WO2009086952A2 - Compositions de traitement de maladies articulaires dégénératives - Google Patents

Compositions de traitement de maladies articulaires dégénératives Download PDF

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WO2009086952A2
WO2009086952A2 PCT/EP2008/062287 EP2008062287W WO2009086952A2 WO 2009086952 A2 WO2009086952 A2 WO 2009086952A2 EP 2008062287 W EP2008062287 W EP 2008062287W WO 2009086952 A2 WO2009086952 A2 WO 2009086952A2
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disease
differentiation
inhibitor
chondrocyte
articular
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PCT/EP2008/062287
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WO2009086952A3 (fr
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Juan Carlos RODRÍGUEZ CIMADEVILLA
Cecilia Lorena Sgarlata
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Projech Science To Technology, S.L.
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Publication of WO2009086952A3 publication Critical patent/WO2009086952A3/fr

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    • 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
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • 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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
    • A61K31/423—Oxazoles condensed with carbocyclic 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • 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
    • A61K31/52—Purines, e.g. adenine
    • A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
    • 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
    • A61K31/54—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 sulfur as the ring hetero atoms, e.g. sulthiame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • 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
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
    • 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
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/91091—Glycosyltransferases (2.4)
    • G01N2333/91097—Hexosyltransferases (general) (2.4.1)
    • G01N2333/91102—Hexosyltransferases (general) (2.4.1) with definite EC number (2.4.1.-)
    • 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 technical field of the present invention relates to the therapeutic uses O- linked N-acetylglucosamine transferase inhibitors in the treatment of diseases which involve terminal cell differentiation, in particular, chondrocyte hypertrophy.
  • cartilage chondrocytes
  • a hydrophilic extracellular matrix composed by collagen mainly Collagen II
  • Proteoglycans mainly aggrecan.
  • Cartilage functions include distributing weight, minimize the stress inflicted on subchondral bone and, in general, providing a platform that supports weight and reduces friction.
  • articular cartilage Compared with other tissues, articular cartilage has low metabolic activity, and, in adult life, it contains neither blood-vessels nor nerve terminals. In order to fulfil its functions, however, articular cartilage needs to maintain intact its complex architecture.
  • Osteoarthritis known in the past as degenerative arthritis, is the most common form of arthritis. Osteoarthritis is a chronic degenerative joint condition, characterized by degeneration of articular cartilage. There is also hypertrophy chondrocytes at the joint margins, and changes in the synovial membrane. Secondary changes in underlying bone cause pain and affect joint function.
  • Osteoarthritis is strongly age-related, with over 50% of people over the age of 70 being treated for this condition. It is also associated with obesity and with over-use injuries. It is currently estimated that in the United States 35 million people- 13 percent of the population-are 65 and older, and that more than half of these people have radiological evidence of osteoarthritis in at least one joint. It is estimated that there are about 103 million osteoarthritis sufferers in the European Union.
  • therapies for osteoarthritis are directed mainly towards treating the symptoms.
  • therapies available to treat osteoarthritis are limited to the use of analgesics and/or anti-inflammatory agents, reduction in pressure across the joint, and weight loss.
  • Most current treatments are designed only to relieve pain and/or inflammation, and to reduce or prevent the disability caused by bone and cartilage degeneration.
  • treatments for osteoarthritis include non-pharmacological and pharmacological interventions.
  • Non-pharmacological interventions can include weight loss, physical therapy, and surgical procedures.
  • Pharmacological treatment can include oral analgesics such as acetaminophen and non-steroidal anti-inflammatory drugs (NSAIDs), topical analgesics such as capsaicin, corticosteroids, and hyaluronate injections. Dietary supplements such as glucosamine and chondroitin are also used to treat osteoarthritis. One of the most common treatments for osteoarthritis is NSAID therapy.
  • NSAIDs non-steroidal anti-inflammatory drugs
  • NSAIDs are believed to work by inhibiting the enzyme cyclooxygenase (COX), which catalyzes the conversion of arachidonic acid to prostaglandins and leukotrienes, thereby reducing their respective biosynthesis.
  • COX-2 selective inhibitors or COX-2 inhibitors can be relatively more specific for inhibiting the COX-2 isoenzyme in different tissues under certain and relative circumstances.
  • COX-2 inhibitors when administered to a mammal for therapeutic purposes, may have the potential to reduce the risk of clinically significant and life-threatening perforations, obstructions and bleedings (POBs) of the upper gastrointestinal system, and a reduced risk of altering platelet function when compared to other less selective and/or specific NSAID COX inhibitors.
  • POBs obstructions and bleedings
  • WO2007013078 discloses a method of treating a disease in which up-regulating glycosaminoglycans (GAGs) is therapeutically beneficial, such as osteoarthritis.
  • the method comprises locally administering to a subject a therapeutically effective amount of an agent capable of down-regulating activity or expression of a component of the renin- angiotensin system.
  • Glucosamine is an endogenous amino monosaccharide that is synthesized intracellularly from glucose.
  • the majority of the glucosamine synthesized in the cell is destined for glycoprotein synthesis.
  • Quantitatively, most glycosylation occurs on those proteins destined for export or the cell surface. This type of glycosylation is initiated as the protein is being translated in the rough endoplasmic reticulum mostly through the N- linkage of complex sugar groups to asparagine residues in the protein.
  • These N-linked complex sugar chains are then modified further in the Golgi apparatus prior to the trafficking of these proteins to the plasma membrane or to exocytotic vesicles.
  • glycosylation involves the 0-linkage of the monosaccharide N- Acetyl glucosamine (GIcNAc) to serine or threonine residues in the protein backbone.
  • This modification is catalyzed by O-linked N-acetylglucosamine transferase (O-GlcNAc transferase or OGT).
  • OGT cDNA has been cloned and the expression of this enzyme is ubiquitous, although it appears to be most highly expressed in the pancreas, brain and pituitary.
  • pancreatic [beta] -cells In cells with high level of OGT expression, blockage of N-acetyl-[beta]-D-glucosaminidase (O- GlcNAcase), which cleaves O-linked N-acetylglucosamine off protein, results in unopposed O-GlcNAc transferase activity that would cause cellular pathology.
  • O- GlcNAcase N-acetyl-[beta]-D-glucosaminidase
  • pancreatic [beta] -cells In situ hybridization showed that pancreatic [beta] -cells have the highest level of O-GicNAc transferase mRNA expression of all known cells. Pancreatic [beta]-cells undergo glucose-dependent apoptosis following blockage of O-GlcNAcase.
  • O-GlcNAc transferase Another site of high expression of O-GlcNAc transferase is the somatotropes (growth hormone (GH) secreting cells) in the pituitary. Blockage of O-GlcNAcase resulted in an immediate blunting of GHrelease from and a marked retention of GH secretory granules in the pituitary. These results suggest that O-GlcNAcase blockade in the endocrine tissues where O-GlcNAc transferase is abundant results in a defect in vesicular traffic.
