Abstract:
The invention provides compositions and methods for preserving, prolonging, or augmenting joint lubrication by contacting a tissue of a joint such as a knee, elbow, or other articulating joint, with a tribonectin and an inhibitor of a proinflammatory mediator.

Description:
RELATED APPLICATIONS 
       [0001]    This patent application claims the benefit of U.S. Provisional Patent Application Ser. No. 60/994,372, filed Sep. 18, 2007, the contents of which are herein incorporated by reference in their entirety. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The present invention relates to the lubrication of mammalian joints. 
       BACKGROUND OF THE INVENTION 
       [0003]    Osteoarthritis (OA) is a common form of joint disease. Factors which contribute to the development of OA include a family history of OA, previous damage to the joint through injury or surgery, and age of the joint, i.e., “wear and tear” of the articulating surfaces of the joint. OA is very common in older age groups, but can affect children as well. 
         [0004]    Current treatment is directed to relieving pain and other symptoms of OA, e.g., by administering analgesics and anti-inflammatory drugs. Other therapeutic approaches include viscosupplementation by administering hyaluronic acid and derivatives thereof to joint tissue to increase the viscosity of synovial fluid. 
       SUMMARY OF THE INVENTION 
       [0005]    The invention provides a method for preserving or augmenting joint lubrication by contacting a tissue of a joint such as a knee, elbow, or other articulating joint, with a tribonectin and an inhibitor of a proinflammatory mediator. For example, the mediator is a cytokine such as interleukin-1β (IL-1β) or tumor necrosis factor-α (TNF-α). Alternatively the mediator is another proinflammatory mediator such as a cathepsin, e.g. cathepsin B. Preferably, the combination leads to a synergistic effect in preserving or augmenting joint lubrication. The inhibitor reduces the expression or function of TNF-α such as an antibody or antibody conjugate that binds to TNF-α Exemplary compositions include ENBREL™. In another example, the inhibitor reduces the expression or function of a proinflammatory IL-1, such as an IL-1 RA, e.g., KINERET™. By reducing inflammation, the methods also prolong joint lubrication, e.g., augmented joint lubrication after administration of a lubricating compound to the joint, by reducing catabolism of endogenously produced and exogenous administered lubricating compositions. 
         [0006]    As an alternative to the combination therapy approach described above, one or more inhibitors that reduce the expression or function of TNF-α are administered alone either subcutaneously at or near an articulating joint or intra-articularly directly to the joint itself. 
         [0007]    The compounds are administered directly to or adjacent to a target joint. For example, the inhibitor is administered (injected or infused) directly into a joint. Alternatively, inhibitor is administered subcutaneously, e.g., into cutaneous tissue adjacent to the target joint tissue of the affected joint. For example, the compounds are injected subcutaneously 5 mm deep (shallow injection just beneath the skin surface) to 1.5 cm deep into the tissue (i.e., approaching or entering the joint) of an adult human. The compounds are injected 3-4 mm deep into the tissue (shallow injection) to approximately 10 mm deep for a child. The tribonectin and inhibitor are administered simultaneously or sequentially. In some cases, the compositions are compounded together for simultaneously delivery. Optionally hyaluronic acid is also administered. Accordingly, a composition containing a tribonectin and an inhibitor of a proinflammatory mediator is within the invention. 
         [0008]    The compositions are administered in the following dose ranges: tribonectin or recombinant lubricin (0.1 μg/ml-1 mg/ml); HA (0.1 mg/ml-50.0 mg/ml); inhibitor of TNF (0.1-10 mg/kg). For example, a TNF inhibitor is administered at 0.5-5 mg/kg. The compositions are administered to a joint in a volume of 0.5-5 ml. For example, the compositions are administered to a joint, e.g., a knee, shoulder, or elbow, in a volume of approximately 1 ml. In the event of a acute injury, which may be characterized by swelling or effusion, an abnormal amount of fluid is present in the joint. In the latter case, up to 5 ml of a solution is administered to the joint. For example, the joint is first aspirated to remove the effusion and then the therapeutic composition containing a lubricating agent and an anti-inflammatory agent is inject or infused into the joint. 
         [0009]    The compositions described herein are purified. For example, tribonectin polypeptides are biochemically purified. The enzyme chymotrypsin cleaves at sites which bracket amino acids encoded by exon 6 (underlined in SEQ ID NO:1) of the MSF gene. Thus, a polypeptide containing amino acids encoded by exon 6 of the MSF gene (but not any other MSF exons) is prepared from a naturally-occurring or recombinantly produced MSF gene product by enzymatic digestion with chymotrypsin. The polypeptide is then subjected to standard biochemical purification methods to yield a substantially pure polypeptide suitable for therapeutic administration, evaluation of lubricating activity, or antibody production. Alternatively, the compositions are synthetically or recombinantly produced. 
         [0010]    Therapeutic compositions are administered in a pharmaceutically acceptable carrier (e.g., physiological saline). Carriers are selected on the basis of mode and route of administration and standard pharmaceutical practice. A therapeutically effective amount of a therapeutic composition (e.g., lubricating polypeptide, anti inflammatory compound, or other composition) is an amount which is capable of producing a medically desirable result, e.g., boundary lubrication of a mammalian joint, in a treated animal. 
         [0011]    The combination therapy described herein confers a clinical benefit such as increased lubrication, reduced inflammation/swelling, increased range of motion/mobility, and/or decrease in pain. A medically desirable result is a reduction in pain (measured, e.g., using a visual analog pain scale described in Peyron et al., 1993, J. Rheumatol.(suppl.39):10-15) or increased ability to move the joint (measured, e.g., using pedometry as described in Belcher et 30 al., 1997, J. Orthop. Trauma 11: 106-109). Lubricity (μ) of synovial fluid is measured before or after treatment by reaspirating a small volume of synovial fluid from the affected joint and testing the lubricating properties in vitro using know methods, e.g., a friction apparatus described in Jay et al., 1992, Conn. Tiss. Res. 28:71-88 or Jay et al., 1998, J. Biomed. Mater. Res. 40:414-418 and U.S. Pat. No. 6, 690,562. 
