesic action (see FIGS. 3 and 7).

Calcium-mobilized purine receptors belonging to the G-protein receptor super-family have been reported to be present on mammalian articular chondrocyte surfaces. ATP has been shown to stimulate a dose-dependent transient rise in calcium ion concentration in differentiated primary chondrocytes. Heterologous desensitization experiments demonstrated that chondrocytes showed no subsequent response to UTP after initial stimulation with ATP. These results are consistent with the fact that P2Y receptors are present on the cell surface of chondrocytes. Purine-induced calcium mobilization in passaged chondrocytes showed the same pharmacological profile for agonist sensitivity. ATP and UTP did not alter cartilage matrix synthesis, as measured by the rate of incorporation of [35S] sulfate into glycosaminoglycans by cartilage explants or primary chondrocytes. Matrix degradation, measured by release of glycosaminoglycans from cartilage explants, was also unchanged by either agonist. The presence of a functional P2Y purine receptor on the surface of primary articular chondrocytes allows the enrichment of extracellular purines, such as ATP, to activate chondrocyte metabolism.

Other studies have defined the expression of both P1 and P2 purine receptor genes by human articular chondrocytes and profiled ligand-mediated prostaglandin E2 release. The P2Y2 receptor agonists ATP and UTP stimulated fire exit of synergistically enhanced PGE2 after pretreatment with human IL-1a. PGE2 release in response to the addition of ATP and UTP together after IL-1 pretreatment was mimicked by phorbol myristate acetate. The function of the P2Y2 receptor is to promote inflammation and pain in the joints by increasing IL-1-mediated PGE2 release. Therefore, the use of P2Y antagonists in the present invention should prevent the activation of inflammatory mediator production by both synovial cells and chondrocytes.

Antagonists of P _2X / ATP purinoceptors suitable for use in the present invention include, but are not limited to, suramin and pyridoxylphosphate-6-azophenyl-2,4-disulfonic acid ("PPADS"). Suitable concentrations for topical delivery of these agents are shown in Table 29. Similarly, the systemic composition according to the present invention will suitably comprise a dosage or loading of the agent sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges. In the case of targeted sustained release delivery systems, formulation dosages or loadings sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges over a predetermined sustained release period are included in the compositions of the present invention.

Therapeutic and preferred concentrations of pain and / or anti-inflammatory agents Formulation Category Topical delivery therapeutic concentration (nanomol) Local delivery preferred concentration (nanomol) P2X and / or P2Y antagonists: Suramin 100-100,000 10,000-100,000 PPADS 100-100,000 10,000-100,000

14. Ca ²⁺ channel antagonist

Calcium channel antagonists are a separate group of drugs that interfere with the transmembrane outflow of calcium ions required for activation of cellular responses that mediate neuroinflammation. Calcium influx into synovial cells and chondrocytes is an important event that mediates response activation in these cells. In addition, the role of bradykinin, histamine, serotonin (SHT ₂ ) and neurokinin receptors (NK ₁ and NK ₂ ) in mediating neuroinflammatory signal transduction pathways includes an increase in intracellular calcium and thus plasma Activation of calcium channels is induced on the membrane. In many tissues, calcium channel antagonists, such as nifedipine, can reduce the release of arachidonic acid, prostaglandins and leukotrienes caused by various stimuli (see Moncada, S. et al., Goodman's and Gilman's Pharmacological Basis of Therapeutics, (7th ed.), MacMillan Publ. Inc., 660-5 (1995).

Finally, calcium channel antagonists and tachykinin, histamine or bradykinin antagonists have a synergistic effect in inhibiting neuroinflammation. The role of neurokinin receptors in mediating neuroinflammatory has been established. The neurokinin ₁ (NK ₁ ) and neurokinin ₂ (NK ₂ ) receptors (members of G-protein coupled super-family) signal transduction pathways include an increase in intracellular calcium, which results in calcium channels on the plasma membrane. Activation is induced. Similarly, activation of bradykinin ₂ (BK ₂ ) receptors is associated with increased intracellular calcium in synovial cells and chondrocytes. Thus, calcium channel antagonists interfere with a common mechanism involved in raising intracellular calcium (some of which enter through L-type channels). This is the basis for synergistic interactions between antagonists and calcium channel antagonists on neurokinin, histamine, P ₂ Y and bradykinin ₂ receptors.

Calcium channel antagonists suitable for the practice of the present invention include nisoldipine, nifedipine, nimodipine, lassidipine, isradipine and amlodipine. Suitable concentrations for topical delivery of these agents are shown in Table 30. Similarly, the systemic composition according to the present invention will suitably comprise a dosage or loading of the agent sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges. In the case of targeted sustained release delivery systems, formulation dosages or loadings sufficient to bring a local concentration at the joint or site of action within the listed therapeutic ranges over a predetermined sustained release period are included in the compositions of the present invention.

Therapeutic and preferred concentrations of anticonvulsant preparations Formulation Category Topical delivery therapeutic concentration (nanomol) Local delivery preferred concentration (nanomol) Calcium Channel Antagonists: Nisoldipine 1-10,000 100-1,000 Nifedipine 1-10,000 100-5,000 Nimodipine 1-10,000 100-5,000 Lacidipine 1-10,000 100-5,000 Isradipine 1-10,000 100-5,000 Amlodipine 1-10,000 100-5,000

VI. Example

The following are several formulations in accordance with aspects of the invention that are suitable for irrigation in certain surgical procedures (Examples 1 to 3) and for systemic delivery, for example intramuscular or subcutaneous injection (Examples 4 to 11). A summary of the three clinical studies utilizing the formulations of the present invention is described below.

