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WO2009026705A1 - Procédé de diagnostic et de traitement de l'ostéoarthrite - Google Patents

Procédé de diagnostic et de traitement de l'ostéoarthrite Download PDF

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Publication number
WO2009026705A1
WO2009026705A1 PCT/CA2008/001526 CA2008001526W WO2009026705A1 WO 2009026705 A1 WO2009026705 A1 WO 2009026705A1 CA 2008001526 W CA2008001526 W CA 2008001526W WO 2009026705 A1 WO2009026705 A1 WO 2009026705A1
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tgfα
antagonist
expression
osteoarthritis
mammal
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PCT/CA2008/001526
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English (en)
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Frank Breier
Thomas Appleton
Shirine E. Usmani
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The University Of Western Ontario
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Publication of WO2009026705A1 publication Critical patent/WO2009026705A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • 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
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • 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
    • A61K31/275—Nitriles; Isonitriles
    • A61K31/277—Nitriles; Isonitriles having a ring, e.g. verapamil
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • 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
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G01N2333/495—Transforming growth factor [TGF]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G01N2800/105—Osteoarthritis, e.g. cartilage alteration, hypertrophy of bone

Definitions

  • the present invention generally relates to a method of diagnosing and treating osteoarthritis. More particularly, the present invention relates to the treatment of osteoarthritis by inhibiting TGF ⁇ .
  • OA osteoarthritis
  • ECM cartilage extracellular matrix
  • chondrocytes are responsible for maintaining the cartilage extracellular matrix (ECM), consisting largely of type II collagen and proteoglycans.
  • ECM extracellular matrix
  • chondrocytes are responsible for maintaining the cartilage extracellular matrix (ECM), consisting largely of type II collagen and proteoglycans.
  • ECM extracellular matrix
  • catabolic factors e.g. matrix metalloproteinases
  • chondrocytes also attempt to repair damaged tissues by chondrocyte proliferation, but repair is generally suboptimal.
  • TGF ⁇ transforming growth factor alpha
  • Osteoarthritis is the most common form of arthritis, and affects about 1 in 10 Canadians.
  • the term "osteoarthritis” refers to a degenerative condition in which there is deterioration of cartilage and overgrowth of bone that may result from traumatic injury to a joint or "wear and tear".
  • Osteoarthritis is vastly different from rheumatoid arthritis, a systemic autoimmune disorder which causes the immune system to attack the joints.
  • treatment protocols for osteoarthritis exist, including administration of non-steroidal anti-inflammatory drugs as well as injection of glucocorticoids or hyaluronon, there is no cure for osteoarthritis. Given the prevalence of osteoarthritis, it would be desirable to develop additional methods of treatment.
  • a method of treating osteoarthritis in a mammal comprising the step of inhibiting, or at least reducing TGF ⁇ activity and/or expression in the articular cartilage in the mammal.
  • composition for treating osteoarthritis comprising a TGF ⁇ antagonist in combination with a pharmaceutically acceptable carrier.
  • an article of manufacture comprising packaging within which is contained a composition comprising a TGF ⁇ antagonist in combination with a pharmaceutically acceptable carrier, wherein said packaging is labelled to indicate that said composition is useful to treat osteoarthritis.
  • a method of diagnosing osteoarthritis in a mammal comprising the step of quantifying in a biological sample obtained from the mammal the level of TGF ⁇ expression in the sample, wherein an increased level of TGF ⁇ expression in the sample in comparison to a healthy control is indicative of osteoarthritis.
  • a method of screening candidate compounds useful to treat osteoarthritis comprises the steps of: i) incubating a candidate compound with TGF ⁇ in the presence of the EGF receptor under conditions suitable for ligand receptor interaction; and ii) determining the level of interaction, wherein a decrease in the level of interaction in comparison to the level of interaction achieved in the absence of the candidate compound indicates that the compound may be useful to treat osteoarthritis.
  • kits for use in the diagnosis of osteoarthritis in a mammal comprises at least one agent useful to detect TGF ⁇ expression in a biological sample from the mammal.