  • GH growth hormone
  • the present invention refers to an inhibitor of the O- linked N-acetylglucosamine transferase for use as a medicament for the treatment of a disease, whereby the disease comprises abnormal cell differentiation.
  • the invention refers to an in vitro method for the identification of compounds for the treatment of a disease, whereby the disease comprises abnormal cell differentiation, said method comprising a) culturing a population of chondrocytes or a polulation of cells capable of undergoing differentiation towards the chondrocyte lineage in conditions allowing them to differentiate; b) bringing into contact a cell population according to step a) with a test compound; c) evaluating the protein glycosylation levels in said cell population and/or evaluating the cell differentiation status of said cell population wherein if said compound is capable of inhibiting protein glycosylation in said cells or it is capable of inhibiting the abnormal differentiation of said cells, indicates that the test compound may be used to treat said disease.
  • the invention refers to an in vitro method for the diagnosis of a disease in a subject or for determining the predisposition of a subject to develop a disease, wherein said disease comprises abnormal cell differentiation, said method comprising detecting and quantifying the protein glycosylation levels in a sample from said subject wherein, if said levels are increased in comparison with reference values, then is indicative that said subject suffers from said disease or is predisposed to develop said disease.
  • Figure 1 shows a microscope photographs of Alizarin Red Staining ATDC5 cell cultures at 42 days after seeding in maintenance medium (panel A) and hypertrophic medium (panel B).
  • Figure 2 shows a Western Blot picture of ATDC5 cell protein extracts incubated with anti-O-linked N-acetylglucosamine (O-GlcNAc) antibody (AbCam) for the detection of O-GlcNAc glycosylated proteins.
  • Figure 2A Lane 1 no differentiation; Lane 2: hypethropic cells.
  • Figure 2B lanes 1, 2, and 3: no differentiation medium + alloxan treatment: lane 1 : 0.1 mM; 2 0.5 mM; 3: 1 mM.
  • Lanes 4, 5, and 6 Alloxan treatment of hypertrophic chondrocytes; lane 1 : 0.1 mM; 2: 0,5 mM; 3: 1 mM.
  • Low panel loading control.
  • Figure 3 shows cell cultures photographs of chondrocytes in maintenance medium (MM) and hypertrophic medium (HM) ( Figure 3A) after alloxan treatment at the indicated concentrations (0,1 mM; 0,5 mM; and 1 mM).
  • Figure 3B shows a graph diagram of the corresponding Alizarin Red staining measurements.
  • the present invention shows that an inhibitor of the OGT enzyme can be used in the treatment of a disease, whereby the disease involves abnormal cell differentiation, more particularly, wherein the disease involves chondrocyte terminal differentiation.
  • O-linked protein glycosylation is altered in hypertrophic chondrocytes.
  • the present invention shows that O-linked protein glycosylation is increased in said cells compared with non differentiated chondrocytes.
  • alloxan treatment results in a decrease in O-linked protein glycosylation levels in said hypertrophic cells and moreover, alloxan treatment reduces deposition of calcium salts in said cells.
  • the invention refers to an inhibitor of the O-linked N- acetylglucosamine transferase (OGT) for use as a medicament for the treatment of a disease, whereby the disease comprises abnormal cell differentiation.
  • OHT O-linked N- acetylglucosamine transferase
  • O-linked protein glycosylation refers to glycosylation that involves the O-linkage of N-acetylglucosamine (GIcNAc) to serine or threonine residues in the protein backbone.
  • the enzyme performing this protein modification is O-linked N-acetylglucosamine transferase (OGT).
  • chondrocytes undergo a multitude of differentiation events, including proliferation, hypertrophy (chondrocytes become enlarged), terminal differentiation and eventually cell death (apoptosis). Terminally differentiated chondrocytes release matrix vesicles, alkaline phosphatase, annexin II and annexin V-containing particles which initiate mineralization of the extracellular matrix.
  • Chondrocytes in healthy human cartilage maintain a stable phenotype and do not proliferate, but are capable of producing matrix components, such as type II, IX, XI collagen and aggrecan to replace damaged matrix molecules, thereby preserving the structural integrity of the cartilage matrix.
  • matrix components such as type II, IX, XI collagen and aggrecan to replace damaged matrix molecules, thereby preserving the structural integrity of the cartilage matrix.
  • chondrocytes in the articular cartilage mimic the differentiation pattern characteristic of fetal skelotogenesis and undergo hypertrophic and terminal differentiation and produce matrix calcification.
  • abnormal cell differentiation refers to an alteration in the normal differentiation program of the cell; in particular, it refers to an alteration in the normal differentiation program of the cells of the articular cartilage, more particularly, it refers to an alteration in the normal differentiation program of chondrocytes present in said articular cartilage and thus, it refers to the process by which chondrocytes alter its normal phenotype in the articular cartilage and undergo a series of phenotype changes including hypertrophy, which finally lead to cartilage degradation.
  • chondrocytes refers to the cells that produce, maintain, remodel and repair the extracellular matrix of articular cartilage.
  • said abnormal cell differentiation is terminal differentiation.
  • said terminal differentiation is chondrocyte terminal differentiation.
  • said chondrocyte terminal differentiation comprises chondrocyte hypertrophy.
  • said disease is an articular disease.
  • said articular disease is a degenerative articular disease.
  • said degenerative articular disease is a degenerative articular cartilage disease.
  • said degenerative articular cartilage disease is osteoarthritis.
  • said disease comprises cartilage degradation.
  • said inhibitor is a compound or agent that inhibits activity or expression of the OGT.
  • An agent capable of down- regulating activity or expression of the OGT refers to a molecule such as a chemical, nucleic acid or proteinacious molecule or a combination thereof which is capable of inhibiting activity or expression of the OGT.
  • inhibitor or “inhibiting”, “neutralize” or “neutralizing”, “down- regulating” and their cognates as used herein refer to a reduction in the activity of OGT by an OGT inhibitor, relative to the activity or expression of OGT in the absence of the same inhibitor.
  • the reduction in activity or expression is preferably at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or higher with respect to reference values as explained below.
  • said inhibitor according to the invention is a compound selected from the group consisting of an antibody, a non- functional derivative, an antisense polinucleotide, a RNA interference oligonucleotide, a DNAzyme, a ribozyme, a triplex forming oligonuclotide (TFO), a compound represented by the chemical formula (I), a compound represented by the chemical formula (II), a 5-substituted-isouric acid, a 4,5- disubstituted-4,5-dihydrouric acid, a 5-substituted-pseudouric acid, N-ethylmeleimide,
  • R 1 and R 2 may be the same or different and are selected from the group consisting of:
  • aryl wherein said aryl is substituted with a methyl or a methoxyl group
  • R 3 and R 4 may be the same or different and are selected from the group consisting of:
  • R 5 is selected from the group consisting of:
  • R a is selected from:
  • R b is selected from:
  • R c is selected from:
  • R d is selected from: (1) Ci-C 5 alkyl
  • said inhibitor is a compound represented by the chemical formula (II).