         [0012]    As is well known in the medical arts, dosage for any one animal depends on many factors, including the animal&#39;s size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Subjects to be treated include humans, companion animals such as dogs, cats as well as horses, oxen, donkey, cow, sheep, pig, rabbit, monkey or mouse. Administration is generally local to an injured or inflamed joint. Alternatively, the compositions are administered subcutaneously in close proximity to the joint or via a timed-release implant placed in close proximity to a joint for slow release at the site of an injured or inflamed joint. Optionally, hyaluronic acid (HA) is co-administered. Administration of a tribonectin and an inhibitor of an anti-inflammatory mediator leads to a synergistic clinical benefit. 
         [0013]    A purified composition such as a protein or peptide (e.g., antibody, fusion protein) is at least 60%, by weight, free from proteins and naturally occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably 90%, and most preferably at least 99%, by weight, the desired composition. A purified antibody may be obtained, for example, by affinity chromatography. By “substantially pure” is meant a nucleic acid, polypeptide, or other molecule that has been separated from the components that naturally accompany it. Typically, the polypeptide is substantially pure when it is at least 60%, 70%, 80%, 90%, 95%, or even 99%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. For example, a substantially pure polypeptide may be obtained by extraction from a natural source, by expression of a recombinant nucleic acid in a cell that does not normally express that protein, or by chemical synthesis. 
         [0014]    By “substantially identical,” when referring to a protein or polypeptide, is meant a protein or polypeptide exhibiting at least 75%, but preferably 85%, more preferably 90%, most preferably 95%, or even 99% identity to a reference amino acid sequence. For proteins or polypeptides, the length of comparison sequences will generally be at least 20 amino acids, preferably at least 30 amino acids, more preferably at least 40 amino acids, and most preferably 50 amino acids or the full length protein or polypeptide. Nucleic acids that encode such “substantially identical” proteins or polypeptides constitute an example of “substantially identical” nucleic acids; it is recognized that the nucleic acids include any sequence, due to the degeneracy of the genetic code, that encodes those proteins or polypeptides. In addition, a “substantially identical” nucleic acid sequence also includes a polynucleotide that hybridizes to a reference nucleic acid molecule under high stringency conditions. 
         [0015]    By “high stringency conditions” is meant any set of conditions that are characterized by high temperature and low ionic strength and allow hybridization comparable with those resulting from the use of a DNA probe of at least 40 nucleotides in length, in a buffer containing 0.5 M NaHPO4, pH 7.2, 7% SDS, 1 mM EDTA, and 1% BSA (Fraction V), at a temperature of 65 oC, or a buffer containing 48% formamide, 4.8XSSC, 0.2 M Tris-Cl, pH 7.6, 1X Denhardt&#39;s solution, 10% dextran sulfate, and 0.1% SDS, at a temperature of 42 oC. Other conditions for high stringency hybridization, such as for PCR, Northern, Southern, or in situ hybridization, DNA sequencing, etc., are well known by those skilled in the art of molecular biology. See, e.g., F. Ausubel et al., Current Protocols in Molecular Biology, John Wiley &amp; Sons, New York, N.Y., 1998, hereby incorporated by reference. 
         [0016]    The term “isolated DNA” is meant DNA that is free of the genes which, in the naturally occurring genome of the organism from which the given DNA is derived, flank the DNA. Thus, the term “isolated DNA” encompasses, for example, cDNA, cloned genomic DNA, and synthetic DNA. 
         [0017]    Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. Publications, U.S. patents and applications, GenBank/NCBI accession numbers, and all other references cited herein, are herby incorporated by reference. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS  
         [0018]      FIG. 1  is a photograph of a histologically stained rat articular cartilage using mab S6.89. A-A representative right knee joint one week following the initiation of the arthritic model showing less lubricin staining in the superficial zone articular chondrocytes. B-A representative contra-lateral left knee joint one week following the initiation of the arthritic model showing presence of lubricin. 
           [0019]      FIG. 2  is a bar graph showing ex-vivo μ of ACL transected (ACLT) (n=6) and contralateral (CL) (n=6) rat joints at 1 and 4 weeks following transection. *Indicates that μ values of ACLT joints at week 4 were significantly (p=0.02) higher than μ values of CL joints at week 4 and higher (p=0.03) than μ values of ACLT joints at week 1. 
           [0020]      FIG. 3  is is a bar graph showing SF lavage sGAG levels of ACL transected (ACLT) (n=6) and contralateral (CL) (n=6) rat joints at 1 and 4 weeks following transaction. * Indicates that sGAG levels of ACLT joints at week 1 and 4 were significantly (p&lt;0.001) higher than sGAG levels of CL joints at week 1 and 4. 
           [0021]      FIG. 4  is a bar graph showing Lubricin synovial fluid (SF) lavage levels in ACL-transected (ACLT), contralateral (CL), sham and contralateral sham (sham-CL) following etanercept-treatment (n=5), no treatment(n=5) or sham surgery (n=4) at 2 weeks following ACLT. *Indicates that lubricin SF lavage levels were significantly (p&lt;0.001) higher in the etanercept-treated ACLT joints compared to non-treated ACLT joints. 
           [0022]      FIG. 5  is a bar graph showing ex-vivo joint coefficient of friction (μ) of ACL-transected (ACLT), contralateral (CL), sham and contralateral sham (sham-CL) following etanercept-treatment (n=5), no treatment (n=5) or sham surgery (n=4) at 2 weeks following ACLT. *Indicates that μ values of etanercept-treated ACLT joints were significantly (p=0.021) lower than the non-treated ACLT joints. 
           [0023]      FIG. 6  is a bar graph showing sGAG SF lavage levels in ACL-transected (ACLT), and contralateral (CL) joints following etanercept-treatment (n=5), or no treatment (n=5) at 2 weeks following ACLT. *Indicates that sGAG SF lavage levels were significantly (p&lt;0.001) lower in the etanercept-treated joints compared to non-treated joints. 
           [0024]      FIG. 7  is a bar graph showing synovial fluid (SF) lavage lubricin levels in ACL-transected (ACLT) and contralateral (CL) joints of animals with ACLT, Etanercept-A, Etanercept-B treatments and sham surgery. *Indicates that SF lavage lubricin levels in treatment-A were significantly (p&lt;0.01) higher than SF lavage lubricin levels in ACLT and treatment-B. Statistical analyses were performed by student&#39;s t-test. 
           [0025]      FIG. 8  is a bar graph showing percentage surface coverage of articular surface by lubricin ACL-transected (ACLT) and contra lateral (CL) joints of animals with ACLT, Etanercept-A, Etanercept-B treatments and sham surgery. *Indicated that percentage surface coverage of articular surface by lubricin in the ACLT joints was significantly (p&lt;0.01) lower than in treatment-A, treatment-B or sham surgery. 
       
