Composition and methods for regulating chondrocyte proliferation and increasing cartilage matrix production

Abstract

To provide improved pharmaceutical formulations for use in treatment of cartilage disease, which induce anabolic stimulation of chondrocytes and a reduction in chondrocyte catabolism, including a reduction in cartilage matrix loss and cartilage degeneration.SOLUTION: Provided is a pharmaceutical composition for use in improving synovial membrane health, comprising a glucagon-like peptide-1 analog, the glucagon-like peptide-1 analog being selected from the group consisting of exenatide, lixisenatide, albiglutide, dulaglutide, semaglutide, and liraglutide.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for treating cartilage disorders, including anabolic and catabolic cytokine regulation. GLP-1 and GLP-1 analogs induce increased chondrocyte regeneration and decreased cartilage degeneration The present invention relates to a novel pharmaceutical composition comprising: [Background technology]

[0002] Osteoarthritis (OA) is the most common chronic joint disease. OA occurs in people over the age of 65. Affects nearly 50% of people, and in young people it occurs after a joint injury. 250 million people worldwide of people suffer from OA, and the disease has significant economic and social consequences for patients and healthcare systems. OA is characterized by structural degradation of the periarticular bone, synovial joint lining and adjacent supporting joints. Destruction of articular cartilage is a disease of the entire joint characterized by a tissue component. This results in an inability to maintain the balance between bone matrix synthesis and degradation. Interleukin-1β (I) produced by phages, monocytes, synovial cells, and chondrocytes Pro-inflammatory cytokines such as IL-1β play an important role in the pathogenesis of the disease.

[0003] Glucagon-like peptide-1 (GLP-1) is a post-translational product of the preproglucagon gene The action of GLP-1 on pancreatic β cells involves the upregulation of glucose transporter 2 expression. These include increased blood glucose levels, increased insulin secretion in response to increased glucose levels, and increased GL P-1 inhibits inflammation-inducing enzymes such as interleukin-6, tumor necrosis factor-α, and interferon-c. It has been shown to reduce the secretion of pro-inflammatory cytokines.

[0004] GLP-1 analogues are commercially available drugs prescribed to patients for the treatment of type 2 diabetes. Patent document 1 discloses some derivatives and analogs of glucagon-like peptide 1 (GLP-1). , their preparation, and their pharmaceutical use. Derivatives and Analogues of GLP-1 for Use in the Prevention and Treatment of Diabetes Mellitus - Patent application Regarding.

[0005] Initially, osteoarthritis was thought to be a disease of the articular cartilage, but recent studies have shown that Symptoms of rheumatoid arthritis have been shown to affect the entire joint.

[0006] Although articular cartilage loss has been considered a primary change, cellular and biomechanical changes have been implicated. The combination of stress and stress can cause subchondral bone remodeling, osteophyte formation, bone marrow lesion development, synovial membrane involvement, and osteoporosis. Several conditions, including changes to the joint capsule, ligaments, and periarticular muscles, as well as meniscal tears and extrusions, can occur. This causes some secondary changes.

[0007] There are two patterns of cartilage growth. One is interstitial growth, which is differentiated into chondrocytes. The cells surrounded by the cartilage matrix proliferate by cell division. Another growth pattern is the expansion of the cartilage tissue by the perichondrium. Cartilage tissue is covered with perichondrium except for the articular surface of the articular cartilage. The perichondrium is composed of fibroblasts, which are similar to the chondrocytes in the inner layer. The difference between fibroblasts and chondrocytes is unclear. The cells of the inner layer of the perichondrium gradually change into a round shape. These cells then grow outward, secreting further cartilage matrix.

[0008] Patent Document 2 discloses an incretin hormone or its derivatives for use in the treatment of osteoarthritis. More specifically, this patent relates to analogs of benzodiazepines for use in the treatment of osteoarthritis. The patent discloses the use of GLP-1 and GLP-1 analogs such as liraglutide. The peptides disclosed in the patent of Reference 2 are administered by systemic (parenteral, intravenous, etc.), oral, rectal The drug may be administered via any known route of administration, including intravenously, topically, or subcutaneously. It does not disclose the role of GLP-1 in bone degradation, nor has it demonstrated an association with chondrocytes. do not have.

[0009] Normal turnover of cartilage matrix is mediated by chondrocytes, which Chondrocytes synthesize proteins and the proteolytic enzymes involved in their breakdown. Growth factors and cytokines, structural and physical stimuli, as well as matrix metalloproteinases, It is influenced by many factors, including body composition.

[0010] Osteoarthritis is a condition in which chondrocytes are unable to balance between anabolism and catabolism of these extracellular matrix components. This is due to the inability to maintain normal function, which initiates an imbalance between cartilage breakdown and repair. The mechanism of action of the enzyme is not well known. The resulting inflammation can allow for the formation of wear particles, which are then transported by resident macrophages. At some point, the production of these wear particles exceeds the system's ability to eliminate them. They act as mediators of inflammation and stimulate chondrocytes to release degradative enzymes. Degradation of collagen and proteoglycans also taken up by synovial macrophages These molecules induce the release of pro-inflammatory cytokines such as TNFα, IL-1, and IL-6. There is a close relationship between cytokine expression and OA. Interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) stimulate synovial cells and carcinomas. Interleukin-6 (IL-6) and interleukin-8 (IL-8) production by bone cells However, all studies have focused on synovial tissue rather than chondrocytes. was.

[0011] Anabolic stimulation of chondrocytes occurs via stimulation of proteoglycan and collagen synthesis. Measured in vitro. GM-CSF, granulocyte-macrophage colony-stimulating factor (non-patented) 1) and CXCL10 / IP10 (Non-patent Document 2) cytokines It has been shown to stimulate the anabolic process of

[0012] Enhances anabolic activity of chondrocytes, including chondrocyte proliferation, and reduces cartilage matrix degradation in OA. There is a need for improved compositions and methods for reducing chondrocyte catabolic activity, including This will be of great interest in the development of new therapeutic strategies in the treatment of osteoarthritis. Deaf.

[0013] In addition, to promote cartilage regeneration and reduce cartilage destruction, soft tissues including chondrocyte differentiation were also investigated. There is a need to enhance bone cell anabolic activity. Such improved compositions and methods are There will be great interest in developing new therapeutic strategies in the treatment of osteoarthritis. [Prior art documents] [Patent documents]

[0014] [Patent Document 1] International Publication No. WO2017 / 149070 Brochure [Patent Document 2] European Patent EP2890390B1 [Non-patent literature]

[0015] [Non-Patent Document 1] Quinetro et al., 2008 Cytokine 44(3):366-72 [Non-patent document 2] Neidlin et al., 2018, annals of biomedical engineering, volume 46, ISSUE 2 pp345~353 Summary of the Invention [Problem to be solved by the invention]

[0016] The present invention provides a method for the treatment of cartilage disorders, comprising administering to the patient a chondrocyte anabolic pathway, including chondrocyte proliferation. This induces a decrease in catabolic processes, including enhanced cartilage metabolism and reduced cartilage matrix loss and degradation. The present invention discloses improved pharmaceutical formulations that lead to

[0017] The present invention further provides a method for the differentiation of chondrocytes for cartilage regeneration for use in the treatment of cartilage disorders. Enhancement of chondrocyte anabolic processes, including chondrocyte cleavage, and reduction of cartilage matrix loss and cartilage degradation The present invention discloses improved pharmaceutical formulations and compositions that induce a decrease in catabolic processes, including:

[0018] More specifically, such compositions are useful for treating osteoarthritis and for relieving joint irritation. It is useful for preventing the reduction or worsening of existing joint inflammation. [Means for solving the problem]

[0019] The present invention provides a method for the anabolism of chondrocytes, including chondrocyte proliferation, for use in the treatment of cartilage disorders. stimulates and induces catabolic activity, including reduction of cartilage matrix loss and cartilage degeneration The present invention relates to a pharmaceutical composition for reducing

[0020] According to a particular aspect, the present invention provides a chondrocyte proliferation and proliferation method for use in the treatment of cartilage diseases. and / or induce anabolic stimulation of chondrocytes, including stem cell differentiation into chondrocytes for cartilage regeneration. and reduced catabolic activity, including reduced cartilage matrix loss and cartilage degeneration. The present invention relates to a pharmaceutical composition comprising:

[0021] According to a particular embodiment, the present invention provides a method for treating a rheumatoid arthritis comprising administering to a subject a therapeutically effective amount of a glucagon-like peptide-1 analogue as an active ingredient. and a pharmaceutical composition for inducing anabolic stimulation of chondrocytes for use in the treatment of cartilage disorders. Regarding.

[0022] According to the present invention, the glucagon-like peptide-1 (GLP-1) analogue is selected from the group consisting of exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, semaglutide or liraglutide.

[0023] According to one aspect of the present invention, a soft gelatin-like substance containing a glucagon-like peptide-1 analogue as an active ingredient is provided. Inducing anabolic stimulation of chondrocytes, including chondrocyte proliferation, for use in the treatment of bone diseases and / or a pharmaceutical composition that reduces catabolic activity, including reducing cartilage matrix loss. Provided.

[0024] According to another aspect of the present invention, the glucagon-like peptide-1 (GLP-1) analog is It is glutide.

[0025] According to yet another aspect of the present invention, the concentration of liraglutide is 1 ng / ml to 10 mg / ml. ml.

[0026] According to yet another embodiment of the present invention, the concentration of liraglutide is 0.1 to 10 mg / ml. be.

[0027] According to further features in preferred embodiments of the invention described below, The pharmaceutical composition for use comprises a therapeutically effective amount of a GLP-1 analogue and a non-ionic surfactant. Surfactants, cellulose, polyether, glucan, glycerophospholipid, polysaccharide, protein and a gel comprising a polymer selected from the group consisting of .

[0028] According to further features in preferred embodiments of the invention described below, The pharmaceutical composition for use comprises a therapeutically effective amount of a GLP-1 analogue and a non-ionic surfactant. Surfactants, cellulose, polyether, glucan, glycerophospholipid, polysaccharide, protein and an excipient comprising a polymer selected from the group consisting of: do.

[0029] The pharmaceutical formulation according to the present invention also contains one or more pharmaceutically acceptable carriers / excipients. obtain.

[0030] The present invention is not limited to gel formulations and includes liquid and semi-solid pharmaceutical forms suitable for topical administration, e.g. For example, liquids, solutions, creams, gels or transdermal patches are preferred, and particularly suitable for intra-articular injection. In various forms, such as liquids, solutions, and semi-solid forms for transdermal applications, such as creams or gels. Pharmaceutical forms may also be used in which some or all of the ingredients are dissolved in water prior to use. Dry, optionally lyophilized, to be reconstituted in solution or other suitable vehicle. It may also be in a dried form.

[0031] The formulation may contain known excipients such as binders, disintegrants, fillers, stabilizers, diluents and colorants. They can also be used to prepare the compounds of the present invention by methods well known in the state of the art. The formulation may include delayed or sustained release forms made of suitable polymers known in the art.

[0032] Pharmaceutically acceptable carriers / excipients, such as solvents, preservatives, such as antioxidants and / or or chelating agents and antimicrobial agents, tonicity adjusting agents, and buffer systems are added to form a liquid suitable for injection use. It is preferred for the preparation of the form.

[0033] The solvent is preferably water, optionally with a cosolvent, such as a glycol or polyalcohol. , for example, ethylene glycol.

[0034] Preservatives or chelating agents (sodium edetate and sodium metabisulfite are preferred) Antibacterial agents, preferably benzyl alcohol, may also be used.

[0035] As the tonicity adjusting agent, sodium chloride or mannitol is particularly preferred.

[0036] A preferred buffer system is a complex of salts of phosphate and citrate buffers, preferably sodium It may be in the form of a ammonium salt or a potassium salt.