Example 1

Irrigation solution for arthroscopy

The following compositions are suitable for use in anatomical joint irrigation during arthroscopic treatment. Each drug is solubilized in a carrier solution containing a physiological electrolyte, such as normal saline or lactated Ringer's solution, as in the residual solution described in the following examples.

Formulation Category drug Concentration (nanomol) MAP Kinase Inhibitor SB203580 200 Matrix Metalloproteinase Inhibitors U-24522 200 TGF-β2 agonists TGF-β2 200

Example 2

Another irrigation solution for irrigation

The following composition is another formulation suitable for use in anatomical joint irrigation during arthroscopic treatment.

Formulation Category drug Concentration (nanomol) MAP Kinase Inhibitor SB203580 200 Nitric oxide synthase inhibitor L-NIL 1,000 Interleukin Receptor Agonists IL-10 100

Example # 3

Another irrigation solution

The following drugs and concentration ranges in solutions in physiological carrier fluids are suitable for use in the present invention.

Formulation Category drug Concentration (nanomol) MAP Kinase Inhibitor SB242235 200 Nitric oxide inhibitor L-NIL 10,000 TGF-β agonist TGF-β2 100

Example # 4

Cartilage protective solution for injection

The following compositions are suitable for injection into anatomical joints. Each drug is solubilized in a carrier solution containing a physiological electrolyte, such as normal saline or lactated Ringer's solution. A dose of 20 ml of this solution is suitable for administration to the patient.

Formulation Category drug Concentration (nanomol) BMP Receptor Agonists BMP-7 100 ng / ml Nitric oxide synthase inhibitor 1,3 PBIT 4.4 μg / ml NFκB agonists Pyrrolidine-dithiocarbamate 16.4 μg / ml

Example 5

Cartilage protective composition for systemic delivery

The cartilage protective composition is then suitable for systemic delivery, eg intramuscular or subcutaneous administration. Each drug is included in the composition at a concentration that will result in a concentration at the next intended site of action and is solubilized in a physiological carrier fluid or delivery system.

Formulation Category drug Concentration at the site of action (nanomol) MAP Kinase Inhibitor SB203580 200 Matrix Metalloproteinase Inhibitors U-24522 200 TGF-β agonist TGF-β2 200

Example # 6

Another cartilage protective composition for systemic delivery

The cartilage protective composition is then suitable for systemic delivery, eg intramuscular or subcutaneous administration. Each drug is included in the composition at a concentration that will result in a concentration at the next intended site of action and is solubilized in a physiological carrier fluid or delivery system.

Formulation Category drug Concentration at the site of action (nanomol) MAP Kinase Inhibitor SB 203580 200 Nitric oxide synthase inhibitor L-NIL 1,000 Interleukin Receptor Agonists IL-10 100

Example # 7

Another cartilage protective composition for systemic delivery

The cartilage protective composition is then suitable for systemic delivery, eg intramuscular or subcutaneous administration. Each drug is included in the composition at a concentration that will result in a concentration at the next intended site of action and is solubilized in a physiological carrier fluid or delivery system.

Formulation Category drug Concentration at the site of action (nanomol) MAP Kinase Inhibitor SB 242235 200 Nitric oxide synthase inhibitor L-NIL 10,000 TGF-β agonist TGF-β2 100

Example # 8

Another cartilage protective composition for systemic delivery

The cartilage protective composition is then suitable for systemic delivery, eg intramuscular or subcutaneous administration. Each drug is included in the composition at a concentration that will result in a concentration at the next intended site of action and is solubilized in a physiological carrier fluid or delivery system.

Formulation Category drug Concentration at the site of action (nanomol) Soluble TNF-α Receptor (sTNFRII: Fc) Etanerocept (Enbrel ^TM , Immunex) 250 MAP Kinase Inhibitor SB 203580 500 TGF-β agonist TGF-β2 200

Example # 9

Another cartilage protective composition for systemic delivery

The cartilage protective composition is then suitable for systemic delivery, eg intramuscular or subcutaneous administration. Each drug is included in the composition at a concentration that will result in a concentration at the next intended site of action and is solubilized in a physiological carrier fluid or delivery system.

Formulation Category drug Concentration at the site of action (nanomol) IL-1 Receptor Antagonist (IL-1Ra) Anakinra (Kineret ^TM , Amgen) 250 MAP Kinase Inhibitor SB 203580 500 TGF-β agonist TGF-β2 200

Example # 10

Another cartilage protective composition for systemic delivery

The cartilage protective composition is then suitable for systemic delivery, eg intramuscular or subcutaneous administration. Each drug is included in the composition at a concentration that will result in a concentration at the next intended site of action and is solubilized in a physiological carrier fluid or delivery system.

Formulation Category drug Concentration at the site of action (nanomol) MAP Kinase Inhibitor SB 203580 500 IGF-1 IGF-1 250 BMP agonists BMP-2 200

Example # 11

Another cartilage protective composition for systemic delivery

The cartilage protective composition is then suitable for systemic delivery, eg intramuscular or subcutaneous administration. Each drug is administered at the doses mentioned and solubilized in a physiological carrier fluid or delivery system.

Formulation Category drug Dose (mg / kg / day) Soluble TNF-α Receptor (sTNFRII: Fc) Etanerocept (Enbrel ^TM , Immunex) 0.5-1.0 MAP Kinase Inhibitor SB 203580 30-60 TGF-β agonist TGF-β2 0.1-10

Example # 12

Another cartilage protective composition for systemic delivery

The cartilage protective composition is then suitable for systemic delivery, eg intramuscular or subcutaneous administration. Each drug is administered at the doses mentioned and solubilized in a physiological carrier fluid or delivery system.