    
    
     DETAILED DESCRIPTION  
       [0026]    Endogeneous tribosupplementation includes a role for anti-inflammatory agents such as TNF-α blockers such as Etanercept or IL-1 inhibitors in the treatment of injured joints or joints that are at risk of degeneration following joint trauma. 
       Joint Injury and Degeneration of Joint Tissue 
       [0027]    Post-traumatic causes of OA involve catabolic cytokines. IL-1 has been shown to down regulate lubricin, which by itself does not lead to OA but may play a degenerative role since IL-1β has also been shown to result in less lubricin secretion in vitro. The SF levels of IL-1β and TNF-α are elevated following an acute ACL-injury, and remain elevated 3 weeks post injury. Both IL-1β and TNF-α stimulate the secretion of latent cysteine proteinases, primarily that of cathepsin B, from synovial fibroblasts. Cathepsin B has been shown to possess a strong ability to proteolytically degrade lubricin and abolish lubricin&#39;s lubricating ability. Inhibition prolongs and augments joint lubrication. 
         [0028]    ACL injury has both immediate and long-term implications on patients&#39; quality of life, long-term disability and risk for OA. Many ACL-injured subjects suffer a severe functional deficit. Although one third of them may improve function with rehabilitation alone, there is substantial evidence that they will manifest articular cartilage degeneration. On average, patients with OA following ACL injury are 15- to 20-years younger than those with primary OA when they seek medical help for their symptoms. Reasons for this profile include the initial inflammatory response, the presence of concomitant injuries, altered kinematics, abnormal contact stresses, and/or the increased incidence of “giving way” following injury producing additional insult to the cartilage and menisci. All of these factors may disrupt the homeostasis of the joint and place the knee at risk for early OA. Therefore, surgeons advocate reconstruction of the ACL to decrease joint laxity, to restore an active lifestyle, and to reduce future knee injuries. The impact of a non-reconstructed ACL injury upon the chondroprotective features of the joint has not been established. The inflammatory response at the time of injury decreases the lubricating ability of the joint therefore sets the stage for post-traumatic arthritis. 
         [0029]    Patients who undergo ACL reconstruction also exhibit progressive articular cartilage damage in the reconstructed knee. In a recent 14-year follow-up study of 205 male atheletes, 78% had degenerative signs in their injured knee compared to 4% in their uninjured knee (Von Porat et al., 20054, Ann. Rheum. Dis. 63:269-273). In a 5-year follow-up, others (Daniel et al., 1994, Am. J. Sports Med.22:632-644) determined that both acute and chronic ACL reconstructed knees had significantly great radiographic evidence of OA as compared to those in a conservatively treated (non-operative) group. These findings were confirmed by another group (Fithian et al., 2002, Orthoped. Clin. N. America 33:621-636) who found that the incidence of chondropathy was 94% and 92% with and without meniscus tears respectively using MRI. Although radiographic signs of OA were present within five years of surgery, the mechanisms of chondropathy were heretofore unknown. 
         [0030]    Studies were carried out to evaluate the association of dysfunction of joint tribologic properties with acute ACL injuries in both an animal model and clinical aspirates. Inhibiting inflammation was found to preserve lubricating properties and prevent chondropathy. 
       Lubrication of Articulating Joints 
       [0031]    Re-establishing the normal lubricating ability of the diarthrodial joint is important following an injury. Catabolism of lubricin eliminates the lubricating ability of synovial fluid. This process occurs both within the synovial fluid as well as the lubricin located on the surface of articular cartilage. Introducing lubricin and hyaluronic acid together (i.e., artificial synovial fluid) is a potential therapy but the half life of this therapy may be short. Combination therapy in which anti-inflammatory treatment is administered simultaneously or sequentially preserves the introduced lubricant and prevent its digestion by activated proteases. Intervention of the inflammatory process is accomplished at multiple stages in the cyclo-oxygenase and lipoxy-oxygenase inflammatory cascades. One method involves treating the injured diarthrodial joint at the same time through the intra-articular administration of IL-1 inhibitors and TNF-beta inhibitors to block the upstream proinflammatory cytokines, which initiate the downward cascade resulting in protease expression. The methods involve the introduction of lubricants intended to protect the articular surface and lengthening of their half life and residence time by the introduction of anti-inflammatory medication and biologics. 
         [0032]    The data described herein shows that such a treatment regimen preserves and augments lubrication of injured joints. Treatment of an inflamed rat joint following an ACL injury with a TNF-α inhibitor etanerept/ENBREL™ (Amgen) reestablishes some of the lubricin layer in the superficial zone. An increase in the appearance of lubricin was observed with ENBREL™ alone. Exogenous tribonectin is administered to protect the articular surface from wearing against one another. A combination of anti-inflammatory therapy in traumatized joints with a boundary lubricant (tribonectin) to restore chondroprotection re-establishes or increases lubricin concentration and decreases one or more symptoms of joint degeneration (e.g., pain, decreased mobility, decreased range of motion). 
       Lubricin 
       [0033]    Lubricin is a mucinous glycoprotein, secreted by synovial fibroblasts (Jay et al, J Ortho Res 2001, 19:677-687) and the superficial zone articular chondrocyte (Flannery CR et al, Biochem Biophys Res Commun 1999, 254:535-541) and is responsible for lubrication of articular surfaces. SF from patients with an acute ACL injury had decreased lubricin at early stage following injury (Elsaid K A et al, Trans Orthop Res Soc 2007; 32:160), which has been associated with increased cartilage damage. 
         [0034]    Endogenous lubricin SF levels were significantly lowered following ACL injury, and were associated with increased cartilage damage. Furthermore, inflammatory cytokines e.g. IL-1β and TNF-α were shown to decrease lubricin synthesis (Jones ARC et al, Eur Cell Mater 2007; 31:40-45). 
       Tribonectins 
       [0035]    A tribonectin is an lubricating polypeptide, which contains at least one repeat of an amino acid sequence which is at least 50% identical to KEPAPTT (SEQ ID NO:3). For example, a tribonectin comprises a polypeptide, the amino acid sequence of which comprises at least one but less than 76 subunits, with each subunit comprising at least 7 amino acids and the amino acid sequence of each subunit being at least 50% identical to SEQ ID NO:3 with a non-identical amino acid being a conservative amino acid substitution. A tribonectin contains at least one O-linked lubricating moiety. Preferably the lubricating moiety is a β(1-3)Gal-GalNAc moiety. Optionally, the β(1-3)Gal-GalNAc is capped with NeuAc. The polypeptide contains an oligosaccharide, which is moiety is N-linked to an asparagine residue, e.g., the oligosaccharide is asialo-, digalactosylated bi-antennary, bisecting N-acteylglucosamine, tri-mannosyl core-fucosylated. Tribonectins are further described in U.S. Pat. Nos. 7,001,881; 6,960,562; 6,743,774; U.S. Patent Application Pub. No. 2004/0229804; and U.S. Patent Application Pub. No. 2004/0072741. 
         [0036]    The amino acid sequence of the protein backbone of a lubricating polypeptide may differ depending on alternative splicing of exons of the human megakaryocyte stimulating factor (MSF) gene (GENBANK® accession number U70136). Tribonectins are purified naturally-occurring polypeptides or synthetically produced or recombinant polypeptides. In one example, the tribonectin has the amino acid sequence of a naturally-occurring full-length MSF; alternatively, the sequence corresponds to an alternative splice variant of the MSF gene. Optionally, the polypeptide compositions do not contain a lipid. For example, less than 10%, preferably less than 5%, and more preferably less than 1% of the lubricating polypeptide composition is a lipid. The polypeptide compositions are purified from fibronectin. For example, less than 10%, preferably less than 5%, and more preferably less than 1% of the lubricating polypeptide composition is a fibronectin. 
         [0037]    The amino acid sequence of the backbone of purified, synthetic, or recombinant tribonectins is at least 50% identical to the amino acid sequence of a naturally-occurring tribonectin and possess at least 50% of the lubricating activity of a naturally-occurring tribonectin. A tribonectin lubricating polypeptide is a purified protein or polypeptide containing the amino acid sequence of human megakaryocyte stimulating factor (MSF) or a fragment thereof. For example, a tribonectin lubricating polypeptide contains amino acids 67-106 and 200-1140 of SEQ ID NO:1 and at least one O-linked oligosaccharide, but lacks amino acids 26-66 of SEQ ID NO:1. In other examples, the polypeptide further lacks amino acids 107-156 of SEQ ID NO:1 or amino acids 107-199 of SEQ ID NO:1, or the polypeptide contains amino acids 1-25, 67-106, and 200-1404 of SEQ ID NO:1. Alternatively, the polypeptide contains amino acids 67-106 and 200-1140 of SEQ ID NO:1 and at least one O-linked oligosaccharide, but lacks amino acids 107-156 of SEQ ID NO:1. In another variation, the lubricating polypeptide contains amino acids 67-106 and 200-1140 of SEQ ID NO:1 and at least one O-linked oligosaccharide, but lacks amino acids 157-199 of SEQ ID NO:1. For example, the polypeptide contains amino acids 1-156 and 200-1404 of SEQ ID NO:1 (lacking amino acids 157-199 of SEQ ID NO:1) or the polypeptide contains amino acids 1-106 and 200-1404 (lacking amino acids 107-199 of SEQ ID NO:1). The amino acid sequences are contiguous to one another or may be separated by intervening unrelated sequences. Preferably, the lubricating polypeptide contains residues encoded by exons 1, 3, and 6-12 of the MSF gene. 
         [0038]    Polynucleotides encoding the polypeptide backbone of a lubricating polypeptide include nucleotides 232-351 and 631-3453 of SEQ ID NO:2 and lacks nucleotides 109-231 of SEQ ID NO:2. In another example, the polynucleotide contains nucleotides 232-351 and 631-3453 of SEQ ID NO:2 and lacks nucleotides 352-501 of SEQ ID NO:2. In yet another examples, the polynucleotide contains nucleotides 232-351 and 631-3453 of SEQ ID NO:2 and lacks nucleotides 502-630 of SEQ ID NO:2. A polynucleotide containing nucleotides 232-351 and 631-3453 of SEQ ID NO:2 and lacking nucleotides 352-630 of SEQ ID NO:2 is also within the invention as well as a polynucleotide containing nucleotides 232-351 and 631-3453 of SEQ ID NO:2 and lacking nucleotides 109-231 and 352-630 of SEQ ID NO:2. For example, the polynucleotide contains nucleotides 34-501 linked in-frame to nucleotides 631-4245 of SEQ ID NO:2 or nucleotides 34-501 linked in-frame to nucleotides 631-4245 of SEQ ID NO:2. The polynucleotide contains a first sequence containing nucleotides 34-501 of SEQ ID NO:2, a second sequence containing nucleotides 232-351 of SEQ ID NO:2, and a third sequence containing nucleotides 631-4245 of SEQ ID NO:2. The first, second and third sequences are linked in-frame. Preferably, the sequences described above are contiguous. 
         [0000]    
       