  • Figure 1 graphically illustrates TGF ⁇ expression in osteoarthritic cartilage as compared with normal controls
  • Figure 2 graphically illustrates the effect of TGF ⁇ on chondrocyte proliferation
  • FIG. 3 illustrates the effect of TGF ⁇ on expression of anabolic genes, including aggrecan (A), type II collagen (B), cartilage link protein (C), as well as the effect on total collagen (D);
  • Figure 4 illustrates the effect of TGF ⁇ on expression of catabolic and inflammatory mediator genes such as Mmpl3 (A), cathepsin C (B), Adamts5 (C), Ccl2 (D) and TNF ⁇ (E);
  • Figure 5 graphically illustrates the effect of TGF ⁇ on chondrocyte cluster formation;
  • Figure 6 graphically illustrates the effect of TGF ⁇ on expression of SOX9 (A) and down-regulation of SOX9 protein levels (B) in OA cartilage.
  • a method of treating osteoarthritis in a mammal comprises the step of inhibiting, or at least reducing, TGF ⁇ activity in the articular cartilage of the mammal.
  • TGF ⁇ refers to any mammalian transforming growth factor-alpha including human TGF ⁇ , for example, as depicted by GenBank NM_003236.2 and as described by Derynck et al. (in Cell 38 (1), 287-297 (1984)), non-human forms of TGF ⁇ from other species and human or non-human functionally equivalent variants or subtypes of TGF ⁇ which exhibit functional equivalence to TGF ⁇ insofar as the increased expression thereof results in degeneration of articular cartilage resulting in osteoarthritis.
  • Non-limiting examples of non-human forms of TGF ⁇ include, but are not limited to, mouse TGF-alpha as described in Vaughan,TJ. et al. Biochim. Biophys. Acta 1132 (3), 322-324 (1992), and rat TGF-alpha as described in Lee et al. Nature 313 (6002), 489-491 (1985).
  • treatment refers to the prophylaxis, amelioration or elimination of osteoarthritis or at least one effect associated with osteoarthritis including, for example, tissue degradation, pain and the like.
  • treatment may also refer to the prevention of osteoarthritis in a mammal that may be predisposed to the disease, for example, as a result of trauma to one or more joints from sports or other activities.
  • TGF ⁇ activity encompasses inhibition of TGF ⁇ activity in whole or in part.
  • inhibition of TGF ⁇ activity includes inhibition of the TGF ⁇ protein per se, inhibition at the EGF receptor, as well as inhibition of the gene and other nucleic acids expressing the protein.
  • TGF ⁇ activity may be inhibited by administration of a TGF ⁇ antagonist or inhibitor, e.g. an entity capable of down-regulating the expression or activity of TGF ⁇ in the articular cartilage, such as a receptor-binding antagonist.
  • TGF ⁇ is known to bind to the EGF receptor, which is known as "ErbBl" (GenBank - NM 005228).
  • the EGF receptor is a single-chain glycoprotein (1186 amino acids) containing three functional domains: 1) an extracellular, glycosylated portion that binds to a ligand such as EGF and TGF ⁇ ; 2) a small transmembrane portion; and 3) a cytoplasmic portion that has the intrinsic tyrosine kinase activity and multiple sites that can be phosphorylated.
  • EGF/TGF ⁇ binds to the receptor its intrinsic tyrosine kinase is activated, resulting in increased phosphorylation of intracellular tyrosine residues both on the receptor (autophosphorylation sites) and on exogenous proteins involved in regulating cellular functions.
  • EGF/TGF ⁇ antagonist compounds suitable for use in the present treatment method include, for example, tyrosine kinase inhibitors, tyrphostins such as tyrphostin AG1478 (4-(3-Chloroanillino)-6,7- dimethoxyquinazoline), gefitinib (N-(3-chloro-4-fluoro-phenyl)-7-methoxy-6-(3- morpholin-4-ylpropoxy)quinazolin-4-amine), erlotinib (N-(3-ethynylphenyl)-6,7- bis(2-methoxyethoxy)quinazolin-4-amine), erbstatin, PD 153035, lapatanib (GW572016), GW583340, BIBX1382BS, and functionally equivalent analogs of any of these, e.
  • tyrosine kinase inhibitors such as tyrphostin AG1478 (4
  • suitable TGF ⁇ antagonists may readily be identified using established protocols, for example cell line screening methods in which cells are modified by transfection to express functional mammalian TGF ⁇ in a detectable manner. The modified cell line is then grown in appropriate media in the presence of a candidate antagonist. Inhibition of TGF ⁇ activity, e.g. receptor binding, can then be monitored to identify candidates which exhibit TGF ⁇ inhibition. Accordingly, a method of screening for TGF ⁇ antagonists is provided comprising incubating TGF ⁇ and a candidate TGF ⁇ antagonist in the presence of TGF ⁇ receptor under conditions suitable to permit ligand receptor interaction, e.g. binding, and determining the extent of interaction, e.g.