[0037] In preparing a liquid form suitable for nebulization, a pharmaceutically acceptable vehicle / excipient is used as the solvent. Preservatives, such as antioxidants and / or chelating agents and antibacterial agents, isotonicity Includes a modifier and a buffer system.

[0038] According to further features in the described preferred embodiments of the pharmaceutical composition, The analogue is liraglutide and the gel contains albumin.

[0039] According to further features in the described preferred embodiments of the pharmaceutical composition, The analog is liraglutide and contains albumin.

[0040] According to further features in the described preferred embodiments of the pharmaceutical composition, The analog is liraglutide, which contains alpha 1 acid glycoprotein (A1AGP).

[0041] Further in the described preferred embodiments of the pharmaceutical composition for use according to the present invention According to a feature, the albumin concentration is about 0.1% to about 10% (wt / wt) of the formulation, preferably or 5% (wt / wt).

[0042] Further in the described preferred embodiments of the pharmaceutical composition for use according to the present invention According to the characteristics, the concentration of alpha-1 acid glycoprotein (A1AGP) is about 0.1% to about 0.1% of the formulation. It is about 10% (wt / wt), preferably 5% (wt / wt).

[0043] According to still further features in the described preferred embodiments, the pharmaceutical composition comprises 1 ng / ml and 10 mg / ml liraglutide, and 5% (wt / wt) albumin include.

[0044] According to still further features in the described preferred embodiments, the pharmaceutical composition comprises 6 mg / ml of liraglutide, and 5% (wt / wt) albumin.

[0045] According to yet another aspect of the present invention, the cartilage disease is caused by trauma or surgical treatment. from cartilage defects, osteochondritis dissecans, osteoarthritis, congenital cartilage diseases and cartilage injuries is selected from the group consisting of:

[0046] According to yet another aspect of the present invention, anabolic cytokine secretion or or a method for increasing production of IL-1, comprising administering to a patient a composition according to the present invention. is provided.

[0047] According to still further features in the preferred embodiments, the anabolic cytokines are GMCSF and and / or CXCL10 / IP10.

[0048] According to yet another aspect of the present invention, catabolic cytokine secretion or a method for reducing the production of IL-1, comprising administering to a patient a composition according to the present invention. is provided.

[0049] According to further features in the preferred embodiments, the catabolic cytokine is MMP3, MMP4, MMP5, MMP6, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP22, MMP23, MMP24, MMP25, MMP2 It is selected from the group consisting of MP13, PGE2, IL7, and MCP1.

[0050] According to yet another aspect of the invention, the composition is administered to the subject via intra-articular injection.

[0051] In accordance with a further aspect of the present invention, a method for enhancing anabolic cytokine secretion or production and promoting chondrocyte proliferation is provided. Treating cartilage disorders by reducing cartilage loss and / or repair through stimulation of cellular proliferation The use of liraglutide for placing a steroid drug is provided.

[0052] In accordance with a further aspect of the present invention, a method for enhancing anabolic cytokine secretion or production and promoting chondrocyte proliferation is provided. Treating cartilage disorders by reducing cartilage loss and / or regeneration through stimulation of cell proliferation The use of liraglutide for placing a steroid drug is provided.

[0053] According to certain aspects of the present invention, a method for treating cartilage disorders by increasing chondrocyte anabolic function is provided. There is provided the use of liraglutide in the manufacture of a medicament for the treatment of

[0054] Cartilage disorders include cartilage defects caused by trauma or surgical procedures, osteochondritis dissecans, , osteoarthritis, congenital cartilage disease and cartilage damage.

[0055] In accordance with a further aspect of the present invention, a method for reducing catabolic cytokine secretion or production and promoting chondrocyte proliferation is provided. Treating cartilage disorders by reducing cartilage loss and / or repair through stimulation of cellular proliferation The use of liraglutide for placing a steroid drug is provided.

[0056] In accordance with a further aspect of the present invention, a method for reducing catabolic cytokine secretion or production and promoting chondrocyte proliferation is provided. Treating cartilage disorders by reducing cartilage loss and / or regeneration through stimulation of cell proliferation The use of liraglutide for placing a steroid drug is provided.

[0057] Cartilage disorders include cartilage defects caused by trauma or surgical procedures, osteochondritis dissecans, , osteoarthritis, congenital cartilage disease and cartilage damage.

[0058] According to yet another aspect of the present invention, at least one chondrocyte is treated with a composition according to the present invention. A method for promoting chondrocyte proliferation is provided, comprising contacting a chondrocyte with a soluble component of a compound of formula (I) with a soluble component of a compound of formula (I). According to further features, the cell is a mammalian cell. According to a further feature, the cell is a human cell.

[0059] According to yet another aspect of the present invention, a subject diagnosed with or suffering from an immunoinflammatory disorder is A method for treating a patient at risk of developing a steroid agonist, comprising administering a composition according to the present invention to the patient. A method is provided that includes providing

[0060] According to yet another aspect of the present invention, there is provided a method of treating an inflammatory condition in a subject, comprising: Methods are provided that include administering a composition according to the present invention to a subject. According to a further characterization of the condition, inflammatory pathologies are inflammatory conditions occurring in the joints and joint cavities, and The main treatment is cartilage matrix degeneration and osteoarthritis. According to a further feature in the embodiment, the composition is administered to the subject via intra-articular injection.

[0061] According to yet another aspect of the present invention, the composition is administered to the fat pad of a joint via intra-articular injection. will be done.

[0062] According to yet another aspect of the present invention, there is provided a method for promoting cartilage matrix repair in a subject. The method comprises administering a composition according to the present invention.

[0063] According to yet another aspect of the present invention, there is provided a method for promoting cartilage matrix regeneration in a subject. The method comprises administering a composition according to the present invention.

[0064] According to yet another aspect of the present invention, there is provided a method for ameliorating a pro-inflammatory condition in a subject. and administering a composition according to the present invention to the subject.

[0065] According to yet another aspect of the present invention, there is provided a method for alleviating or reducing joint irritation in a mammalian subject. or as an active ingredient in the manufacture of pharmaceutical formulations for the reduction of the exacerbation of existing joint inflammation Use of liraglutide is provided.

[0066] According to yet another aspect of the present invention, there is provided a method for alleviating or reducing joint irritation in a mammalian subject. or as an active ingredient for use in a method for reducing the exacerbation of existing joint inflammation Use of liraglutide in a patient with a rheumatoid arthritis, the composition of which is administered via intra-articular injection into the fat pad of a joint. The use of

[0067] According to yet another aspect of the present invention, there is provided a method for alleviating or reducing joint irritation in a mammalian subject. or an active ingredient in the manufacture of an injectable pharmaceutical formulation for reducing the exacerbation of existing joint inflammation Use of liraglutide as a therapeutic agent, The second therapeutic agent is an anti-inflammatory agent, an antioxidant, a vitamin, a polyol, or any of these. Uses including combinations are provided.

[0068] According to yet another aspect of the present invention, there is provided a method for treating mesenchymal stem cells comprising administering liraglutide as an active ingredient to a patient. The present invention provides a composition for use as an agent for differentiating cells into chondrocytes.

[0069] According to yet another aspect of the present invention, the concentration of liraglutide is 1 ng / ml to 10 mg / ml. ml.

[0070] According to yet another aspect of the present invention, there is provided a method for differentiating mesenchymal stem cells into chondrocytes, There was, a) adding a composition according to the present invention to a cell culture medium containing mesenchymal stem cells; b) differentiating the mesenchymal stem cells into chondrocytes. According to yet another aspect of the present invention, there is provided a method for differentiating mesenchymal stem cells into chondrocytes, There was, c) adding a GLP-1 analog to a cell culture medium containing mesenchymal stem cells; and d) differentiating the mesenchymal stem cells into chondrocytes.

[0071] According to another aspect of the present invention, the glucagon-like peptide-1 (GLP-1) analog is It is glutide.

[0072] According to yet another embodiment of the present invention, the concentration of liraglutide is 0.1 nM to 625 μM. be.

[0073] According to yet another aspect of the present invention, the cell culture medium further comprises MesenPRO RS-enriched Contains growth aids and 1% L-glutamine.

[0074] According to yet another aspect of the present invention, there is provided a method for enhancing SOX9 expression for treating or preventing arthropathy. Use of a SOX9 expression-enhancing peptide in the manufacture of a medicament containing the peptide, Use wherein the expression-enhancing peptide is a GLP-1 analogue that selectively targets the SOX9 gene According to another aspect of the present invention, the GLP-1 analogue is liraglutide.

[0075] According to yet another aspect of the present invention, the concentration of liraglutide is 1 ng / ml to 10 mg / ml. ml.

[0076] According to yet another aspect of the present invention, there is provided a method for the treatment or prevention of inflammation using a SOX9 expression enhancing peptide. Use of a SOX9 expression-enhancing peptide in the manufacture of a medicament containing the peptide, The potentiating peptide is a GLP-1 analog that selectively targets the SOX9 gene. According to another aspect of the present invention, the GLP-1 analogue is liraglutide.

[0077] According to yet another aspect of the present invention, the concentration of liraglutide is 1 ng / ml to 10 mg / ml. ml.

[0078] According to yet another aspect of the present invention, a medicament for use in the treatment or prevention of arthropathy. A composition comprising a pharmaceutically acceptable carrier and a SOX9 expression-enhancing peptide, X9 expression-enhancing peptide is a GLP-1 analog that selectively targets the SOX9 gene In a preferred embodiment, the SOX9 expression enhancing peptide is SOX9. Liraglutide selectively targets the OX9 gene. Another preferred embodiment of the present invention In this case, the concentration of liraglutide is 1 ng / ml to 10 mg / ml.

[0079] According to yet another aspect of the present invention there is provided a pharmaceutical composition for use in the treatment or prevention of inflammation. A composition comprising a pharmaceutically acceptable carrier and a SOX9 expression-enhancing peptide, 9The expression-enhancing peptide is a GLP-1 analog that selectively targets the SOX9 gene. In a preferred embodiment, the SOX9 expression enhancing peptide is a SOX9 peptide. Another preferred embodiment of the present invention is liraglutide, which selectively targets the X9 gene. In this case, the concentration of liraglutide is 1 ng / ml to 10 mg / ml.

[0080] According to yet another aspect of the present invention, a medicament for use in the treatment or prevention of arthropathy. A composition comprising a pharmaceutically acceptable carrier and a SOX9 expression enhancing peptide according to the present invention. The pharmaceutical composition comprises a therapeutically effective amount of liraglutide, a non-ionic surfactant, cellulose , polyethers, glucans, glycerophospholipids, polysaccharides, proteins, and combinations thereof and a gel comprising a polymer selected from the group consisting of a combination of The gel contains albumin, and the albumin concentration is about 0.1% to about 10% of the formulation. % (wt / wt), preferably 5% (wt / wt).

[0081] In another preferred embodiment, the gel comprises alpha-1 acid glycoprotein and The monoacid glycoprotein concentration is about 0.1% to about 10% (wt / wt) of the formulation, preferably 5%. % (wt / wt).

[0082] According to yet another aspect of the present invention there is provided a pharmaceutical composition for use in the treatment or prevention of inflammation. A composition comprising a pharmaceutically acceptable carrier and the SOX9 expression enhancing peptide of the present invention. The pharmaceutical composition comprises a therapeutically effective amount of liraglutide, a nonionic surfactant, cellulose, Polyethers, glucans, glycerophospholipids, polysaccharides, proteins, and their combinations and a gel comprising a polymer selected from the group consisting of: wherein the gel contains albumin, and the albumin concentration is about 0.1% to about 10% of the formulation. (wt / wt), preferably 5% (wt / wt).