         
               
             
               
               
             
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 MSF amino acid sequence 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 (SEQ ID NO:1) 
                   
               
             
          
           
               
                 (GENBANK ® U70136) 
                   
               
               
                 MAWKTLPIYLLLLLSVFVIQQVSSQDLSSCAGRCGEGYSRDATCNCDYNC 
               
               
                   
               
               
                 QHYMECCPDFKRVCTAELSCKGRCFESFERGRECDCDAQCKKYDKCCPDY 
               
               
                   
               
               
                 ESFCAEVHNPTSPPSSKKAPPPSGASQTIKSTTKRSPKPPNKKKTKKVIE 
               
               
                   
               
               
                 SEEITEEHSVSENQESSSSSSSSSSSSTIWKIKSSKNSAANRELQKKLK V   
               
               
                   
               
               
                 
                   KDNKKNRTKKKPTPKPPVVDEAGSGLDNGDFKVTTPDTSTTQHNKVSTSP 
                 
               
               
                   
               
               
                 
                   KITTAKPINPRPSLPPNSDTSKETSLTVNKETTVETKETTTTNKQTSTDG 
                 
               
               
                   
               
               
                 
                   KEKTTSAKETQSIEKTSAKDLAPTSKVLAKPTPKAETTTKGPALTTPKEP 
                 
               
               
                   
               
               
                 
                   TPTTPKEPASTTPKEPTPTTIKSAPTTPKEPAPTTTKSAPTTPKEPAPTT 
                 
               
               
                   
               
               
                 
                   TKEPAPTTPKEPAPTTTKEPAPTTTKSAPTTPKEPAPTTPKKPAPTTPKE 
                 
               
               
                   
               
               
                 
                   PAPTTPKEPTPTTPKEPAPTTKEPAPTTPKEPAPTAPKKPAPTTPKEPAP 
                 
               
               
                   
               
               
                 
                   TTPKEPAPTTTKEPSPTTPKEPAPTTTKSAPTTTKEPAPTTTKSAPTTPK 
                 
               
               
                   
               
               
                 
                   EPSPTTTKEPAPTTPKEPAPTTPKKPAPTTPKEPAPTTPKEPAPTTTKKP 
                 
               
               
                   
               
               
                 
                   APTAPKEPAPTTPKETAPTTPKKLTPTTPEKLAPTTPEKPAPTTPEELAP 
                 
               
               
                   
               
               
                 
                   TTPEEPTPTTPEEPAPTTPKAAAPNTPKEPAPTTPKEPAPTTPKEPAPTT 
                 
               
               
                   
               
               
                 
                   PKETAPTTPKGTAPTTLKEPAPTTPKKPAPKELAPTTTKEPTSTTSDKPA 
                 
               
               
                   
               
               
                 
                   PTTPKGTAPTTPKEPAPTTPKEPAPTTPKGTAPTTLKEPAPTTPKKPAPK 
                 
               
               
                   
               
               
                 
                   ELAPTTTKGPTSTTSDKPAPTTPKETAPTTPKEPAPTTPKKPAPTTPETP 
                 
               
               
                   
               
               
                 
                   PPTTSEVSTPTTTKEPTTIHKSPDESTPELSAEPTPKALENSPKEPGVPT 
                 
               
               
                   
               
               
                 
