WO2008143816A1 - Compositions et procédés pour la réduction de la friction entre la surface des tendons et d'autres tissus mous - Google Patents

Compositions et procédés pour la réduction de la friction entre la surface des tendons et d'autres tissus mous Download PDF

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Publication number
WO2008143816A1
WO2008143816A1 PCT/US2008/006049 US2008006049W WO2008143816A1 WO 2008143816 A1 WO2008143816 A1 WO 2008143816A1 US 2008006049 W US2008006049 W US 2008006049W WO 2008143816 A1 WO2008143816 A1 WO 2008143816A1
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composition
tendon
soft tissue
tribonectin
gelatin
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PCT/US2008/006049
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English (en)
Inventor
Peter C. Amadio
Gregory D. Jay
Kai-Nan An
Chunfeng Zhao
Yu Long Sun
Mark Zobitz
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Mucosal Therapeutics
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Publication of WO2008143816A1 publication Critical patent/WO2008143816A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/4015—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil having oxo groups directly attached to the heterocyclic ring, e.g. piracetam, ethosuximide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]

Definitions

  • Tendon injuries are often encountered in daily life.
  • restoration of digital function following flexor tendon injury continues to challenge the surgery and therapy communities.
  • tissue adhesions represent a serious problem to the patient, the surgeon, and society. Therefore, there is a need for improved methods for treating joint, tendon, and soft-tissue injury and disease conditions.
  • the methods of the invention are directed to treating a soft tissue, tendon, or joint by administration of a first composition that includes collagen, l-ethyl-3-[3- dimethylaminopropyl] carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to an injured or inflamed joint, tendon, or soft tissue surface.
  • a first composition that includes collagen, l-ethyl-3-[3- dimethylaminopropyl] carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS)
  • the first composition is mixed in a ratio of about 10% denatured collagen (gelatin), about 1% EDC, and about 1% NHS, with or without about 1% hyaluronic acid (HA), and at a temperature sufficient to denature the collagen (e.g., in the range of about 37.5 °C to about 60 0 C; more preferably greater than about 41.5°C).
  • the method further includes removing the first composition from the joint, tendon, or soft tissue surface by rubbing or washing.
  • the method further includes contacting the joint, tendon, or soft tissue surface with a second composition comprising a tribonectin (e.g., lubricin, superficial zone protein, or any one or more of the tribonectins disclosed in U.S.
  • the tribonectin is recombinantly produced, naturally occurring, or chemically synthesized.
  • the tribonectin is present in the second composition at a concentration between about 1 ⁇ g/ml and about 1 mg/ml.
  • the tribonectin is present in the second composition at a concentration between about 250 and about 500 ⁇ g/ml.
  • the second composition is contacted to the joint, tendon, or soft tissue surface for at PATENT ATTORNEY DOCKET NO.: 50047/035WO2 least about 1 minute, preferably about 10 minutes, more preferably at least about 30 minutes.
  • the second composition can be removed from the joint, tendon, or soft tissue surface by washing or rubbing, or it can remain on the joint, tendon, or soft tissue surface without being removed.
  • the first and second compositions are mixed prior to administration to the soft tissue, tendon, or joint of the patient.
  • the first and second compositions of the present invention can be administered, e.g, intra-articularly, or by any other methods known in the art, as is discussed in detail below.
  • the treatment is in vivo, in situ, or in vitro.
  • compositions of the invention can be administered to a joint (e.g., the knee, shoulder, wrist, ankle, or elbow) or to a connective tissue (e.g., tendons, ligaments, or cartilage) within the body of a mammal.
  • a joint e.g., the knee, shoulder, wrist, ankle, or elbow
  • a connective tissue e.g., tendons, ligaments, or cartilage
  • the present invention further features compositions for the repair or treatment of joints, tendons, or soft tissue, which include collagen, l-ethyl-3-[3- dimethylaminopropyl] carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and a tribonectin.
  • the compositions further include HA.
  • the soft tissue is a tendon and the tribonectin is lubricin.
  • the compositions of the invention include hyaluronic acid and a tribonectin at a molar ratio of from about 2:1 to about 4:1, respectively.
  • the hyaluronic acid and tribonectin are present in the compositions of the invention at a molar ratio of from about 10:1 to about 50:1, respectively.
  • the hyaluronic acid and tribonectin are present in the compositions of the invention at a molar ratio of from about 100:1 to about 500:1, respectively.
  • a composition for tendon repair or grafting comprises preparation of a tendon from a non-autologous source.
  • the source can be isogeneic, allogeneic, or xenogeneic.
  • the tendon tissue is from a cadaver.
  • Another aspect of the invention features a cadaveric tendon composition that is prepared by contacting a cadaveric tendon tissue surface with a first composition PATENT ATTORNEY DOCKET NO.: 50047/035WO2 that includes collagen, EDC and NHS.
  • the first composition includes 10% denatured collagen (gelatin), 1% EDC, and 1% NHS.
  • the tendon tissue is immersed in the first composition for at least about 1 minute, preferably about 10 minutes, 20 minutes, 30 minutes, or 1 hour, and more preferably about 5 hours, 12 hours, or up to 24 hours or more.
  • immersion of the tendon tissue in the first composition is for a period of more than five seconds and less than one minute. Most preferably, immersion of the tendon tissue in the first composition is for at least thirty seconds.
  • the tendon tissue is then removed from the first composition and allowed to cure for at least about 1 minute, preferably about 10 minutes, 20 minutes, 30 minutes, or 1 hour, and more preferably about 5 hours, 12 hours, or up to 24 hours or more.
  • the tendons are cured in an insulating material (e.g., a smooth rubber sheet).
  • the insulating material further includes a cloth moistened by a liquid (e.g., to keep the tendon hydrated until grafting), which is placed adjacent to the tendon tissue.
  • the cloth is a towel and the liquid is saline.
  • the curing process may last more than about five minutes, but less than about fifteen minutes. Most preferably the curing process lasts for about ten minutes, hi an embodiment, the curing process occurs at a temperature of 20°C or less, preferably 10°C or less, and more preferably 4°C or less. Following the curing process, the first composition is then removed from the tendon surface by rubbing or washing.
  • the first composition is removed by a number of cycles of tendon motion.
  • the cadaveric tendon tissue surface is contacted with a second composition that includes a tribonectin (e.g., lubricin, superficial zone protein, or any one or more of the tribonectins disclosed in U.S. Patent No. 7, 001,881, which is incorporated herein by reference in its entirety).
  • a tribonectin e.g., lubricin, superficial zone protein, or any one or more of the tribonectins disclosed in U.S. Patent No. 7, 001,881, which is incorporated herein by reference in its entirety.
  • contact of the cadaveric tendon with the second composition containing a tribonectin occurs for at least about 1 minute, 5 minutes, or 10 minutes, preferably about 20 minutes, more preferably about 1 hour, 2 hours, or more.
  • the tribonectin is present in the second composition at a concentration between about 250 and about 500 ⁇ g/ml.
  • the tribonectin is applied to the cadaveric tendon tissue for at least about 1 minute, 5 PATENT ATTORNEY DOCKET NO.: 50047/035WO2 minutes, or 10 minutes, and more preferably at least about 30 minutes.
  • the resulting cadaverous tendon composition can be used for the repair of a tendon in a subject in need thereof.