Formulation Category drug Dose (mg / kg / day) IL-1 Receptor Antagonist (IL-1Ra) Anakinra (Kineret ^TM , Amgen) 2.0 MAP Kinase Inhibitor SB 203580 30-60 TGF-β agonist TGF-β2 0.1-10

Example 13

Targeted Cartilage Protective Drug Delivery System

The following cartilage protective compositions are suitable for systemic delivery, eg, intravenous, intramuscular, subcutaneous or inhaled administration. The drug is encapsulated within DL-lactide / glycolide copolymer (PLGA) nanospheres, coupled with anti-human type II collagen monoclonal antibodies. The antibody targets epitopes on human type II collagen in articular cartilage. Each drug is included in the composition at a concentration sufficient to yield a mean concentration at the next intended site of action upon degradation of the nanospheres and release of the agent during the sustained release period.

Formulation Category Concentration at the site of action (nanomol) Growth factor IGF-1 250 nM MAP Kinase Inhibitor SB220025 1000 nM MMP inhibitor BB2516 (marimar start) 200 nM

Example # 14

Targeted Cartilage Protective Drug Delivery System

The following cartilage protective compositions are suitable for systemic delivery, eg, intravenous, intramuscular, subcutaneous or inhaled administration. The drug is encapsulated in biodegradable PLA / PLGA copolymeric nanospheres coupled with anti-human agrecan monoclonal antibodies. The antibody targets neoepitopes on human aggrecan in articular cartilage. Each drug is included in the composition at a concentration sufficient to bring the concentration at the next intended site of action upon degradation of the nanospheres and release of the agent during the sustained release period.

Formulation Category Concentration at the site of action (nanomol) Growth factor IGF-1 250 nM MAP Kinase Inhibitor SB220025 1000 nM MMP inhibitor BB2516 (Marima start) 200 nM

Example 15

Targeted Cartilage Protective Drug Delivery System

The following cartilage protective compositions are suitable for systemic delivery, eg, intravenous, intramuscular, subcutaneous or inhaled administration. The drug is encapsulated in chitosan / gelatin nanospheres, coupled with anti-human type II collagen monoclonal antibodies. The antibody targets neoepitopes on human type II collagen. Each drug is included in the composition at a concentration sufficient to bring the concentration at the next intended site of action upon degradation of the nanospheres and release of the agent during the sustained release period.

Formulation Category Concentration at the site of action (nanomol) BMP Receptor Agonists BMP-7 200 MAP Kinase Inhibitor SB203580 500

Example 16

Targeted Cartilage Protective Drug Delivery System

The following cartilage protective compositions are suitable for systemic delivery, eg, intravenous, intramuscular, subcutaneous or inhaled administration. The drug is encapsulated in albumin nanospheres, coupled with anti-human type II collagen monoclonal antibodies. The antibody targets neoepitopes on human type II collagen. Each drug is included in the composition at a concentration sufficient to bring the concentration at the next intended site of action upon degradation of the nanospheres and release of the agent during the sustained release period.

Formulation Category Concentration at the site of action (nanomol) BMP Receptor Agonists BMP-7 200 MMP inhibitor BB2516 200

Example # 17

Targeted Cartilage Protective Drug Delivery System

The following cartilage protective compositions are suitable for systemic delivery, eg, intravenous, intramuscular, subcutaneous or inhaled administration. The drug is encapsulated within poly (lactide-co-glycolide) / poly (ethyleneglycol) copolymer nanospheres, coupled with anti-human type II collagen monoclonal antibodies. The antibody targets neoepitopes on human type II collagen in articular cartilage. Each drug is included in the composition at a concentration sufficient to bring the concentration at the next intended site of action upon degradation of the nanospheres and release of the agent during the sustained release period.

Formulation Category Concentration at the site of action (nanomol) BMP Receptor Agonists BMP-7 200 MAP Kinase Inhibitor SB203580 500

Example 18

Targeted Cartilage Protective Drug Delivery System

The following cartilage protective compositions are suitable for systemic delivery, eg, intravenous, intramuscular, subcutaneous or inhaled administration. BMP-7, a BMP receptor agonist, is encapsulated within poly (lactide-co-glycolide) (PLGA) nanospheres, coupled with anti-human type II collagen monoclonal antibodies. IGF-1, an IGF receptor agonist, is encapsulated separately in chondroitin-6-sulfate / gelatin nanospheres coupled with anti-human agrecan monoclonal antibodies. Antibodies used to target each type of nanosphere bind to neoepitopes on human type II collagen and neoepitopes on agrecan in articular cartilage. Each drug is included in the composition at a concentration sufficient to yield a mean concentration at the next intended site of action upon degradation of the nanospheres and release of the agent during the sustained release period.

Formulation Category Concentration at the site of action (nanomol) BMP Receptor Agonists BMP-7 200 IGF Receptor Agonists IGF-1 500

Example # 19

Targeted Cartilage Protective Drug Delivery System

The following cartilage protective compositions are suitable for systemic delivery, eg, intravenous, intramuscular, subcutaneous or inhaled administration. The drug is encapsulated in albumin nanospheres, coupled with an anti-human F (ab ') ₂ fragment that binds a type II collagen monoclonal antibody. Such F (ab ') ₂ antibodies target neoepitopes on human type II collagen. Each drug is included in the composition at a concentration sufficient to bring the concentration at the next intended site of action upon degradation of the nanospheres and release of the agent during the sustained release period.