                   TKTPAATKPEMTTTAKDKTTERDLRTTPETTTAAPKMTKETATTTEKTTE 
                 
               
               
                   
               
               
                 
                   SKITATTTQVTSTTTQDTTPFKITTLKTTTLAPKVTTTKKTITTTEIMNK 
                 
               
               
                   
               
               
                 
                   PEETAKPKDRATNSKATTPKPQKPTKAPKKPTSTKKPKTMPRVRKPKTTP 
                 
               
               
                   
               
               
                 
                   TPRKMTSTMPELNPTSRIAEAMLQTTTRPNQTPNSKLVEVNPKSEDAGGA 
                 
               
               
                   
               
               
                   EGETPHML LRPHVFMPEVTPDMDYLPRVPNQGIIINPMLSDETNICNGKP 
               
               
                   
               
               
                 VDGLTTLRNGTLVAFRGHYFWMLSPFSPPSPARRITEVWGIPSPIDTVFT 
               
               
                   
               
               
                 RCNCEGKTFFFKDSQYWRFTNDIKDAGYPKPIFKGFGGLTGQIVAALSTA 
               
               
                   
               
               
                 KYKNWPESVYFFKRGGSIQQYIYKQEPVQKCPGRRPALNYPVYGEMTQVR 
               
               
                   
               
               
                 RRRFERAIGPSQTHTIRIQYSPARLAYQDKGVLHNEVKVSILWRGLPNVV 
               
               
                   
               
               
                 TSAISLPNIRKPDGYDYYAFSKDQYYNIDVPSRTARAITTRSGQTLSKVW 
               
               
                   
               
               
                 YNCP 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                 MSF cDNA 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 (SEQ ID NO:2) 
                   
               
             
          
           
               
                    1 
                 gcggccgcga ctattcggta cctgaaaaca acgatggcat ggaaaacact tcccatttac 
                   
               
               
                   61 
                 ctgttgttgc tgctgtctgt tttcgtgatt cagcaagttt catctcaaga tttatcaagc 
               
               
                  121 
                 tgtgcaggga gatgtgggga agggtattct agagatgcca cctgcaactg tgattataac 
               
               
                  181 
                 tgtcaacact acatggagtg ctgccctgat ttcaagagag tctgcactgc ggagctttcc 
               
               
                  241 
                 tgtaaaggcc gctgctttga gtccttcgag agagggaggg agtgtgactg cgacgcccaa 
               
               
                  301 
                 tgtaagaagt atgacaagtg ctgtcccgat tatgagagtt tctgtgcaga agtgcataat 
               
               
                  361 
                 cccacatcac caccatcttc aaagaaagca cctccacctt caggagcatc tcaaaccatc 
               
               
                  421 
                 aaatcaacaa ccaaacgttc acccaaacca ccaaacaaga agaagactaa gaaagttata 
               
               
                  481 
                 gaatcagagg aaataacaga agaacattct gtttctgaaa atcaagagtc ctcctcctcc 
               
               
                  541 
                 tcctcctctt cctcttcttc ttcaacaatt tggaaaatca agttttccaa aaattcagct 
               
               
                   
               
               
                   
                                                  EXON 6 
               
               
                  601 
                 gctaatagag aattacagaa gaaactcaaa  gtaaaagata  acaagaagaa cagaactaaa 
               
               
                  661 
                   aagaaaccta   cccccaaacc   accagttgta   gatgaagctg   gaagtggatt   ggacaatggt   
               
               
                  721 
                   gacttcaagg   tcacaactcc   tgacacgtct   accacccaac   acaataaagt   cagcacatct   
               
               
                  781 
                   cccaagatca   caacagcaaa   accaataaat   cccagaccca   gtcttccacc   taattctgat   
               
               
                  841 
                   acatctaaag   agacgtcttt   gacagtgaat   aaagagacaa   cagttgaaac   taaagaaact   
               
               
                  901 
                   actacaacaa   ataaacagac   ttcaactgat   ggaaaagaga   agactacttc   cgctaaagag   
               
               
                  961 
                   acacaaagta   tagagaaaac   atctgctaaa   gatttagcac   ccacatctaa   agtgctggct   
               
               
                 1021 
                   aaacctacac   ccaaagctga   aactacaacc   aaaggccctg   ctctcaccac   tcccaaggag   
               
               
                 1081 
                   cccacgccca   ccactcccaa   ggagcctgca   tctaccacac   ccaaagagcc   cacacctacc   
               
               
                 1141 
                   accatcaagt   ctgcacccac   cacccccaag   gagcctgcac   ccaccaccac   caagtctgca   
               
               
                 1201 
                   cccaccactc   ccaaggagcc   tgaacccacc   accaccaagg   agcctgcacc   caccactccc   
               
               
                 1261 
                   aaggagcctg   cacccaccac   caccaaggag   cctgcaccca   ccaccaccaa   gtctgcaccc   
               
               
                 1321 
                   accactccca   aggagcctgc   acccaccacc   cccaagaagc   ctgccccaac   tacccccaag   
               
               
                 1381 
                   gagcctgcac   ccaccactcc   caaggagcgc   acacccacca   ctcccaagga   gcctgcaccc   
               
               
                 1441 
                   accaccaagg   agcctgcacc   caccactccc   aaagagcttg   cacccactgc   ccccaagaag   
               
               
                 1501 
                   cctgccccaa   ctacccccaa   ggagcctgca   cccaccactc   ccaaggagcc   tgcacccacc   
               
               
                 1561 
                   accaccaagg   agccttcacc   caccactccc   aaggagcctg   cacccaccac   caccaagctc   
               
               
                 1621 
                   gcacccacca   ctaccaagga   gcctgcaccc   accactacca   agtctgcacc   caccactccc   
               
               
                 1681 
                   aaggagcctt   cacccaccac   caccaaggag   cctgcaccca   ccactcccaa   ggagcctgca   
               
               
                 1741 
                   cccaccaccc   ccaagaagcc   tgccccaact   acccccaagg   agcctgcacc   caccactccc   
               
               
                 1801 
                   aaggaacctg   cacccaccac   caccaagaag   cctgcagcca   ccgctcccaa   agagcctgcc   
               
               
                 1861 
                   ccaactaccc   ccaaggagac   tgcacccacc   acccccaaga   agctcacgcc   caccaccccc   
               
               
                 1921 
                   gagaagctcg   cacccaccac   ccctgagaag   cccgcaccca   ccacccctga   ggagctcgca   
               
               
                 1981 
                   cccaccaccc   ctgaggagcc   cacacccacc   acccctgagg   agcctgctcc   caccactccc   
               
               
                 2041 
                   aaggcagcgg   ctcccaacac   ccctaaggag   cctgctccaa   ctacccctaa   ggagcctgct   
               
               
                 2101 
                   ccaactaccc   ctaaggagcc   tgctccaact   acccctaagg   agactgctcc   aactacccct   
               