  • said inhibitor is a compound represented by the chemical formula (II) wherein R 1 and R 2 are hydrogen. More preferably, said inhibitor is a compound as mentioned before, i.e. a compound represented by the chemical formula (II) wherein R 1 and R 2 are hydrogen and wherein R 5 is oxygen, i.e. said inhibitor is alloxan.
  • the compounds of formula (I) and (II) can form salts which form part of the present invention.
  • said compounds and pharmaceutical salts thereof can be used as a medicament for the treatment of a disease, whereby the disease comprises abnormal cell differentiation.
  • appropriate pharmaceutically acceptable salts of said compounds include salts of organic acids, especially carboxylic acids, including but not limited to acetate, trifluoroacetate, lactate, gluconate, citrate, tartrate, maleate, malate, pantothenate, isethionate, adipate, alginate, aspartate, benzoate, butyrate, digluconate, cyclopentanate, glucoheptanate, glycerophosphate, oxalate, heptanoate, hexanoate, fumarate, nicotinate, palmoate, pectinate, 3- phenylpropionate, picrate, pivalate, proprionate, tartrate, lactobionate, pivolate, camphor
  • the compounds of formula (I) and (II) may in some cases be isolated as the hydrate. Hydrates are typically prepared by recrystallisation from an aqueous/organic solvent mixture using organic solvents such as dioxin, tetrahydrofuran or methanol.
  • the non-cyclic carbohydrate groups e.g. alkyl, alkenyl, alkynyl, alkoxy, aralkyl, alkaryl, alkylamine, etc
  • the substituent of the compounds of the present invention are either branched or unbranched, preferably containing from one or two to twelve carbon atoms.
  • said inhibitor is an antibody.
  • the antibody specifically binds to at least one epitope of the protein.
  • epitope refers to any antigenic determinant on an antigen to which the paratope of an antibody binds.
  • Epitopic determinants usually consist of chemically active surface groups of molecules such as amino acids or carbohydrate side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics.
  • antibody as used in this invention includes intact molecules as well as functional fragments thereof, such as Fab, F(ab')2, and Fv that are capable of binding to the antigen presented by the macrophages.
  • Fab the fragment which contains a monovalent antigen-binding fragment of an antibody molecule
  • Fab' the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain
  • two Fab' fragments are obtained per antibody molecule
  • (Fab')2 the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction
  • F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bridges
  • Fv defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains
  • SCA Single Chain Antibody
  • Antibody fragments according to the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment.
  • Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5 S fragment denoted F(ab')2.
  • This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5 S Fab ⁇ l> monovalent fragments.
  • a thiol reducing agent and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5 S Fab ⁇ l> monovalent fragments.
  • an enzymatic cleavage using pepsin produces two monovalent Fab ⁇ l> fragments and an Fc fragment directly.
  • Fv fragments comprise an association of VH and VL chains. This association may be noncovalent.
  • the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde.
  • the Fv fragments comprise VH and VL chains connected by a peptide linker.
  • sFv single-chain antigen binding proteins
  • sFv single-chain antigen binding proteins
  • the structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli.
  • the recombinant host cells synthesize a single peptide chain with a linker peptide bridging the two V domains.
  • Methods for producing sFvs are described, for example, by [Whitlow and Filpula [(1991), Methods 2: 97-105]; Pack et al, [(1993), BioTechnology 11 :1271-77]; and U.S. Pat. No. 4,946,778.
  • CDR peptides (“minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry [(1991) Human Antibodies and Hybridomas, 2:172-189 and U.S. Pat. No. 6,580,016].
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab ⁇ l>, F(ab').sub.2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues form a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementary determining region
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non- human residues.
  • Humanized antibodies may also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non- human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Riechmann et al, Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol, 2:593-596 (1992)].
  • Fc immunoglobulin constant region
  • a humanized antibody has one or more amino acid residues introduced into it from a source that is non- human. These non- human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method well known in the state of the art, by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies, wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • Human antibodies can also be produced using various techniques known in the art, including phage display libraries.
  • human antibodies can be made by introduction of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire.
  • another proteinaceous agent capable of down-regulating the activity of the OGT can be a non- functional derivative thereof (i.e. dominant negative).
  • the agent of this aspect of the present invention may be an agent capable of down regulating the expression of the OGT gene.
  • Gene expression is a process that involves transcription of the DNA code into mRNA, translocation of mRNA to ribosomes, and translation of the RNA message into proteins. Additionally there are other factors that contribute to the great variation in gene expression levels and in the penetrance of gene activity: 1) the mRNA molecule can be more or less stable thus contributing to changes in mRNA levels. 2) there is a precursor of the mature mRNA molecule that can be alternatively spliced, adding complexity to the mechanisms of mRNA expression regulation.
  • mRNAs can be degraded by endogenous cells mechanisms like the existence of RNAses or other more complex systems.
  • translation into proteins can also be regulated at different levels (i.e. initiation of translation, etc.) and, finally, 5) Proteins can be postranslationally modified, thus changing their activity, their molecular stability, etc.
  • Current methods to suppress a gene include, for example, the use of antisense, co-suppression, and RNA interference.
  • down-regulation of OGT can be effected by using an antisense polynucleotide capable of specifically hybridizing with an mRNA transcript encoding OGT.
  • the first aspect is delivery of the oligonucleotide into the cytoplasm of the appropriate cells, while the second aspect is design of an oligonucleotide that specifically binds the designated mRNA within cells in a way that inhibits translation thereof.
  • antisense oligonucleotides suitable for the treatment of cancer have been successfully used [Homlund et ah, Curr Opin MoI Ther 1 :372-85 (1999)], while treatment of hematological malignancies via antisense oligonucleotides targeting c-myb gene, p53 and Bcl-2 had entered clinical trials and had been shown to be tolerated by patients [Gerwitz Curr Opin MoI Ther 1 :297-306 (1999)].
  • antisense-mediated suppression of human heparanase gene expression has been reported to inhibit pleural dissemination of human cancer cells in a mouse model [Uno et al, Cancer Res 61 :7855-60 (2001)].
  • small interfering RNA refers to an RNA construct that contains one or more short sequences that are at least partially complementary to and can interact with a polynucleotide sequence of the OGT gene.
  • Interaction may be in the form of a direct binding between complementary (antisense) sequences of the small interfering RNA and polynucleotide sequences of the target, or in the form of an indirect interaction via enzymatic machinery ⁇ e.g., a protein complex) that allows the antisense sequence of the small interfering RNA to recognize the target sequence.