  • tribonectin an articular boundary lubricant which contains at least one repeat of an amino acid sequence which is at least 50% identical to
  • 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 KEPAPTT, 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 KEPAPTT.
  • the tribonectin may also contain one or more repeats of the amino acid sequence XXTTTX.
  • a tribonectin can be formulated for administration to a mammalian joint, soft tissue or tendon.
  • the tribonectin is isolated from a natural source, but may also include a recombinant or chemically-synthesized lubricating polypeptide.
  • Lubricating polypeptides for use in the compositions of the invention are at least about 10 amino acids (containing at least one KEPAPTT or XXTTTX repeat sequence), 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.
  • the polypeptide is approximately 500 amino acids in length and contains 76 repeats of KEPAPTT.
  • 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.
  • proteolytic cleavage of a recombinant MSF protein de novo synthesis
  • genetic engineering e.g., cloning and expression of at least exon 6, 7, 8, and/or 9 of the MSF gene.
  • Tribonectin polypeptides for use in the compositions of the invention are also biochemically purified or isolated.
  • the enzyme chymotrypsin cleaves at sites which bracket amino acids encoded by exon 6 of the MSF gene.
  • a polypeptide containing amino acids encoded by exon 6 of the MSF gene may be 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.
  • a tribonectin may include an O-linked oligosaccharide, e.g., an N- acetylgalactosamine and galactose in the form ⁇ (l-3)Gal-GalNAC.
  • O-linked oligosaccharide e.g., an N- acetylgalactosamine and galactose in the form ⁇ (l-3)Gal-GalNAC.
  • ' KEPAPTT and XXTTTX repeat domains are glycosylated by ⁇ (l-3)Gal-GalNAC (which may at times be capped with NeuAc in the form of ⁇ (l-3)Gal-GalNAC- NeuAc.
  • the lubricating moieties of human joints are produced by superficial zone articular chondrocytes (Jay et al., J. Orthop. Res. 19:9-19, 2001 ; Flannery et al.
  • glycosylated with respect to a polypeptide (e.g., a tribonectin) 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.
  • a tribonectin can contain a substantially pure fragment of megakaryocyte stimulating factor (MSF).
  • MSF megakaryocyte stimulating factor
  • the molecular weight of a substantially pure tribonectin having an amino acid sequence of a naturally-occurring tribonectin is in the range of, e.g., about 206-345 kDa, more preferably about 220-280 kDa.
  • 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 PATENT ATTORNEY DOCKET NO.: 50047/035WO2 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, see GenBank Accession No. Q92954).
  • the tribonectin may contain a polypeptide, the amino acid sequence of which is at least 50%, preferably 60%, 70%, 80%, and more preferably 90%, 95%, 99% or more 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.
  • the polypeptide contains an amino acid sequence that is at least 50%, preferably 60%, 70%, 80%, and more preferably 90%, 95%, 99% or more 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%, preferably 60%, 70%, 80%, and more preferably 90%, 95%, 99% or more 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%, preferably 60%, 70%, 80%, and more preferably 90%, 95%, 99% or more 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.
  • 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.
  • Any tribonectin known in the art can be used in the present invention (e.g., those described in U.S. Patent No. 6,743,774).
  • a fragment of human lubricin includes an amino acid sequence that has less than the naturally occurring 1 ,404 amino acids set for in SEQ ID NO: 1.
  • Polypeptides or other compounds described herein are said to be "substantially pure" when they have been separated from at least 60% to 75% or more of the components that naturally accompany them.
  • polypeptides or other compounds described herein are substantially pure when they are separated from at least about 85 to 90% of the components that naturally accompany them, more preferably at least about 95%, and most preferably about 99% or more.
  • PATENT ATTORNEY DOCKET NO.: 50047/035WO2 purity is measured on a chromatography column, polyacrylamide gel, or by HPLC analysis.
  • 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.
  • 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 contiguous 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.
  • 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.
  • allogeneic or "allograft” refer to a graft taken from another person, such as a live donor or human cadaver.
  • xenogeneic or "xenograft” refer to a graft taken from another species, e.g. a pig.
  • crosslink or “crosslinking” is meant the process of forming covalent bonds (or cross-links) either directly between free active moieties on or within the tissue or between the free active moieties of the tissue and one or more compounds (or cross-linking agents).
  • the EDC and NHS are crosslinkers that bind the gelatin to the surface of a soft tissue, joint, or tendon.
  • a “crosslinking agent”, as used herein, is a compound capable of binding to the free active moieties of soft tissue, tenond or joint, or to other cross-linking agents in such a manner as to result in cross-linking between and within the molecules of the soft tissue, tendon, or joint and between the molecules of the soft tissue, tendon, or joint and the agent.
  • the cross-linking agent(s) is selected in such a way as to maximize fixation of the tissue being treated while minimizing the risk of damage to the soft tissue, tendon, or joint during treatment and minimizing the risks, such as of toxicity, inflammation, calcification, etc. to the host animal in whom the treated soft tissue, tendon, or joint is to be applied or grafted.
  • cross-linking agents are preferably water-soluble so that aqueous buffers may be utilized thereby minimizing the risk of damage to the soft tissue, tendon, or joint during the fixation process.
  • crosslinking agents of the invention are l-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS).
  • hyaluronic acid means hyaluronic acid, a cross-linked form of HA, or a non-crosslinked salt form. Examples are sodium hyaluronate, potassium hyaluronate, magnesium hyaluronate, and calcium hyaluronate.
  • soft tissue is meant tissues that connect, support, or surround other structures and organs of the body. Soft tissue includes muscles, tendons, fibrous tissues, fat, blood vessels, nerves, synovial tissues, ligament, cartilage, or a visceral surface (e.g., intrathoracic or intraabdominal viscera).
  • FIGURE 1 is a schematic illustration of binding lubricant molecules on the tendon surface (A) and cross-linking loose structures on the tendon surface (B).
  • FIGURE 2 illustrates the physical and chemical interactions to cross-link biopolymers.
  • the physical interactions include (1) ionic interaction, (2) hydrogen bond, and (3) hydrophobic interaction.
  • the chemical interactions include the PATENT ATTORNEY DOCKET NO.: 50047/035WO2 formation of chemical bonds between chemical groups.
  • the cross-links provide both inter-molecular and intra-molecular bonds.
  • FIGURES 3 A and 3B are graphs showing the gliding resistance of peroneus longus tendon with (FIG. 3A) or without (FIG. 3B) paratenon.
  • the results of gliding resistance of PL tendon without paratenon were similar to that with paratenon.
  • the mean gliding resistance of chemical HA treated PL tendon was significantly lower than the unmodified HA treated tendon from 1 to 100 cycles (p ⁇ 0.01).
  • FIGURE 4 is a graph showing the gliding resistance of PL tendons treated with saline (C), 10% gelatin/0.25%EDC/0.25% NHS (GE) or 10% gelatin/1% HA/0.25%EDC/0.25% NHS (GHE) at different cycles of simulated flexion/extension.
  • FIGURES 5A and 5B show that extrasynovial tendons treated with cd-HA- gelatin did not show any significant changes either in compression modulus (FIG. 5A) or in tensile modulus (FIG. 5B) as compared to extrasynovial tendon. Both compression and tensile modulus of intrasynovial tendon were higher than the extrasynovial tendon.