               
                 2161 
                   aaagggactg   ctccaactac   cctcaaggaa   cctgcaccca   ctactcccaa   gaagcctgcc   
               
               
                 2221 
                   tccaaggagc   ttgcacccac   caccaccaag   gagcccacat   ccaccacctc   tgacaagccc   
               
               
                 2281 
                   gctccaacta   cccctaaggg   gactgctcca   actaccccta   adgadcctgc   tccaactacc   
               
               
                 2341 
                   cctaaggagc   ctgctccaac   tacccctaag   gggactgctc   caactaccct   caaggaacct   
               
               
                 2401 
                   gcacccacta   ctcccaagaa   gcctgccccc   aaggagcttg   cacccaccac   caccaagggg   
               
               
                 2461 
                   cccacatcca   ccacctctga   caagcctgct   ccaactacac   ctaaggagac   tgctccaact   
               
               
                 2521 
                   acccccaagg   agcctgcacc   cactaccccc   aagaagcctg   ctccaactac   tcctgagaca   
               
               
                 2581 
                   cctcctccaa   ccacttcaga   ggtctctact   ccaactacca   ccaaggagcc   taccactatc   
               
               
                 2641 
                   cacaaaagcc   ctgatgaatc   aactcctgag   ctttctgcag   aacccacacc   aaaagctctt   
               
               
                 2701 
                   gaaaacagtc   ccaaggaacc   tggtgtacct   acaactaaga   ctcctgcagc   gactaaacct   
               
               
                 2761 
                   gaaatgacta   caacagctaa   agacaagaca   acagaaagag   acttacgtac   tacacctgaa   
               
               
                 2821 
                   actacaactg   ctgcacctaa   gatgacaaaa   gagacagcaa   ctacaacaga   aaaaactacc   
               
               
                 2881 
                   gaatccaaaa   taacagctac   aaccacacaa   gtaacatcta   ccacaactca   agataccaca   
               
               
                 2941 
                   ccattcaaaa   ttactactct   taaaacaact   actcttgcac   ccaaagtaac   tacaacaaaa   
               
               
                 3001 
                   aagacaatta   ctaccactga   gattatgaac   aaacctgaag   aaacagctaa   accaaaagac   
               
               
                 3061 
                   agagctacta   attctaaagc   gacaactcct   aaacctcaaa   agccaaccaa   agcacccaaa   
               
               
                 3121 
                   aaacccactt   ctaccaaaaa   gccaaaaaca   atgctcagag   tgagaaaacc   aaagacgaca   
               
               
                 3181 
                   ccaactcccc   gcaagatgac   atcaacaatg   ccagaattga   aaccctacctc   aagaatagca   
               
               
                 3241 
                   gaagccatgc   tccaaaccac   caccagacct   aaccaaactc   caaactccaa   actagttgaa   
               
               
                 3301 
                   gtaaatccaa   agagtgaaga   tgcaggtggt   gctgaaggag   aaacacctca   tatgcttctc   
               
               
                 3361 
                   aggccccatg   tgttcatgcc   tgaagttact   cccgacatgg   attacttacc   gagagtaccc   
               
               
                 3421 
                   aatcaaggca   ttatcatcaa   tcccatgctt   tcc gatgaga ccaatatatg ccatggtaag 
               
               
                 3481 
                 ccagtagatg gactgactac tttgcgcaat gggacattag ttgcattccg aggtcattat 
               
               
                 3541 
                 ttctggatgc taagtccatt cagtccacca tctccagctc gcagaattac tgaagtttgg 
               
               
                 3601 
                 ggtattcctt cccccattga tactgttttt actaggtgca actgtgaagg aaaaactttc 
               
               
                 3661 
                 ttctttaagg attctcagta ctggcgtttt accaatgata taaaagatgc agggtacccc 
               
               
                 3721 
                 aaaccaattt tcaaaggatt tggaggacta actggacaaa tagtggcagc gctttcaaca 
               
               
                 3781 
                 gctaaatata agaactggcc tgaatctgtg tattttttca agagaggtgg cagcattcag 
               
               
                 3841 
                 cagtatattt ataaacagga acctgtacag aagtgccctg gaagaaggcc tgctctaaat 
               
               
                 3901 
                 tatccagtgt atggagaaat gacacaggtt aggagacgtc gctttgaacg tgctatagga 
               
               
                 3961 
                 ccttctcaaa cacacaccat cagaattcaa tattcacctg ccagactggc ttatcaagac 
               
               
                 4021 
                 aaaggtgtcc ttcataatga agttaaagtg agtatactgt ggagaggact tccaaatgtg 
               
               
                 4081 
                 gttacctcag ctatatcact gcccaacatc agaaaacctg acggctatga ttactatgcc 
               
               
                 4141 
                 ttttctaaag atcaatacta taacattgat gtgcctagta gaacagcaag agcaattact 
               
               
                 4201 
                 actcgttctg ggcagacctt atccaaagtc tggtacaact gtccttagac tgatgagcaa 
               
               
                 4261 
                 aggaggagtc aactaatgaa gaaatgaata ataaattttg acactgaaaa acattttatt 
               
               
                 4321 
                 aataaagaat attgacatga gtataccagt ttatatataa aaatgttttt aaacttgaca 
               
               
                 4381 
                 atcattacac taaaacagat ttgataatct tattcacagt tgttattgtt tacagaccat 
               
               
                 4441 
                 ttaattaata tttcctctgt ttattcctcc tctccctccc attgcatggc tcacacctgt 
               
               
                 4501 
                 aaaagaaaaa agaatcaaat tgaatatatc ttttaagaat tcaaaactag tgtattcact 
               
               
                 4561 
                 taccctagtt cattataaaa aatatctagg cattgtggat ataaaactgt tgggtattct 
               
               
                 4621 
                 acaacttcaa tggaaattat tacaagcaga ttaatccctc tttttgtgac acaagtacaa 
               
               
                 4681 
                 tctaaaagtt atattggaaa acatggaaat attaaaattt tacactttta ctagctaaaa 
               
               
                 4741 
                 cataatcaca aagctttatc gtgttgtata aaaaaattaa caatataatg gcaataggta 
               
               
                 4801 
                 gagatacaac aaatgaatat aacactataa cacttcatat tttccaaatc ttaatttgga 
               
               
                 4861 
                 tttaaggaag aaatcaataa atataaaata taagcacata tttattatat atctaaggta 
               
               
                 4921 
                 tacaaatctg tctacatgaa gtttacagat tggtaaatat catctgctca acatgtaatt 
               
               
                 4981 
                 atttaataaa actttggaac attaaaaaaa taaattggag gcttaaaaaa aaaaaaaaaa 
               
               
                 5041 
                 a 
               
               
                   
               
             
          
         
       
     