  • enzymatic machinery e.g., a protein complex
  • recognition of the target sequence by the small interfering RNA results in cleavage of OGT sequences within or near the target site that is recognized by the recognition (antisense) sequence of the small interfering RNA.
  • the small interfering RNA can exclusively contain ribonucleotide residues, or the small interfering RNA can contain one or more modified residues, particularly at the ends of the small interfering RNA or on the sense strand of the small interfering RNA.
  • the term "small interfering RNA” as used herein encompasses shRNA and siRNA, both of which are understood and known to those in the art to refer to RNA constructs with particular characteristics and types of configurations.
  • shRNA refers to an RNA sequence comprising a double-stranded region and a loop region at one end forming a hairpin loop.
  • the double-stranded region is typically about 19 nucleotides to about 29 nucleotides in length on each side of the stem, and the loop region is typically about three to about ten nucleotides in length (and 3'- or 5 '-terminal single-stranded overhanging nucleotides are optional).
  • siRNA refers to an RNA molecule comprising a double- stranded region with a 3' overhang of nonhomologous residues at each end.
  • the double- stranded region is typically about 18 to about 30 nucleotides in length, and the overhang may be of any length of nonhomologous residues, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.
  • the siRNA can also comprise two or more segments of 19-30 base pair separated by unpaired regions.
  • RNAi molecules suitable for use with the present invention can be carried out as follows. First, the mRNA sequence target is scanned downstream of the AUG start codon for AA dinucleotide sequences. Occurrence of each AA and the 3' adjacent 19 nucleotides is recorded as potential siRNA target sites. Preferably, siRNA target sites are selected from the open reading frame, as untranslated regions (UTRs) are richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNA endonuclease complex.
  • UTRs untranslated regions
  • siRNAs directed at untranslated regions may also be effective, as demonstrated for GAPDH wherein siRNA directed at the 5' UTR mediated about 90 % decrease in cellular GAPDH mRNA and significantly reduced protein level (www.ambion.com/techlib/tn/91/912.html) Second, potential target sites are compared to an appropriate genomic database
  • target sites that exhibit significant homology to other coding sequences are filtered out. Qualifying target sequences are selected as template for siRNA synthesis. Preferred sequences are those including low G/C content as these have proven to be more effective in mediating gene silencing as compared to those with G/C content higher than 55 %. Several target sites are preferably selected along the length of the target gene for evaluation. For better evaluation of the selected siRNAs, a negative control is preferably used in conjunction.
  • Negative control siRNA preferably include the same nucleotide composition as the siRNAs but lack significant homology to the genome. Thus, a scrambled nucleotide sequence of the siRNA is preferably used, provided it does not display any significant homology to any other gene.
  • DNAzyme molecule capable of specifically cleaving an mRNA transcript or a DNA sequence of the target.
  • DNAzymes are single- stranded polynucleotides which are capable of cleaving both single and double stranded target sequences.
  • a general model (the "10- 23" model) for the DNAzyme has been proposed.
  • "10-23" DNAzymes have a catalytic domain of 15 deoxyribonucleotides, flanked by two substrate-recognition domains of seven to nine deoxyribonucleotides each. This type of DNAzyme can effectively cleave its substrate RNA at purine :pyrimidine junctions. Examples of construction and amplification of synthetic, engineered DNAzymes recognizing single and double- stranded target cleavage sites have been disclosed in U.S. Pat. No. 6,326,174.
  • Ribozyme molecule capable of specifically cleaving an mRNA transcript encoding OGT.
  • Ribozymes are being increasingly used for the sequence- specific inhibition of gene expression by the cleavage of mRNAs encoding proteins of interest.
  • the possibility of designing ribozymes to cleave any specific target RNA has rendered them valuable tools in both basic research and therapeutic applications.
  • ribozymes have been exploited to target viral RNAs in infectious diseases, dominant oncogenes in cancers and specific somatic mutations in genetic disorders. Most notably, several ribozyme gene therapy protocols for HIV patients are already in Phase 1 trials.
  • ribozymes have been used for transgenic animal research, gene target validation and pathway elucidation.
  • Several ribozymes are in various stages of clinical trials.
  • ANGIOZYME was the first chemically synthesized ribozyme to be studied hi human clinical trials.
  • ANGIOZYME specifically inhibits formation of the VEGF-r
  • HEPTAZYME Hepatitis C Virus
  • TFOs triplex forming oligonuclo tides
  • oligonuclo tides such as the introduction of intercalators and backbone substitutions, and optimization of binding conditions (pH and cation concentration) have aided in overcoming inherent obstacles to TFO activity such as charge repulsion and instability, and it was recently shown that synthetic oligonucleotides can be targeted to specific sequences.
  • the triplex-forming oligonucleotide has the sequence correspondence: oligo 3'-A G G T duplex 5' ⁇ A G C T duplex 3' ⁇ T C G A
  • the A-AT and G-GC triplets have the greatest triple helical stability.
  • Triplex- forming oligonucleotides preferably are at least 15, more preferably 25, still more preferably 30 or more nucleotides in length, up to 50 or 100 bp.
  • Transfection of cells for example, via cationic liposomes
  • TFOs Transfection of cells (for example, via cationic liposomes) with TFOs, and formation of the triple helical structure with the target DNA induces steric and functional changes, blocking transcription initiation and elongation, allowing the introduction of desired sequence changes in the endogenous DNA and resulting in the specific downregulation of gene expression.
  • Examples of such suppression of gene expression in cells treated with TFOs include knockout of episomal supFGl and endogenous HPRT genes in mammalian cells, and the sequence- and target-specific downregulation of expression of the Ets2 transcription factor, important in prostate cancer etiology, the pro -inflammatory ICAM-I gene.
  • TFOs designed according to the abovementioned principles can induce directed mutagenesis capable of effecting DNA repair, thus providing both downregulation and upregulation of expression of endogenous genes.
  • Detailed description of the design, synthesis and administration of effective TFOs can be found in U.S. Patent Application Nos. 2003017068 and 20030096980 and U.S. Pat. No.5,721,138.
  • therapeutic oligonucleotides may further include base and/or backbone modifications, which may increase bioavailability, therapeutic efficacy and reduce cytotoxicity. Such modifications are described in Younes (2002) [Current Pharmaceutical Design 8:1451-1466].
  • the oligonucleotides of the present invention may comprise heterocylic nucleosides consisting of purines and the pyrimidines bases, bonded in a 3' to 5 ⁇ 1> phosphodiester linkage.
  • Preferably used oligonucleotides are those modified in backbone, internucleoside linkages or bases, as is broadly described herein below.
  • oligonucleotides useful according to this aspect of the present invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages.
  • Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates including 3'- alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3 '-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are
  • modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
  • morpholino linkages formed in part from the sugar portion of a nucleoside
  • siloxane backbones sulfide, sulfoxide and sulfone backbones
  • formacetyl and thioformacetyl backbones methylene formacetyl and thioformacetyl backbones
  • alkene containing backbones sulfamate backbones
  • sulfonate and sulfonamide backbones amide backbones; and others having mixed N, O, S and CH2 component parts.
  • oligonucleotides which can be used according to the present invention, are those modified in both sugar and the internucleoside linkage, i.e. the backbone, of the nucleotide units are replaced with novel groups.
  • the base units are maintained for complementation with the appropriate polynucleotide target.
  • An example for such an oligonucleotide mimetic includes peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • a PNA oligonucleotide refers to an oligonucleotide where the sugar-backbone is replaced with an amide containing backbone, in particular an aminoethylglycine backbone.
  • the bases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,
  • Oligonucleotides of the present invention may also include base modifications or substitutions.
  • "unmodified” or “natural” bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • Modified bases include but are not limited to other synthetic and natural bases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2- thiouracil, 2- thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4- thiouracil, 8- halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5- substituted
  • Recombinant agents or oligonucleotide agents of the present invention can be administeretd to the subject employing any suitable mode of administration, described hereinbelow (i.e. in vivo gene therapy).
  • the nucleic acid construct can be introduced into a suitable cell using an appropriate gene delivery vehicle/method (transfection, transduction, etc.) and an appropriate expression system.
  • the modified cells are subsequently expanded in culture and returned to the individual (i.e. ex vivo gene therapy).
  • suitable constructs include, but are not limited to, pcDNA3, [rho]cDNA3.1 (+/-), pGL3, PzeoSV2 (+/-), pDisplay, pEF/myc/cyto, pCMV/myc/cyto each of which is commercially available from Invitrogen Co. (www.invitrogen.com).
  • retroviral vector and packaging systems are those sold by Clontech, San Diego, Calif, including Retro-X vectors pLNCX and pLXSN, which permit cloning into multiple cloning sites and transcription of the transgene is directed from the CMV promoter.
  • Vectors derived from Mo-MuLV are also included such as pBabe, where the transgene will be transcribed from the 5'LTR promoter.
  • nucleic acid agents of the present invention can be can be introduced to the subject using the well known "gene knock-in strategy" which will result in the formation of a non- functional protein [see e.g., Matsuda et aL, Methods MoI Biol. 2004; 259:379-90].
  • treatment refers to preventing, curing, reversing, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of the disease.
  • treatment comprises treating said degenerative articular disease to reverse disease's symptoms, more particularly, preventing the development of said disease, as well as managing and/or ameliorating said disease or one or more symptoms thereof.
  • treatment refers to administering a therapeutically effective amount of the inhibitor of the invention to achieve a desired therapeutic effect.
  • the desired therapeutic effect may include, without being limited thereto, improving motility of the subject, decrease in swelling and tenderness of the joints, slowing or preventing the deterioration of the joints and the surrounding tissue, slowing any irreversible damage caused by a chronic stage of said degenerative articular disease, more particularly, osteoarthritis, increasing the time period of the remission between acute attacks of the disease, lessening of the severity of or curing said disease, or providing more rapid recovery from said disease, as well as decreasing any one of the following symptoms: stiffness, pain and joint deformity, joint edema, hot flashes and abnormal enlargement of joints or preventing the manifestation of such symptoms before they occur.
  • treatment also includes prevention of the development of the disease (e.g. in subjects having high disposition of developing the disease, such as athletes) as well as reversal of damage caused to cartilage as a result of the disease.
  • the active agent i.e. the inhibitor
  • a pharmaceutical formulation or composition comprising as active ingredient an effective amount of an inhibitor according to the invention.
  • the pharmaceutical formulation or composition in the context of the invention is intended to mean a combination of the active agent(s), together or separately, with a pharmaceutically acceptable carrier as well as other additives.
  • pharmaceutically acceptable carrier in the context of the present invention denotes any one of inert, non-toxic materials, which do not react with the compound of the invention and which can be added to formulations as diluents, carriers or to give form or consistency to the formulation.
  • the carrier may at times have the effect of the improving the delivery or penetration of the active ingredient to the target tissue, for improving the stability of the drug, for slowing clearance rates, for imparting slow release properties, for reducing undesired side effects etc.
  • the carrier may also be a substance that stabilizes the formulation (e.g. a preservative), for providing the formulation with an edible flavor, etc.
  • stabilizers and adjuvants see E. W. Martin, REMINGTON'S PHARMACEUTICAL SCIENCES, MacK Pub Co (June, 1990).
  • compositions of the present invention are administered and dosed in accordance with good medical practice, taking into account the clinical condition of the individual patient, the site and method of administration, scheduling of administration, patient age, sex, body weight and other factors known to medical practitioners.
  • the choice of carrier will be determined in part by the particular active ingredient, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable pharmaceutical compositions of the present invention.
  • said inhibitors are oligonucleotides that recognize and hybridize with the promoter region of the OGT gene thereby inhibiting OGT gene expression.
  • said oligonucleotides may be delivered using any suitable method.
  • naked DNA is administered.
  • lipofection is utilized for the delivery of nucleic acids to a subject.
  • oligonucleotides are modified with phosphothiolates for delivery as described, for example, in U.S. Pat. No.6, 169, 177.
  • nucleic acids for delivery are compacted to aid in their uptake (See e.g., U.S. Pat. Nos. 6,008,366, 6,383,811).
  • compacted nucleic acids are targeted to a particular cell type (e.g., chondrocytes)
  • oligonucleotides are conjugated to other compounds to aid in their delivery.
  • nucleic acids are conjugated to polyethylene glycol to aid in delivery.
  • oligonucleotides are conjugated to protected graft copolymers, which are chargeable drug nano-carriers (Pharmaln).
  • the transport of oligonucleotides into cells is facilitated by conjugation to vitamins.
  • oligonucleotides are conjugated to nanoparticles (e.g., NanoMed Pharmaceuticals; Kalamazoo, Mich.). In other embodiments, oligonucleotides are enclosed in lipids (e.g., liposomes or micelles) to aid in delivery. In still further embodiments, oligonucleotides are complexed with additional polymers to aid in delivery.
  • nanoparticles e.g., NanoMed Pharmaceuticals; Kalamazoo, Mich.
  • oligonucleotides are enclosed in lipids (e.g., liposomes or micelles) to aid in delivery.
  • oligonucleotides are complexed with additional polymers to aid in delivery.
