Patent Application
20080287369
The invention relates to lubrication of mammalian joints.
Osteoarthritis (OA) is the one of the most 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.
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 (HA) and derivatives thereof to joint tissue to increase the viscosity of synovial fluid. Despite the useful properties of HA, such as biocompatibility, (bio)degradability, resorption, non-immunogenicity, very low and rare pyrogenicity, it is a highly viscous material, with poor lubricating properties. Still needed are improved methods and compositions for viscosupplementation.
Accordingly, in a first aspect, the present invention features a viscosupplementation composition that includes hyaluronic acid, or a polymer thereof, a concentration of from 1.0 mg/mL to 5 mg/mL and tribonectin at a concentration of from 10 μg/mL to 250 μg/mL. As described in U.S. Pat. No. 6,743,774, a tribonectin is an artificial boundary lubricant which contains at least one repeat of an amino acid sequence which is at least 50% identical to KEPAPTT (SEQ ID NO:3). A tribonectin is formulated for administration to a mammalian joint. Preferably, the tribonectin is a recombinant or chemically-synthesized lubricating polypeptide. For example, a tribonectin includes a substantially pure polypeptide the amino acid sequence of which includes at least one but less than 76 subunits. Each subunit contains at least 7 amino acids (and typically 10 or fewer amino acids). The amino acid sequence of each subunit is at least 50% identical to SEQ ID NO:3, and a non-identical amino acid in the reference sequence is a conservative amino acid substitution. For example, one or both of the threonine residues are substituted with a serine residue. Preferably, the amino acid sequence of the subunit is identical to SEQ ID NO:3. The tribonectin may also contain one or more repeats of the amino acid sequence XXTTTX (SEQ ID NO:4). Polypeptides or other compounds described herein are said to be “substantially pure” when they are within preparations that are at least 60% by weight (dry weight) the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. Purity can be measured by any appropriate standard method, for example, by column chromatography, polyacrylaminde gel electrophoresis, or HPLC analysis.
Where a particular polypeptide is said to have a specific percent identity to a reference polypeptide of a defined length, the percent identity is relative to the reference polypeptide. Thus, a peptide that is 50% identical to a reference polypeptide that is 100 amino acids long can be a 50 amino acid polypeptide that is completely identical to a 50 amino acid long portion of the reference polypeptide. It can also be a 100 amino acid long polypeptide which is 50% identical to the reference polypeptide over its entire length.
A polypeptide which is “substantially identical” to a given reference polypeptide or nucleic acid molecule is a polypeptide having a sequence that has at least 85%, preferably 90%, and more preferably 95%, 98%, 99% or more identity to the sequence of the given reference polypeptide sequence or nucleic acid molecule. The term, “identity” has an art-recognized meaning and is calculated using well known published techniques, e.g., Computational Molecular Biology, 1988, Lesk A. M., ed., Oxford University Press, New York; Biocomputing: Informatics and Genome Projects, 1993, Smith, D. W., ed., Academic Press, New York; Computer Analysis of Sequence Data, Part I, 1994, Griffin, A. M. and Griffin, H. G., eds., Humana Press, New Jersey; Sequence Analysis in Molecular Biology, 1987, Heinje, G., Academic Press, New York; and Sequence Analysis Primer, 1991, Gribskov, M. and Devereux, J., eds., Stockton Press, New York). A tribonectin is characterized as reducing the coefficient of friction (μ) between bearing surfaces. For example, reduction of friction is measured in vitro by detecting a reduction in friction in a friction apparatus using latex:glass bearings. Reduction of friction is also measured in vivo, e.g., by measuring reduction of patient pain. Tribonectins of the invention are lubricating substances or components of compositions. Polypeptides that have at least 50% (but less than 100%) amino acid sequence identity to a reference sequence are tested for lubricating function by measuring a reduction in the μ between bearing surfaces.
A tribonectin may include an O-linked oligosaccharide, e.g., an N-acetylgalactosamine and galactose in the form β(1-3)Gal-GalNAC. For example, KEPAPTT (SEQ ID NO:3) and XXTTTX (SEQ ID NO:4) repeat domains are glycosylated by β(1-3)Gal-GalNAC (which may at times be capped with NeuAc in the form of β(1-3)Gal-GalNAC-NeuAc. The term “glycosylated” with respect to a polypeptide means that a carbohydrate moiety is present at one or more sites of the polypeptide molecule. For example, at least 10%, preferably at least 20%, more preferably at least 30%, and most preferably at least 40% of the tribonectin is glycosylated. Up to 50% or more of the tribonectin can be glycosylated. Percent glycosylation is determined by weight.