[0083] According to another aspect of the present invention, there is provided a method for the treatment of cartilage regeneration comprising administering to the patient a therapeutically effective amount of a glucagon-like peptide-1 analogue. Compositions for use in

[0084] According to another embodiment of the composition for use in cartilage regeneration, glucagon-like peptide-1 The analogue is selected from the group consisting of exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, semaglutide or liraglutide.

[0085] According to another embodiment of the composition for use in cartilage regeneration, glucagon-like peptide-1 An analogue is liraglutide.

[0086] According to yet another embodiment of the composition for use in cartilage regeneration, a glucagon-like peptide The concentration of Do-1 is approximately 0.1 nM to 625 μM.

[0087] According to yet another aspect, the composition for use in cartilage regeneration is The composition further comprises at least 5% by weight or more of a pharmaceutically acceptable formulation vehicle.

[0088] According to yet another embodiment of the composition for use in cartilage regeneration, In the composition for use, the pharmaceutically acceptable formulation vehicle is albumin or alpha 1 Acid glycoproteins are selected from the group consisting of:

[0089] According to yet another aspect of the composition for use in cartilage regeneration, a pharmaceutically acceptable The concentration of the formulation vehicle is about 0.1% to about 10% (wt / wt) of the formulation, preferably 5%. % (wt / wt).

[0090] According to yet another aspect of the composition for use in cartilage regeneration, a pharmaceutically acceptable The concentration of the formulation vehicle is 5% (wt / wt) of the formulation.

[0091]

[0092] According to yet another aspect of the composition for use in cartilage regeneration, a pharmaceutically acceptable The formulation vehicle is albumin.

[0093] According to yet another aspect of the composition for use in cartilage regeneration, a pharmaceutically acceptable The formulation vehicle is alpha-1 acid glycoprotein.

[0094] According to yet another aspect of the present invention, the composition for use in cartilage regeneration is administered orally, dermally or intravenously. It is intended that the compound be administered intravenously, intravenously or intraarticularly.

[0095] According to yet another aspect of the present invention, the composition for use in cartilage regeneration comprises cartilage damage It is administered by intra-articular injection into the

[0096] According to yet another aspect of the present invention, the composition for use in cartilage regeneration comprises chondrocytes Induce anabolic stimulation of chondrocytes, including proliferation and / or stem cell differentiation into chondrocytes.

[0097] The present invention relates to the proliferation or growth of chondrocytes from mesenchymal stem cells or cartilage repair, including cartilage repair. It has utility where stimulation of cell formation is deemed desirable.

[0098] Thus, the present invention provides a method for the proliferation or growth of chondrocytes, including cartilage repair and / or regeneration. It has utility in any application where long stimulation is deemed desirable.

[0099] The present applicants have demonstrated that the effective concentration of liraglutide in chondrocytes of OA patients can be increased. has been shown to stimulate anabolic cytokines and reduce catabolic cytokines in chondrocytes. I found that...

[0100] The present inventors have demonstrated that intra-articular injection (acute or repeated) of a pharmaceutical composition formulation and liraglutide The use of sustained release of into the synovial fluid reduces and delays the fibrotic process induced after cartilage injury, and demonstrated that it induces actual functional and histological improvements in the joint after joint injury. Ta.

[0101] Furthermore, a specific dose regimen (i.e., It has been demonstrated that multiple injections (i.e., several injections spaced one week apart) are necessary.

[0102] According to the present invention, the term regeneration refers to the formation of fibers that result in functional and histological improvement of the joint. This includes chondrocyte anabolic function / proliferation in cartilage in the absence of disease.

[0103] Using a chemically induced OA model, the inventors demonstrated that intra-articular administration of a pharmaceutical composition formulation Injection and sustained release of liraglutide into synovial fluid induces SOX9 expression. was demonstrated.

[0104] According to its principal aspect, broadly stated, the present invention provides a glucagon-like peptide-1 analogue. Contains the active ingredient, cartilage defects and transections caused by trauma or surgical procedures. cartilage damage, cartilage damage, osteochondritis, congenital cartilage disease, osteoarthritis, osteoarthritis, congenital cartilage disease, cartilage damage, Enhanced chondrocyte proliferation, increased cartilage repair and cartilage matrix formation for use in the treatment of bone diseases. The present invention provides pharmaceutical compositions that induce a decrease in vasopressin.

[0105] According to another principal aspect, the present invention, broadly stated, provides a glucagon-like peptide-1 analog. Contains the active ingredient, cartilage defects and transections caused by trauma or surgical procedures. cartilage damage, cartilage damage, osteochondritis, congenital cartilage disease, osteoarthritis, osteoarthritis, congenital cartilage disease, cartilage damage, Enhanced chondrocyte proliferation, increased cartilage regeneration and cartilage matrix formation for use in the treatment of bone diseases The present invention provides pharmaceutical compositions that induce a decrease in ATP loss. [Brief explanation of the drawings]

[0106] [Figure 1] FIG. 1 shows the calculated mean concentration differential expression levels of secreted cytokines in chondrocytes of OA patients after treatment with several doses of Victoza®. [Figure 2]FIG. 1 shows the release profiles of formulations 6, 8, 14, 17, 19, and 20. [Figure 3] FIG. 1 shows the percent weight bearing change (R / L) in surgically induced OA over the course of the study. [Figure 4] FIG. 1 shows histological findings in surgically induced OA during the study. [Figure 5] FIG. 1 shows the dose response of combined liraglutide IA on knee measurements at endpoint. [Figure 6] 1 is a representative photograph of a right knee section stained with hematoxylin and eosin showing fibrous synovial chronic proliferation and tibial plate fibrosis, particularly in an animal injected subcutaneously with Victoza®. [Figure 7] 10 is a representative photograph of a right knee section stained with hematoxylin and eosin showing the absence of fibrosis and chondrocyte nests in the combined liraglutide-injected animals. [Figure 8] FIG. 1 shows the long-term reduction in cartilage matrix loss in formulated liraglutide-injected animals compared to vehicle animals. [Figure 9] FIG. 1 shows the long-term reduction in cartilage degeneration scores in formulated liraglutide-injected animals compared to vehicle animals. [Figure 10] 10 is a representative photograph of right knee sections stained with toluidine blue showing the difference in synovial thickening between formulated liraglutide-injected and vehicle animals. [Figure 11] FIG. 1 illustrates medial capsule repair. [Figure 12] 10 is a representative photograph of a right knee section stained with hematoxylin and eosin showing chondrocyte nests in Group 8M. [Figure 13] FIG. 1 shows the assessment of the total number and density of chondrocyte nests. [Figure 14] FIG. 1 shows the effect of liraglutide on the sphere formation process, showing positive Alcian blue staining of chondrocytes formed from mesenchymal stem cells. [Figure 15] FIG. 1 shows the effect of various doses of liraglutide on lactate dehydrogenase secretion by chondrocytes into the culture medium. [Figure 16] FIG. 1 shows SOX9 RTqPCR analysis of knee joint structures in monoiodoacetate-injected mice treated with liraglutide. [Figure 17] FIG. 1 shows total joint scores (histology) of injected knees of animals from Group 5M treated with A1AGP vehicle and Group 6M treated with A1AGP formulated liraglutide. [Figure 18] Representative photographs of toluidine blue stained right knee sections of animals from group 5M treated with vehicle (A) and group 6M treated with liraglutide formulated with A1AGP (B). Detailed Description of the Invention

[0107] One embodiment of the present invention is a soft gelatinized gel for use in the treatment of cartilage disorders and osteoarthritis. Inducing enhanced osteocyte differentiation and chondrocyte proliferation and increased cartilage matrix production Such compositions are preferably administered by direct injection ( It can be administered directly into the affected joint by intra-articular injection.

[0108] Before describing the present invention in detail, it is to be understood that unless otherwise specified, the present invention does not involve specific materials or manufacturing processes. It should be understood that the terms "coefficient of variation" are not limited to the above and may therefore vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Please understand that this is not the case.

[0109] The articles "a" and "an" are used herein to refer to one or two of the grammatical objects of the article. It is used to refer to more than one (i.e., at least one). For example, an "element" refers to one "protein" means an element or more than one element, or "protein" means more than one protein It means quality.

[0110] The term "about" as used herein means approximately, within the range of, roughly, When the term "about" is used in conjunction with a numerical range, it means at or around , which qualifies the range by extending the boundaries above and below the stated numerical values. In general, the term "about" is used herein to mean a number above and below the stated value by a 10% approximation. Therefore, approximately 50% means a range of 45% to 55%. Numerical ranges recited herein by endpoints include any number of values within that range. Includes all numbers and fractions (e.g., 1-5 becomes 1, 1.5, 2, 2.75, 3, 3. 90, 4, and 5), and all such numbers and fractions are subject to the term "about." It should also be understood that the term is presumed to be modified accordingly.

[0111] The term "chondrocyte" refers to cells isolated from cartilage.

[0112] The terms "cartilage" or "articular cartilage" or "cartilage matrix" are used in reference to human and other Cartilage refers to the elastic, translucent connective tissue of mammals, including the various species of mammals. Cartilage is composed primarily of chondrocytes, type II collagen Proteins, small amounts of other collagen types, other non-collagenous proteins, proteoglycans and and water, usually containing type I and II collagen and other proteolipids. It is surrounded by a perichondrium composed of fibroblasts in a matrix of glycans. Most cartilage matures into bone, but some cartilage remains in the bones of the nose, ears, knees, and other joints. Cartilage does not have a blood or nerve supply, and chondrocytes are found in this tissue. It is the only cell type in the

[0113] The terms "active agent," "active excipient," "active ingredient," and "pharmacologically active excipient" is used herein to refer to a chemical substance or compound that induces a desired pharmacological or physiological effect. These terms are used interchangeably in the literature and include therapeutically active and prophylactically active agents. including, but not limited to, salts, esters, amides, prodrugs, active metabolites, inclusion complexes, and analogs pharmaceutically acceptable pharmacologically active compounds of the active agents specifically mentioned herein, including, This includes derivatives and analogs thereof.

[0114] The use of "effective amount" or "therapeutically effective amount" of a pharmacologically active agent or active excipient The term refers to a non-toxic but sufficient amount of drug or excipient to provide a desired therapeutic effect. The "effective" amount varies from subject to subject. However, it is not always possible to specify an exact "effective amount" in any individual case. An appropriate "effective" amount may be determined by one of ordinary skill in the art using routine experimentation. The precise "effective" amount of an active agent incorporated into the compositions or dosage forms of the present invention is within a therapeutically effective range. in a range sufficient to allow easy application of the formulation to deliver an amount of active agent to As long as it is the concentration, it is not important.

[0115] The preferred route of administration is via a hydrogel or collagen matrix or artificial scaffold (matrix). the damaged cartilage site or the cartilage or soft tissue, preferably in fluid form, in a This includes local or applied locally to the site of (surgical) intervention close to the bone.

[0116] The present invention also relates to pharmaceutical compositions comprising the compounds of the present invention. The compounds may be formulated with standard pharmaceutically acceptable carriers, fillers, solubilizers and Stabilizers may be used to formulate the pharmaceutical composition.

[0117] The present invention relates to a pharmaceutical comprising, as an active ingredient, a compound useful for treating a disease disclosed herein. Such pharmaceutical compositions are in a form suitable for administration to a subject. The pharmaceutical composition may consist of the active ingredient alone in a pharmaceutical form, or the pharmaceutical composition may consist of the active ingredient in combination with one or more other drugs. a pharmaceutical acceptable carrier, one or more additional ingredients, or any combination thereof. It may include.