  • compositions or medicaments may be administered or coadministered by a wide variety of routes, including for example, orally, parenterally, intraperitoneally, intravenously, intraarterially, transdermally, sublingually, intramuscularly, rectally, transbuccally, intranasally, liposomally, via inhalation, vaginally, intraoccularly, via local delivery (for example by catheter or stent), subcutaneously, intraadiposally, intraarticularly, or intrathecally.
  • the compositions may also be administered or coadministered in slow release dosage forms. Dosage forms known to those of skill in the art are suitable for delivery of the compounds of the invention.
  • compositions that contain therapeutically effective amounts of the inhibitor according to the invention.
  • one or more inhibitors of the invention are mixed with a suitable pharmaceutically acceptable carrier.
  • the resulting mixture may be a solution, suspension, emulsion, or the like.
  • Liposomal suspensions may also be suitable as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art.
  • the form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle.
  • the effective concentration is sufficient for lessening or ameliorating at least one symptom of the disease, disorder, or condition treated and may be empirically determined.
  • compositions are preferably formulated in a unit dosage form.
  • unit dosage from refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.
  • the active materials can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action, or have another action.
  • the compounds, i.e. the inhibitor may be formulated as the sole pharmaceutically active ingredient in the composition or may be combined with other active ingredients.
  • solubilizing may be used. Such methods are known and include, but are not limited to, using cosolvents such as dimethylsulfoxide (DMSO), using surfactants such as Tween.RTM., and dissolution in aqueous sodium bicarbonate. Derivatives of the compounds, such as salts or prodrugs may also be used in formulating effective pharmaceutical compositions.
  • the inhibitor of the invention may be prepared with carriers that protect them against rapid elimination from the body, such as time-release formulations or coatings. Such carriers include controlled release formulations, such as, but not limited to, microencapsulated delivery systems.
  • the active compound is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the subject treated.
  • Such carriers include controlled release formulations, such as, but not limited to, implants and microencapsulated delivery systems, and biodegradable, biocompatible polymers such as collagen, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid, and the like. Methods for preparation of such formulations are known to those skilled in the art.
  • Compounds of the invention may also be advantageously delivered in a nano crystal dispersion formulation. Preparation of such formulations is described, for example, in U.S. Pat. No. 5,145,684.
  • the nano crystalline formulations typically afford greater bioavailability of drug compounds.
  • the inhibitors and compositions of the invention can be enclosed in multiple or single dose containers.
  • the enclosed compounds and compositions can be provided in kits, for example, including component parts that can be assembled for use.
  • a therapeutic compound in lyophilized form and a suitable diluent may be provided as separated components for combination prior to use.
  • a kit may include an inhibitor according to the present invention and a second therapeutic agent for coadministration.
  • the inhibitor of the invention and second therapeutic agent may be provided as separate component parts.
  • a kit may include a plurality of containers, each container holding one or more unit dose of the inhibitor of the invention.
  • the containers are preferably adapted for the desired mode of administration, including, but not limited to tablets, gel capsules, sustained-release capsules, and the like for oral administration; depot products, pre-filled syringes, ampoules, vials, and the like for parenteral administration; and patches, medipads, creams, and the like for topical administration.
  • the inhibitor of the invention is administered in amounts which are sufficient to achieve the desired effect, in a preferred embodiment, an anti-osteoarthritic effect. As will be appreciated, the amount of the compound will depend on the severity of the disease, the intended therapeutic regiment and the desired therapeutic dose.
  • an amount effective to achieve the desired effect is determined by considerations known in the art.
  • the effective amount or concentration depends on a variety of factors including the distribution profile of the compound within the body, a variety of pharmacological parameters such as half life in the body, on undesired side effects, if any, on factors such as age and gender of the subject to be treated, etc.
  • the therapeutically effective amount or concentration may be determined empirically by testing the compounds in known in vitro and in vivo model systems for the treated disorder. The effective amount is typically tested in clinical studies having the aim of finding the effective dose range, the maximal tolerated dose and the optimal dose. The manner of conducting such clinical studies is well known to a person versed in the art of clinical development.
  • An amount may also at times be determined based on amounts shown to be effective in animals. It is well known that an amount of X mg/Kg administered to rats can be converted to an equivalent amount in another species (notably humans) by the use of one of possible conversions equations well known in the art.
  • concentration of active compound in the drug composition will depend on absorption, inactivation, and excretion rates of the active compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art.
  • the inhibitor may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.
  • the inhibitor should be provided in a composition that protects it from the acidic environment of the stomach.
  • the composition can be formulated in an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine.
  • the composition may also be formulated in combination with an antacid or other such ingredient.
  • Oral compositions will generally include an inert diluent or an edible carrier and may be compressed into tablets or enclosed in gelatin capsules.
  • the active compound or compounds can be incorporated with excipients and used in the form of tablets, capsules, or troches. Pharmaceutically compatible binding agents and adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches, and the like can contain any of the following ingredients or compounds of a similar nature: a binder such as, but not limited to, gum tragacanth, acacia, corn starch, or gelatin; an excipient such as microcrystalline cellulose, starch, or lactose; a disintegrating agent such as, but not limited to, alginic acid and corn starch; a lubricant such as, but not limited to, magnesium stearate; a gildant, such as, but not limited to, colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; and a flavoring agent such as peppermint, methyl salicylate, or fruit flavoring.
  • a binder such as, but not limited to, gum tragacanth, acacia, corn starch, or gelatin
  • an excipient such as microcrystalline cellulose, starch, or lactose
  • a disintegrating agent such as, but not limited to, alg
  • dosage unit form When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as fatty oil.
  • dosage unit forms can contain various other materials, which modify the physical form of the dosage unit, for example, coatings of sugar and other enteric agents.
  • the compounds can also be administered as a component of an elixir, suspension, syrup, wafer, chewing gum or the like.
  • a syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings, and flavors.
  • the active materials can also be mixed with other active materials for the treatment of the disease that do not impair the desired action, or with materials that supplement the desired action.
  • said medicament additionally comprises another compound for the treatment of said disease for simultaneous, separate or sequential use in the treatment of said disease.
  • said other active materials or compounds are other drugs used to treat arthritis, more particular, said drugs are drugs commonly used to treat osteoarthritis.
  • said drugs are analgesics such as acetaminophen, non-steroidal anti-inflammatory drugs (NSAIDs), capsaicin, corticosteroids, and hyaluronate injections. Dietary supplements such as glucosamine and chondroitin are also used to treat osteoarthritis.
  • Other illustrative drugs are methotrexate (MTX) and steroids. In such a combination treatment the other drug and the compound of the invention may be given to patients at the same time or at different times, depending on the dosing schedule of each of the drugs.
  • MTX for example, is typically given to patients once weekly at doses ranging between 5 and 25 mg, each weekly dose, either orally or parenterally.