A tribonectin can contain a substantially pure fragment of megakaryocyte stimulating factor (MSF). For example, the molecular weight of a substantially pure tribonectin having an amino acid sequence of a naturally-occurring tribonectin is in the range of 220-280 kDa. Preferably, the apparent molecular weight of a tribonectin is less than 230 kDa, more preferably less than 250 kDa, and most preferably less than 280 kDa. A protein or polypeptide fragment is defined as a polypeptide which has an amino acid sequence that is identical to part, but not all, of the amino acid sequence of a naturally-occurring protein or polypeptide from which it is derived, e.g., MSF. The tribonectin may contain a polypeptide, the amino acid sequence of which is at least 50% identical to the sequence of residues 200-1140, inclusive, of SEQ ID NO:1 (see Table 1), e.g., it contains the amino acid sequence of residues 200-1140, inclusive, of SEQ ID NO:1. In another example, the polypeptide contains an amino acid sequence that is at least 50% identical to the sequence of residues 200-1167, inclusive, of SEQ ID NO:1, e.g., one having the amino acid sequence identical to residues 200-1167, inclusive, of SEQ ID NO: 1. The polypeptide contains an amino acid sequence that is at least 50% identical to the sequence of residues 200-1212, inclusive, of SEQ ID NO: 1, e.g., the amino acid sequence of residues 200-1212, inclusive, of SEQ ID NO:1, or the polypeptide contains an amino acid sequence that is at least 50% identical to the sequence of residues 200-1263, inclusive, of SEQ ID NO: 1, e.g., an amino acid sequence identical to residues 200-1263, inclusive, of SEQ ID NO:1. Preferably, the sequence of the polypeptide lacks the amino acid sequence of residues 1-24, inclusive, of SEQ ID NO:1 and/or the amino acid sequence of residues 67-104, inclusive of SEQ ID NO:1.
In one embodiment, the hyaluronic acid is at a concentration of from 2.5 mg/mL to 5.0 mg/mL, or at a concentration of from 3.0 mg/mL to 4.0 mg/mL. In another embodiment, a HA/tribonectin composition of the invention includes hyaluronic acid and tribonectin are at a molar ratio of from 2:1 to 4:1, respectively.
In another aspect, the invention features a method of lubricating a mammalian joint by contacting the joint with a composition of the invention. The mammal is preferably a human, horse, dog, ox, donkey, mouse, rat, guinea pig, cow, sheep, pig, rabbit, monkey, or cat, and the joint is an articulating joint such as a knee, elbow, shoulder, hip, or any other weight-bearing joint. The compositions of the present invention can be administered intra-articularly.
In yet another aspect, the invention features a method of increasing the elasticity of a viscosupplement for the lubrication and chondroprotection of a mammalian joint by adding a tribonectin to the viscosupplement. In one embodiment, the viscosupplement also includes hyaluronic acid. In another embodiment, the tribonectin is added to a final concentration of 10 μg/mL to 250 μg/mL. In another embodiment, the ratio of hyaluronic acid to tribonectin is from 2:1 to 4:1, respectively, after the addition of the tribonectin. The mammal is preferably a human, horse, dog, ox, donkey, mouse, rat, guinea pig, cow, sheep, pig, rabbit, monkey, or cat, and the joint is an articulating joint such as a knee, elbow, shoulder, hip, or any other weight-bearing joint. The viscosupplement can be administered intra-articularly. Alternatively, the mammalian joint can be treated first with a viscosupplement and then subsequently treated separately with the tribonectin, which is added to the viscosupplement in vivo.
a through 7d are graphs showing the viscoelastic behavior of glycerol, bovine synovial fluid (BSF), trypsinized BSF, and synovial fluid from a human patient with camptodactyly-arthropathy-coxa vara-pericarditis syndrome, respectively.