Formulation Category Concentration at the site of action (nanomol) BMP Receptor Agonists BMP-7 200 MMP inhibitor BB2516 200

Example 20

Targeted Cartilage Protective Drug Delivery System

The following cartilage protective compositions are suitable for systemic delivery, eg, intravenous, intramuscular, subcutaneous or inhaled administration. The drug is encapsulated within albumin nanospheres, coupled with an anti-human single chain minimal binding domain (scFV) of an immunoglobulin molecule that binds type II collagen monoclonal antibodies. Such scFV antibodies target neoepitopes on human type II collagen. Each drug is included in the composition at a concentration sufficient to bring the concentration at the next intended site of action upon degradation of the nanospheres and release of the agent during the sustained release period.

Formulation Category Concentration at the site of action (nanomol) BMP Receptor Agonists BMP-7 200 MMP inhibitor BB2516 200

Study 1

Synergistic Stimulation of Rapid PGE2 Explosion Upon Exposure to IL-1 and GPCR Agonists.

Fibroblast-like synovial cells characterize inflammatory cells and are believed to be determinants of joint inflammation and cartilage degradation. A synovial cell culture model system was used to characterize synergistic interactions between IL-1 and non-cytokine inflammatory mediators, which are important in coordinating joint tissue destruction, including damage that occurs as a result of tissue damage during arthroscopic surgery. . Investigate G-protein coupled receptor (GPCR) agonists (histamine, bradykinin and isoproteronol) for prostanoid production and cytokine regulation in cultured human synovial fibroblasts and in these systems An experiment was conducted to confirm the activity of ketoprofen. The kinetics in which prostaglandin E2 (PGE ₂ ), interleukin-6 (IL-6) and interleukin-8 (IL-8) are induced in response to stimulation with interleukin-1 (IL-1) are described. The ability of GPCR ligands to enhance cytokine production after IL-1 priming was studied.

In studies 1 to 3, the following experimental methods and materials were used unless otherwise stated.

Cell culture. Acquire synovial tissue from patients with osteoarthritis undergoing joint replacement surgery through the MacNeal Hospital's Clinical Research Center, which has penicillin (100 units / ml), streptomycin (100 μg / ml), and punzione Transported to the laboratory in Dulbecco's Modified Eagle's Medium (DMEM) containing (0.25 μg / ml). The synovial membrane was excised and chopped and plated as explants in culture media consisting of L-glutamine (2 mM), heat inactivated fetal bovine serum (10% v / v), and DMEM containing antibiotics. Cultures were incubated at 37 ^° C. under a hygroscopic atmosphere of 5% CO ₂ . Sticky synovial cells grew from the explanted tissue within 2 to 3 weeks and were passaged by trypsinization. Seed cultures were fed twice weekly and passaged densely. Experiments were performed on cells from passage 3-8. Experimental cultures were plated in 35 mm dishes at a density of 7.5 × 10 ³ cells / cm ² in 2 ml culture medium. The culture grew almost close to the experimental density, which contained 2.3 + 0.3 X 10 ⁵ cells (mean + SEM, n = 3), and 104 + 13 μg protein (n = 10). The growth medium is replaced twice a week.

2. Experimental treatment. On the day before initiating the experimental treatment, the medium is changed to an experimental growth medium consisting of DMEM containing 2% heat inactivated fetal bovine serum, L-glutamine and antibiotics as described above, bringing the cells to sleep. The following day, the cultures were primed by adding a specified concentration of IL-1 or additional ligand to the conditioned growth medium at 12-24 hour intervals as indicated. In some experiments, conditioned growth medium was collected for analysis after priming with IL-1. After this priming interval, acute experimental treatment was performed as follows. The cultures were withdrawn from the incubator, and 2 ml aliquots of rocker physiological buffer (LB composition (mM): NaCl, 154; KCl, 2.6; KH ₂ PO ₄ , 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), followed by an additional aliquot containing the specified ligand for 10 minutes on a 37 ° C. bath. Equilibrate with the amount of LB. This solution is removed by aspiration and replaced with a fresh buffer aliquot containing the indicated ligands at the specified time intervals at 37 ° C. When pharmacological inhibitors were typically added during the 10 minute preincubation period, specific inhibitors and agonists were present during the 3 minute challenge period.

3. Determination of prostaglandin E2. After performing the indicated treatment protocol, an aliquot of the culture supernatant (1 ml) is collected and quickly frozen in liquid nitrogen. Samples were stored at −80 ° C. until processing. Aliquots of culture supernatants were analyzed by competitive binding radioimmunoassay as specified by the manufacturer (Sigma Chemical Co.) using antibodies that showed equivalent reactivity to prostaglandins E2 and E1. For quantification, standard curves were prepared for each assay using fixed concentrations of [ ³ H] prostaglandin E2 and increasing concentrations of the original competitive prostaglandin E2.

4. Measurement of IL-6. The production of cytokine IL-6 was also measured in aliquots of supernatant culture medium frozen at -80 ° C. IL-6 was measured by sandwich ELISA using alkaline phosphatase detection as described by the manufacturer (Pharmingen) and quantified using standard curves prepared with each pure recombinant human cytokine. Experimental determinations were made under double culture.

Study 2

[ ³ H] Thymidine incorporation and assay for MTT

Synovial cell lines were routinely assessed for their ability to proliferate in response to IL-1, which was measured as incorporation of [ ³ H] thymidine (Kimball & Fisher, 1988). In such formulations, the maximum effective concentration of IL-1 stimulated [ ³ H] thymidine incorporation by 10 to 20 times more compared to dormant cultures maintained in 2% serum (data not shown).

1. Data Analysis. Immunoassays were routinely performed in double aliquots from each culture. Experimental determinations were made on double or triple cultures. Each demonstration was repeated in at least two cell lines. Using Graph-PAD Prism software (San Diego, CA), non-linear regression curve fixation and statistical analysis were performed.