  • FIGURE 6 is a graph showing that the change in gliding resistance over 500 cycles showed similar patterns in each of the three groups tested. During the first 5 cycles there was a decrease in gliding resistance, followed by an increase in gliding resistance, and then, finally, stabilization of the gliding resistance.
  • FIGURES 7A-7D show treatment of PL tendons with experimental gelatin groups in comparison to controls. Surface features of PL tendons by scanning electron microscopy are examined showing untreated, untested PL tendon has a smooth surface (FIG. 7A).
  • FIGURES 8A-8C show treatment of PL tendons with experimental lubricin groups in comparison to controls. Experimental groups are outlined in FIG. 8A. Gliding resistance of PL tendon before treatment and after treatment at 1000 cycles. Tendons were treated with saline (saline), 260 ⁇ g/ml lubricin (lubricin), 10% gelatin/1% EDC/1%
  • FIGURES 9A-9F are photomicrographs showing the histology of the PL tendon surface with varying lubricin treatments in comparison to controls. Histology of the tendon surface treated with (FIG. 9A) saline, (FIG. 9B) lubricin, (FIG. 9C) cd- gelatin, (FIG. 9D) cd-HA-gelatin and (FIG. 9E) cd-gelatin+lubricin after 1000 cycles of tendon motion.
  • Normal PL tendon (FIG. 9F), hematoxylin and eosin staining; original magnification XlOO.
  • FIGURES 1OA and 1OB are graphs showing that the friction associated with extrasynovial tendons increased more significantly than did the friction associated with intrasynovial tendons as the load (FIG. 10A) and gliding cycles (FIG. 10B) increased.
  • FIGURES HA and HB are photographs showing the effect of cd-HA-gelatin on the reduction of adhesion formation in extrasynovial tendons in vivo.
  • FIG. 1 IA shows the treated tendon
  • FIG. 11 B shows a saline-treated control tendon.
  • FIGURES 12A-12D are photographs showing the immunohistochemical detection of lubricin in PL tendon after treatment.
  • the tendon surface of the saline control (FIG. 12A) and the lubricin only control (FIG. 12B) groups appears to be PATENT ATTORNEY DOCKET NO.: 50047/035WO2 rough; lubricin is clearly present on the tendon surface (FIG. 12B).
  • FIGURES 13A and 13B are photographs showing canine FDP tendons from a dog forepaw that was repaired with the use of a modified Pennington technique (locking Modified Kessler) with 3-0 Ethibond and with a circumferential simple running suture of 6-0 prolene.
  • FIG. 13 A shows the tendon prior to treatment;
  • FIG. 13B shows the tendon after treatment with cd-HA-gelatin.
  • FIGURE 14 is a graph showing the effect of saline, cd-HA-gelatin, cd- gelatin+lubricin and cd-HA-gelatin+lubricin treatment on tendon gliding resistance after 1000 cycles of canine tendon motion.
  • FIGURE 15 is a graph showing the effect of saline, cd-HA-gelatin, cd- gelatin+lubricin, and cd-HA-gelatin+lubricin treatment on canine tendon gliding resistance change.
  • FIGURES 16A-16D are scanning electron micrographs of random fibers on the surface of repaired FDP tendons treated with saline (FIG. 16A), cd-HA-gelatin (FIG. 16B), cd-gelatin+lubricin (FIG. 16C), and cd-HA-gelatin+lubricin (FIG. 16D) after 1000 cycles of tendon motion.
  • FIGURES 17A-17D are scanning electron micrographs of the proximal pulley surface from tendons treated with saline (FIG. 17A), cd-HA-gelatin (FIG. 17B), cd-gelatin+lubricin (FIG. 17C), and cd-HA-gelatin+lubricin (FIG. 17D) after 1000 cycles of tendon motion.
  • FIGURE 18 is a graph showing the effect of saline, cd-HA-gelatin, cd- gelatin+lubricin and cd-HA-gelatin+lubricin treatment on human palmaris longus (hPL) tendon gliding resistance after 1000 cycles of tendon motion.
  • hPL human palmaris longus
  • FIGURE 19 is a graph showing the effect of saline, cd-HA-gelatin, cd- gelatin+lubricin, and cd-HA-gelatin+lubricin treatment on human palmaris longus (hPL) tendon gliding resistance change after 1000 cycles of tendon motion.
  • FIGURES 20A-20D are scanning electron micrographs of random fibers on the surface of repaired hPL tendons treated with saline (FIG. 20A), cd-HA-gelatin (FIG. 20B), cd-gelatin+lubricin (FIG. 20C), and cd-HA-gelatin+lubricin (FIG. 20D) after 1000 cycles of tendon motion.
  • the present invention features methods and compositions for reducing friction between tissues, inhibiting adhesion formation, and improving healing following surgical procedures.
  • the methods and compositions of the present invention which reduce friction at sites of tendons, joints, and other soft tissues, can be used to treat arthritic joint surfaces, tendon adhesions (e.g., adhesions in the hand, elbow, shoulder, foot, hip, knee, and viscera that develop after surgery), and similar conditions, such as tendon grafts, tendon transfers, trigger finger, carpal tunnel syndrome, and other common problems in which adhesion formation or restoration of tissue gliding is a problem.
  • tendon adhesions e.g., adhesions in the hand, elbow, shoulder, foot, hip, knee, and viscera that develop after surgery
  • tendon grafts tendon transfers, trigger finger, carpal tunnel syndrome, and other common problems in which adhesion formation or restoration of tissue gliding is a problem.
  • compositions of the current invention which are administered to a joint, a soft tissue surface, or a tendon surface, contain a tribonectin,.
  • a tribonectin e.g., proteoglycan 4 (PRG4), articular cartilage superficial zone protein (SZP), megakaryocyte stimulating factor precursor (MSF), and 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 lubricin, also characterized by the NCBI as MSF (SEQ ID NO:1, GenelD: 10216) 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 an O-glycosylated mucin domain.
  • a polypeptide encoded by nucleotides 631-3453 of SEQ ID NO:2 provides boundary lubrication of articular cartilage (See Table 2, bold sequence).
  • Tribonectins provide 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 tribonectins 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).
  • Lubricin is a product of the PRG4 gene (GenBank accession number NM_005807) and is expressed by synovial fibroblasts (Jay et al., J. Rheum. 27:594-600, (2000).
  • Lubricin is a mucinous glycoprotein responsible for the boundary lubrication of articular cartilage (Swann et al., Biochem J. 161(3): 473-85 (1977); Jay et al., Connect Tissue Res. 28(1-2): 71-88 (1992)). It has the same lubricating ability as normal synovial fluid in vitro.
  • lubricin may play an important role in controlling adhesion-dependent synovial growth (Rhee et al., J Clin Invest. 115(3): 622-31 (2005)), preventing protein deposition onto cartilage from synovial fluid, and inhibiting the adhesion of synovial cells to the cartilage surface (Rees et al., Matrix Biol. 21(7): 593-602 (2002), Flannery et al., Biochem Biophys Res Commun. 254: 535-41 (1999), and Marcelino et al., Nature Gene. 23(3): 319-22 (1999)).
  • lubricin provides the lubrication for normal joint function (Swann et al., Biochem J.