Synovial fluid is a semi-dilute solution of hyaluronate (HA) with additional constituents that play a wide variety of biological roles, which may include the regulation of the molecular structure of the fluid. Hyaluronic acid is a naturally-occurring polysaccharide containing alternating N-acetyl-D-glucosamine and D-glucuronic acid monosaccharide units linked with beta 1-4 bonds and the disaccharide units linked with beta 1-3 glycoside bonds with molecular weight range of about 50,000 to 8×106 Synovial hyaluronate is a long linear negatively charged polyelectrolyte molecule with rotational bonds, usually occurring as the sodium salt (sodium hyaluronate). Intra-articular (injection) administration of high-molecular-weight HA to the patients is described as an effective procedure in the treatment of traumatized arthritic joints (Kikuchi et al., Osteoarthritis and Cartilage 4:99, 1996). The average molecular weight of synovial HA of healthy humans lies in the range (1.6-10.9)×106 Da; while its concentration equals 2˜4 mg/mL (Balazs et al., Arthritis Rheum. 10:357, 1967). Molecular weight values of commercially available HA preparations obtained from various (natural) sources such as, e.g., bacteria Streptococcus zooepidemicus or Streptococcus equii, rooster combs, etc., vary in the range from hundreds of thousands to ca. 1-2 million Da. High-molecular-weight HA binds up to 1000 times more water than is its own mass and forms pseudoplastic, clastoviscous solutions, that behave as soft gels that reveal so-called shear-dependent viscosity and frequency-dependent elasticity (Larsen and Balazs, Adv. Drug Delivery Rev. 7:279, 1991). At the low magnitude of the shear tension, solutions of high-molecular-weight HA reveal high viscosity and low elasticity; while at the increasing values of shear tension the solutions become more elastic (Simon, Osteoarthritis 25:345, 1999). Such non-Newtonian behavior of synovial fluid is essential for the lubrication of joints during the (fast) movement. The cartilage surface is covered by a thin film of SF that smoothens (fine) unevenness of the articular structure. Deficiency of this layer leads to increased friction coefficient between the moving parts of the joint which results in strong pain (Nishimura et al., Biochim. Biophys. Acta 1380:1, 1998). Ultrapure (ready for injection application) preparations of the elastoviscous solutions of the hyaluronan sodium salt (HEALON™; Pharmacia, Uppsala, Sweden), obtained from the rooster combs, have found extended application especially in opthalmology (viscosurgery) (Nimrod et al, J. Ocular Pharmacol. 8:161, 1992], as well as in rheumatology (viscosupplementation) (Peyron, J. Rheumatology 20 Suppl. 39:10, 1993; T. Kikuchi et al, Osteoarthritis and Cartilage 4:99, 1996).
Recently another preparation for the intra-articular administration to OA patients was approved in the USA and some other countries. This product named HYLAN™ (Biomatrix Inc., Ridgefield, N.J., USA), contains high-molecular-weight HA originating from the rooster combs, and includes additionally cross-linked HA (L. S. Simon, Osteoarthritis 25:345, 1999). The water-soluble HYLANs with ultra-high molecular weight (on average around 6×106 Da) that were prepared by chemical cross-linking of HA with formaldehyde reveal a significantly longer biological half-life period (Simon, Osteoarthritis 25:345, 1999). See also Larsen and Balazs, Adv. Drug Delivery Rev. 7:279, 1991; Al-Assaf et al, Radiat. Phys. Chem. 46:207, 1995; and Wobig et al., Clin. Ther. 20:41, 19980 for summaries of pre-clinical and clinical trials involving injections of HYLAN™ solutions. Other HA-based viscosupplements are known as, Hyalgan™, Artzal™, Suplazyn™, BioHy™, Orthovisc™, and Synvisc™. As used herein, the term hyaluronic acid, abbreviated as HA, means hyaluronic acid, a cross-linked form of HA, or its salts of hyaluronic acid, such as, for example, sodium hyaluronate, potassium hyaluronate, magnesium hyaluronate, and calcium hyaluronate.
Hyaluronic acid was once thought to add viscoelastic effects to synovial fluid to enable hydrodynamic lubrication during periods of fast joint reciprocation. Under these circumstances some of the load from locomotion is borne by wedges of fluid between the articular surfaces. This effect restores ‘shock absorber’ characteristics to the diseased synovial fluid. Naturally occurring hyaluronate from human umbilical cord and rooster comb were used. Transformation into hyalns was performed by cross-linking hydroxyl groups creating high molecular weight polymer networks (Pelletier and Martel-Pelletier, J Rheumatol (suppl 39)20:19-24, 1993.
An unintended consequence of meshed polymers is the creation of excluded volume which inhibit small molecule movement. For example, a 0.3 mg/mL solution of cross-linked hyaluronate requires 1 liter of aqueous solvent in order to be fully solvated. This concentration is 10 times less than normal synovial hyaluronate concentration, with the result that, in synovial fluid, each HA polymer is touching another. Understandably, injection of 0.3 mg of a viscosupplement into a confined knee joint would have significant effects on the rheological properties of a patient's synovial fluid and arrest small molecule movement by utilization of all available solvent. For example, solvation requirements would exclude cytokines and nociceptive mediators from triggering pain while restoring viscoelasticity.