  • TGF ⁇ antagonist by measuring a resulting activity such as tyrosine kinase activity or another downstream event.
  • a resulting activity such as tyrosine kinase activity or another downstream event.
  • antibody antagonists may be utilized directed either to TGF ⁇ per se, or directed to a TGF ⁇ receptor such as the EGF receptor, which are effective to block TGF ⁇ activity. Details of methods of making antibodies for this purpose are included herein in the description that follows.
  • a TGF ⁇ antagonist may be administered directly into the articular cartilage by injection, or by intramuscular or intraperitoneal injection; however, as one of skill in the art will appreciate, other modes of administration may also be used including, for example, oral administration either alone or in conjunction with a carrier compound that selectively targets the articular cartilage; intravenous administration; topical administration in which the antagonist is combined with a carrier suitable to penetrate the skin and carry the inhibitor to the desired target site; and by intranasal administration.
  • Suitable dosages of TGF ⁇ antagonists for use in treating osteoarthritis can readily be determined using established in vivo protocols as well as by implementing appropriately controlled trials as one of skill in the art will appreciate. Appropriate dosages for administration to a mammal in need of treatment will depend on many factors including, for example, the mammal being treated, the extent of the condition being treated, the mode of administration and the TGF ⁇ antagonist being utilized. In one embodiment, a TGF ⁇ antagonist dosage in a range of about 1-1000 mg/kg may be used to treat osteoarthritis.
  • a genetic approach for treating osteoarthritis may also be applied, for example, to inhibit expression of TGF ⁇ in articular cartilage, thereby preventing or at least reducing TGF ⁇ activity in the articular cartilage.
  • Oligonucleotides such as antisense, siRNA and other suitable oligonucleotides, that may be used to selectively target the TGF ⁇ gene in the articular cartilage and block TGF ⁇ expression in the articular cartilage resulting in reduced TGF ⁇ activity in the articular cartilage and, thus, decreased degeneration of the articular cartilage.
  • Such blocking occurs when a selected oligonucleotide binds to the TGF ⁇ gene thereby preventing transcription or translation of the gene to yield functional protein.
  • oligonucleotide refers to an oligomer or polymer of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars, and intersugar (backbone) linkages.
  • the term also includes modified or substituted oligomers comprising non-naturally occurring monomers or portions thereof, which function similarly. Such modified or substituted oligonucleotides may be preferred over naturally occurring forms because of properties such as enhanced cellular uptake, or increased stability in the presence of nucleases.
  • the term also includes chimeric oligonucleotides which contain two or more chemically distinct regions.
  • chimeric oligonucleotides may contain at least one region of modified nucleotides that confer beneficial properties (e.g. increased nuclease resistance, increased uptake into cells), or two or more oligonucleotides of the invention may be joined to form a chimeric oligonucleotide.
  • antisense oligonucleotide as used herein means a nucleotide sequence that is complementary to a target TGF ⁇ -encoding nucleic acid sequence.
  • the antisense oligonucleotides of the present invention may be ribonucleic or deoxyribonucleic acids and may contain naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil.
  • the oligonucleotides may also contain modified bases such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines, 8-amino guanine, 8-thiol guanine, 8-thiolalkyl guanines, 8-hydrodyl guanine and other 8- substituted guanines, other aza and deaza uracils, thymidines, cytosines, adenines, or guanines, 5-tri-fluoromethyl urac
  • Other antisense oligonucleotides of the invention may contain modified phosphorous, oxygen heteroatoms in the phosphate backbone, short chain alkyl or cycloalkyl intersugar linages or short chain heteroatomic or heterocyclic intersugar linkages.
  • the antisense oligonucleotides may contain phosphorothioates, phosphotriesters, methyl phosphonates, and phosphorodithioates.
  • phosphorothioate bonds may link only the four to six 3 '-terminal bases, may link all the nucleotides or may link only 1 pair of bases.
  • the antisense oligonucleotides of the invention may also comprise nucleotide analogs that may be better suited as therapeutic or experimental reagents.
  • An example of an oligonucleotide analogue is a peptide nucleic acid (PNA) in which the deoxyribose (or ribose) phosphate backbone in the DNA (or RNA), is replaced with a polymide backbone which is similar to that found in peptides (P.E. Nielson, et al Science 1991, 254, 1497).