2. Material. Cell Culture: Cell culture medium was obtained from a source (Sigma or Gibco / BRL). Fetal bovine serum was obtained from a source (; Drug: Recombinant human interleukin-1 was obtained from a source (Genzyme; Cambridge, MA). Ketoprofen was provided by the source (; Amitriptyline, Forskolin, 5 -Hydroxytryptamine, isoproterenol, bradykinin, histamine and prostaglandin E2 were obtained from the source (Sigma) Radiochemical: [ ³ H] prostaglandin E2 was obtained from the source (American Radiolabeled Chemicals, Inc .; Louis, MO) All other formulations were obtained in the highest purity available from standard suppliers.

Common pharmacological effects through these different classes of receptors, measured by the effect of GPCR agonists, histamine and bradykinin on PGE2 production in human synovial cells, with or without prior stimulation for IL-1 The functional interactions between agonists that mediate them were evaluated. Exposure of cultured human synovial fibroblasts to IL-1 (10 U / ml) overnight significantly enhanced sustained (4 hours) PGE2 production, which was significantly enhanced by immunoassay with increased PGE2 in the culture supernatant. Can be measured. The gradual increase in PGE2 production during prolonged IL-1 treatment (16-24 hours) was found to result from equivalent upregulated expression of cPLA2 and COX-2 (Crofford, 1984, Hulkower et al., 1984). Cultures primed by overnight exposure to IL-1 produce PGE2 additionally quickly (in minutes), with subsequent challenge to maximum effective concentrations of histamine (100 μM) or bradykinin (1 μM) Reacts to Representative data on the time course of PGE2 production in response to histamine or bradykin stimulation are shown in FIG. 7. Under these conditions, histamine increases PGE2 production by 5-10 fold as compared to IL-1 primed cells with no GPCR agonist added. Bradykinin is increased 10 to 15 times. The absolute amount of PGE2 produced during the brief agonist challenge for 2 minutes is close to or exceeds the amount produced cumulatively over a total of 18 hours of IL-1 priming time. This is surprising as long as FIG. 7 shows that the majority of histamine-induced PGE2 production explosions occur within the initial 2 minutes because minimal additional accumulation is observed over the next 60 minutes. Bradykinin-stimulated PGE2 responses continue to increase (twice) over the same time period. In the absence of IL-1 priming, synovial cells that did not undergo specific experiments showed no detectable PGE2 production in response to stimulation with GPCR agonists alone. Under IL-1 priming conditions, both histamine and bradykinin synergistically enhance release.

Using synovial fibroblasts cultured from osteoarthritis patients, we found a time-dependent synergistic interaction between pro-inflammatory cytokine IL-1 and G-protein coupled receptors on physiologically related PGE2 production. And the action of the targeted treatment was evaluated. GPCR agonists acting through endogenous synovial cell receptors, inositol phosphate and PKC signaling pathways associated with increased intracellular calcium, amplify PGE2 production rapidly and rapidly in cells pre-primed by IL-1. COX inhibitors effectively attenuate both the agonist-induced rapid PGE2 explosion and long term accumulation of PGE2. Thus, different and pathways for intracellular signal transduction interact synergistically with long-term control of the response either faster or slower.

The synergy between calcium-regulated GPCRs and IL-1 in synovial cells causing a rapid PGE2 explosion can be partly explained by the rapid increase in arachidonic acid release, which is a measure of cPLA2 activation in many cell types. In addition to inducing COX-2 expression, IL-1 increases the expression of cPLA2 (Hulkower et al., 1994). These two proteins work together to provide a free arachidonic acid substrate for COX-2. Thus, upregulation of important eicosonoid metabolizing enzymes induced by IL-1, in combination with the ability of GPCR ligands to activate arachidonate release, is expected to increase overall substrate aberration through prostanoid synthesis. can do. cPLA2 is the only PLA2 known to exhibit functional properties that direct receptor regulation, which is expected to be involved in eicosonoid production and intracellular signaling. cPLA2 is activated by increasing calcium concentration for full activity, and because bradykinin B2 and histamine H1 receptor activation is linked to intracellular calcium mobilization, it is an excellent factor in controlling rapid agonist-stimulated explosions in PGE2 production. It is estimated. Finally, the extremely rapid and transient increase in cytoplasmic calcium triggered by B2 or H1 receptor activation is similar to the known kinetics for cPLA2 activation, arachidonic acid release, and the observed PGE2 explosion.

Study 3

Inhibition of PGE2 Explosion Formation by Cyclooxygenase Inhibitor

The action of the cyclooxygenase inhibitor ketoprofen, which attenuates PGE2 formation, is determined by co-incubation with IL-1 for prolonged exposure (16 hours), as shown in FIG. 8; Determination by simple pre-incubation prior to subsequent GPCR agonist challenge intervals. Adding specific concentrations of ketoprofen during overnight priming with IL-1 eliminates PGE2 formation, with IC ₅₀ = 4.5 + 0.8 nM as determined by non-linear regression analysis (mean + SEM, n = 4 synovial cell lines). Indicates. Using cyclooxygenase inhibitor etodolak (IC ₅₀ = 15.2 + 4.6 nM, n = 4), ketorolac (2.2 + 0.4 nM, n = 4), and indomethacin (3.2 + 1.5 nM, n = 2) Similar decisions were made (data not shown).