Chondroprotection is served by a very different mechanism in synovial fluid. Synovial fluid is present to provide for lubrication of apposed and pressurized cartilaginous surfaces and to also nourish chondrocytes, as these highly specialized cells have no supportive blood supply. Digesting synovial fluid with hyaluronidase results in a non-viscous fluid which continues to lubricate (McCutchen, Wear 5:412-15, 1962). Synovial fluid digested with trypsin results in a viscous fluid which fails to lubricate (McCutchen, Fed Proc Fed Am Soc Exp Bio 25:1061-68, 1966 and Jay, Conn Tiss Re 28:71-88, 1992). The phenomenon of lubricating in the absence of viscosity is termed “boundary lubrication”.
The modicum of therapeutic value in the intra-articular administration of viscosupplements may be appropriate for those patients unable to tolerate NSAIDs (Lo et al., J. Am. Med. Assoc. 290:3115-21, 2003). However, the routine use of these devices in treating OA effectively is not well established as their mechanism of action is unclear. Multiple injections are required and therapeutic value is typically not seen until 3-6 months later, but can last longer than intra-articular steroid administration (Caborn et al., J. Rheumatol. 31:333-43. It should be appreciated that the HA-based viscosupplements that are currently commercially available are not articular lubricants and more likely work as retardants of pro-inflammatory factors. This effect may be more pronounced as the molecular weight of the hyaluronate is increased.
The human joint disease group most closely aligned with race horses that are treated with viscosupplements are active patients with inflammatory joint conditions (Vad et al., Sports Medicine 32:729-39, 2002) and not those with advanced OA. Deficient lubricating ability among patients with synovitis stands paradoxically in contrast to synovial fluid aspirated from joints of patients afflicted with OA (Jay et al., J Rheumatol 31:557-64, 2004). These former patients demonstrate absent lubricating ability. By contrast, patients with OA have normal lubricating ability. These intriguing observations are partly explained by the fact that the lubricating moiety is produced by superficial zone articular chondrocytes (Flannery et al. Biochem. Biophys. Res. Comm. 234:535-41, 1999) and synovial fibroblasts (Jay et al., J. Rheumatol. 27:594-600, 2000), secreting superficial zone protein (SZP) and lubricin respectively. Both are highly homologous protein products of megakaryocyte stimulating factor gene expression. Patients with advanced OA undoubtedly may lack superficial zone chondrocytes and yet continue to have normal synovial fluid lubricating ability, suggesting that the synovial fibroblast contribution continues. Disease states such as traumatic synovitis and RA, exemplified by synovial fluid deficient in lubricating ability, have both cell types affected. The histopathologic appearance of traumatic synovitis is similar to RA but less intense and extensive (Brit. J. Rheum.; 29:422-25, 1990). Inflammatory processes can lead to IL-1α expression which in the case of superficial zone articular chondrocytes, down regulates expression of SZP/lubricin and can ultimately lead to proteolysis. Arresting this process while at the same time restoring some of the mechanical features of synovial fluid (even the viscoelasticity by itself) may be of some importance. The implication is that an unlubricated joint will result in cartilage injury and premature wear, consequently leading to the fibrillation of cartilage and appearance OA.
The rheology of hyaluronate depends on aggregates and proteins present in the fluid (see Gribbon et al., Biochem. 350:329-35, 2000; Krause et al. Biomacromol. 2:65-9, 2001; and Pelletier et al., J. Biomed. Res. 54:102-8, 2001). The transport of nutrients and factors is greatly influenced by the molecular structure of the fluid. As noted above, two products of the gene PRG4, lubricin expressed by synovial fibroblasts (Jay et al., Conn. Tiss. Res. 28:245-55, 1992) and superficial zone protein expressed by surface chondrocytes (Jay et al., J. Rheum. 27:594-600, 2000) participate in the boundary lubrication of cartilaginous joints. Hyaluronate and lubricin synergistically reduce friction under high loads, although hyaluronate alone does not have lubrication ability (Flannery et al. Biochem. Biophys. Res. Comm. 234:535-41, 1999).