  • PNA analogues have been shown to be resistant to degradation by enzymes and to have extended lives in vivo and in vitro.
  • oligonucleotide analogues may contain nucleotides containing polymer backbones, cyclic backbones, or acyclic backbones.
  • the nucleotides may have morpholino backbone structures (U.S. Pat. No. 5,034,506).
  • Oligonucleotide analogues may also contain groups such as reporter groups, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an antisense oligonucleotide.
  • Antisense oligonucleotides may also incorporate sugar mimetics as will be appreciated by one of skill in the art.
  • Antisense nucleic acid molecules may be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art based on TGF ⁇ amino acid sequence information such as that provided.
  • the antisense nucleic acid molecules of the invention, or fragments thereof, may be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed with mRNA or the native gene, e.g. phosphorothioate derivatives and acridine substituted nucleotides.
  • the antisense sequences may be produced biologically using an expression vector introduced into cells in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense sequences are produced under the control of a high efficiency regulatory region, the activity of which may be determined by the cell type into which the vector is introduced.
  • the antisense oligonucleotides may be introduced into tissues or cells using well-established techniques in the art including via vectors (retroviral vectors, adenoviral vectors and DNA virus vectors) or physical techniques such as microinjection.
  • the antisense oligonucleotides may be directly administered in vivo or may be used to transfect cells in vitro which are then administered in vivo.
  • siRNA technology may be applied to prevent expression of TGF ⁇ .
  • Application of nucleic acid fragments such as siRNA fragments that correspond with regions in the TGF ⁇ gene and which selectively target the TGF ⁇ gene may be used to block TGF ⁇ expression thereby reducing the cartilage degeneration associated with osteoarthritis. Such blocking occurs when the siRNA fragments bind to the TGF ⁇ thereby preventing translation of the gene to yield functional protein.
  • RNA molecules small interfering RNA molecules, corresponding to TGF ⁇ may be made using well-established methods of nucleic acid syntheses including automated systems. Since the structure of the TGF ⁇ gene is known, fragments of RNA that correspond therewith can readily be made. Reference may be made, for example, to Derynck et al. Cell 38 (1), 287-297 (1984) for TGF ⁇ nucleic acid sequence data. The effectiveness of selected siRNA to block TGF ⁇ activity can be confirmed using TGF ⁇ -expressing cell lines as described above.
  • SiRNA fragments may be designed according to the Tuschl design rule in which target sites begin with AA, include 3'- UU overhangs for both the sense and antisense SiRNA strands, have approximately 50% G/C content and are 50-100 nucleotides downstream of the start codon.
  • SiRNA designed based on the Tuschl rule may be more efficient in inhibition of targeted mRNA expression.
  • siRNA fragments useful in the present method may be derived from native siRNA fragments, i.e. siRNA fragments corresponding with a region of the gene such as those identified above.
  • Modifications that may be made to native siRNA fragments to render a functionally equivalent variant siRNA strand include, for example, addition, deletion or substitution of one or more of the nucleotide bases therein, provided that the modified siRNA retains it ability to bind to the target gene.
  • Selected siRNA fragments may additionally be modified to incorporate desired properties such as increased stability.
  • siRNA fragments may be modified to include terminal protecting groups such as poly-A tails.
  • Synthetic siRNA can be administered to a patient for delivery to a target site using techniques established in the art including cation lipid-mediated transfection.
  • suitable cationic lipids include compounds such as oligofectamine and DOTAP.
  • SiRNA may also be administered using a polycation-based gene delivery system such as a PEI conjugate.
  • protocol associated with the administration of siRNA to block TGF ⁇ expression can be determined using techniques known in the art such as those described in Tan et al. Gene Therapy (2005) 12, 59-66.
  • TGFa antagonists may be administered to a mammal being treated for osteoarthritis either alone or in combination with one or more pharmaceutically acceptable adjuvants or carriers to form a composition which facilitates administration and delivery of the antagonist.
  • pharmaceutically acceptable carrier means acceptable for use in the pharmaceutical and veterinary arts, i.e. not being unacceptably toxic or otherwise unsuitable for administration to a mammal.
  • pharmaceutically acceptable adjuvants include any and all solvents, dispersion media, coatings, flavouring agents, wetting and emulsifying agents, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the antagonist is formulated for administration by infusion, or by injection either subcutaneously or intravenously, and is accordingly utilized as an aqueous solution in sterile and pyrogen-free form and optionally buffered or made isotonic.