8 also shows 100 μM histamine (IC ₅₀ = 3.4 + 0.2 nM, n = 3) or 1 μM bradykinin (IC ₅₀ = 9.5 + 2.0) in synovial cells primed overnight with IL-1 (10 U / ml) nM, n = 3) depicts ketoprofen concentration-dependent inhibition of agonist-induced PGE2 explosion in response to challenge. These values are comparable to those observed for ketoprofen during overnight IL-1 induction of PGE2. These results indicate that initiation of inhibition by COX inhibitors occurs within a 10 minute pretreatment interval prior to GPCR agonist addition, which is consistent with direct reversible inhibition of COX activity and is linked to changes in the expression level of prostanoid regulatory enzymes. Prove that it is not due. This immediate inhibitory effect also provides a basis for the immediate efficacy of the drug when delivered locally into the joint in irrigation solution during arthroscopic surgery.

Study 4

Induction of IL-6 production by IL-1 and GPCR agonists and inhibition by ketoprofen.

The kinetics that interleukin-6 is induced in response to stimulation with IL-1 are described. Synovial cell cultures are directed to histamine for activating signaling through the inositol triphosphate (InsP3) / protein kinase C pathway, or to isoproterenol-added IL-1 for activating an increase in intracellular cAMP. To the treated process. Production of PGE ₂ , IL-6, and IL-8 was measured in culture supernatants after 1, 2, 4, 6, and 24 hours treatment. In this experiment, each treatment interval was performed in a separate culture. In this treatment regimen, production of IL-6 was significantly increased by IL-1 after 24 hours of exposure, but no IL-6 was detected within the initial 6 hour interval. IL-6 production in response to IL-1 was not further increased by histamine addition, and histamine alone did not stimulate IL-6 production. IL-1 also resulted in a significant elevation of IL-8 (2000 pg / ml), which could be measured for the first time at 6 hours of treatment. IL-8 production increased significantly over 24 hours of exposure to IL-1.

The effect of ketoprofen on the induction of cytokine production by IL-1 and GPCR agonists was examined. The protocol also tested the effect of IL-1 concentration dependent on induction of IL-6 stationary state. Synovial cell cultures are exposed to the indicated concentrations of IL-1 and GPCR agonists. The culture supernatant is collected and replaced with fresh medium aliquots containing the same agonist adduct at 8 hour intervals. PGE ₂ , IL-6 and IL-8 in this supernatant were assayed as described.

Data for IL-6 production is shown in FIG. 9, which shows that IL-6 is present at 16 hours (corresponding to a treatment interval of 8-16 hours) in the presence of the indicated concentration of IL-1 + added ligand. Shows that it was created. The addition of histamine or isoproterenol does not enhance IL-6 production compared to IL-1 alone. At 1.0 pg / ml IL-1, ketoprofen causes a partial ( < 50%) inhibition of IL-1-induced IL-6 production. In addition, ketoprofen inhibited IL-6 production in histamine or isoproterenol / IL-1 costimulated samples.

A synovial cell culture model system was used to characterize synergistic interactions between IL-1 and non-cytokine inflammatory mediators, which are important in coordinating joint tissue destruction, including damage that occurs as a result of tissue damage during arthroscopic surgery. . These results can be summarized as follows: (1) IL-1 induces a significant increase in PGE ₂ , IL-6, and IL-8 in cultured synovial cells, while resting cultures have Fail to produce a detectable amount; (2) the induction of PGE ₂ occurred most rapidly, with PGE ₂ released into the culture supernatant at 4 hours, IL-8 released at 8 hours, and IL-6 released at longer intervals; (3) All three mediators remain elevated in the culture supernatant after 24 h IL-1 exposure.

In contrast to these actions on PGE ₂ production, GPCR agonists do not enhance IL-1 induction of IL-6 or IL-8, but also increase IL-6 and IL-8 release after priming to IL-1. I don't even let you. IL-1 induction of IL-6 and IL-8 is believed to be enhanced by simultaneous induction of PGE ₂ because ketoprofen degrades the production of these cytokines in response to IL-1. This result indicates that ketoprofen could provide a therapeutic cartilage protective effect when delivered to the joint during surgical treatment.

Taken together, these results demonstrate the interaction between the activation of synovial cells by pro-inflammatory stimulation with IL-1 and specific G-coupled receptor signaling pathways. It is believed that similar mechanisms may work in chondrocytes. These interactions provide a means of integrating and modulating the pro-inflammatory response of synovial cells and chondrocytes according to the input from other autocooid or neurotransmitter receptor systems in the joint. These findings suggest a therapeutic intervention that mediates signaling through calcium mobilization, phosphoinositide hydrolysis, and PKC activation and targets the inhibition of G-protein coupled receptors associated with increased PGE ₂ production in arthroscopic surgery. It is the background for theoretical criteria and potential clinical benefits. These receptors on synovial cells and chondrocytes include histamine H ₁ , bradykinin, substance P, 5HT2, and purinacting P2Y receptors.

While preferred embodiments of the invention have been illustrated and described, it will be appreciated that various changes can be made to the solutions and methods described without departing from the spirit and scope of the invention. For example, other cartilage protectors, antibodies, and delivery vehicles may be found that can increase or replace agents, targeting antibodies, and delivery vehicles described in accordance with the present disclosure. Accordingly, the scope of this patent should be limited only by the terms of the appended claims.