Tribonectin, similar to proteoglycan 4 (PRG4), articular cartilage superficial zone protein (SZP), megakaryocyte stimulating factor precursor, or lubricin (Ikegawa et al., Cytogenet. Cell. Genet. 90:291-297, 2000; Schumacher et al., Arch. Biochem. Biophys. 311:144-152, 1994; Jay and Cha, J. Rheumatol., 26:2454-2457, 1999; and Jay, WIPO Int. Pub. No. WO 00/64930) is a mucinous glycoprotein found in the synovial fluid (Swann et al., J. Biol. Chem. 256:5921-5925, 1981). The amino acid sequence of MSF (SEQ ID NO:1) is shown in Table 1. The gene encoding naturally-occurring full length MSF (SEQ ID NO:2) contains 12 exons, and the naturally-occurring MSF gene product contains 1404 amino acids with multiple polypeptide sequence homologies to vitronectin including hemopexin-like and somatomedin-like regions. Centrally-located exon 6 contains 940 residues and encodes a O-glycosylated mucin domain. A polypeptide encoded by nucleotides 631-3453 of SEQ ID NO:2 provides boundary lubrication of articular cartilage.
Tribonectin provides boundary lubrication of congruent articular surfaces under conditions of high contact pressure and near zero sliding speed (Jay et al., J. Orthop. Res. 19:677-87, 2001). These lubricating properties have also been demonstrated in vitro (Jay, Connect. Tissue Res. 28:71-88, 1992). Cells capable of synthesizing tribonectin have been found in synovial tissue and within the superficial zone of articular cartilage within diarthrodial joints (Jay et al., J. Rheumatol. 27:594-600, 2000).
In U.S. Pat. No. 6,743,774 and in U.S. patent application Ser. Nos. 09/897,188 and 10/038,694 are described methods of promoting lubrication between two juxtaposed biological surfaces using tribonectin, or fragments thereof. In PCT Publication No. WO 00/64930 are described tribonectin analogs and methods for lubricating a mammalian joint.
The synovial fluid of an inflamed or injured joint contains proteolytic enzymes that degrade lubricating proteins or polypeptides. For example, infiltrating immune cells such as neutrophils secrete trypsin and/or elastase. Even a minor injury to an articulating joint or an inflammatory state can result in cellular infiltration and proteolytic enzyme secretion resulting in traumatic synovitis. Synovitis for a period of a few days or weeks can result in the loss of the cytoprotective layer of a joint, which in turn leads to the loss of cartilage. Non-lubricated cartilaginous bearings may experience premature wear which may initiate osteoarthritis. Individuals who clinically present with a traumatic effusion (e.g., “water on the knee”) are predisposed to developing osteoarthritis; the elaboration of proteolytic enzymes degrades and depletes naturally-occurring lubricating compositions in the synovial fluid. Depletion of natural lubricating compositions occurs in other inflammatory joint diseases such as rheumatoid arthritis. Replacing or supplementing the synovial fluid of such injured joints with the lubricating compositions of the invention prevents the development of osteoarthritis in the long term (e.g., years, even-decades later) and immediately lubricates the joint to minimize short term damage.
Analogs, homologs, or mimetics of tribonectins which are less susceptible to degradation in vivo can also be used in the present invention. Tribonectin analogs can differ from the naturally-occurring peptides by amino acid sequence, or by modifications which do not affect the sequence, or both. Modifications (which do not normally alter primary sequence) include in vivo or in vitro chemical derivatization of polypeptides, e.g., acetylation or carboxylation. Also included are modifications of glycosylation, e.g., those made by modifying the glycosylation patterns of the polypeptide during its synthesis and processing or in further processing steps, e.g., by exposing the polypeptide to enzymes which affect glycosylation, e.g., mammalian glycosylating or deglycosylating enzymes.
Where proteolytic degradation of the peptidyl component of a composition of the present invention following injection into the subject is a problem, replacement of a particularly sensitive peptide bond with a noncleavable peptide mimetic bond renders the resulting peptide more stable, and thus more useful as a therapeutic. To render the therapeutic peptidyl component less susceptible to cleavage by peptidases such as trypsin or elastase, the peptide bonds of a peptide may be replaced with an alternative type of covalent bond (a “peptide mimetic”). Trypsin, elastase, and other enzymes may be elaborated by infiltrating immune cells-during joint inflammation. Trypsin cleaves a polypeptide bond on the carboxy-side of lysine and arginine; elastase cleaves on the carboxy-side of alanine, glycine. Thrombin, a serine protease which is present in hemorrhagic joints, cleaves a peptide bond on the carboxy-side of arginine. Collagenases are a family of enzymes produced by fibroblasts and chondrocytes when synovial metabolism is altered (e.g., during injury). These enzymes cut on the carboxy-side of glycine and proline. One or more peptidase-susceptible peptide bonds, e.g., those which appear in the KEPAPTT (SEQ ID NO:3) repeat sequence, can be altered (e.g., replaced with a non-peptide bond) to make the site less susceptible to cleavage, thus increasing the clinical half-life of the therapeutic formulation.