  • the antagonist may be administered in distilled water or, more desirably, in saline, phosphate-buffered saline or 5% dextrose solution.
  • compositions for oral administration via tablet, capsule or suspension are prepared using adjuvants including sugars, such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and derivatives thereof, including sodium carboxymethylcellulose, ethylcellulose and cellulose acetates; powdered tragancanth; malt; gelatin; talc; stearic acids; magnesium stearate; calcium sulfate; vegetable oils, such as peanut oils, cotton seed oil, sesame oil, olive oil and corn oil; polyols such as propylene glycol, glycerine, sorbital, mannitol and polyethylene glycol; agar; alginic acids; water; isotonic saline and phosphate buffer solutions.
  • sugars such as lactose, glucose and sucrose
  • starches such as corn starch and potato starch
  • Creams, lotions and ointments may be prepared for topical application using an appropriate base such as a triglyceride base. Such creams, lotions and ointments may also contain a surface active agent. Topical application may also be via a patch incorporating the antagonist in a suitable form. Aerosol formulations, for example, for nasal delivery, may also be prepared in which suitable propellant adjuvants are used. Other adjuvants may also be added to the composition regardless of how it is to be administered, for example, anti-microbial agents may be added to the composition to prevent microbial growth over prolonged storage periods.
  • Inhibition of TGFoc may be used to treat existing osteoarthritis as indicated, but may also be used to prevent osteoarthritis from occurring.
  • utilizing a treatment protocol as described e.g. administration of a TGF ⁇ antagonist or an oligonucleotide which inhibits TGF ⁇ expression, to the joint(s) of a mammal at risk of developing osteoarthritis as a result of injury, or to the joint of a mammal otherwise pre-disposed to osteoarthritis, may be utilized as a preventative measure to inhibit or at least reduce the degenerative osteoarthritic events that may occur.
  • administration of the selected treatment may be oral, by injection, intravenously or topically.
  • an article of manufacture comprises packaging and a composition suitable to inhibit TGF ⁇ activity in articular cartilage.
  • the packaging is labeled to indicate that the composition is useful to treat osteoarthritis.
  • the packaging may be any container suitable to retain the composition including, for example, a carton, bottle, wrap, tube or box.
  • the composition suitable to inhibit TGF ⁇ activity may comprise a TGF ⁇ antagonist.
  • the composition may comprise nucleic acid directed to the TGF ⁇ gene and capable of inhibiting the expression thereof.
  • a method of diagnosing osteoarthritis in a mammal comprises the step of quantifying the expression of one of TGF ⁇ expression or activity in a biological sample obtained from the mammal. Detection of increased TGF ⁇ expression/activity in the sample in comparison with a standard, e.g. a comparable sample known to be healthy and free of osteoarthritis, is indicative of osteoarthritis.
  • biological sample as it is used herein is meant to refer to any sample that may be obtained from a mammal to be diagnosed containing articular cartilage.
  • Quantification of TGF ⁇ expression and/or activity may be conducted using established techniques such as those described in the specific examples that follow, including, for example, quantification of TGF ⁇ expression using nucleic acid probing techniques and quantification of TGF ⁇ activity using established assays such as immunoassays.
  • antibodies to TGF ⁇ may be prepared and effectively utilized.
  • Conventional methods may be used to prepare the antibodies including polyclonal antisera or monoclonal antibodies.
  • a mammal e.g. a mouse, hamster, or rabbit
  • an immunogenic form of the protein which elicits an antibody response in the mammal.
  • Techniques for conferring immunogenicity on a peptide are well known in the art and include, for example, conjugation to carriers.
  • the peptide can be administered in the presence of adjuvant.
  • the progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassay procedures can be used with the immunogen as antigen to assess antibody levels.
  • antisera can be obtained and, if desired, polyclonal antibodies isolated from the sera.
  • antibody-producing cells are harvested from an immunized animal and fused with myeloma cells by standard somatic cell fusion procedures to form immortal hybridoma cells.
  • Such techniques are well known in the art, (e.g., the hybridoma technique originally developed by Kohler and Milstein (Nature 256, 495-497(1975)) as well as other techniques such as the human B-cell hybridoma technique (Kozbor et al., Immunol. Today 4, 72 (1983)), the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., Monoclonal Antibodies in Cancer Therapy (1985) Allen R.
  • Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with TGF ⁇ and the monoclonal antibodies can be isolated.