[0118] The active ingredient may be, as is well known in the art, for example, a physiologically acceptable cation or in pharmaceutical compositions in the form of a physiologically acceptable ester or salt in combination with an anion may exist in

[0119] As used herein, the term "physiologically acceptable" ester or salt , compatible with any other ingredients of the pharmaceutical composition and not harmful to the subject to which the composition is administered. It refers to the ester or salt form of the active ingredient.

[0120] Active ingredient, pharmaceutically acceptable carrier, and any additional ingredients in the pharmaceutical composition of the present invention The relative amounts of the compounds will depend on the identity, size, and condition of the subject being treated, and on the amount of the composition administered. Varies depending on the route taken.

[0121] By way of example, the composition may contain from 0.1% to 100% (w / w) active ingredient. In addition, the pharmaceutical compositions of the present invention may further comprise one or more additional pharmaceutically active agents. .

[0122] As used herein, "additional ingredients" include, but are not limited to, one or more of the following: Contains: excipients; surfactants; dispersing agents; inert diluents; granulating and disintegrating agents; binders; lubricants sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; Buffers; salts; thickeners; bulking agents; emulsifiers; antioxidants; antibiotics; antifungals; stabilizers; and a pharmaceutically acceptable polymeric or hydrophobic material.

[0123] The composition may also contain one or more substances used in the treatment of osteoarthritis, particularly preferably cysteine. Tagliptin, saxagliptin, vildagliptin, alogliptin and linagliptin one or more inhibitors of the dipeptidyl peptidase IV enzyme selected from the group consisting of: Alternatively, analgesics, nonsteroidal anti-inflammatory drugs, steroidal anti-inflammatory drugs and slow-acting anti-inflammatory drugs Arthritis medications, painkillers including paracetamol; acetylsalicylic acid, acetylsalicylic acid lysate phenylbutazone, sulindac, diclofenac potassium or sodium, acetonitrile Clofenac, tiaprofenic acid, ibuprofen, ketoprofen, aluminoprofen phenoprofen, naproxen, flurbiprofen, indomethacin, mefena niflumic acid, tenoxicam, meloxicam, piroxicam, and celecoxib selective cyclooxygenase-2 inhibitors such as cyclobutanol and etoricoxib, betamethasone, oxamethasone, prednisolone, prednisone, tixocortol or triamcinolone Chondroitin, Chondroitin Sulfate (Structum, Chondrosul f), glucosamine or glucosamine sulfate, diacerein (Art50, Zonda r), or other unsaponifiable extracts of avocado and soybean (piascledine) The material may include:

[0124] Other "additional ingredients" that may be included in the pharmaceutical compositions of the present invention are known in the art.

[0125] The formulations of the pharmaceutical compositions described herein may be formulated in any manner known or hereafter developed in the art of pharmacology. The preparation can be carried out by any method. Generally, such a preparation method involves the addition of the active ingredient and then bringing into association with the carrier or one or more other accessory ingredients, if necessary or desired. Forming or packaging the product into a desired single or multiple dosage unit.

[0126] Such pharmaceutical compositions are generally suitable for administration to any type of animal. As will be understood by those skilled in the art, in a preferred embodiment, the subject or patient to be treated is The subject to be treated is an animal, preferably a mammal. According to one embodiment, the subject to be treated is a dog, a cat, The animal is selected from the group consisting of: horses, cattle, sheep, pigs and non-human primates.

[0127] According to a preferred embodiment, the subject to be treated is a human, preferably an adult, particularly preferably a Most are adults over 50 years old.

[0128] The compositions according to the invention may be administered in particular systemically (parenterally, intravenously, etc.), orally, rectally, topically or dermally. It can be administered via any known route of administration, including: The composition may also be administered by intra-articular injection, preferably into an arthritic joint. Other topical agents such as hyaluronic acid, albumin, alpha-1 glycoprotein, and analgesic substances It may be administered in combination with

[0129] The compound may be administered to the animal several times daily, or less frequently, e.g. For example, once a day, once a week, once every two weeks, once a month, or even less frequently. The dose may be administered, for example, once every few months, or even once a year or less.

[0130] The frequency of dosing will be readily apparent to one skilled in the art and will depend on, but not be limited to, the condition being treated or Dose will depend on any number of factors, such as the type and severity of the disease, the species and age of the animal, etc.

[0131] The pharmaceutical compositions of the present invention may be administered in bulk, as a single unit dose, or as a plurality of single unit doses. It may be prepared, packaged, or sold as such.

[0132] The term "unit dose" refers to a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the ingredient is generally the dosage of the active ingredient that would be administered to a subject, or the dosage of such an ingredient. A convenient fraction of the dose, for example equal to one-half or one-third of such a dose.

[0133] The present invention also relates to a method of administering the compounds of the present invention to a subject, in one embodiment, comprising: The present invention provides a method for treating a subject by administering a compound identified using the methods of the present invention. This provides a way to

[0134] As used herein, the terms "treatment" or "therapy" refer to the treatment of a disease or condition. This refers to any action that makes it possible to reduce, suppress or delay the symptoms of a disease. Curative treatment includes both curative and prophylactic treatment of a disease. reduces, improves, and / or eliminates the symptoms of a disease or the burden it causes Prophylactic treatment is defined as treatment that prevents, reduces, and / or stabilizes disease. and treatments that reduce and / or delay the incidence of disease or the risk of it occurring. This includes both treatments that

[0135] In particular, in the context of the present invention, the term "treatment" refers more specifically to the treatment of arthritic destruction of cartilage. refers to the inhibition or suppression of

[0136] As used herein, the term "therapeutically effective dose" refers to a dose that is effective in treating or preventing osteoarthritis. is the amount necessary to observe preventive activity, particularly to observe inhibition or suppression of arthritic cartilage destruction. The amount of peptide administered and the duration of treatment will depend on the individual being treated. The physiological condition will be evaluated by one skilled in the art according to the nature of the joint being treated.

[0137] In some embodiments, the compositions according to the present invention are used to treat primary osteoarthritis (anatomical It can also be used to treat osteoarthritis (with or without traumatic causes) or secondary osteoarthritis The osteoarthritis to be treated may be of any joint, particularly the hip joint (osteoarthritis), knee joint (osteoarthritis), knee osteoarthritis), ankle, foot, hand, wrist, elbow, shoulder or spinal joints, preferably hip, knee It can affect the joints of the hands and spine.

[0138] The present invention also provides a method for alleviating or reducing joint irritation or treating existing arthritis in a mammalian subject. US201301229633A1 - Use of liraglutide as an active ingredient in the manufacture of pharmaceutical compositions for reducing the exacerbation of ... - Google Patents Regarding.

[0139] The present invention also provides a method for increasing chondrocyte proliferation in a patient, comprising administering to the patient a dose of 1 ng / ml to 10 ml of chondrocyte proliferation-increasing ... The therapeutic efficacy of a composition comprising liraglutide and 0.1% to 10% albumin is administering the dose to a patient.

[0140] According to one embodiment, a method for treating an inflammatory condition in a subject in need thereof is provided. The method comprises administering to a subject a pharmaceutical composition comprising at least one compound of the present invention. and administering the compound identified by the method of the present invention to a subject in need thereof. may be administered in conjunction with known compounds or other pharmaceutical agents.

[0141] All references mentioned herein are incorporated by reference into this application. Other characteristics and advantages of the present invention will become more apparent upon reading the following examples, which are provided by way of non-limiting illustration. It will become clear. [Example]

[0142] Example 1: VICTOZA on cytokine release in chondrocytes of OA patients Trademark) effect

[0143] This study investigated the effects of IL-1β-stimulated human chondrocytes isolated from cartilage of osteoarthritis patients. Effect of Victoza®, a GLP-1 analogue, on the release of inflammatory regulators The purpose was to evaluate the following.

[0144] material and method Test substance: Victoza® (Novo Nordisk). Reference substance: Water for injection. Cell culture materials: DMEM, fetal bovine serum, penicillin-streptomycin, phosphate buffer Cholesterol saline, Liberase Breast 3, IL-1β. Test system: MMP3 ELISA kit, MMP13 ELISA kit, PGE2 ELISA kit, cytokine 30-plex panel assay. ELISA assay and and multiplex assays were performed according to the manufacturer's instructions. Preparation of test and reference substance formulations: Victoza stock solution is 6 mg / ml The molecular weight is 3751.202 g / mol.

[0145] For each patient, 5, 25, 50, 125 and 625 nM of Victoza (registered trademark) For this purpose, 4 ml of medium containing 5, 25, 50, 125 and and 625 μM solutions were prepared as follows: 5 μM (18.756 μg / ml): 1.56 μl of 6 mg / ml stock solution, 500 Sterile water, enough to make 1 μl 25 μM (93.78 μg / ml): 1.56 μl of 6 mg / ml stock solution, total 100 Sterile water, enough to make 1 μl 50 μM (187.56 μg / ml): 1.56 μl of 6 mg / ml stock solution, 50 Sterile water, enough to make 1 μl 125 μM (468.2 μg / ml): 1.56 μl of 6 mg / ml stock solution, 20 Sterile water, enough to make 1 μl 625 μM (2344.5 μg / ml): 1.95 μl of 6 mg / ml stock solution, 5 Sterile water, enough to make 1 μl.

[0146] These solutions were prepared for each patient and diluted 1:1000 with culture medium (4 μl in 4 ml). Diluted to final concentration. Vehicle consisted of 4 μl of sterile water in 4 ml of culture medium. there was.

[0147] Liberase solution formulation: A 26U / ml stock solution of Liberase is diluted with 1% P / S and 2% Gluconate. The digestion was carried out in DMEM containing glutamine and stored at -20°C. For the final step of digestion, a 0.52 U / ml solution was extemporaneously prepared in DMEM. In the case of the 0.52 U / ml solution, a 1:4 dilution yields a 0.13 U / ml solution. A solution was prepared.

[0148] Cell culture medium formulation: 15% FBS, 2% in DMEM with 4.5 g / L glucose L-glutamine, 1% penicillin / streptomycin.

[0149] Experimental design and conditions Patients with osteoarthritis undergoing prosthetic knee surgery at Saint Antoine Hospital Cartilage was isolated from four patients with osteoarthritis. Chondrocyte isolation, seeding, culture, and activation were performed. Sample preparation was performed as follows: ELISA and multiplex analysis were then performed.

[0150] The day the human articular cartilage was isolated was designated "day 1," and the end of the test was designated "day 14."

[0151] Isolation of chondrocytes from human cartilage: from osteoarthritis patients undergoing total knee replacement surgery Cartilage was isolated. Cartilage from one patient was processed at a time. Freshly isolated cartilage was placed in a 2 mm diameter The cartilage pieces were cut into ~3 mm pieces, placed in a 50 ml tube, and rinsed with PBS. The mixture was incubated in 40 ml of Liberase at 0.52 U / ml for 45 minutes. Then, Liberase was removed and a fresh Liberase solution of 0.52 U / ml was added for 45 minutes. Then, the solution was removed and the cartilage pieces were incubated overnight in 40 ml of Liberase (0.13 U / ml). Incubated.

[0152] Chondrocyte seeding: 16 hours after chondrocyte isolation, pipette the solution up and down to seed the cells. The solution was then homogenized. The solution was then filtered through a 100 μm cell strainer. The solution was centrifuged at 1600 rpm for 6 minutes at room temperature. The pellet was then resuspended in 15 ml of complete medium (D The cells were resuspended in MEM + 15% FBS + 2% glutamine + 1% P / S. Count and count 200,000-250,000 cells / well in a 12-well culture plate. Cultures were incubated under sterile conditions (37°C, 5% CO2).

[0153] Chondrocyte culture: 48 hours after seeding, the medium was replaced with fresh medium. The medium was renewed every 2 days until the onset of the disease (days 12–13). At 100°C, the medium was replaced with medium containing 0.1% BSA without FBS.