  • Lubricin has also been identified in tendons, including the surface of FDP tendon (Rees et al., Matrix Biol. 21(7): 593-602 (2002), Sun et al., J Orthop Res. 24(9): 1861-8 (2006)). Failure of lubricin secretion causes camptodactyly-arthropathy-coxa vara-pericarditis syndrome (CACP; see, e.g., Marcelino et al., Nature Gene. 23(3): 319-22 (1999); Ochi et al., Arthritis Rheum. 26: 896-900 (1983)). A role for lubricin has also been identified in tendon gliding based on the observation that patients diagnosed with PATENT ATTORNEY DOCKET NO.: 50047/035WO2
  • CACP syndrome exhibit a number of tendon abnormalities within the tenosynovial sheaths and adhesion formation (see, e.g., Ochi et al., Arthritis Rheum. 26: 896-900 (1983)).
  • lubricin is a preferable tribonectin for use in the methods and compositions of the current invention.
  • Tribonectin analogs can differ from the naturally-occurring peptides by amino acid sequence, 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.
  • 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.
  • Trypsin cleaves a polypeptide bond on the carboxy-side of lysine and arginine; elastase cleaves on the carboxy-side of alanine, glycine, and valine.
  • 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.
  • PATENT ATTORNEY DOCKET NO.: 50047/035WO2 peptidase-susceptible peptide bonds 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.
  • 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.
  • 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.
  • tribonectin peptide mimetic is also included as a tribonectin peptide mimetic.
  • Peptoids or N-substituted glycines, are a specific subclass of peptidomimetics. They are closely related to their natural peptide counterparts, but differ chemically in that their sidechains are appended to nitrogen atoms along the molecule's backbone, rather than to the ⁇ -carbons (as they are in amino acids; see, e.g., Simon et al., P.N.A.S. USA 89:9367-9371, 1992).
  • the tribonectin peptoids of the invention can be designed to mimic any of the tribonectin polypeptide sequences, or fragments thereof, disclosed herein.
  • compositions of the current invention for example, a first composition including collagen, l-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS), can also include hyaluronic acid.
  • 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 x 10 6 .
  • 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 PATENT ATTORNEY DOCKET NO.: 50047/035WO2 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 to 10.9 x 10 6 Da; its concentration equals approximately 2 to 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 to 2 million Da.
  • High-molecular- weight HA binds up to 1000 times more water than its own mass and forms pseudoplastic, elastoviscous 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).
  • 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 (fast) movement.
  • the cartilage surface is covered by a thin film of synovial fluid 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 a marked increase in 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 obtained from the rooster combs (HEALONTM; Pharmacia, Uppsala, Sweden) have found extended application especially in ophthalmology (viscosurgery) (Nimrod et al, J. Ocular Pharmacol.
  • Hyaluronic acid for use in the methods and compositions of the current invention include, but are not limited to HYLANTM (Biomatrix Inc., Ridgefield, N. J., USA), which contains high-molecular-weight HA originating from rooster combs, and includes additionally cross-linked HA (L. S. Simon, Osteoarthritis 25:345, 1999).
  • HYLANTM Biomatrix Inc., Ridgefield, N. J., USA
  • the water-soluble HYLANs with ultra-high molecular weight (on average around 6 x 10 6 Da) that are prepared by chemical cross-linking of HA with formaldehyde reveal PATENT ATTORNEY DOCKET NO.: 50047/035WO2 a significantly longer biological half-life period (Simon, Osteoarthritis 25:345, 1999).
  • HA-based viscosupplements that can be used in the methods and compositions of the invention include, HYLAGELTM, HYALGANTM (sodium hyaluronate), ARTZTM, SUPLASYNTM, BIOHYTM, ORTHO VISCTM (high-molecular weight hyaluronan), and SYNVISCTM (hylan G-F 20), and EUFLEXATM (1% sodium hyaluronate).
  • crosslinking agents include l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS).
  • EDC l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
  • NHS N-hydroxysuccinimide
  • An example is sulfo-N-hydroxysuccinimide added to the EDC crosslinking agent, as is described by Staros, Biochem. 21 :3950-3955, 1982, and as described in Example 1.
  • Additional crosslinkers for use with the methods and compositions of the invention can be selected based on their biocompatability or on the basis of their chemical reactivity. These may include, but are not limited to, those known in the art, including, e.g., n- hydroxysuccinimide-esters (NHS-Esters), imidoesters, malemides, haloacetyls, pyridyl disulfides, hydazides, carbodiimides, aryl azides, and arginine-specific crosslinkers, such as glyoxals.
  • Other crosslinking agents for use with the methods and compositions of the invention include those described in, e.g., U.S. Patent No. 4,101,380.
  • the treated soft tissue or tendon tissue of the present invention is prepared by incubating a soft tissue or tendon tissue with a first composition that includes, e.g., collagen (e.g., 10% collagen, such as denatured collagen (gelatin)), EDC (e.g., 1% EDC), and NHS (e.g., 1% NHS).
  • the first composition can also include HA.
  • the PATENT ATTORNEY DOCKET NO.: 50047/035WO2 treated soft tissue or tendon tissue can be used to repair or replace damaged or defective soft tissue or tendon tissue in a mammal.
  • the soft tissue or tendon tissue is wrapped, enfolded, or encompassed in a material suitable for incubation to create an environment that facilitates coating of the tissue with the first composition and prevents dehydration of the soft tissue or tendon.
  • the soft tissue or tendon is incubated in a material that includes an outer material and an inner material.
  • the outer material may be rubber, plastic, or any other material that is suitable to create an environment where the soft tissue or tendon tissue may be incubated without loss of the first composition (e.g., by evaporation).
  • the outer material is smooth rubber.
  • the incubation environment is further maintained by an inner insulating material placed between the soft tissue or tendon tissue and the outer material.
  • the inner material further prevents dehydration of the soft tissue or tendon during the curing, or incubation, step.
  • the inner material may be any material capable of maintaining hydration of the soft tissue or tendon tissue, but is preferably an absorbent material, for example, a cloth, fabric, or towel.
  • the inner material is a towel.
  • the absorbent inner material may be moistened with any solution that maintains the hydration of the soft tissue or tendon tissue during the curing, or incubation, process.
  • the solution is a saline solution (e.g., phosphate buffered saline (137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl, pH 7.4)).
  • the amount of time necessary for completion of the curing, or incubation, process may vary depending on the source or type of soft tissue or tendon tissue to be cured, the outer and inner materials used during the curing process, or the solution applied to the inner material for maintenance of the soft tissue or tendon tissue during the curing process.
  • the incubation time is also dependent upon the level of crosslinking necessary to prepare the soft tissue or tendon tissue prior to contact with a second composition that includes a tribonectin (e.g., lubricin).
  • the incubation time is between about one and about fifteen minutes. More preferably, the incubation time is for about five or ten minutes.
  • the soft tissue or tendon tissue is treated with the second composition of the PATENT ATTORNEY DOCKET NO.: 50047/035WO2 invention that includes a tribonectin.
  • the first composition may be removed from the soft tissue or tendon tissue (e.g., by rubbing or washing) prior to contact with the second composition.
  • the soft tissue or tendon tissue can be contacted with the second composition for at least about one minute and up to about five, ten, twenty, or thirty minutes or more.
  • the tissue can be implanted into a patient, or it can be further treated with other beneficial compositions (e.g., a composition containing a growth factor, an antimicrobial agent, or other therapeutic agent).