         [0039]    In addition to the tribonectins described above, recombinant lubricin molecules and other compositions described in U.S. Patent Application Pub. No. 2007/0191268 are used in combination with tribonectins, HA, and/or inhibitors of proinflammatory mediators to preserve, prolong, or augment joint lubrication. 
       p38 and Inflammation 
       [0040]    A strong link has been established between the p38 pathway and inflammation. Inhibition of inflammation via the p38 pathway leads to enhanced lubricin/tribonectin production. The activation of the p38 pathway plays essential roles in the production of proinflammatory cytokines (IL-1, TNF- and IL-6); induction of enzymes such as COX-2 which controls connective tissue remodeling in pathological conditions; expression of intracellular enzymes such as iNOS, a regulator of oxidation; induction of VCAM-1 and other adherent proteins along with other inflammatory related molecules. In addition, a regulatory role for p38 in the proliferation and differentiation of immune system cells such as GM-CSF, EPO, CSF and CD-40 has been established. Inhibition of elements of this pathway together with administration of a tribonectin lead to improved joint lubrication over prolonged periods of time. 
       Inhibitors of TNF-Alpha 
       [0041]    Exemplary functional blockers of TNF-α include, but are not limited to, recombinant and/or soluble TNFα receptors, monoclonal antibodies, and small molecule antagonists and/or inverse agonists. Exemplary commercial TNF-α blocking agents include, etanerept/ENBREL™, infliximab/Remicade, and adalimumab/Humira. 
         [0042]    Etanercept (ENBREL™, co-marketed by Amgen and Wyeth) is a recombinant human soluble TNF-α receptor (DrugBank BTD0052). It is a small protein (75 kDa) that binds TNFα and decreases its role in inflammation. ENBREL™ is a dimeric fusion protein comprised of the extracellular ligand-binding portion of the human 75 kilodalton (p75) tumor necrosis factor receptor (TNFR) linked to the Fc portion of human IgG1 . The Fc component of etanercept contains the CH2 domain, the CH3 domain and hinge region, but not the CH1 domain of IgG1. Etanercept is produced by recombinant DNA technology in a Chinese hamster ovary (CHO) mammalian cell expression system and is comprised of 934 amino acids (GenBank M32315). Etanercept binds specifically to tumor necrosis factor (TNF) and blocks its interaction with cell surface TNF receptors. The biological activity of TNF is dependent upon binding to either cell surface receptor (p75 or p55). Etanercept is a dimeric soluble form of the p75 TNF receptor that can bind to two TNF molecules, thereby effectively removing them from circulation. 
       Inhibitors of IL-1  
       [0043]    Inhibitors or antagonists of proinflammatory cytokine IL-1 include anakinra/KINERET™ (recombinant human IL-1Ra, rhIL-1Ra). IL-1Ra is an endogenous receptor antagonist, which is primarily produced by activated monocytes and tissue macrophages and inhibits the activities of the proinflammatory forms of IL-1 by competitively binding to IL-1 receptor. IL-1Ra is an inducible gene that is often upregulated in inflammatory conditions. Although the binding affinity of natural IL-1Ra is similar to that of IL-1, it lacks IL-1 agonist activity. 
         [0044]    Exemplary IL-1Ra compositions include NCBI Accession No. NM — 173842 (Human IL-1Ra, transcript 1), which encodes amino acid sequence NCBI Accession No. NM — 173842; Human IL-1Ra, transcript 2 NCBI Accession No. NM — 173841 which encodes amino acid sequence NCBI Accession No. NM — 173841; and Human IL-1Ra, transcript 3 NCBI Accession No. NM — 000577 which encodes amino acid sequence (NCBI Accession No. NM — 000577). Anakinra/KINERET™ is encoded by transcript sequence NCBI Accession No. M55646, and the amino acid sequence of Anakinra/KINERET™ is recorded at DrugBank Accession No. BTD00060. 
         [0045]    Inhibitory antibodies include anti-IL-1α (U.S. Patent No. 20030026806), anti-IL-1β (U.S. Patent No.20030026806) and humanized monoclonal anti-IL-1R (Amgen, U.S. Patent No. 2004022718 and Roche, U.S. Patent No. 2005023872). 
       EXAMPLE 1 
     Reversible Rise in Joint Ex-Vivo μ is a Result of Decreased Synovial Lubricin Expression in an Antigen-Induced Arthritis Model 
       [0046]    Data obtained with an antigen-induced arthritis rat model using limbs studied ex vivo showed that a reversible rise in joint ex-vivo μ was attributable to decreased synoviocyte lubricin expression. The ex-vivo μ of arthritic joints was significantly (P&lt;0.001) higher than the μ of contra-lateral joints at 7, 21 and 24 days following the induction of arthritis. Lubricin mRNA expression in the synovial tissues of the arthritic joints relative to those of the contralateral joints was significantly (P&lt;0.001) lower at days 4 and 7 compared to control. At day 28 following arthritis induction, there was no significant difference between relative lubricin mRNA expression in the arthritic and control joints. Decreased lubricin expression in the superficial zone of articular chondrocytes was demonstrated by decreased lubricin staining in the arthritic joints at 7 days following the initiation of arthritis ( FIG. 1 ). 
       EXAMPLE 2 
     ACL Injury Results in Loss of Joint Lubricating Ability and Damage to the Articular Surface in Rats 
       [0047]    The method described above was applied to a rat ACL injury model. 12 rats underwent ACL transection (ACLT) of the right rear limb knee joints. Following 7 and 28 days both the right and left rear limbs from 6 animals at each time point were harvested and underwent pendulum studies to determine ex-vivo μ. Following pendulum studies, joints were lavaged and SF lavages were analyzed for sulfated glycosaminoglycan (sGAG) levels using alcian blue binding assay. Immunohistochemical staining for surface damage and presence of lubricin, using a lubricin-specific monoclonal antibody was also performed. The ex-vivo μ values of ACLT joints were significantly elevated at 4 weeks following injury compared to either contralateral (CL) joints at week 4 or ACLT joints at 1 week following the injury ( FIG. 2 ). This time-dependent elevation of friction was associated with increased sGAG SF lavage levels ( FIG. 3 ). At 1 week following the injury, sGAG levels dramatically increased in the ACLT joints compared to the contra-lateral joints. At 4 weeks following the injury, the ACLT joints demonstrated elevated SF lavage sGAG levels compared to the contralateral joints. However, sGAG levels of SF lavages at week 4 were significantly (p&lt;0.001) lower than sGAG levels of SF lavages at week 1. 
       EXAMPLE 3 
     Administration of Etanercept Blunts the Catabolic Decrease in Synovial Fluid Lubricin Concentration Following ACLT 
       [0048]    Studies were carried out to investigate the effects of blocking TNF-α at an early stage after injury on SF lubricin levels, joint lubrication and cartilage damage. Inhibition of TNF-α was performed using etanercept, a TNF-α soluble receptor that is approved for treatment of rheumatoid arthritis. 
         [0049]    The following reagents and methods were used to evaluate inhibition of the effects of TNF-α by etanercept and the preservation of joint lubrication and decrease cartilage damage following an ACL injury. ACL-transection (ACLT) model and entanercept administration: Male Lewis rats 7-8 weeks were assigned to either an ACL-transection group (n=5), etanercept-treated ACL-transection group (n=5) or a sham-group (n=4). Etanercept was given subcutaneously at 0.5 mg/kg on days 1, 3, 5, 7, 9, 11, and 13 following ACLT. At 14 days following surgery, ACLT, contralateral (CL) and sham (S) joints were harvested and lavaged. Lavaging was performed by injecting 100 μl in the joint capsule, flexing and extending the joint for ten times and aspirating 50 μl of fluid. SF Lubricin levels: Lubricin levels were determined using sandwich ELISA employing a lubricin-specific monoclonal antibody, and peanut agglutinin (PNA). Ex-vivo joint μ: Articular joints&#39; μ values were determined using modified Stanton pendulum (Elsaid K A et al, Arthritis Rheum 2007; 56:108-116). sGAG levels in SF lavages: Total sGAG levels were determined by Alcian blue binding assay. 
         [0050]    The concentrations of lubricin in the SF lavages were found to be significantly (p&lt;0.001) higher in the etanercept-treated ACLT joints compared to the non-treated joints ( FIG. 4 ). The ex-vivo joints μ valves were significantly (p=0.021) lower in the etanercept-treated ACLT compared to the non-treated joints ( FIG. 5 ). sGAG levels in SF were significantly (p&lt;0.001) lower in the etanercept-treated group compared to the non-treated group ( FIG. 6 ). At an early stage following an acute ACL injury, inhibition of the effects of TNF-α leads to a significant increase in SF lubricin levels and decrease in joint μ. This effect is associated with decreased cartilage damage as determined by a decrease in sGAG turnover. Early intervention following ACL injury by blocking the effects of TNF-α preserved articular joint&#39;s chondroprotective mechanisms and decreased the extent of cartilage-injury. 
         [0051]    ENBREL™ administered subcutaneously led to an upregulation of lubricin, leading to effective chondroprotection and prevention/reduction of cartilage wear. The data indicate that a TNF-α inhibitor (e.g., ENBREL™) is effective in increasing lubricin levels. A combination of recombinant lubricin and ENBREL™ leads to a greater clinical response in the cartilage preservation endpoint by the inhibition of wear. Early intervention is beneficial in retarding long-term cartilage damage and development of secondary osteoarthritis following ACL injury. 
       EXAMPLE 4 
     Comparison of Early and Late Inhibition of TNF-α in Restoring Chondroprotection by Lubricin in the ACL Transection Injury Model 
       [0052]    Etanercept was administered to animals, and condroprotection was evaluated using an ACL-transection model. Male Lewis rats 7-8 weeks were assigned to either an ACL-transection group (n=5), 2 etanercept-treated ACLT groups (n=12, 6 in each group) or a sham surgery group (n=4). Etanercept was given subcutaneously at 0.5 mg/kg on days 8, and 15 following transection (Treatment A) or on days 15 and 22 (Treatment B). At 28 days following transection, ACL-transected (ACLT), contralateral (CL) and sham joints were harvested and lavaged. Lavaging was performed by injecting 50 μl of normal saline in the joint capsule, flexing and extending the joint for ten times and then aspirating 30 μl of fluid. 
         [0053]    SF lubricin levels were determined using a sandwich ELISA employing a lubricin-specific monoclonal antibody such as 9G3, and peanut agglutinin. Surface-associated lubricin coverage was calculated from 9G3-stained histological specimens. Quantitiation of lubricin staining intensity was calculated using Image Pro-Plus software (Media Cybernetics, MD, USA) with pre-determined threshold parameters and expressed as percentage of lubricin surface coverage. An average of lubricin surface coverage of the medial and lateral regions of femur and tibial cartilage surfaces was calculated. Articular joints&#39; μ values were determined ex vivo using modified Stanton pendulum (Elsaid K A et al., Arthritis Rheum., 2007,56:108-116).The SF lavage lubricin levels in treatment-A ACLT joints were found to be significantly (p&lt;0.01) higher than in control or treatment B joints ( FIG. 7 ). SF lavage lubricin levels in CL joints were comparable across the different groups. Lubricin coverage of articular surfaces was significantly (p&lt;0.01) lower in the ACLT joints compared to treatments A, B and sham surgery. Lubricin coverage in the CL joints was comparable across the different groups. The joints&#39; μ values were comparable in treatments A, and B and were lower than the μ values of ACLT joints, but approximated the μ values of sham surgery joints (Table 3). 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                 TABLE 3 
               