[0154] The next day, the chondrocytes were cultured in five aliquots according to the study design (Table 1) and schedule (Table 2). Doses of Victoza® (5 nM, 25 nM, 50 nM, 125 nM, 625 nM) or vehicle for 2 h, followed by IL-1β (5 ng / m l) for 24 hours.

[0155] [Table 1]

[0156] [Table 2]

[0157] Testing and Evaluation: ELISA and Multiplex Assays

[0158] Sample preparation: At the end of each test, the medium was collected, centrifuged, and the supernatant was frozen. , shipped on dry ice within the testing facility and stored at −80°C upon receipt until analysis.

[0159] Detection assay PGE2 Assay: The assay is performed using a limited amount of PGE2 monoclonal antibody. Free PGE2 and PGE2-acetylcholinesterase conjugate (PGE2 tracer) The concentration of the PGE2 tracer was kept constant, but the free PGE 2 varied in each sample. The amount of PGE2 tracer bound to the monoclonal antibody was The amount of free PGE2 in the blood was inversely proportional to the amount of acetylcholinesterase substrate involved in the enzymatic reaction. Therefore, the color was not very strong in the high PGE2 concentration sample and was not very strong in the low PGE2 concentration sample. It was very strong.

[0160] Concentrations were calculated after determining %B / B0 according to the manufacturer's instructions, where B0 is the PGE2 transporter. The absorbance obtained from the reading of the well with the maximum amount of bound PGE2 (non-free PGE2) A represents the absorbance obtained for each standard or sample well. To obtain results, supernatants from IL-1β-treated wells were diluted 1:100 before proceeding with the test. The PGE2 measurement range was 7.8-1000. pg / ml.

[0161] MMP3 assay: The assay is a classical MMP3 assay combined with colorimetric peroxidase-based detection. It was based on sandwich ELISA. To get accurate results, before proceeding with the test , supernatants from IL-1β-treated wells were diluted 1:1000, and others were diluted 1:500. The measurement range of MMP3 was 0.156 to 10 ng / ml.

[0162] MMP13 Assay: The assay is coupled with colorimetric biotin-streptavidin-based detection. The original method was based on a classical sandwich ELISA, which combined the Before proceeding, the supernatants from the IL-1β-treated wells were diluted 1:100 and the others 1: The MMP13 measurement range was 8.23 to 6000 pg / ml.

[0163] Multiplex assay: Luminex technology is a combination of ELISA sandwich technology and It is based on binding to a mixture of fluorescent polystyrene beads that represent a solid phase for detection. The antibodies are conjugated with specific antibodies and different fluorescent dyes. This assay utilizes minimal volume. This allows for the quantification of various cytokines in the same sample. Aologies Human Cytokine Magnetic 30-plex Using EGF, eotaxin, FGF basic, GCSF, GMCSF, HGF, I FN-α, IFN-γ, IL-1RA, IL-1β, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p40 / p 70 ), IL-13, IL-15, IL-17, IP-10, MCP1, MIG, MIP1, MIP1β, RANTES, TNF-α and VEGF were quantified.

[0164] Samples were assayed undiluted according to the manufacturer's instructions.

[0165] At the end of the incubation, the culture medium was collected and centrifuged, and the supernatant was then washed with water. ELISA (MMP3 ELISA kit, MMP13 ELISA kit, PGE2 ELISA kits and cytokine 30-plex panel assays Ta.

[0166] result The concentration of each cytokine was calculated according to the manufacturer's instructions.

[0167] The basal inflammatory profile of each patient was calculated using the calculated concentrations of each detected cytokine. The evaluation was carried out by accumulating the means.

[0168] The results showed that the basal inflammatory profile was consistent with high levels of inflammatory cytokines in two patients and The results were highly variable, including two patients with high and low cytokine levels. .

[0169] Each patient's Victoza® response inflammation profile was determined The results were evaluated by comparing the ratio of the calculated mean concentrations of each cytokine before and after treatment with TM. The general results show that each patient responds differently. Equivalent responses to IL-1β were observed in mice treated with Victoza® in relation to IL-1β. Four cytokines were detected in three of the four patients' chondrocytes; The calculated mean concentrations of MCSF and CXCL10 / IP-10 (anabolic cytokines) increased. However, IL7 and MCP1 (catabolic cytokines) were decreased (Figure 1).

[0170] conclusion We demonstrated different response profiles in each of the four patients tested. Liraglutide inhibits some anabolic cytokine secretion in chondrocytes from OA patients. Increases vasopressin levels and reduces catabolic cytokine secretion to some extent.

[0171] Example 2: Preparation of intra-articular formulations of liraglutide with sustained release - Determination of release profile

[0172] We have tested 20 different formulations of viscous hydrogels containing liraglutide for intra-articular injection. To test the compounds and select the three best formulations for further in vivo preclinical testing. The release profile of each formulation into artificial synovial fluid was then determined.

[0173] material and method Test material: Liraglutide.

[0174] Materials for Hydrogel Formulations Dextran 70EP (70 kDa), Poloxamer 407: Kolliphor® P407, oxyethylene 71 ,5-74,9%, Polyethylene glycol (PEG) 3350 Alginic acid sodium salt, (Hydroxypropyl) methylcellulose (HPMC), viscosity 2,600-5,600 cP, 2% (literature value) in H2O (20°C), Albumin bovine fraction V, pH 7.0, Mr 67.000,00, Polysorbate 80: Tween® 80 Lecithin derived from soybeans, Polyethylene glycol (PEG) 400, Chitosan 95 / 500, high viscosity, Sodium hyaluronate 1.9MDa, PBS pH 7.4

[0175] Materials for artificial synovial fluid compounds Sodium Hyaluronate 1.9 MDa, Wellcos-Markus Grauel Albumin Bovine Fraction V, pH 7.0, Mr 67.000,00, SERVA γ-globulin bovine, Mr150.000,00, SERVA PBS pH 7.4, Panreac AppliChem

[0176] Test systems for determining release profiles Semipermeable membrane used: dialysis tubing Visking, cellulose, thickness 0.023mm, MWCO 12~14kDa Liraglutide concentration measurement ELISA kit

[0177] Artificial synovial fluid formulation: To formulate artificial synovial fluid, 3.5 mg of sodium hyaluronate, 9 mg of albumin and 3.5 mg of γ-globulin per ml of PBS pH 7. The volume of artificial synovial fluid prepared was 10 ml for each experiment.

[0178] The composition of artificial synovial fluid is available from many sources, e.g., Biological Performance ce of Materials:Fundamentals of Biocompa tibility. Fourth Edition, Jonathan Black, CRC Press, 20 gru2005;Synovial Fluid Composition and F unctions.Dr Arun Pal Singh, http: / / bonean dspine.com / synovial-fluid / ;Concentration of Hyaluronic Acid in Synovial Fluid.Ba Ry Decker et al., Clinical Chemistry 1959, 5(5): Selection was based on 465-469.

[0179] Liraglutide formulation for stability testing: For stability testing, 1.02 ml of PBS 1 mg of liraglutide in the capsule was mixed with 10 ml of artificial synovial fluid.

[0180] Test Procedure Determination of release profile: For each formulation prepared, the release profile was determined. On day 0, 10 ml of liraglutide hydrogel formulation (1 mg / ml - 1.02 ml) was administered. The released molecules were placed in a semipermeable membrane test system immersed in artificial synovial fluid at 37°C. This membrane should allow free diffusion of liraglutide monomers into the artificial synovial fluid. However, the physical barriers of liraglutide oligomers and hydrogel formulations are Synovial fluid samples (0.2 ml each) were collected after 1, 2, 4, 7, 10 and 14 days. The collected sample was replaced with fresh artificial synovial fluid to maintain the same volume of fluid outside the membrane. A sample of the fluid was taken from the test tube for further ELISA analysis to determine the liraglutide concentration. It was stored at 2-8°C until completion.

[0181] Stability Testing: To determine the stability of liraglutide under test conditions, Solutions of liraglutide in artificial synovial fluid were prepared at appropriate concentrations and administered in the same manner as the liraglutide hydrogel formulation. Processed.

[0182] ELISA procedure: Quantification of liraglutide concentration was performed with an ELISA kit. The procedure was performed according to the kit supplier's recommendations with one exception: the calibration curve used The standards had higher concentrations than suggested, ranging from 0.977 to 1000 ng / ml. The samples were diluted in EIA buffer. Standards and controls were prepared from a single stock solution of liraglutide. The assay was carried out on several EIA plates. All plates contained the same standard The expected concentration of liraglutide in the diluted samples was 5-22%. Expected concentration ranges for samples: 15, 100, and 200 ng / ml. Three quality controls were used at concentrations covering 100 ml. Each point was measured twice. Measured at 450 nM with a 96-well plate reader.

[0183] Experimental design and conditions Length of the experimental period: The day of formulation preparation and membrane loading was considered as "day 0", and the end of the experiment was considered as "day 1". It was considered the fourth day.

[0184] Group design and study schedule: 20 different groups containing 1 mg / ml liraglutide The hydrogel formulations were tested according to the study design (Table 3) and study schedule (Table 4). Three classes of formulations were included:

[0185] Standard formulation (1, 2, 3, 4, 5, 7, 8, 10, 11, 12, 13, 14, 15, 1 6), potential active excipient formulations (17, 18, 19, 20) and albumin-based formulations compounds 6, 9).

[0186] [Table 3]

[0187] [Table 4]

[0188] result

[0189] Liraglutide was analyzed accurately and reliable results were obtained. The data are shown in Appendix II. All plates passed two of the three quality controls. The standard was met, i.e., the concentration was within ±20% of the theoretical concentration. Guidelines on bioanalytical method validation tion(EMEA / CHMP / EWP / 192217 / 2009Rev.1Corr. 2 ** ) was accepted.

[0190] Liraglutide concentration: Samples from artificial synovial fluid were diluted to 1:400 before proceeding with ELISA staining. All samples collected from the first five time points were analyzed. These eight samples were also analyzed based on the expected consistency of the formulation at the start of the study.

[0191] Determination of release profiles: Maximum expected liraglutide concentrations for all hydrogel formulations The results were calculated as a percentage of the observed values. The amount of liraglutide was not included in the calculation. When glutide is released from the semipermeable membrane, the concentration is 90.74 μg / ml (corresponding to 100%). )

[0192] The calculated percentages for the six best formulations are shown below (Table 5).

[0193] [Table 5]

[0194] To determine the release profile of 20 different formulations containing liraglutide Experiments performed as a group demonstrated the variability among the formulations tested. Of these, six produced liraglutide concentrations greater than 1% after 10 days. These are shown in Figure 2. The formulations 6, 8, 14, 17, 19 and 20 have release profiles similar to those described above.

[0195] Example 3: Three liraglutide bases using a surgically induced model of osteoarthritis in rats Efficacy testing of formulations

[0196] The purpose of this study was to investigate the effects of three surgically induced models of osteoarthritis in rats. The objective of this study was to evaluate the efficacy of a liraglutide-based formulation of

[0197] material and method

[0198] Test substance: liraglutide Vehicle: PBS Liraglutide Formulations: Three viscous hydrogel formulations were tested: Formulation 6: High release Formulation 8: Medium release Formulation 20: Low release

[0199] Unformulated liraglutide corresponds to liraglutide dissolved in PBS.

[0200] The formulations were prepared on the day of treatment for each of the three cycles. For each formulation, 4 mg of liraglutide in 2 ml of the compounded solution or PBS (for uncompounded liraglutide) This resulted in a dose level of 0.18 mg / kg in 25 μl for intra-articular injection. Assuming 0g, the dose administered to each rat was 50µg. It was used to dose the animals within one hour of preparation.