Such mimetics, and methods of incorporating them into polypeptides, are well known in the art. Similarly, the replacement of an L-amino acid residue with a D-amino acid is useful for rendering the a peptidyl component of a composition of the invention less sensitive to proteolysis. Also useful are amino-terminal blocking groups such as t-butyloxycarbonyl, acetyl, theyl, succinyl, methoxysuccinyl, suberyl, adipyl, azelayl, dansyl, benzyloxycarbonyl, fluorenylmethoxycarbonyl, methoxyazelayl, methoxyadipyl, methoxysuberyl, and 2,4-dinitrophenyl.
Clinical formulations of compositions of the present invention may also contain peptidase inhibitors such as N-methoxysuccinyl-Ala-Ala-Pro-Val chloromethylketone (an inhibitor of elastase). Other clinically acceptable protease inhibitors (e.g., as described in Berling et al., Int. J. Pancreatology 24:9-17, 1998) such as leupeptin, aprotinin, α-1-antitrypsin, α-2-macroglobulin, α-1-protease inhibitor, antichymotrypsin (ACHY), secretory leukocyte protease inhibitor (PSTI) can also be co-administered with a composition of the invention to reduce proteolytic cleavage and increase clinical halflife. A cocktail of two or more protease inhibitors can also be coadministered.
Compositions of that include tribonectin polypeptides can be formulated in standard physiologically-compatible excipients known in the art., e.g., phosphate-buffered saline (PBS). Other formulations and methods for making-such formulations are well known and can be found in, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000, Philadelphia or Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 2002, Marcel Dekker, New York).
Standard methods for delivery of peptides are used. Such methods are well known to those of ordinary skill in the art. For intra-articular administration, tribonectin is delivered to the synovial cavity at a concentration in the range of 20-500 .mu.g/ml in a volume of approximately 0.1-2 ml per injection. For example, 1 ml of a tribonectin at a concentration of 250 .mu.g/ml is injected into a knee joint using a fine (e.g., 14-22 gauge, preferably 18-22 gauge) needle. The compositions of the invention are also useful for parenteral administration, such as intravenous, subcutaneous, intramuscular, and intraperitoneal.
For prevention of surgical adhesions, the tribonectins described herein are administered in the form of gel, foam, fiber or fabric. A tribonectin formulated in such a manner is placed over and between damaged or exposed tissue interfaces in order to prevent adhesion formation between apposing surfaces. To be effective, the gel or film must remain in place and prevent tissue contact for a long enough time so that when the gel finally disperses and the tissues do come into contact, they will no longer have a tendency to adhere. Tribonectins formulated for inhibition or prevention of adhesion formation (e.g., in the form of a membrane, fabric, foam, or gel) are evaluated for prevention of post-surgical adhesions in a rat cecal abrasion model (Goldberg et al., In Gynecologic Surgery and Adhesion Prevention. Willey-Liss, pp. 191-204, 1993). Compositions are placed around surgically abraded rat ceca, and compared to non-treated controls (animals whose ceca were abraded but did not receive any treatment). A reduction in the amount of adhesion formation in the rat model in the presence of the tribonectin formulation compared to the amount in the absence of the formulation indicates that the formulation is clinically effective to reduce tissue adhesion formation.
Tribonectins are also used to coat artificial limbs and joints prior to implantation into a mammal. For example, such devices are dipped or bathed in a solution of a tribonectin, e.g., as described in U.S. Pat. Nos. 5,709,020 or 5,702,456.