  • antibody as used herein is intended to include fragments thereof which also specifically react with TGF ⁇ according to the invention.
  • Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above. For example, fragments can be generated by treating an antibody with pepsin. The resulting fragment can be further treated to reduce disulfide bridges.
  • Chimeric antibody derivatives i.e., antibody molecules resulting from the combination of a variable non-human animal peptide region and a constant human peptide region are also contemplated within the scope of the invention.
  • Chimeric antibody molecules can include, for example, the antigen binding domain from an antibody of a mouse, rat, or other species with a constant human peptide region.
  • Conventional methods may be used to make chimeric antibodies containing the immunoglobulin variable region which recognizes TGF ⁇ (see, for example, Morrison et al., Proc. Natl Acad. Sci. U.S.A. 81,6851 (1985); Takeda et al., Nature 314, 452(1985), Cabilly et al., U.S. Pat. No. 4,816,567; Boss et al., U.S. Pat. No. 4,816,397; Tanaguchi et al., European Patent Publication EP171496; European Patent Publication EPO 173494).
  • Monoclonal or chimeric antibodies specifically reactive with TGF ⁇ may be further humanized by producing human constant region chimeras, in which parts of the variable regions, particularly the conserved framework regions of the antigen- binding domain, are of human origin and only the hypervariable regions are of non- human origin.
  • Such immunoglobulin molecules may be made by techniques known in the art, (e.g., Teng et al, Proc. Natl. Acad. Sci. U.S.A.., 80, 7308-7312 (1983); Kozbor et al., Immunology Today, 4, 7279 (1983); Olsson et al., Meth. Enzymol., 92, 3-16 (1982)), and PCT Publication WO92/06193 or EP 0239400). Humanized antibodies can also be commercially produced (Scotgen Limited, 2 Holly Road, Twickenham, Middlesex, Great. Britain).
  • the kit comprises at least one agent useful to detect and quantify the level of TGF ⁇ expression in a biological sample from the mammal.
  • the kit may comprise an oligonucleotide probe or probes directed to TGF ⁇ -encoding nucleic acid.
  • a biological sample obtained from a mammal is probed using the nucleic acid probe(s) of the kit to quantify TGF ⁇ mRNA which may then be compared to an accepted standard or healthy control (e.g. TGF ⁇ expression in the articular cartilage of healthy mammals).
  • An increase in the level of TGF ⁇ expression in the sample as compared with the level of TGF ⁇ expression in a healthy control is indicative of osteoarthritis, for example, an increase of at least about 100% in TGF ⁇ expression, e.g. a 1-fold increase, is indicative of osteoarthritis.
  • a diagnostic kit may comprise an antibody directed to TGF ⁇ .
  • the antibody may be pre-labeled with a detectable label, such as a fluorescent or other suitable label, or can be labeled prior to use.
  • the kit may comprise a primary antibody directed to TGF ⁇ , and a secondary antibody, either labeled or not, directed to the primary antibody.
  • Cell and tissue culture media reagents were purchased from Invitrogen (Burlington, ON, Canada) or Sigma (Oakville, ON, Canada).
  • Total RNA was isolated from articular cartilage samples from each knee as previously described (Appleton, in press) using a QIAgen RNeasy Lipid Tissue Mini Kit, and from primary cell cultures using a QIAgen RNeasy Mini Kit according to the manufacturer's protocols.
  • Real-time PCR analysis was performed using the Applied Biosystems 7900HT Real-Time PCR System, the TaqMan ® One-step Mastermix Kit, and TaqMan ® Gene Expression Assays for each gene assessed relative to Gapdh expression.
  • Real-time PCR data are an average of 4 or 5 independent samples assayed in quadruplicate. Means were quantified relative to Gapdh and normalized to controls on day 0 of culture (set to 1). An unpaired t-test was used to compare between sham and OA Tgfa and Kitl expression means determined by real-time PCR and between sham and OA relative protein levels determined by densitometry. All other real-time PCR experiments, cell number, collagen production and organ culture cell counting data were analyzed by two-way analysis of variance (ANOVA) with a Tukey's post- hoc test. P ⁇ 0.05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism 4.0 software (GraphPad Software, San Diego, CA, USA).
  • TGFa expression is increased in degenerating cartilage
  • IHC for phospho-EGFR in degrading cartilage determined that phospho-EGFR levels were higher in osteoarthritic cartilage compared to shams, indicating that increased expression of TGF ⁇ leads to increased EGF receptor activation in degrading cartilage.