[0201] Experimental model Animal species / strain: Rat / Sprague Dawley (SD) Gender / number / weight average: male / 60 / 6-8 weeks old at the start of the test Diet: Animals were fed commercial rodent chow ad libitum.

[0202] Experimental design and conditions Rats were assigned to one of five stratified study groups according to body weight.

[0203] OA induction by medial ligament transection (MLT) procedure followed by resection of the medial meniscus (MMx) Each rat was anesthetized by the chamber induction technique using inhalation anesthesia (isoflurane 4.0%). During surgery, animals were maintained with isoflurane at a level of 1.5–2.5% and oxygen The flow rate was 1-2 liters / min. Ophthalmic ointment was applied to the eyes to prevent drying of the tissue during the anesthesia period. After induction of anesthesia, the hair on the surface of the skin of the right leg was removed using an electric animal clipper. After shaving the knee joint, the skin was disinfected with iodine and a parapatellar skin incision was made on the medial side of the joint. The medial side of the ganglion cavity was incised. The medial ligament was cut and the medial meniscus was excised using a microsurgical knife. The wound was closed with vicryl 5 / 0 braided absorbable sutures. All surgical procedures were performed under a surgical microscope. Group assignments are shown in Table 6, and the study timeline is shown in Table 7.

[0204] [Table 6]

[0205] [Table 7]

[0206] Testing and Evaluation The weight-bearing changes of OA rats were measured using an incapacity tester. Postural imbalance is reduced, with each hind leg reported to be incapacitated. Each rat was placed on a separate force plate on the apparatus, with each hind paw supported. The weight was measured for 5 seconds. The ratio of the load weight of the right hind paw to the left hind paw was calculated. The average value of consecutive measurements was recorded. Weight-bearing function (incapacity test) was measured at baseline ( The experiment was conducted four times in total on days 1, 14, 28, and 35. I didn't have the knowledge.

[0207] On day 36, rats were sacrificed by CO2 asphyxiation. Knee joint structures were examined for further histological analysis. The contralateral (uninjured) knee was also fixed in 4% buffered formalin. The tissue was fixed in a solution of acetone.

[0208] Numerical results were given as mean ± SD after Grubbs' test analysis at α = 5%. Outlier data points (marked with an asterisk) were identified and not included in the group mean calculation. Where applicable, statistical analysis was performed using two-way ANOVA followed by Bonferroni post hoc tests. ) or one-way ANOVA (followed by Dunnett's multiple comparison post-hoc test). A probability of 0.05% (p≦0.05) was considered significant. In the figure, the degree of statistical significance between groups is indicated by * p ≤ 0.05, ** p<0.01 and *** Shown as p<0.001.

[0209] result Weight Load Test Weight bearing changes in rats with OA were assessed by measuring the weight the animals distribute to each hind paw independently. All animals were evaluated using a disability meter. Before OA induction, the weights of both hind paws were equalized. On day 14, there was a significant increase in the weight loading difference (R / L weight percentage) compared with the control group. This was observed between group 3M and group 3M, accompanied by a prolonged release of liraglutide (Figure 3).

[0210] Histological evaluation Slides were examined by a single pathologist blinded to the treatment groups. The cross-section of the knee was evaluated for the data. Cartilage matrix loss width (0% cartilage intact, 100% is the highest point reached, 50% is the intermediate region) ) Cartilage degeneration score (score 0-5, see section 9.1). Total cartilage degeneration width (0% cartilage intact, 100% is the highest point, 50% is the intermediate region). includes all possible degenerative changes. Significant cartilage degeneration width. Measurements of more than 50% of the thickness are significantly impaired (+ / -). Lesion zonal depth ratio (microns). Osteophytes (score 0–4, see section 9.2). Calcified cartilage and subchondral bone damage score (score 0-5, see section 9.3) stomach). Synovial reaction (score 0-4, see section 9.4). Medial capsule repair (measured in μm) 10. Increase in plate thickness (measured in μm)

[0211] The percent cartilage matrix loss was 43.6% compared to the vehicle-treated control group (1M). The lowest was Group 3M (35.8%). Total cartilage degeneration width (%): This was also the same as in the vehicle-treated group. The incidence was lowest in Group 3M (37.5%) compared to Group 1M (43.2%) in the control group. A Bonferroni post-hoc comparison followed by a one-way ANOVA revealed that the control group A statistically significant difference in cartilage degeneration between the 3M and liraglutide-treated animals was revealed. It became ( * P<0.05). Mean + / -SD, n=11~12.

[0212] conclusion The purpose of the study was to evaluate the efficacy of three liraglutide-containing steroids in the treatment of osteoarthritis in rats using a surgically induced model of osteoarthritis. The objective of this study was to evaluate the effectiveness of the ointment-based formulation.

[0213] The results of this study showed that liraglutide in formulation 6 (group 3) corresponds to a high release of liraglutide. M) showed a significant improvement in the incapacity meter compared to vehicle-treated control animals 14 days after OA induction. The results showed that the weight-bearing test induced a statistically significant reduction in OA damage. The Von Frey test did not reveal any statistically significant differences between all animal groups. Cartilage degeneration was also significantly reduced in the relaxants treated with formulation 6 compared to the control and other formulations. Weight-bearing tests and histological evaluation clearly demonstrated that the OA rat model were susceptible to testing new therapeutic treatments in the

[0214] Liraglutide formulated in formulation 6 exhibited a significant improvement in efficacy compared with other formulations or unformulated liraglutide. and has chondroprotective effects in vivo.

[0215] Example 4: Albumin-dependent effects of liraglutide using a surgically induced model of osteoarthritis in rats Dose-response study using a methicone-based formulation

[0216] The principle of the test is to evaluate the efficacy of albumin as a therapeutic agent for measuring disease parameters in a rat OA model. The evaluation was based on the combined use of liraglutide.

[0217] material and method

[0218] The test substance was liraglutide, and the positive control was dexamethasone (and Novo No Unformulated liraglutide Victoza® (6 mg / ml injection) manufactured by Rdisk )) is.

[0219] compound The vehicle (formulation excipient) was phosphate-buffered saline for intra-articular injection into groups 1M and 7M. It consisted of albumin resuspended in saline (25 μl).

[0220] Albumin-based formulations of liraglutide (high, medium, and low doses) were administered as follows: Prepared to:

[0221] Liraglutide (supplied as a powder) was dissolved in an appropriate volume of vehicle for intra-articular injection. 0.18 mg / kg (groups 2M and 8M), 0.06 mg / kg (group 3M) in 25 μl or 0.02 mg / kg (Group 4M). The formulation was administered in each of three cycles. were prepared on the day of treatment.

[0222] The positive control used in Group 5M was dexamethasone. The human clinical dose for knee treatment was 4 mg / injection, which is equivalent to 0.4 mg / kg in rats. It was supplied "ready to use" at a concentration of 4 mg / ml. The volume was 25-50 μl depending on the rat's average BW on each treatment day.

[0223] Unformulated liraglutide (Victoza®) is available as a 6 mg / ml stock solution. The human clinical "starting" dose for diabetic patients was 0.6 mg / day (as repeated SC injections). (supplied), which is equivalent to 0.06 mg / kg in rats. A stock solution of Benzyl Alcohol (R) was prepared at a final concentration of 0.006 mg / ml for SC injection in Group 6M. The solution was diluted 1000-fold with physiological saline for injection to achieve a concentration of 10 ml / kg.

[0224] Experimental model Animal: Rat / Strain: SD Gender: Male / Number: 72 / Age: 6-8 weeks old at the start of the test Supplier: Janvier Labs, France Initial body weight: The average body weight at the start of the study (day 1) was 260 g. Minor and maximum weights were within ±20% of the group mean. Diet: Animals were fed commercial rodent chow (Safe ref#A04) ad libitum. Animals had free access to filtered drinking osmotic water. Contaminants: Contaminants in food and water supplies that may affect the results of this test It wasn't there.

[0225] Experimental design and conditions Definition of study start and end: The day of OA induction was defined as "Day 1." The end of the study was set at "Day 36." In the satellite group study, the end of the study was set at "Day 57." It was decided.

[0226] Allocation to treatment groups: Rats were randomly assigned to one of eight groups according to body weight. Ta.

[0227] Study design and timeline: The study was conducted as follows: This was carried out in three cycles according to Table 2.

[0228] OA induction by medial ligament transection (MLT) procedure followed by resection of the medial meniscus Anesthesia was induced by the chamber induction technique using inhalation anesthesia (isoflurane 5.0%). During surgery, the animals were kept under isoflurane at a level of 1.5-3.5% and airflow at 1-30°C. The flow rate was 2 liters / min. Ophthalmic ointment was applied to the eyes to prevent drying of the tissue during the anesthesia period. After induction of anesthesia, the hair on the surface of the skin of the right leg was removed using an electric animal clipper. After hair removal, the skin was disinfected with iodine and a parapatellar skin incision was made medial to the joint. This incision provides access to the exposed joint. The medial ligament is cut and a microsurgical knife is inserted. The medial meniscus was excised using a surgical knife. The wound was closed with vicryl 5-0 sutures. Surgical procedure All procedures were performed using a surgical microscope. Group assignments are shown in Table 8, and the study timeline is shown in Table 9. Shown below.

[0229] [Table 8]

[0230] [Table 9]

[0231] Testing and Evaluation Joint swelling: Measurements of knee diameters were taken and joint swelling was inferred as an index of inflammation. Measurements were performed using a digital caliper while the rats were anesthetized. The experiment was conducted on the day after surgery (for baseline), and then once a week until the end of the study. I had no knowledge about it.

[0232] Weight-bearing test: The weight-bearing changes of rats after OA induction were monitored using an incapacity test system. Postural imbalance, which has been reported to show changes in pain threshold and weight distribution in the extremities, was observed. Each rat was tracked so that each hind paw was placed on a separate force plate on the incapacitation device. The weight supported by each hind paw was measured for 5 seconds. The ratio was calculated. The average of three consecutive measurements for each rat was recorded. Function (incapacity test) was assessed at baseline (day 1), days 14, 28, and 35. A total of four trials were performed on each eye. The experimenter was blinded to the groups.

[0233] Animal sacrifice and tissue fixation: On day 36 (the end day of groups 1M-6M), all animals were bled. The rats were euthanized with a lethal dose of Euthasol vet. Knee joint structure The contralateral side was harvested and fixed in 4% buffered formalin solution for further histological analysis. The (uninjured) knees were also fixed in 4% buffered formalin solution. On day 57 (satellite groups 7M-8), On the final day of the study (the end of the study period), rats were euthanized. Knee joint structures were collected for further histological analysis. The contralateral (uninjured) knee was also fixed in 4% buffered formalin. The tissue was fixed in a solution of acetone.

[0234] Histological analysis: Histological analysis was performed. Knee joint sections were stained with hematoxylin-eosin or The extent of pathological lesions was assessed by staining with toluidine blue. erwin et al., Osteoarthritis and Cartilage18(20 10) Scored as S24-S34.

[0235] Histological analysis was performed on: Right knee (affected): All groups, 10 animals / group, collected on day 36. Sample size: n=60 Left knee (healthy control): Vehicle group 1M, 5 animals / group. Sample size: n=5 Right knee (affected): All animals in groups 7M-8M, 6 animals / group, collected on day 57. Sample size: n =12 Total sample size: n=77 Statistical analysis: Numerical results were presented as mean ± standard deviation (SD). Outliers or excluded data were excluded. Data points (marked with $) were not included in the group mean calculation. or one-way ANOVA (followed by Dunnett's multiple comparison post-hoc test) to evaluate statistical significance. A statistical analysis was performed. A probability of 5% (p≦0.05) was considered significant. In the figures, the results are shown as mean ± S The degree of statistical significance between groups is shown as EM. * p ≤ 0.05, ** p<0.01 and *** Shown as p<0.001.