  • beneficial compositions e.g., a composition containing a growth factor, an antimicrobial agent, or other therapeutic agent.
  • compositions (first and second) of the present invention may also contain peptidase inhibitors such as N-methoxysuccinyl-Ala-Ala- Pro-Val chloromethylketone (an inhibitor of elastase).
  • 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, ⁇ -1 -antitrypsin, ⁇ -2-macro globulin, ⁇ -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 halfiife.
  • a cocktail of two or more protease inhibitors can also be coadministered.
  • compositions of the invention that include a tribonectin polypeptide or a fragment thereof can be formulated in standard physiologically-compatible excipients known in the art, e.g., phosphate-buffered saline (PBS).
  • PBS phosphate-buffered saline
  • 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
  • Treatment of Soft Tissue, Tendon, and Joints It is envisioned that the methods and compositions of the invention are applicable for the treatment of soft tissue to promote healing and/or engraftment of PATENT ATTORNEY DOCKET NO.: 50047/035WO2 soft tissue at the site of soft tissue damage in a patient. Treatment involves reducing friction at the site of damage, or at the site of subsequent damage repair.
  • the methods and compositions of the present invention can be used to treat tendon adhesions, e.g., in the hand, elbow, shoulder, foot, hip, and knee, visceral adhesions, which result after abdominal or thoracic surgery, arthritic joint surfaces, and other conditions, such as tendon grafts, tendon transfers, trigger finger, carpal tunnel syndrome, and other common problems in which adhesion formation occurs or in which restoration of tissue gliding is required.
  • compositions of the invention can also be administered to alleviate, inhibit, relieve, or treat soft tissue injuries including ligament, tendon, muscle, cartilage, connective tissue injuries, subluxation, repetitive stress injury, sprains (partial tear or complete tear), strains (partial tear or complete tear), contusions, tendonitis, bursitis, stress injuries (including stress fracture), cervical stain injuries, epicondylitis, lumbar strain injuries, ileotibial band syndrome, oro-facial soft tissue injuries, ACL (Anterior Cruciate Ligament) injuries, MCL (Medial Collateral Ligament) injuries, PCL (Posterior Cruciate Ligament) injuries.
  • soft tissue injuries including ligament, tendon, muscle, cartilage, connective tissue injuries, subluxation, repetitive stress injury, sprains (partial tear or complete tear), strains (partial tear or complete tear), contusions, tendonitis, bursitis, stress injuries (including stress fracture), cervical stain injuries, epicondylitis, lumbar strain
  • compositions of the invention can also be administered to alleviate, inhibit, relieve, or treat arthritic conditions associated with spondylitis, including ankylosing spondylitis, reactive arthritis (Reiter's syndrome), arthritis associated with chronic inflammatory bowel disease and AIDS-related seronegative spondyloarthropathy.
  • arthritic conditions associated with spondylitis including ankylosing spondylitis, reactive arthritis (Reiter's syndrome), arthritis associated with chronic inflammatory bowel disease and AIDS-related seronegative spondyloarthropathy.
  • compositions of the invention can also be administered to alleviate or inhibit symptoms of rheumatic disease and disorders, e.g., systemic sclerosis and forms of scleroderma, polymyositis, dermatomyositis, necrotizing vasculitis and other vasculopathies, hypersensitivity vasculitis (including Henoch-Schonlein purpura),
  • rheumatic disease and disorders e.g., systemic sclerosis and forms of scleroderma, polymyositis, dermatomyositis, necrotizing vasculitis and other vasculopathies, hypersensitivity vasculitis (including Henoch-Schonlein purpura),
  • compositions of the invention are also useful for alleviating or inhibiting the symptoms of diseases with rheumatic states, including, e.g., gout, pseudogout, chondrocalcinosis, amyloidosis, scurvy, speicific enzyme deficiency states (including Fabry's disease, alkaptonuria, ochonosisi, Lesch-Nyhan syndrome, and Gaucher' s disease), hyperlipoproteinemias (types II, Ha, IV), Ehlers-Danlos syndrome, Marfan' s syndrome, pseudoxanthoma elasticum, and Wilson's disease.
  • diseases with rheumatic states including, e.g., gout, pseudogout, chondrocalcinosis, amyloidosis, scurvy, speicific enzyme deficiency states (including Fabry's disease, alkaptonuria, ochonosisi, Lesch-Nyhan syndrome, and Gaucher' s disease), hyperlipoprotein
  • compositions of the invention can be administered to the joint (e.g., the knee, shoulder, wrist, ankle, or elbow) or connective tissue of a mammal.
  • the first composition of the invention can be delivered in combination with, or delivered separately from, the second composition of the invention containing a tribonectin.
  • the second composition when administered, preferably contains a tribonectin at a concentration in the range of 20-500 ⁇ g/ml.
  • the compositions of the invention are preferably topically administered, e.g., directly to the surface of the soft tissue, tendon or joint to be treated (e.g., during open surgery).
  • the compositions of the invention are administered by intra-articular injection.
  • the compositions of the invention can be also be administered by parenteral routes, such as intravenous, subcutaneous, intramuscular, and intraperitoneal administration.
  • compositions of the invention can be administered in the form of, e.g., a liquid, gel, foam, film, fiber or fabric.
  • compositions of the invention formulated in such a manner is placed over and between damaged or exposed tissue interfaces in order to prevent adhesion formation between apposing surfaces.
  • the liquid, gel, foam, film, fiber, or fabric remains in place and prevents tissue contact at the interface for a time sufficient to prevent adherence of the tissue when they are again allowed to come into contact.
  • compositions formulated for administration to soft tissue, tendons, or joints to reduce friction or improve soft tissue gliding ability can be evaluated for improved soft-tissue surface modification and for the ability to improve soft-tissue gliding capability using a canine extrasynovial tendon.
  • a composition e.g., a carbodiimide- derivatized-gelatin polymer solution (with or without HA)
  • HA extrasynovial tendon
  • compositions containing a tribonectin may preferentially adhere to a tendon surface pre-treated with a composition containing cd-gelatin (e.g., see Example 10).
  • a composition containing cd-gelatin e.g., see Example 10
  • This combination administration of a first composition (cd-gelatin) and a subsequent second composition (lubricin) improves tendon lubrication, and thus may significantly impact therapeutic adjuncts for soft tissue or tendon injury.
  • compositions of the invention which are formulated for administration to inhibit or prevent adhesion formation (e.g, in the form of a liquid, gel, foam, film, fiber or fabric), can be evaluated for prevention of post-surgical adhesions using a rat cecal abrasion model (Goldberg et al., In Gynecologic Surgery and Adhesion Prevention. Willey-Liss, pp. 191-204, 1993).
  • Compositions of the invention can be placed around surgically abraded rat ceca and compared to non-treated controls
  • compositions of the invention can also be used to coat artificial limbs and joints prior to implantation into a mammal.
  • such devices are dipped or bathed in a solution of the collagen-containing composition of the invention and a tribonectin-containing composition of the invention (either separately or the compositions can be combined), according to the methods described in e.g., U.S. Pat. Nos. 5,709,020 and 5,702,456.
  • 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 of the invention e.g., one containing a lubricating polypeptide, such as lubricin
  • a medically desirable result is a reduction of friction and improvement in gliding resistance of tendons in response to repetitive motion (Zhao et al., J. Bone Joint Surg. Am.