Lubricating polypeptides are at least about 10 amino acids ((containing at least one KEPAPTT (SEQ ID NO:3)) or XXTTTX (SEQ ID NO:4) repeat), usually about 20 contiguous amino acids, preferably at least 40 contiguous amino acids, more preferably at least 50 contiguous amino acids, and most preferably at least about 60 to 80 contiguous amino acids in length. For example, the polypeptide is approximately 500 amino acids in length and contains 76 repeats of KEPAPTT (SEQ ID NO:3). The polypeptide is less than 1404 residues in length, e.g., it has the amino acid sequence of naturally-occurring MSF (SEQ ID NO:1) but lacks at least 5, 10, 15, 20, or 24 amino acids at the N-terminus of naturally-occurring MSF. Such peptides are generated by methods known to those skilled in the art, including proteolytic cleavage of a recombinant MSF protein, de novo synthesis, or genetic engineering, e.g., cloning and expression of at least exon 6, 7, 8, and/or 9 of the MSF gene.
Tribonectin polypeptides are also biochemically purified. The enzyme chymotrypsin cleaves at sites which bracket amino acids encoded by exon 6 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.
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) is an amount which is capable of producing a medically desirable result, e.g., boundary lubrication of a mammalian joint, in a treated animal. 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. 20 (suppl. 39):10-15) or increased ability to move the joint (measured, e.g., using pedometry as described in Belcher et al., 1997, J. Orthop. Trauma 11: 106-109). Another method to measure lubricity of synovial fluid after treatment is to reaspirate a small volume of synovial fluid from the affected joint and test the lubricating properties in vitro using a friction apparatus as described herein.
As is well known in the medical arts, dosage for any one animal depends on many factors, including the animal'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. Administration is generally local to an injured or inflamed joint. Alternatively, the polypeptides are administered via a timed-release implant placed in close proximity to a joint for slow release at the site of an injured or inflamed joint.
Canine osteoarthritis is a prevalent clinical disorder that is treated using the methods described herein. Osteoarthritis afflicts an estimated one in five adult dogs; an estimated 8 million dogs suffer from this degenerative, potentially debilitating disease. Yet, many owners do not recognize the signs of chronic canine pain. While any dog can develop osteoarthritis, those most at risk are large breeds, geriatric dogs, very active dogs (such as working or sporting animals), and those with inherited joint abnormalities such as hip or elbow dysplasia.
Equine degenerative joint disease such as osteoarthritis is a cause of lameness and impaired performance in horses. As with humans and other mammals, degenerative joint diseases which affect horses are progressive disorders of synovial joints characterized by articular cartilage degeneration and joint effusion. Acute or chronic trauma, overuse, developmental disease, joint instability and old age leads to synovitis, impaired chondrocyte metabolism, and the formation of fissures in the joint cartilage. Destructive enzymes such as trypsin, elastase, stromelysin and hyaluronidase are released into the joint where they degrade synovial fluid and cartilage components, resulting in decreased synovial fluid viscosity, poor lubrication, depressed cartilage metabolism and enhanced wear resulting in pain and cartilage erosion. Current therapeutic approaches include medications for pain relief and anti-inflammatory drugs. The compositions and methods described herein are useful to replenish the lubricating capabilities of the affected joint.
The effects of tribonectin upon synovial fluid's viscosity was studied in a novel multiple particle tracking technique which studies random walk behavior of particles introduced into synovial fluid. Viscosity is calculated from mean squared displacement (MSD) of tracked particles via the Einstein-Stokes relation. The advantage of this technique is that very small samples volumes are required; suitable for the study of clinical aspirates.
Fluorescent microspheres (Duke Scientific Corp., Palo Alto, Calif.) of 200 nm mean-diameter were added to the solutions being tested (0.3% volume fraction). A drop (˜2-5-μl) of the sample was deposited in a hydrophobic multi-well slide (Erie Scientific, Portsmouth, N.H.). This static condition of the fluid was confirmed by observing the relative motion of tracers over an extended amount of time (t>20 s). The slide was covered and placed on the stage of an inverted light microscope, (Nikon TE 200) and a peltier chip (MELCOR, Trenton, N.J.) temperature set up was placed on top of the slide to stabilize the temperature (˜295 K). The temperature of the sample was set using a thermoelectric controller (Oven Industries, Mechanicsburg, Pa.), which varies the amount current through the chip. An objective (Nikon) of 100×, 1.4 NA was used for magnification. The fluorescent beads were tracked with a 1500-EX charged-coupled digital (CCD) camera (IDT, Tallahassee, Fla.) of 6.45 μm×6.45 μm pixel resolution and 12 bit of dynamic range with 1×1 binning, for an effective 64.5 nm×64.5 nm per pixel resolution for the optical system.