[0236] result In this study, six groups (1M-6M, n = 9-10 per group) were assigned from day 1 to day 36. I tracked it.

[0237] Histological analysis revealed no lesions in the right knee joint, so the animals from Group 2M were Subject 2 was excluded from the entire study.

[0238] Knee Measurements (KM): Knee measurements were recorded preoperatively, the day after surgery, and weekly thereafter. For each group, the mean diameters of the left and right knees in two dimensions were calculated.

[0239] At termination, rats from group 2M treated IA with high doses of liraglutide (width and thickness ), in rats from group 3M treated IA with medium dose liraglutide (thickness), A response is observed. As shown in Figure 5, the group treated SC with Victoza® Rats from 6M (thickness) also showed a significant decrease in knee measurements compared to the vehicle-treated group 1M. A little.

[0240] Weight bearing test: Changes in weight bearing in rats with OA were assessed by measuring the weight the animals distributed to each hind paw. The incapacity test was performed on Day 1 (baseline) ) and on days 14, 28, and 35. On day 14, Victoza® A significant increase in the R / L ratio in % was observed for group 6M treated with . Although not significant, it was also observed during the other measurement periods (days 28 and 35). Control animals and those receiving either a medium dose (14 and 35 days after OA induction) or a high dose (35 days) were treated with OA. A small, non-significant trend for weight bearing differences was observed between liraglutide IA-treated animals and those treated with liraglutide IA. was done.

[0241] Histological analysis The left hind limb (from animals 1M1, 1M9, 1M26, 1M31, and 1M57) served as a control. As expected, the knee joint showed no lesions.

[0242] In all groups, the lesions observed ranged from marked to severe. The observed pattern is a localized widespread tear of the cartilage, generally involving the entire medial tibial plate. It was.

[0243] The ulcerative margins are generally characterized by fibrillation of the cartilage and / or complete destruction of the proteoglycan matrix. The subchondral bone was generally characterized by necrosis with complete loss of bone. The osteochondral interface (highest attained) including the area beyond the friction zone was confounded with fibrotic bundles (fibrosis). At this point, the subchondral bone showed necrosis (eosinophilia with surrounding fibrin, karyorrhexis, cells showing pyknotic nuclei).

[0244] The dexamethasone IA treatment group (5M group) and the Victoza® SC treatment group ( 6M group) tended to show lower cartilage loss. However, in some individuals, fibrous tissue forming adhesions between the medial meniscus remnant, femoral cartilage, and synovium, as shown in Near complete replacement of the tibial plate was observed.

[0245] The observed repair changes were characterized by fibrous collagen bundles within the synovial capsule, which were associated with dextromethorphan. Metasone IA treatment group (5M) and Victoza® SC treatment group (6M) shows large fibrous papillary-like highly vascularized processes, and other structures (inner Adhesions may form in all joints (meniscal remnants, articular cartilage, ligamentous remnants) In the liraglutide IA treatment group, no fibrosis was observed and no pathological differences were observed. In the glutide IA-treated group, chondrocyte nests were observed in the cartilage with a dose-response pattern (Figure 7): 3 / 9 in the high dose group, 2 / 8 in the medium dose group, and 1 / 9 in the low dose group In the Victoza® SC treatment group, only 1 out of 7 animals had chondrocyte nests. showed proliferation in the 10 mice but not in the vehicle-treated group (0 / 10 mice).

[0246] These observed chondrocyte proliferations suggest attempts at cartilage regeneration.

[0247] Satellite group study In the satellite group study, two groups (7M-8M, group equivalent) were used from day 1 to day 57. The histological parameters were measured as previously in this study. The percentage of recovery 3 weeks after treatment cessation was assessed in the liraglutide high-dose IA treatment group. and vehicle groups were compared and analyzed.

[0248] [Table 10]

[0249] Overall, the lesions observed were significant for both groups. Thus, the typically observed pattern is a localized dislocation of the cartilage involving most of the medial tibial plate. The ulceration margin was covered with cartilage fibrillation and / or proteoglycans. It was characterized by necrosis with complete loss of matrix.

[0250] Animals treated IA with high doses of liraglutide (Group 8M) were significantly higher than vehicle-treated animals (Group 7M) ) and lower matrix loss in the liraglutide IA-treated group compared with placebo. There is a tendency for losses to decrease over time (Figure 8).

[0251] No differences were observed in degeneration scores and total degeneration width. Throughout the course of the study, degeneration scores were significantly higher in the liraglutide IA-treated group at Z1 and Z3. was lower (Fig. 9).

[0252] There was no significant difference in osteophyte formation between the two groups. However, the course of the study after treatment cessation Overall, the percentage of osteophytes tended to be lower in the liraglutide IA-treated group.

[0253] The high-dose treatment group showed more significant synovial repair as observed by membrane thickening compared to the control group. (Figures 10 and 11).

[0254] An example of a nest diagram is shown in Figure 12. As assessed in Figure 13, the vehicle group (2 / 5; 8 0.1±4.1 foci / μm 2 ), compared with the high-dose test substance treatment group (5 / 6; 30 0.6±7.0 foci / μm 2 ) significantly more chondrocyte nests were observed within the chondrocytes.

[0255] conclusion As previously observed during this study, significant differences in each group regarding cartilage, subchondral bone, and synovial membrane were observed. Histological changes were observed.

[0256] Synovial repair changes were significantly greater in the IA treatment group with high-dose liraglutide compared to the vehicle group. Furthermore, cartilage degenerative changes were more pronounced in the liraglutide-treated group compared to the vehicle group. Although the difference was not significant, it was not statistically significant.

[0257] In particular, more chondrocyte nests were observed in the liraglutide IA treatment group, indicating intracartilage regeneration. Attempts have been proven.

[0258] Under the test conditions, dexamethasone IA and Victoza® SC treatment significantly reduced several OA-related defects (knee joint swelling, incapacity, tissue loss associated with cartilage loss) Although the IA treatment group was able to reduce the fibrosis (i.e., the fibrosis was not observed in the IA treatment group), repair was not observed in the IA treatment group. accompanied.

[0259] Interestingly, histological analysis showed that the endochondral chondrocytes were significantly smaller in the liraglutide-treated group than in the control group. showed alveoli.

[0260] These figures show chondrocyte proliferation demonstrating attempts at cartilage repair. In the IA liraglutide-treated group, the presence of foci was not associated with changes in synovial repair. However, in the satellite study (day 57), IA treatment with high doses of liraglutide The chondrocyte nests seen in the animal group were associated with more pronounced synovial repair changes (thickening).

[0261] Overall study results suggest that liraglutide may play a role in the inflammatory and regenerative processes associated with OA. This indicates that the targeting mechanism is related to the

[0262] Example 5: Reactivation of chondrogenesis in an in vitro differentiation model of human mesenchymal stem cells The effects of Chido

[0263] The present inventors have demonstrated that cartilage formation occurs in an in vitro differentiation model of human mesenchymal stem cells (hMSCs). To test the effect of liraglutide on chondrogenesis and determine whether liraglutide promotes chondrogenesis. was evaluated.

[0264] material and method Test material: Liraglutide. Test system: Human mesenchymal stem cells (StemPro BM, Cat A15652, The rmoFisher Scientific). Basal medium: MesenPRO RS growth supplement, L-glutamine (1%) and Genta MesenP supplemented with 100 ml of medium containing 10 mg / ml of cephalosporin (50 μl per 100 ml of medium). RO RS basal medium (ThermoFisher Scientific). Differentiation medium (used as positive control): StemPRO chondrogenic differentiation supplement and Gentamax StemPR supplemented with isin (10 mg / ml, 50 μl per 100 ml of medium) O Chondrogenic Differentiation Medium (ThermoFisher Scientific)

[0265] procedure Mesenchymal stem cells were used at a confluence of 60-80%. and 1.6 × 107 cells were cultured in basal medium (MesenPRO RS basal medium + supplements). A cell suspension of 100 cells / ml was prepared.

[0266] 5 μl of this suspension was placed in the center of a well of a 24-well plate. The plate was incubated at 37°C for 1 hour. The plates were incubated in a humidified incubator for 2 hours. After 2 hours, the plates were incubated in a humidified incubator for 2 hours. 11. For the test timeline, follow Table 12 and add either (negative control) or 1 ml of basal medium containing the test substance was added. As a positive control, differentiation medium (StemPR 1 ml of chondrocyte differentiation basal medium + supplements was used. The plate was incubated at 37°C in 5% CO2. The cells were returned to the incubator for 7, 14, or 21 days. During the differentiation phase, the medium was changed every 3–4 days. I replaced it.

[0267] [Table 11]

[0268] [Table 12]

[0269] At the end of each test period (8, 15, or 22 minutes), alcian blue staining and microscopic analysis were performed. The plates were harvested for analysis. The medium was removed and 1 ml of PBS was added to gently incubate the cells. The PBS was removed and 1 ml of 4% formaldehyde was added for 30 minutes at room temperature. .

[0270] Then, remove the 4% formaldehyde and gently rinse the fixed cells twice with 1 ml of distilled water. The distilled water was removed and 1 ml of 1% Alcian blue (prepared in 0.1 N HCl) was added. The staining solution was removed and the cells were soaked in 1 ml of 0.1% PBS (prepared by manufacturer) for 2 hours at room temperature and protected from light. The plate was washed twice or three times with N HCl. The HCl solution was removed, and 1 ml of distilled water was added to each well. The cells were observed under a microscope and photographed.

[0271] result The effect of liraglutide on sphere formation was assessed by microscopic observation 5 days per week. Then, Alcian blue staining was performed at three time points (e.g., 7, 14, and 21 days after treatment). This dye uptake reflects the presence of sulfated glycosaminoglycans (GAGs), Demonstrates the formation of chondrocyte spheroids.

[0272] [Table 13]

[0273] As shown in Table 13, sphere formation was observed for vehicle-treated cells in basal medium throughout the study. No spherogenesis was observed with a dose-response for the two test doses of liraglutide. Indeed, in cells treated with 10 nM and 100 nM liraglutide, On day 22, sphere formation was observed in 67% and 100% of treated wells, respectively. The enriched medium induces hMSCs to commit to the chondrogenic pathway and generate chondrocytes. As expected, the spheres were not spheroidized for vehicle-treated cells in this medium. Formation was observed (83% of treated wells on day 22). The liraglutide-induced spheroids were confirmed to be chondrocyte spheroids. This indicates that chondrocyte differentiation can be induced by spheroid formation and positive adenosine monophosphate (SA) alone. An example of Lucian blue staining is shown in FIG.

[0274] conclusion In this study, an in vitro assay for chondrogenesis was used to assess the role of chondrogenesis in this process. The effect of raglutide was tested.

[0275] We found that liraglutide induced sphere formation in a dose-response manner in the presence of basal medium. The results demonstrated that chondrocyte sphere formation was observed in the vehicle-treated cells. This was confirmed by positive Lucian blue staining (a marker of cartilage matrix synthesis).

[0276] Under the conditions tested, our data demonstrate that liraglutide alone commits hMSCs to the chondrogenic pathway. This indicates that liraglutide induces the production of chondrocytes. The ability to target resident stem cell populations in the joint area and stimulate cartilage repair through chondrocyte differentiation This allows stimulation of the OA, which is considered a promising approach for the treatment of OA.

[0277] Example 6: Effect of liraglutide on mouse primary chondrocyte viability

[0278] The purpose of this study was to investigate the effect of liraglutide on cell viability using mouse primary chondrocytes. The purpose was to evaluate the effectiveness.