  • Administration is generally local to an injured or inflamed soft tissue, tendon or joint, or connective tissue associated with a joint.
  • a timed- release formulation of the composition of the invention can be prepared, in which one or more of the components (e.g., the cd-gelatin or the tribonectin) are slowly released (e.g., released over the course of 1-5 hours, 1-24 hours, 1-2 days, or 1-2 weeks) at the site of an injured or inflamed soft tissue, tendon, joint or a connective tissue associated with a joint, following administration of the composition.
  • the components e.g., the cd-gelatin or the tribonectin
  • Example 2 Chemical modification of PL tendon with gelatin/EDC/NHS Thirty peroneus longus (PL) tendons from thirty canine hind paws were used.
  • the paratendon was carefully removed, so as not to injure the tendon surface.
  • the PL tendons were immersed for 30 seconds in 10% gelatin, 0.25% EDC, 0.25% NHS, 0.9% NaCl, 0.1 M Mes pH 6.0. Tendons were placed in an incubator at 100% humidity at 37°C for 1.25 hours.
  • Example 3 Chemical modification of PL tendon with gelatin/HA/EDC/NHS
  • PL peroneus longus
  • Flexor digitorum profundus (FDP) tendons from the digits of canine hindpaw were used for testing.
  • a complete laceration to the FDP tendon was made at a level 6 mm distal to the proximal tendon marker, in order to allow the repair site to travel the full length of the proximal pulley during normal excursion.
  • the tendon was repaired using a modified Kessler technique with 4/0 looped Supramid suture (S Jackson, Arlington, VA).
  • the core suture was reinforced with a circumferential epitendon simple running suture of 6-0 prolene so that the repaired tendon edge would not lock or catch on the pulley edge. (Ethicon, Inc. Somerville, NJ).
  • the gliding resistance test was performed between the PL tendon and the ipsilateral proximal pulley of the 2 nd a digit in a testing jig.
  • the actuator was positioned at an angle of 30° and the distal load-transducer was positioned at 20°.
  • the load was 4.9 N. All tests were executed in a saline bath at 37°C.
  • the gliding resistance of all tendons was measured before any tendons were modified.
  • the treated tendons were then tested for 100-500 cycles of simulated flexion/extension.
  • the excursion distance was 14 mm, an average distance for digital flexor tendon.
  • the rate of movement was 2 mm/second. Gliding resistance between the tendon and proximal pulley was recorded at the selected cycles (FIGS. 3 and 4).
  • the extrasynovial tendon modified by cd-HA gelatin decreases the friction and improves the surface smoothness based upon our previous study.
  • the purpose of this study was to measure the tendon compression and tensile modulus before and after treatment with cd-HA gelatin.
  • Indentation Test Flexor digitorum profundus (FDP) tendons, peroneus longus (PL) tendons from canine model were harvested and dissected into 7mm length pieces. The tendon was mounted on the cell culture dish using double tape to avoid tendon slipping during the indentation test, then emerged in the PBS. The dish was mounted on the Dynamic Mechanical Analyzer (DMA) (TA Instruments, New Castle, DE) for compressive testing to measure the hardness of the tendons. The diameter of the indentor was 3 mm, which is smaller than the tendon diameter. Controlled force with a ramp rate of 10 N/min was applied to tendon.
  • DMA Dynamic Mechanical Analyzer
  • FDP tendon and PL tendons were obtained from canine model that were sacrificed for other proposed study.
  • the tendons were further dissected into 15 mm length and preserved at -80°C before processing, then embedded in Tissue-Tek (O. CT.) (Sakura Finetek, Torrance, CA, USA).
  • the specimens were mounted on the Cryostat (JUNG CM 3000, Leica Instrument, GmbH, Nussloch, Germany) and sectioned into 400 ⁇ m slice. Both ends of tendon slice were clamped by two pieces of plastic glass and fixed with super glue.
  • Embedded specimens were mounted on the DMA for tensile testing. The sections were collected on charged glass slides (Fisher Scientific, Pittsburgh, PA, USA) and dried over night at room temperature.
  • the proximal phalanx (with PIP joint, proximal pulley, and FDP) was mounted on a custom jig with the palmar side upward.
  • the measurement system consists of one mechanical actuator with a linear potentiometer, two tensile load transducers, and a mechanical pulley.
  • the load transducers were connected to the proximal and distal ends of the FDP tendon using a nylon cord.
  • a 500-gram weight was attached to the distal end of the FDP tendon through the mechanical pulley to maintain tension on the FDP tendon.
  • the distal transducer (Fl) was connected to the weight and the proximal load transducer (F2) was connected to the mechanical actuator.
  • the actuator was positioned at an angle ⁇ , of 30°, between the horizontal plane and the proximal cable extension.
  • the tendon was pulled proximally by the actuator against the weight at a rate of 2.0 mm/sec.
  • This movement PATENT ATTORNEY DOCKET NO.: 50047/035WO2 of the tendon toward the actuator was regarded as flexion.
  • the actuator movement was then reversed, causing the tendon to move distally under the pull of the 50Og weight, to simulate extension.
  • the tendon gliding resistance over the total excursion was summarized by averaging the results from flexion and extension and was calculated as (F2fl e ⁇ ion - F2 ex tension)/2. For each flexion/extension cycle the peak value of gliding resistance was calculated. Prior to the surface treatment, the normal tendon gliding resistance was measured for 1 cycle. The peak gliding resistance for the subsequent 500 cycles following treatment was normalized to this initial value by subtracting the initial untreated result. The gliding resistance data were reduced and reported at cycles 1, 5, 10, 50, 100, 200, 300, 400, and 500 (FIG. 6).
  • the surface of untreated PL tendon and PL tendon samples obtained after the gliding resistance test were studied by scanning electron microscopy. Tendon pieces were fixed in Trump's EM fixative and dehydrated. The specimens were mounted on a specimen stub and coated with gold/palladium. The tendon surface was then evaluated using a Hitachi S-4700 Field Emission Scanning Electron Microscope (FIG 7 A and 8).
  • Example 9 Normalized Work of Flexion and Gliding Resistance of Tendon Graft
  • the gliding resistance of the PL tendons in saline control, gelatin and cd-HA groups showed similar trends and there was no significant difference in gliding resistance among these three groups (FIG 7B).
  • the gliding characteristic of the PL tendon was significantly improved by the treatment with cd-gelatin and cd- HA-gelatin, with the cd-HA-gelatin group having the lowest gliding resistance (FIG 7B).
  • the normalized work of flexion of the grafted digits in both the cd-HA-treated and the control group were significantly higher than that of the normal digits at one, three, and six weeks (p ⁇ 0.05).
  • the normalized work of flexion of the cd-HA-treated grafts was significantly lower than that of the saline-solution-treated grafts at all three time points (p ⁇ 0.05). There was no significant difference among the findings at one, three, and six weeks in the normal digits. However, the normalized work of flexion in the cd-HA-treated and saline solution-treated groups at three weeks and six weeks was significantly increased compared with the normalized work of flexion at one week in the respective groups (p ⁇ 0.05). There was no significant difference in the normalized work of flexion between the three and six-week grafts in either the cd-HA or the saline-solution-treated group (FIG 7C).