               
                   
                   
               
               
                   
                 ACLT 
                 Treatment A 
                 Treatment B 
                 Sham 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                 ACLT 
                 8.9*10 −4  ± 
                 6.5*10 −4  ± 
                 6.6*10 −4  ± 
                 6.1*10 −4  ± 
               
               
                   
                 4.6*10 −5   
                 6.1*10 −5   
                 8.6*10 −5   
                 1.6*10 −5   
               
               
                 CL 
                 6.6*10 −4  ± 
                 4.7*10 −4  ± 
                 4.9*10 −4  ± 
                 6.2*10 −4  ± 
               
               
                   
                 1.5*10 −5   
                 1.5*10 −5   
                 8.6*10 −5   
                 1.6*10 −5   
               
               
                   
               
             
          
         
       
     
         [0054]    Table 3 shows the mean coefficient of friction (μ)±standard error of the mean (SEM) of ACLT and CL joints of different groups. Inhibition of the effects of TNF-α by etanercept treatment re-establishes lubricin coverage on the surface of articular cartilage and is associated with a reduction in joints&#39; coefficient of friction compared to non-treated joints. Initiating TNF-α inhibition early (treatment A) results in higher SF lavage lubricin levels compared to later intervention (treatment B). Differences in SF lavage lubricin levels did not correspond to changes in surface-associated lubricin or changes in ex-vivo μ values. Articular cartilage surface coverage with lubricin was associated with a lowering of μ values in etanercept-treated joints compared to no treatment. These results indicate that surface-associated lubricin is more important than SF lubricin in modulating articular lubrication ability. Two early administrations of etanercept was found to be more effective than later treatment in re-establishing high SF lubricin which may serve as a reservoir of lubricin for articular surfaces. This administration approach is useful as an alternative or adjunct to intra-articular lubricin supplementation. TNF-α inhibitory compositions either alone or in combination with a tribonectin confer a clinical benefit by increasing the amount of surface-associated lubricin and reducing the co-efficient of friction in the joint. 
       OTHER EMBODIMENTS 
       [0055]    While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.