Claims (48)

A number of cartilage protectors, including one or more anabolic cartilage protectors and one or more cartilage catabolic inhibitors, each contained in a delivery vehicle coupled with an antibody or antibody fragment specific for an antigenic determinant localized in the joint, each of which A plurality of cartilage protectors are included in a therapeutically effective amount to inhibit cartilage catabolism and simultaneously promote cartilage assimilation. A number of cartilage protectors comprising at least one anabolic cartilage protector and at least one cartilage catabolism inhibitor (each of these cartilage protectors is treated to enhance cartilage assimilation while simultaneously inhibiting cartilage catabolism. Included in a pharmaceutically effective amount, wherein at least one of the cartilage protectors is contained within a joint-targeted delivery vehicle. The method of claim 2, A targeted drug delivery system in which a targeted delivery vehicle is coupled with an antibody or antigen fragment specific for an antigenic determinant localized in a joint. The method of claim 3, wherein Targeted drug delivery system wherein the targeting antigenic determinant is on or in articular cartilage. The method of claim 4, wherein A targeting drug delivery system wherein the targeting antigenic determinant is on type II collagen of articular cartilage. The method of claim 3, wherein Targeted drug delivery system wherein the targeting antigenic determinant is on cartilage collagen or cartilage proteoglycans. The method of claim 3, wherein Collagen, wherein the targeting antigenic determinant comprises collagen type II and minor collagen types V, VI, IX, X and XI; Proteoglycans, including large cohesive proteoglycans, aggrecans, decorin, bigglycans, fibromodulin and lumicans; Cartilage oligomeric matrix protein, glycoprotein-39; Proteoglycan chondroitin-sulfate and glycosaminoglycans; Macrophage synovial cells and fibroblast synovial cells; And a targeting drug delivery system found on or within a cell, molecule or structure selected from the group consisting of chondrocytes. The method of claim 3, wherein Targeted drug delivery system wherein the targeting antigenic determinant is on or in the synovial membrane. The method of claim 3, wherein Targeted drug delivery system wherein the targeting antigenic determinant is an epitope or neoepitope associated with articular cartilage degradation. The method of claim 9, A targeting drug delivery system wherein the targeting epitope or neoepitope is immunolocalized to the articular cartilage superficial layer in a patient diagnosed with osteoarthritis, rheumatoid arthritis or other degenerative joint disease. The method of claim 9, A targeting drug delivery system wherein the targeting antigenic determinant is a neoepitope on a type II collagen or type II collagen fragment of articular cartilage. The method of claim 11, Targeted neoepitopes are generated by individual or combination actions of enzymes selected from the group consisting of matrix metalloproteinases (MMP) -1, MMP-3, MMP-8 and MMP-13, and other members of the MMP family Targeted drug delivery system immunolocalized to the cleavage site. The method of claim 9, Targeted drug delivery system wherein a targeting epitope or neoepitope is present on aggrecan, aglycan, or decorin of articular cartilage. The method of claim 9, Targeted drug delivery system wherein a targeting epitope or neoepitope is present on an aggrecan or aggrecan fragment of articular cartilage. The method of claim 14, Targeted drug delivery system wherein the targeted neoepitope is immunolocalized to a cleavage site caused by the action of an enzyme belonging to a group consisting of a disintegrin and metalloproteinase (ADAMTS) family and / or a MMP family with a thrombospondin motif . The method of claim 15, Targeted drug delivery system wherein the targeting neoepitope is immunolocalized to a cleavage site generated by the individual or combined action of ADAMTS-4 and / or ADAMTS-5 / 11 enzyme. The method of claim 3, wherein Targeted drug delivery system wherein the targeting antibody or antibody fragment is a human adaptation, chimeric, or human monoclonal antibody. The method of claim 2, A targeted drug delivery system wherein an anabolic cartilage protectant is contained within the targeted cleavage vehicle. The method of claim 2, Interleukin (IL) agonists in which anabolic cartilage protectors enhance cartilage anabolic action; Members of the transforming growth factor-β super-family, including TGF-β agonists and bone morphogenic protein (BMP) agonists, which enhance cartilage anabolic action; Insulin-like growth factors that promote cartilage anabolic action; And a fibroblast growth factor that promotes cartilage anabolic action. The method of claim 2, Anabolic cartilage protectors include IL-4, IL-10, IL-13, TGFβ1, TGFβ2, TGFβ3, BMP-2, BMP-4, BMP-6, BMP-7, IGF-1, bFGF, and natural preparations. Targeted drug delivery system selected from the group consisting of fragments, deletions, adducts, amino acid substitutions, mutations and modifications possessing biological characteristics. The method of claim 2, Members of the transforming growth factor-β super-family, including TGF-β agonists and bone morphogenic protein (BMP) agonists, wherein the anabolic cartilage protectors enhance cartilage anabolic action; Insulin-like growth factors that promote cartilage anabolic action; And a fibroblast growth factor that promotes cartilage anabolic action. The method of claim 2, A targeted drug delivery system wherein a cartilage catabolic inhibitor is contained within the targeted delivery vehicle. The method of claim 2, IL-1 receptor antagonists in which cartilage catabolism inhibitors inhibit cartilage catabolism; TNF-α receptor antagonists that inhibit cartilage catabolism; Cyclooxygenase-2 specific inhibitors that inhibit cartilage catabolism; MAP kinase inhibitors that inhibit cartilage catabolism; Nitric oxide synthase inhibitors that inhibit cartilage catabolism; And a nuclear factor kappa B inhibitor that inhibits cartilage catabolism. The method of claim 2, Inhibitors of matrix metalloproteinases in which the cartilage catabolism inhibitor inhibits cartilage catabolism; Cell adhesion molecules, including integrin agonists and integrin antagonists, which inhibit cartilage catabolism; Intracellular signaling inhibitors including protein kinase C inhibitors and protein tyrosine kinase inhibitors that inhibit cartilage catabolism; And an inhibitor of the SH2 domain that inhibits cartilage catabolism. The method of claim 2, IL-1 receptor antagonists in which cartilage catabolism inhibitors inhibit