  • the gliding resistance of the saline- solution-treated grafts was significantly higher than that of the normal flexor digitorum profundus tendons as well as the cd-HA-treated tendon grafts at three and six weeks (p ⁇ 0.05). There was no significant difference in the gliding resistance between the normal flexor digitorum profundus tendons and the cd-HA treated tendon grafts at any time point. The gliding resistance of the saline-solution-treated tendon grafts at three and six weeks was significantly higher than that at one week (p ⁇ 0.05). PATENT ATTORNEY DOCKET NO.: 50047/035WO2
  • Example 10 Superiority ofLubricin in Surface Modification of Tendons After 1000 cycles of tendon motion, the gliding resistance of the PL tendon in saline, lubricin, cd-gelatin, cd-HA-gelatin and cd-gelatin+lubricin groups was 0.91 ⁇ 0.11 N, 0.91 ⁇ 0.13 N, 0.34 ⁇ 0.17 N, 0.21 ⁇ 0.07 N and 0.11 ⁇ 0.01 N, respectively (FIG. 8B). There was no significant difference in gliding resistance after 1000 cycles between the tendon treated with saline control and lubricin 260 ⁇ g/ml alone.
  • the gliding resistance of the PL tendons in cd-gelatin, cd-HA-gelatin and cd- gelatin+lubricin groups was significantly lower than that of saline control after 1000 cycles (p ⁇ 0.05).
  • the tendon treated with cd-gelatin+lubricin had the lowest gliding resistance after 1000 cycles, which decreased 18.7 % compared with the PL tendon prior to treatment, while that of the saline control increased 418.3%.
  • the gliding resistance of the PL tendon treated with cd-gelatin+lubricin was significantly lower than that of the cd-gelatin treated tendons after 1000 cycles (p ⁇ 0.05).
  • the trend of gliding resistance in each group is shown in (FIG. 8C).
  • the gliding resistance of the PL tendons treated with saline and lubricin alone increased dramatically over first 400 cycles and then increased at a gradual rate in the next 600 cycles.
  • the gliding resistance of the PL tendons in the cd-gelatin, cd-HA-gelatin and cd-gelatin+lubricin groups increased at a much more gradual rate over the 1000 cycles.
  • the gliding resistance of the PL tendon treated with cd-gelatin+ lubricin was the most stable over 1000 cycles.
  • Tendon grafts play an important role in reconstruction to restore finger function in cases such as delayed primary repair, severe trauma, and also primary repair failure, e.g., rupture after tendon repair.
  • Smooth digital flexion requires an intact and efficient flexor apparatus.
  • the flexor tendon must glide unimpeded through the flexor sheath and must be positioned by the flexor sheath at the center of joint rotation, this allows for full range of motion and economic tendon excursion.
  • Critical to normal digital flexion are the position and length of the annular pulleys, the integrity and size of the flexor tendon, and the dynamics of the tendon-pulley interface, including surface histology and the presence of boundary lubrication to lessen the friction of flexor tendon gliding.
  • most tendon grafts are obtained from extrasynovial tendon sources that are easily harvested without any risk of important functional loss.
  • Extrasynovial tendon grafts are known to develop adhesions to a greater extent than intrasynovial tendon grafts.
  • a significant increase in resistance to gliding and a significantly greater coefficient of friction is commonly observed in these postrepair tendons.
  • a change in the pattern for the coefficient of friction during tendon excursion is also often observed, suggesting that a change in the gliding surface and shape of the tendon resulted in an alteration of its functional properties.
  • Recent evidence shows that extrasynovial tendons generate more friction under an intact annular pulley than do intrasynovial grafts.
  • intrasynovial tendon grafts demonstrate a significantly lower resistance to tendon excursion as compared to extrasynovial tendon grafts, suggesting that the surface morphology of the tendon is an important factor in addressing the kinematics of tendon gliding after reconstruction. From our previous studies, we found that the friction of extrasynovial tendon increases more significantly than the friction of intrasynovial tendon with an increase PATENT ATTORNEY DOCKET NO.: 50047/035WO2 of the load and gliding cycles (FIGS. 1OA and 10B). High friction of extrasynovial tendon is associated with increased adhesion formation.
  • Example 12 Tendon Surface Modification using cd-gelatin-lubricin
  • Lubricin is a mucinous glycoprotein responsible for the boundary lubrication of articular cartilage. Recent studies indicate that lubricin may play an important role in inhibiting the adhesion of synovial cells to the cartilage surface, controlling adhesion-dependent synovial growth, and preventing protein deposition onto cartilage from synovial fluid in addition to providing articular lubrication, which is necessary for normal joint function.
  • the purpose of this study was to investigate the effect of exogenously applied lubricin on the gliding of the canine extrasynovial tendon graft (i.e., peroneus longus (PL) tendons) in vitro.
  • FIGS. 12A-12D show immunohistographic photographs of the treated PL tendons.
  • the tendon surface of the saline control and lubricin groups appears to be rough (FIGS. 12A and 12B); lubricin is clearly present on the tendon surface (FIG. 12B).
  • cd-gelatin group In the cd-gelatin group (FIG.
  • the gliding resistance of the PL tendons in the cd-gelatin, cd-HA-gelatin, and cd-gelatin+lubricin groups increased at a much more gradual rate than the gliding resistance observed in the saline control (see FIG. 8C). Especially, the gliding resistance of cd-gelatin+lubricin treated tendon, which was almost as stable as a normal flexor tendon over 1000 cycles.
  • Lubricin does, though, preferentially adhere and remains strongly adhered to a surface treated with carbodiimide-activated gelatin. In this case, the lubricin fills up the gaps and irregularities on the PL tendon surface and provides effective tendon lubrication.
  • 13B shows the intrasynovial PATENT ATTORNEY DOCKET NO.: 50047/035WO2 tendon post treatment with cd-HA-gelatin.
  • the gliding resistance of repaired FDP tendon was measured before and after treatment in each group.
  • the gliding resistance in the cd-HA- gelatin, cd-gelatin+lubricin, and cd-HA-gelatin+lubricin groups was significantly lower than the gliding resistance observed in the saline control group.
  • the gliding resistance of the saline control increased 0.25 N compared with the gliding resistance exhibited by the tendon before treatment, the gliding resistance exhibited by the cd-HA-gelatin, cd-gelatin+lubricin, and cd-HA-gelatin+lubricin groups was still lower than that exhibited by the tendons before treatment; the gliding resistance also increased in these groups at a much more gradual rate than that observed in the saline control group (FIG.15).
  • the proximal pulley surface of saline control tendons appeared to be rough, however the pulley surface of cd-HA-gelatin, cd- gelatin+lubricin, and cd-HA-gelatin+lubricin treated tendons were still smooth (FIG.17).
  • the chemical coating creates a smoother tendon surface and prevents abrasion of the surface by the suture material, thus reducing surface roughness of the repaired tendon.
  • cd-HA-gelatin, cd-gelatin+lubricin, and cd-HA-gelatin+lubricin tendon treatments all improved the gliding resistance of canine repaired FDP tendon compared with controls.
  • the effect of cd-HA-gelatin+lubricin was significantly greater than that of cd-HA-gelatin, suggesting that lubricin is a more effective lubricant for tendon gliding.
  • the gliding resistance of the human palmaris longus (hPL) tendon which is a common source for clinical grafts, is higher than that of canine peroneus longus (cPL) tendon.