  • NSAIDs can include, but are not limited to, the following examples: ibuprofen, naproxen, ketoprofen, oxaprozin, diclofenac, indomethacin, sulindac, piroxicam, meclofenamate, mefanamic acid, nabumetone, etoldolac, nimesulide, ketorolac, choline magnesium trisalicylate, aspirin, diflunisal, salsalate, fenoprofen, flurbiprofen, pirprofen, tiaprofenic acid, loxoprofen, indoprofen, fenbufen, carprofen, suprofen, celecoxib, valdecoxib, rofecoxib, parecoxib, deracoxi
  • Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include any of the following components: a sterile diluent such as water for injection, saline solution, fixed oil, a naturally occurring vegetable oil such as sesame oil, coconut oil, peanut oil, cottonseed oil, and the like, or a synthetic fatty vehicle such as ethyl oleate, and the like, polyethylene glycol, glycerine, propylene glycol, or other synthetic solvent; antimicrobial agents such as benzyl alcohol and methyl parabens; antioxidants such as ascorbic acid and sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, and phosphates; and agents for the adjustment of tonicity such as sodium chloride and dextrose.
  • a sterile diluent such as water for injection, saline solution, fixed oil, a naturally occurring vegetable oil
  • parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass, plastic, or other suitable material. Buffers, preservatives, antioxidants, and the like can be incorporated as required.
  • suitable carriers include physiological saline, phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents such as glucose, polyethylene glycol, polypropyleneglycol, and mixtures thereof.
  • Liposomal suspensions including tissue-targeted liposomes may also be suitable as pharmaceutically acceptable carriers. These may be prepared according to methods known for example, as described in U.S. Pat. No. 4,522,811.
  • the compounds of the invention can be administered intranasally.
  • the appropriate dosage forms are a nasal spray or dry powder, as is known to those skilled in the art.
  • the compounds of the invention can be administered intrathecally.
  • the appropriate dosage form can be a parenteral dosage form as is known to those skilled in the art.
  • the compounds of the invention can be administered topically.
  • the appropriate dosage form is a cream, ointment, or patch.
  • the compounds of the invention can be administered rectally by suppository as is known to those skilled in the art.
  • the compounds of the invention can be administered by implants as is known to those skilled in the art. When administering a compound of the invention by implant, the therapeutically effective amount is the amount described above for depot administration.
  • the compounds of the invention can be used in combination, with each other or with other therapeutic agents or approaches used to treat or prevent the osteo-articular conditions that are the subject of this patent. There is nothing novel about the route of administration nor the dosage forms for administering the therapeutic compounds. Given a particular therapeutic compound, and a desired dosage form, one skilled in the art would know how to prepare the appropriate dosage form for the therapeutic compound.
  • 0-linked protein glycosylation is altered in hypertrophic chondrocytes.
  • the present invention shows that 0-linked protein glycosylation is increased in said cells compared with non differentiated chondrocytes.
  • alloxan treatment results in a decrease in 0-linked protein glycosylation levels in said hypertrophic cells and moreover, alloxan treatment reduces deposition of calcium salts in said cells (see Example 1 accompanying the present invention).
  • the invention refers to an in vitro method for the identification of compounds for the treatment of a disease, whereby the disease comprises abnormal cell differentiation, said method comprising a) culturing a population of chondrocytes or a polulation of cells capable of undergoing differentiation towards the chondrocyte lineage in conditions allowing them to differentiate, wherein said differentiation is terminal differentiation; b) bringing into contact a cell population according to step a) with a test compound; c) detecting and quantifying the protein glycosylation levels in said cell population and/or evaluating the cell differentiation status of said cell population; wherein if said compound is capable of inhibiting protein glycosylation in said cells and/or it is capable of inhibiting the differentiation of said cells, indicates that the test compound may be used to treat said disease.
  • said abnormal cell differentiation is terminal differentiation, more particularly, chondrocyte terminal differentiation.
  • said differentiation of the chondrocyte population or of said polulation of cells capable of undergoing differentiation towards the chondrocyte lineage according to step a) is terminal differentiation.
  • said terminal differentiation comprises cell hypertrophy, more particularly, chondrocyte hypertrophy.
  • said disease is an articular disease.
  • said articular disease is a degenerative articular disease.
  • said degenerative articular disease is a degenerative articular cartilage disease.
  • said degenerative articular cartilage disease is osteoarthritis.
  • said disease comprises cartilage degradation.
  • the inventors have found that the glycosylation pattern, in particular, the O-N Acetyl glycosylation pattern, in said differentiated cells, in particular, in said hypertrophic cells, is increased in comparison with non-differentiated cells. Additionally, inventors have demonstrated that after treatment with an OGT inhibitor, said glycosylation is inhibited. Thus, according to the present invention, a decrease in said glycosylation levels in more than a 10% with respect to reference values is indicative that said test compound may be used to treat said disease.
  • the term "reference values” or "control values” refers to the protein glycosylation levels in non differentiated cells, more particularly, in cells which do not undergo terminal differentiation, even more particularly, in non-hypertrophic cells.
  • Said decrease is preferably at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or higher with respect to said reference values.
  • said glycosylation is O- glycosylation.
  • said glycosylation is O-N Acetyl glycosylation.
  • Methods for determining protein glycosylation levels in cultured cells are well known in the state of the art. For example, it can be carried out by immunological techniques such as e.g. ELISA, Western Blot or immunofluorescence.
  • Western blot is based on the detection of proteins previously resolved by gel electrophoreses under denaturing conditions and immobilized on a membrane, generally nitrocellulose by the incubation with an antibody specific and a developing system (e.g. chemoluminiscent).
  • the analysis by immunofluorescence requires the use of an antibody specific for the target protein for the analysis of the expression and subcellular localization by microscopy.
  • the cells under study are previously fixed with paraformaldehyde and permeabilised with a non-ionic detergent.
  • ELISA is based on the use of antigens or antibodies labelled with enzymes so that the conjugates formed between the target antigen and the labelled antibody results in the formation of enzymatically-active complexes. Since one of the components (the antigen or the labelled antibody) are immobilised on a support, the antibody-antigen complexes are immobilised on the support and thus, it can be detected by the addition of a substrate which is converted by the enzyme to a product which is detectable by, e.g. spectrophotometry or fluorometry. This technique does not allow the exact localisation of the target protein or the determination of its molecular weight but allows a very specific and highly sensitive detection of the target protein in a variety of biological samples.
  • any antibody or reagent known to bind with high affinity to the target protein can be used for detecting the amount of target protein. It is preferred nevertheless the use of antibody, for example polyclonal sera, hybridoma supernatants or monoclonal antibodies, antibody fragments, Fv, Fab, Fab' y F(ab')2, ScFv, diabodies, triabodies, tetrabodies and humanised antibodies.