  • Human knee cartilage was obtained from 13 normal individuals (mean age of 71.7 years) and 12 late-stage OA (mean age of 64.7 years), classified according to American College of Rheumatology criteria as described (Altman. 1991. J Rheumatol Suppl 27:10-12).
  • Microarray analysis of RNA extracts from cartilage samples was performed at the M ⁇ nster University (FRG) Genomics Center using the Affymetrix GeneChip ® Human Genome U133 Plus 2.0 Array for more than 47,000 transcripts. Array experiments were performed according to protocols supplied by Affymetrix.
  • cDNA fragments were hybridized with a pre-equilibrated Affymetrix chip, washed, stained and scanned in the HP ChipScanner (Affymetrix Inc., Santa Clara, CA, USA).
  • OA samples could be separated into two groups; 7 out of 12 samples showed similar TGF ⁇ mRNA levels as control samples, but expression was markedly increased in 5 of 12 OA samples. These data confirm that TGF ⁇ transcript levels are increased in a subset of human OA patients.
  • media was replaced daily with serum-free media and vehicle (0.1 % bovine serum albumin in PBS) or 10 ng/ml recombinant human transforming growth factor alpha (TGF ⁇ ) (Biosource - Medicorp, Montreal, QC, Canada) or 10 ng/ml recombinant human kit ligand (Biosource - Medicorp, Montreal, QC, Canada).
  • TGF ⁇ human transforming growth factor alpha
  • Biosource - Medicorp Biosource - Medicorp, Montreal, QC, Canada
  • Cultures were maintained at 37 C in a humidified environment with 5 % CO 2 .
  • chondrocyte morphology was assessed using phase contrast microscopy.
  • chondrocytes were permeabilized and probed with anti-cleaved caspase-3 primary antibody (Cell Signaling Technologies, Danvers, MA, USA) or anti-phospho (S 181 ) SOX9 antibody, followed by secondary antibodies conjugated to FITC.
  • nuclei were counterstained with Toto-3 iodide (Molecular Probes, Burlington, ON, Canada). Coverslips were mounted with Vectashield ® (Vector Laboratories, Burlingame, CA, USA), and images were acquired using a Zeiss LSM510 META confocal microscope and Axiovision LE image processing software (Carl Zeiss, Toronto, ON, Canada).
  • Total soluble collagen was measured in culture supernatants on each day of culture using the SircolTM Collagen Assay (Accurate Chemical & Scientific Corp., Wesbury, NY, USA). One ml of SircolTM dye was added to 200 ⁇ l of each sample supernatant (assayed in duplicate). Samples and dye were mixed at room temperature for 30 min. Collagen-dye complexes were precipitated by centrifugation at 15,000 X g for 5 min. The pelleted material was washed twice with ethanol and re-suspended in 0.5 M NaOH. Absorbance at 540 nm was measured using a spectrophotometer. Collagen concentrations (mg/ml) were interpolated from a standard curve set up using collagen standards provided by the manufacturer.
  • TGFa signaling reduces anabolic activity in primary chondrocytes
  • articular chondrocytes One of the primary functions of articular chondrocytes is to maintain and repair the cartilage extracellular matrix (ECM).
  • ECM cartilage extracellular matrix
  • Primary chondrocyte anabolic gene expression in response to TGF ⁇ stimulation was investigated.
  • Treatment of chondrocytes with TGF ⁇ resulted in significantly attenuated expression levels of transcripts for aggrecan (Agcl), type II collagen (Col2al), and cartilage link protein (Crtll) (Figure 3A-C). These reductions were significant after 24 hours of treatment and more pronounced after 3 days.
  • Spectrophotometric analysis of collagen-dye complexes demonstrated a significant reduction in total soluble collagen in the media on the second and third days of TGF ⁇ treatment (Figure 3D).
  • TGFa induces the expression of genes known to play a role in human OA
  • Ctsc cathepsin C
  • Ctss cathepsin S
  • chemokines trigger a cascade of degenerative events in OA.
  • the chemokine Ccl2 (encoding chemokine (C-C motif) ligand 2) has been shown to be highly induced in degrading cartilage. High levels of Ccl2 have also been identified in human OA chondrocytes.
  • Real-time PCR determined that TGF ⁇ induced the expression of Ccl2 in primary chondrocytes more than 4-fold ( Figure 4D).
  • TGF ⁇ also induced a transient increase in transcripts of the inflammatory mediator C0X2 (encoded by Ptgs2).