  • hPL human palmaris longus
  • cPL canine peroneus longus
  • the mean gliding resistance of human palmaris longus tendon and canine peroneus longus tendon were reported to be 0.52N and 0.09N, respectively.
  • We tested the cd-HA-gelatin and lubricin formulations to determine whether they would reduce the gliding resistance of human palmalis longus tendon. Thirty two fresh-frozen human cadavaric fingers and sixteen Palmaris Longus
  • hPL hPL tendons were obtained from 16 different human cadavers. The third and fourth fingers of each hand were randomly assigned to four different treatment groups. The experimental groups in this study were the same as in Examples 12 and 13. The gliding resistance of human PL tendon was measured before and after treatment in each group.
  • HA-gelatin, cd-gelatin+lubricin, and cd-HA-gelatin+lubricin was still smooth after 1000 cycles of tendon motion (FIG. 20).
  • the cd-HA-gelatin, cd- gelatin+lubricin, and cd-HA-gelatin+lubricin all exhibit improved gliding resistance when contacted to human PL tendon graft, as compared to treatment with a saline control.
  • the difference was not significant, the cd-HA-gelatin+lubricin formulation appeared to be the most effective at reducing the gliding resistance of tendons when examined over 1000 repetitive cycles of tendon motion.
  • Tendon surface modification with lubricants such as HA and lubricin improve the gliding ability of extrasynovial and intrasynovial tendon grafts and repaired tendons and, as a result, treatment of tendons in situ during repair or grafting or in vivo during treatment using this tendon modification method is expected to promote improved outcomes for restoring function during tendon surgeries (e.g., full finger function after injury or repair).

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Abstract

La présente invention concerne les compositions et procédés pour la réduction de la friction aux interfaces entre tissus mous. Les compositions et procédés lient les molécules lubrifiantes naturelles ou synthétiques, l'acide hyaluronique, la lubricine, ou autres tribonectines à la surface des tissus, et réduisent les irrégularités normales de surface par la réticulation de collagène sur la surface des tissus. Ces modifications réduisent la résistance au glissement et facilitent le mouvement des surfaces lubrifiées, en particulier après une blessure ou une opération, et améliorent ainsi les résultats du traitement. Les compositions et procédés peuvent également être utilisés pour limiter la formation d'adhésion dans les tendons et les tissus mous.
PCT/US2008/006049 2007-05-15 2008-05-13 Compositions et procédés pour la réduction de la friction entre la surface des tendons et d'autres tissus mous WO2008143816A1 (fr)

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EP2381957A2 (fr) * 2009-01-13 2011-11-02 Singularis Inc. Modulation thérapeutique de la lubrification limite de l'épithélium vaginal
WO2015061488A1 (fr) * 2013-10-22 2015-04-30 Lubris Llc Production de lubricine recombinée
EP3078388A1 (fr) * 2010-03-22 2016-10-12 Allergan, Inc. Hydrogels réticulés pour l'augmentation des tissus mous
US10500250B2 (en) 2013-11-26 2019-12-10 Lubris Llc Compositions and methods for inhibiting intercellular interactions
US10967048B2 (en) 2015-01-26 2021-04-06 Lubris Llc Use of PRG4 as an anti-inflammatory agent
CN115154667A (zh) * 2022-06-27 2022-10-11 天津大学 一种基于重组润滑素蛋白修饰的胶原蛋白基质的制备方法
US11666529B2 (en) 2015-05-19 2023-06-06 Lubris Llc Use of PRG4 to improve dynamic visual acuity and higher order aberrations

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JP2012515170A (ja) * 2009-01-13 2012-07-05 シンギュラリス，インク． 膣上皮境界潤滑の治療的調節
EP2381957A4 (fr) * 2009-01-13 2013-12-25 Lubris Llc Modulation thérapeutique de la lubrification limite de l'épithélium vaginal
JP2015180620A (ja) * 2009-01-13 2015-10-15 ルブリス，エルエルシー． 膣上皮境界潤滑の治療的調節
EP2381957A2 (fr) * 2009-01-13 2011-11-02 Singularis Inc. Modulation thérapeutique de la lubrification limite de l'épithélium vaginal
EP3520827A1 (fr) * 2010-03-22 2019-08-07 Allergan, Inc. Hydrogels réticulés pour l'augmentation des tissus mous
EP4062950A1 (fr) * 2010-03-22 2022-09-28 Allergan, Inc. Seringue comprenant un agent de remplissage dermique pour l'augmentation des tissus mous
EP3078388A1 (fr) * 2010-03-22 2016-10-12 Allergan, Inc. Hydrogels réticulés pour l'augmentation des tissus mous
EP3520827B1 (fr) 2010-03-22 2022-05-25 Allergan, Inc. Hydrogels réticulés pour l'augmentation des tissus mous
US10905797B2 (en) 2010-03-22 2021-02-02 Allergan, Inc. Polysaccharide and protein-polysaccharide cross-linked hydrogels for soft tissue augmentation
US10111984B2 (en) 2010-03-22 2018-10-30 Allergan, Inc. Polysaccharide and protein-polysaccharide cross-linked hydrogels for soft tissue augmentation
EP3060577A4 (fr) * 2013-10-22 2017-05-17 Lubris LLC Production de lubricine recombinée
EP3971203A1 (fr) * 2013-10-22 2022-03-23 Lubris LLC Production de lubricine recombinée
US10125180B2 (en) 2013-10-22 2018-11-13 Lubris Llc Recombinant lubricin composition
WO2015061488A1 (fr) * 2013-10-22 2015-04-30 Lubris Llc Production de lubricine recombinée
JP2016535590A (ja) * 2013-10-22 2016-11-17 ルブリス，エルエルシー． 組換えラブリシンの産生
JP2020039348A (ja) * 2013-10-22 2020-03-19 ルブリス，エルエルシー． 組換えラブリシンの産生
US10723773B2 (en) 2013-10-22 2020-07-28 Lubris Llc Recombinant lubricin
US9982027B2 (en) 2013-10-22 2018-05-29 Lubris Llc Control of rheological properties of mixed hyaluronate/lubricin solutions
US11485764B2 (en) 2013-10-22 2022-11-01 Lubris Llc Production of recombinant lubricin
AU2014340080B2 (en) * 2013-10-22 2018-12-06 Lubris Llc Production of recombinant lubricin
US10960047B2 (en) 2013-11-26 2021-03-30 Lubris Llc Compositions and methods for inhibiting intercellular interactions
US10500251B2 (en) 2013-11-26 2019-12-10 Lubris Llc Compositions and methods for inhibiting intercellular interactions
US10500250B2 (en) 2013-11-26 2019-12-10 Lubris Llc Compositions and methods for inhibiting intercellular interactions
US11752194B2 (en) 2013-11-26 2023-09-12 Lubris Llc Compositions and methods for inhibiting intercellular interactions
US10967048B2 (en) 2015-01-26 2021-04-06 Lubris Llc Use of PRG4 as an anti-inflammatory agent
US11717557B2 (en) 2015-01-26 2023-08-08 Lubris Llc Use of PRG4 as an anti-inflammatory agent
US11666529B2 (en) 2015-05-19 2023-06-06 Lubris Llc Use of PRG4 to improve dynamic visual acuity and higher order aberrations
CN115154667A (zh) * 2022-06-27 2022-10-11 天津大学 一种基于重组润滑素蛋白修饰的胶原蛋白基质的制备方法

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