The solutions studied were:
Particles in different locations of the middle plane of the sample were tracked separately and a region of interest (ROI) of approximately 8 μm×8 μm was used to confirm the same particle was being tracked frame after frame. The time-dependent ensemble-average mean squared displacement (MSD) of each particle was measured and analyzed over a ranged of frequencies using a MATLAB code. The code fits a Gaussian distribution to the Airy disks formed by the intensity of the light emitted from the flourophores in each particle. The center of this distribution is taken as the center of the particle and is tracked to measure the time-dependent mean-squared displacement (MSD). Subpixel interpolation is done and in this manner particles are tracked with ˜5 nm spatial resolution. To avoid particle-on-particle interaction artifacts, only particle probes with approximately ten diameters distance from the next probe were tracked. Approximately 80 particles were tracked for these experiments for a time of 12 s each at a rate of 16 Hz. The time-dependent MSD ensemble-average was used to study the time-dependent diffusivity of the tracers in the fluid preparations and in this way a description of the macroscopic behavior of the complex fluid was derived from microscopic measurements. The time-dependent ensemble-average diffusion coefficient was extracted from the two dimensional random walk model, using the formula I (Berg, Random Walks in Biology, Expanded Edition, Princeton University Press, pp. 5-12, Berg, 1993).
D(τ)=Δr2(τ)
/4τ. (1)
The structural and mechanical heterogeneity of the network in the dilute solutions was probed by observing the time-dependent distribution of MSD of individual particle at different locations throughout the sample (Apgar et al., Multiple-Particle Tracking Measurements of Heterogeneities in Solutions of Actin Filaments and Actin Bundles, Biophysical J. 79:1095-1106, 2000; Xu et al., Microheterogeneity and Microrheology of Wheat Gliadin Suspensions Studies by Multiple-Particle Tracking, Biomacromolecules 3:92-99, 2002). The time-dependent complex modulus |G*(ω)| along with its components, the elastic Gs(ω), and loss Gd(ω) moduli for the samples was calculated for the samples bywith the method described by Gardel et al., and developed by Mason et al. [5, 6], using formula 2, where kh is the Boltzmann constant, T is the temperature in Kelvins, a is the radius of the probes, Δr2(τ)
is the MSD with respect to the frequency (ω) of interest, Γ is the gamma function and d ln
r2(τ)
/d ln τ|r=1/ω is the slope of the MSD,
Δr2(τ)
, with respect to the time lag (τ) between measurements.
It is assumed that the fluid being probed is isotropic and incompressible around the sphere, which is acceptable at these low Renumbers. Also the characteristic mesh size of the network in the complex fluid is smaller that the diameter of the particle.
In order to test the system, BSF was compared to and subsequently diluted with a mixture of glycerol, a Newtonian fluid of known viscosity (1P), and DDIW. The time-dependent ensemble-average MSD of probes embedded in polymeric viscoelastic fluids adopts a power law, (Δr2(τ)
˜τa), behavior, where α is the slope of the natural logarithmic curve. The slope over the range of time scales probed sheds light on the viscoelastic behavior of the fluid. A 4:1 glycerol to DDIW (GDDIW) solution was used in this experiment to match the viscous behavior of BSF at lower frequencies. At these frequencies BSF shows a mostly diffusive behavior which is evident by the slope (α≈1), of the time-dependent ensemble-average MSD. As shown in
Apgar et al., vide supra, demonstrated the effects of regulatory protein on the network formation and overall viscoelasticity of complex fluids. Similarly, the influence of Purified Synovial Lubricating Factor (PSLF) on the structural and mechanical properties of Synovial Fluid were studied using multiple-particle-tracking microrheology (MPTM). In
The structural heterogeneity of the networks was assessed by looking at the time-dependent distribution of the MSD for individual particles, as shown in Δr2(τ)
is measured at a time-lag of 0.12 s and normalized by the corresponding ensemble-averaged mean. As expected, the distribution of the
Δr2(τ)
of the glycerol solution was symmetric about the mean and exhibited the normal distribution of a Newtonian fluid. The other distributions show signs of deviating from this normal distribution, but more particles (˜250) at more locations across the sample need to be observed at different time-lags to make a better statistical assessment of heterogeneity of the complex fluids and how it changes under different conditions.
The complex modulus for the fluids tested, as shown in
The viscoelastic behavior is shown in
All publications and patents cited in this specification are hereby incorporated by reference herein as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US05/26004 | 7/22/2005 | WO | 00 | 1/23/2007 |
| Number | Date | Country | |
|---|---|---|---|
| 60590766 | Jul 2004 | US |