  • antibody for example polyclonal sera, hybridoma supernatants or monoclonal antibodies, antibody fragments, Fv, Fab, Fab' y F(ab')2, ScFv, diabodies, triabodies, tetrabodies and humanised antibodies.
  • western blot analysis can be performed by means of using an anti-glycosylated protein antibody as described in the Example accompanying the present invention.
  • said hypertrophic differentiation status can be measured by means of measuring deposition of calcium salts by said cells.
  • Methods for measuring deposition of calcium salts are well known in the art.
  • Illustrative, non limitative, methods for measuring deposition of calcium salts include, for example, Alizarin Red staining (see Example 1 accompanying the present invention). Said dye has high affinity for Ca 2+ , therefore it is an appropriate dye for the identification of hypertrof ⁇ c chondrocytes and matrix thereof promoting the deposition of calcium salts.
  • the invention refers to an in vitro method for the diagnosis of a disease in a subject or for determining the predisposition of a subject to develop a disease, wherein said disease comprises abnormal cell differentiation, said method comprising detecting and quantifying the protein glycosylation levels in a sample from said subject wherein, if said levels are increased in comparison with said reference values, then is indicative that said subject suffers from said disease or is predisposed to develop said disease.
  • said abnormal cell differentiation is terminal differentiation.
  • said terminal differentiation is chondrocyte terminal differentiation.
  • said chondrocyte terminal differentiation comprises chondrocyte hypertrophy.
  • said disease is a degenerative articular disease. More particularly, said degenerative articular disease is a degenerative articular cartilage disease. In a preferred embodiment, said degenerative articular cartilage disease is osteoarthritis.
  • said disease comprises cartilage degradation.
  • sample as used herein, relates to any sample which can be obtained from the subject.
  • the present method can be applied to any type of biological sample from a subject, such as a biopsy sample, tissue, cell or fluid.
  • said sample is a cartilage tissue sample.
  • said sample is an articular cartilage tissue sample.
  • the method of the invention comprises detecting and quantifying the protein glycosylation levels in a sample from said subject wherein, if said levels are increased in comparison with said reference values, then is indicative that said subject suffers from said disease or is predisposed to develop said disease.
  • said glycosylation is O-glycosylation.
  • the term "reference values” or "control values” refers to the protein glycosylation levels in a sample of a healthy subject, i.e. a subject not suffering from said disease.
  • an increase in said glycosylation levels in more than a 10% with respect to said reference values is indicative that said subject suffers from said disease or is predisposed to develop said disease.
  • Said increase is preferably at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or higher.
  • the murine ATDC5 cell line (Riken cell Bank, Japan) has been selected after long term culture of a teratocarcinoma. These cells undergo differentiation towards the chondrocyte lineage when treated with insulin. Thus, this cell line is an established surrogate model for chondrocyte differentiation.
  • This process can be divided into different phases from the undifferentiated state to the terminally differentiated hypertrophic chondrocytes:
  • Undifferentiated phase To maintain the undifferentiated phenotype, ATDC5 cells are cultured in basic maintenance medium (DMEM/F12 supplemented with 5% fetal bovine serum (FBS), 4 mM Glutamine, 10 ⁇ g/ml transferrin, 3x10 "8 Sodium Selenite and antibiotics, all from Sigma-Aldrich).
  • DMEM/F12 supplemented with 5% fetal bovine serum (FBS), 4 mM Glutamine, 10 ⁇ g/ml transferrin, 3x10 "8 Sodium Selenite and antibiotics, all from Sigma-Aldrich).
  • Differentiated chondrocytes To reach full chondrocyte differentiation, ATDC5 cells are cultured at 6x10 3 cells/cm 2 in maintenance medium supplemented with 10 ⁇ g/ml insulin (Sigma-Aldrich) for 21 days. The culture medium is kept fresh, being replaced every other day.
  • Terminal differentiation (hypertrophic chondrocytes): After 21 days in culture, the medium is replaced by a hypertrophic medium (ccMEM supplemented with
  • the Alizarin Red dye has high affinity for Ca 2+ , therefore it is an appropriate dye for the identification of hypertrofic chondrocytes and matrix thereof promoting the deposition of calcium salts.
  • the staining protocol that has been used is one among others known in the art.
  • Alloxan Sigma- Aldrich
  • O-glycosylation is a dynamic process that occurs in many intracellular proteins (generally nuclear or cytoplasmic proteins). As a rule, O-Glycosylation occurs as an alternative to O-Phosphorylation on specific Serine and Threonine residues within proteins.
  • ATDC5 cells (with or without alloxan treatment) are lysed and cell protein extracts are analysed by Western-Blot, using an anti-O-linked N-acetylglucosamine (O- GIcNAc) antibody (CTDl 10.6, AbCam) for the detection of O-GlcNAc glycosylated proteins.
  • the inventors have demonstrated that the compound Alloxan, an OGT inhibitor, is able to inhibit the terminal differentiation of chondrocytes in a murine model of chondrocyte differentiation (ATDC5 cell line). Indeed, Alizarin Red staining results ( Figure 3) show that after alloxan treatment, the deposition of calcium salts is inhibited in hypertrophic medium.

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Abstract

La présente invention porte sur les utilisations thérapeutiques d'inhibiteurs de la N-acétylglucosamine transférase à O lié dans le traitement de maladies qui mettent en jeu une différenciation cellulaire terminale. En particulier, la présente invention porte sur l'utilisation desdits inhibiteurs pour le traitement de maladies articulaires, notamment pour le traitement de maladies articulaires du cartilage.
PCT/EP2008/062287 2008-01-07 2008-09-16 Compositions de traitement de maladies articulaires dégénératives WO2009086952A2 (fr)

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WO2013006758A1 (fr) * 2011-07-06 2013-01-10 President And Fellows Of Harvard College Mimétiques de diphosphate et leurs utilisations
US8524444B2 (en) 2007-06-15 2013-09-03 President And Fellows Of Harvard College Methods and compositions for detections and modulating O-glycosylation
US8957075B2 (en) 2009-06-01 2015-02-17 President And Fellows Of Harvard College O-GlcNAc transferase inhibitors and uses thereof

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8524444B2 (en) 2007-06-15 2013-09-03 President And Fellows Of Harvard College Methods and compositions for detections and modulating O-glycosylation
US8993718B2 (en) 2007-06-15 2015-03-31 President And Fellows Of Harvard College Methods and compositions for detecting and modulating O-glycosylation
US8957075B2 (en) 2009-06-01 2015-02-17 President And Fellows Of Harvard College O-GlcNAc transferase inhibitors and uses thereof
WO2013006758A1 (fr) * 2011-07-06 2013-01-10 President And Fellows Of Harvard College Mimétiques de diphosphate et leurs utilisations
US9573911B2 (en) 2011-07-06 2017-02-21 President And Fellows Of Harvard College Diphosphate mimetics and uses thereof

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