  • C0X2 functions in prostaglandin synthesis and is known for its inducible role in cartilage inflammation.
  • kits ligand [KUI) activate the c-kit RTK transcripts were previously determined to increase approximately 5-fold compared to normal cartilage. This was confirmed using real-time PCR. The effects of kit ligand on primary chondrocytes was also determined. Over 3 days in culture, kit ligand had no effect on either the number of primary chondrocytes or on the levels o ⁇ Col2al, Agcl, Crtll or Mmpl3 gene expression.
  • TGFa stimulates articular chondrocyte cluster formation and catabolic factor expression
  • chondrocyte clusters are the features of degrading OA cartilage. Having determined that TGF ⁇ stimulates chondrocyte proliferation, induction of chondrocyte cluster formation was investigated in the authentic three-dimensional context of articular cartilage using osteochondral explants treated with 10 ng/ml TGF ⁇ . Explant histology with safranin- O and fast green staining revealed that TGF ⁇ induced the presence of chondrocyte clusters, starting after 3 days of treatment, with greater effects after 7 days.
  • TGFa diminishes phosphorylated SOX9 levels andSox9 expression
  • TGFa contributes to molecular events influencing cartilage degeneration.
  • Molecular events in which TGF ⁇ plays a role that result in onset of OA include the following.
  • Activation of EGFR levels are concomitantly increased in degrading articular cartilage. Higher levels of activated EGFR correlate well with the increased expression of TGF ⁇ .
  • kit ligand which activates the c-kit receptor, another tyrosine kinase receptor
  • kit ligand was also found to be increased in degrading cartilage, but did not affect chondrocytes according to the parameters tested herein.

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Abstract

La présente invention concerne un procédé de traitement de l'ostéoarthrite chez un mammifère comprenant une étape d'inhibition de l'expression et/ou de l'activité de TGFα dans le cartilage articulaire du mammifère. L'invention concerne également un procédé de diagnostic de l'ostéoarthrite chez un mammifère comprenant l'identification de l'expression et/ou de l'activité accrue de TGFα dans le cartilage articulaire d'un mammifère.
PCT/CA2008/001526 2007-08-24 2008-08-22 Procédé de diagnostic et de traitement de l'ostéoarthrite WO2009026705A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011132182A1 (fr) 2010-04-18 2011-10-27 Yeda Research And Development Co. Ltd. Molécules et méthodes d'utilisation de celles-ci pour le traitement des maladies associées aux ligands erbb/erbb

Citations (3)

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Publication number Priority date Publication date Assignee Title
US6552066B1 (en) * 1995-09-11 2003-04-22 Thomas R. Sharpe Protein tyrosine kinase inhibitors for treating osteoarthritis
WO2006063042A2 (fr) * 2004-12-07 2006-06-15 Genentech, Inc. Selection de patients pour une therapie avec un inhibiteur de her
CA2604985A1 (fr) * 2006-10-02 2008-04-02 The University Of Western Ontario Methodes de diagnostic d'osteoarthrite

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
US6552066B1 (en) * 1995-09-11 2003-04-22 Thomas R. Sharpe Protein tyrosine kinase inhibitors for treating osteoarthritis
WO2006063042A2 (fr) * 2004-12-07 2006-06-15 Genentech, Inc. Selection de patients pour une therapie avec un inhibiteur de her
CA2604985A1 (fr) * 2006-10-02 2008-04-02 The University Of Western Ontario Methodes de diagnostic d'osteoarthrite

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Title
APPLETON, C. T. ET AL.: "Transforming Growth Factor Alpha Suppression Of Articular Chondrocyte Phenotype And Sox9 Expression In A Rat Model Of Osteoarthritis", ARTHRITIS RHEUM, vol. 56, November 2007 (2007-11-01), pages 3693 - 3705, XP055354485, ISSN: 0004-3591 *
HALLBECK, A. L. ET AL.: "TGF-alpha And ErbB2 Production In Synovial Joint Tissue: Increased Expression In Arthritic Joints", SCAND J RHEUMATOL, vol. 34, May 2005 (2005-05-01), pages 204 - 211, ISSN: 0300-9742 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011132182A1 (fr) 2010-04-18 2011-10-27 Yeda Research And Development Co. Ltd. Molécules et méthodes d'utilisation de celles-ci pour le traitement des maladies associées aux ligands erbb/erbb

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