cartilage catabolism; And an agent selected from TNF-α receptor antagonists that inhibit cartilage catabolism. The method of claim 2, Targeted drug delivery system wherein the anabolic cartilage protector and cartilage catabolic inhibitor each comprise a protein. The method of claim 2, A targeted drug delivery system wherein both anabolic cartilage protectors and cartilage catabolic inhibitors are contained within a targeted delivery vehicle. The method of claim 2, A targeted drug delivery system comprising a plurality of targeted delivery vehicles in which anabolic cartilage protectors and cartilage catabolic inhibitors are contained separately in the first and second plurality of targeted delivery vehicles, respectively. The method of claim 28, The targeted drug delivery system, wherein the first and second targeted delivery vehicles are selected such that transient release kinetics occur temporarily for the anabolic cartilage protector contained therein and the cartilage catabolic inhibitor contained therein. The method of claim 2, Targeted drug delivery system wherein the targeted delivery vehicle comprises a targeting immunoparticle. The method of claim 30, Targeted drug delivery system wherein the targeting immunoparticle comprises nanoparticles. The method of claim 31, wherein Targeted drug delivery system wherein the nanoparticles range in diameter from 5 nanometers to 750 nanometers. The method of claim 31, wherein Targeted drug delivery system wherein the nanoparticles range in diameter from 10 nanometers to 500 nanometers. The method of claim 31, wherein Targeted drug delivery system wherein the nanoparticles range in diameter from 20 nanometers to 200 nanometers. The method of claim 31, wherein Targeted drug delivery system wherein the nanoparticles are formed from a polymer selected from the group consisting of hyaluronan, chitosan, collagen, gelatin, alginate, polylactic acid (PLLA), polyglycolic acid (PGA) and PLGA. The method of claim 31, wherein Targeted drug delivery system in which the nanoparticles provide sustained release of cartilage protectors over a period of 1 to 4 weeks. The method of claim 2, A targeted drug delivery system further comprising a carrier suitable for intravenous, intramuscular, subcutaneous or inhaled administration. The method of claim 2, Targeted drug delivery system further comprising one or more additional therapeutic agents. The method of claim 2, A targeted drug delivery system further comprising one or more pain or inflammation inhibitory agents. The method of claim 39, Pain or inflammation inhibitory agents include serotonin receptor antagonists, serotonin receptor agonists, histamine receptor antagonists, bradykinin receptor antagonists, kallikrein inhibitors, tachykinin receptor antagonists, calcitonin gene-related peptide (CGRP) receptor antagonists, interleukin receptor antagonists , Inhibitors of enzymes active in the synthetic route to arachidonic acid metabolites, prostanoid receptor antagonists, leukotriene receptor antagonists, opioid receptor agonists, purinoceptor agonists and antagonists, adenosine triphosphate (ATP) -sensitive potassium A targeted drug delivery system selected from the group consisting of channel openers, and calcium channel antagonists. A number of cartilage protectors comprising at least one anabolic cartilage protector and at least one cartilage catabolism inhibitor (each of these cartilage protectors is treated to enhance cartilage assimilation while simultaneously inhibiting cartilage catabolism. In a pharmaceutically effective amount, wherein at least one of the cartilage protectors is contained in a delivery vehicle targeted to molecules, cells, or structures of the chondrocytes. Cartilage, comprising a therapeutically effective amount of one or more cartilage protectors, either anabolic cartilage protectors or cartilage catabolic inhibitors, contained in a targeting immunoparticle coupled with an antibody or antibody fragment specific for an antigenic determinant localized in the joint Targeted drug delivery system for protection. A targeted drug delivery system for cartilage protection comprising a therapeutically effective amount of anabolic cartilage protectant contained in a delivery vehicle targeting the joint. A targeted drug delivery system for cartilage protection, comprising a therapeutically effective amount of anabolic cartilage protectant contained in a delivery vehicle that targets molecules, cells, or structures of the chondrocytes. A number of cartilage protectors, including one or more anabolic cartilage protectors and one or more cartilage catabolic inhibitors, each contained in a delivery vehicle coupled with an antibody or antibody fragment specific for an antigenic determinant localized in the joint, each of which Including delivering a targeted drug delivery system to a patient in need thereof, wherein a number of cartilage protectors are included in a therapeutically effective amount to inhibit cartilage catabolism and simultaneously promote cartilage assimilation. How to protect cartilage in such patients. A number of cartilage protectors comprising at least one anabolic cartilage protector and at least one cartilage catabolism inhibitor (each of these cartilage protectors is treated to enhance cartilage assimilation while simultaneously inhibiting cartilage catabolism. Contained in a pharmaceutically effective amount, wherein at least one of the cartilage protectors is contained in a delivery vehicle targeted to the joint), including simultaneously administering to the patient in need thereof. . A number of cartilage protectors comprising at least one anabolic cartilage protector and at least one cartilage catabolism inhibitor (each of these cartilage protectors is treated to enhance cartilage assimilation while simultaneously inhibiting cartilage catabolism. In a pharmaceutically effective amount, wherein at least one of the cartilage protectors is contained in a delivery vehicle targeted to a neoepitope associated with degeneration of the articular cartilage) to the patient in need thereof. How to protect cartilage in such patients. A plurality of cartilage protectors comprising at least one anabolic cartilage protector and at least one cartilage catabolic inhibitor (each of these cartilage protectors is treated to enhance cartilage assimilation while simultaneously inhibiting cartilage catabolic activity. Included in a pharmaceutically effective amount, wherein at least one of the cartilage protectors is contained in a delivery vehicle targeted to the molecule, cell or structure of the chondrocytes) to the patient in need thereof. How to protect cartilage from patients