[0279] material and method Test material: liraglutide Test system: Mouse primary chondrocytes Cell culture media formulation DMEM containing 2 mM L-glutamine, 10% fetal bovine serum (FBS), 1% penicillin Phosphorus / streptomycin was used for cell culture from day 1 to day 7. On day 7, 2 m DMEM containing M L-glutamine, 0.1% bovine serum albumin (BSA), and 1% Penicillin / streptomycin (P / S) was used in the absence of FBS. .

[0280] Experimental design and conditions Definition of test start The day the cells were plated into the wells was considered "Day 1," and the end of the test was considered "Day 9." Everyone agreed.

[0281] procedure Isolation of mouse articular cartilage Immature mouse chondrocytes were derived from newborn mice (5-6 day old C57Bl / 6). All procedures were carried out in a sterile flow hood. The mice were then decapitated with scissors to ensure their safety. After death, the animals were fixed in a prone position and the front legs were pinned with needles. The hind legs were then cut with scissors and pliers. The skin was removed from the hind limbs along the spine. The skin and muscle residues were removed from the limbs. The foot is flattened with curved forceps to release the small, translucent, hard sphere that corresponds to the femoral head. When the ball was separated, it was placed in 30 ml of 1x PBS and the muscle was removed from the rest of the leg. The bones were then removed from the bones and other tissues. The bones appeared reddish-brown and the cartilage appeared white. The cut is made on both sides, which is the joint (forming two balls). After removing the tissue, the sphere was cut in half to separate the two spheres, and then cut in half again. This allows for easier digestion. The femoral condyles and tibial plateaus were also placed in 30 ml of 1x PBS. I put it in.

[0282] Isolation of immature mouse chondrocytes The cartilage pieces were placed in a 100 mm Petri dish with 10 ml of digestion solution (DMEM, 2 mM L-Glutamine). Incubate at 37°C in 5% CO2 in a medium containing glutamic acid, 1% P / S, and 3 mg / ml collagenase. Between the two digestions, the cartilage pieces were incubated in beta for 45 minutes. After two digestions, the contents were collected using a 25 ml pipette and placed in a new Petri dish. The cartilage fragments were placed in 10 ml of DMEM, 2 mM L-glucose, and in a solution of tamoxifen + 1% P / S and 0.5 mg / ml (diluted 1 / 6) collagenase D The cells were incubated overnight in an incubator at 37°C and 5% CO2.

[0283] Chondrocyte seeding After overnight digestion, 10 ml of DMEM, 2 mM L-glutamine + 10% FBS was added to each petri dish. The collagenase D was added to the dish to stop the action of collagenase D. The medium and residual cartilage were collected and Place in a 0 ml Falcon tube. Disperse the aggregates using a reduced size pipette. This was carried out to obtain a suspension of separated cells, which was then filtered through a sterile 70 μm cell strainer. The cells were centrifuged at 400 g for 10 minutes at 20°C. The medium was removed and the pellet was collected in 5 ml of The cells were washed by resuspending in PBS. The cells were centrifuged at 400 g for 10 minutes at 20°C. Remove the PBS and add 15 ml of DMEM 2 mM L-glutamine + 10% FBS + 1% P / S. The chondrocytes were counted using a Neubauer hemocytometer, and the viability of the extracted cells was determined. The chondrocytes were cultured in 2 ml of DMEM 2 mM in a 12-well plate. 40 x 10 in L-glutamine + 10% FBS + 1% P / S / well 3 Cells seeded at a density The cultures were maintained under inorganic conditions in an incubator at 37°C and 5% CO2.

[0284] Chondrocyte culture Immature mouse articular chondrocytes reached confluence after 6-7 days of culture. On day 7, the DMEM medium containing 10% FBS was removed and the wells were filled with 1 ml 1 ml of PBS and rinse twice with 1 ml of DMEM, 2 mM L-glutamine + 1% P / S +0.1% BSA was added. On day 8, the medium was removed and 500 μl of DMSO was added per well. EM, 12 different concentrations of L-glutamine in 2 mM L-glutamine + 1% P / S + 0.1% BSA Treatment with raglutide was performed (Table 14). The plates were incubated at 37°C + 5% CO2 for 24 hours. The study timeline is shown in Table 15.

[0285] [Table 14]

[0286] Each treatment condition was performed in triplicate. [Table 15]

[0287] Testing and Evaluation At the end of the test (day 9), the culture medium from each well (±500 μl) was transferred to a 1.5 ml tube. Collect the supernatant (1 tube per well) and centrifuge at 4000 rpm for 10 minutes at room temperature. The supernatant was placed in a new 1.5 ml tube. Samples were frozen at -70°C until dosing.

[0288] LDH assay Lactate dehydrogenase secretion into the culture medium was measured by LDH assay (Abcam) 100 μl of the supernatant was used to measure the lactate dehydrogenase levels secreted by injured cells. The LDH assay was used as detailed in the description of the specific LDH assay kit. Follow the instructions in the Plate Reader (96 wells) The absorbance was measured using a fluorometric analyzer (Fiberscope FC, Thermo Fisher Scientific). The wavelength of the reading was 450 nM. The average optical density (OD) of the reading blank wells was calculated for each reading. was subtracted from the value.

[0289] result Lactate dehydrogenase is present in all cell types and is secreted into cell culture media upon plasma membrane damage. LDH enzyme is a stable enzyme that is rapidly released into the blood. Detected by SkanIt software for microplate readers, ThermoFi LDH oxidizes lactate to generate NADH, which is then It reacts with the WST substrate to produce a yellow color, the intensity of which directly correlates with the number of cells lysed. LDH activity was quantified by spectrophotometry at OD450nM. After 24 hours of incubation with acetaldehyde (1.7 nM to 300 μM), LDH activity was measured. A positive control was used, where 5 μl of LDH enzyme was added directly to the wells. % Cytotoxicity was calculated by the formula ((test sample-low control) / (high control-low control))×100. As shown in Figure 15, the presence of the lowest tested dose of liraglutide (up to 11.1 μM) Presence induced the release of small amounts of lactate dehydrogenase in the medium. There was no significant difference compared to treated cells. In the presence of In fact, the calculated % mortality was as follows: Kuru: 0.0% ± 0.008 and liraglutide 33.3 μM: 8.5% ± 0.006; Liraglutide 100 μM: 11.1% ± 0.051 and liraglutide 300 μM: 11 0.5% ± 0.069, p<0.001). The use of a positive control (marked in yellow) All reagents were confirmed to be functioning properly.

[0290] conclusion This study demonstrated the 24-hour impact of liraglutide on chondrocytes depending on the dose tested. It is shown that mortality can be observed after incubation.

[0291] Example 6: Monoiodoacetate after intra-articular administration of an albumin-based formulation of liraglutide SOX9 expression in the knee joints of mice injected with methadone

[0292] SOX9 is a crucial transcription factor in developing and adult cartilage. is expressed from the multipotent skeletal progenitor cell stage and is active throughout chondrocyte differentiation. This is suppressed in hypertrophic chondrocytes of the growth plate, but not in permanent chondrocytes of healthy articular cartilage. is expressed throughout life. SOX9 is required for chondrogenesis: chondrocyte lineage commitment ensures cell viability, promotes cell survival, and transduces genes for many cartilage-specific structural components and regulatory factors. Photographically activated.

[0293] The objective of this study was to evaluate the efficacy of monoiodopa in the treatment of patients with liraglutide after intra-articular administration of an albumin-based formulation. The purpose of this study was to examine SOX9 expression in the knee joints of mice injected with miR-1224 acetate (MIA). Ta.

[0294] material and method compound Monoiodoacetate (MIA): MIA powder was resuspended in injection saline and administered to groups 2M, 3M, 4M, and 5M. 0.75 mg in 5 μl per mouse was injected into the knee joint. Treatment formulation: Vehicle (formulation excipient) was administered intra-articularly (5 μl) to groups 1M and 2M. It consisted of 5% human albumin resuspended in phosphate-buffered saline (PBS). -Albumin-based formulations of liraglutide: Liraglutide (supplied as powder) was dissolved in an appropriate volume of vehicle in Group 3M, 10 μg, 20 μg or 30 μg in 5 μl per mouse for 4M and 5M, respectively g was injected into the knee joint.

[0295] Experimental model Animal species / strain Mouse / C57Bl / 6 Gender / Age Male / 12 weeks old on day 1 supplier Janvier Labs, France

[0296] diet Animals were fed commercial rodent chow (Safe ref#A04) ad libitum. had free access to filtered drinking percolate water.

[0297] Experimental design and conditions Definition of test start In this study, the day of MIA induction was defined as "Day 1" and the end of the study was defined as "Day 11." Ta.

[0298] OA induction by intra-articular (IA) injection of MIA Animals were anesthetized by chamber induction technique using inhalation anesthesia (isoflurane 5%). During the procedure, the animals were maintained under isoflurane at a level of 1.5-3% and an air flow rate of 1-2 liters. The area around the knee joint was wiped with alcohol. MIA was administered in a volume of 0.75 mg. 5 μl of the solution was injected intra-articularly (IA) through the patellar tendon. Using a 30-gauge 0.5-inch needle attached, only a 2-3 mm needle can puncture the joint. After injection, the knee was massaged to ensure uniform distribution of the solution. The mice were injected once on day 1 (groups 2M, 3M, 4M, and 5M). Group 1M (sham control) 5 μl of saline for injection was injected into the knee joint.

[0299] Study Design and Timeline The study was divided into three subgroups according to Table 16 for study design and Table 17 for study timeline. I went with Ikuru. [Table 16]

[0300] Sham mice were randomly assigned to group 1M on day 1. MIA-injected mice were Group allocation was made on day 3 based on BW. [Table 17]

[0301] Testing and Evaluation Animal sacrifice and tissue collection On day 11 (the end date), the mice were euthanized. The knee joint structures (including the synovium) were collected. The samples were rapidly frozen in liquid nitrogen. RNA was extracted using the SV Total RNA Isolation System Kit (Promega). ega) according to the manufacturer's recommendations. PCR analysis was performed. SOX9 is the first transcription factor essential for chondrocyte differentiation and cartilage formation. has been identified as a factor.

[0302] result SOX9 RTqPCR analysis of knee joint structures As shown in Figure 16, the vehicle administered 0.75 mg of MIA to the knee joint on day 1 Mice (Group 2M) had significantly higher SOX9 relative expression on day 11 compared to vehicle sham controls (Group 1M). On day 3, MIA-injected mice were treated with albumin-containing liraglutide. When the mice received intra-articular injection of liraglutide, SOX9 expression was restored, and the expression of SOX9 was significantly higher in group 3M (liraglutide 10 μg). and Group 4M (liraglutide 20 μg) were the same as the sham control Group 1M. In the group 5M receiving 30 μg of methicillin, the relative expression of SOX9 was 5.5 compared with the sham control group 1M. It has increased by 5%.

[0303] conclusion The purpose of the study was to evaluate the efficacy of intra-articular administration of compounded liraglutide in the treatment of monoiodoacetate injection mice. The objective of this study was to perform RTqPCR of SOX9 in the knee joint of rats.

[0304] The monoiodoacetate (MIA) model is a deformable model in both rats and mice. It has become the standard for modeling joint destruction in osteoarthritis (OA). delivered a single injection of MIA into the knee joint and administered glyceraldehyde-3-phosphatase dehydrogenase (Glyceraldehyde-3-phosphatase dehydrogenase). It inhibits glycolysis in chondrocytes by inhibiting glycogenase, and specifically induces chondrocyte death. Chondrocytes differentiated after condensation of mesenchymal stem cells produce collagen and proteoglycans. The transcription factor SOX9 is responsible for the secretion of extracellular matrix molecules. Using this animal model of OA, we found that compared with sham healthy controls, Intra-articular injection of compounded liraglutide restored or increased SOX9 relative expression. On the other hand, we showed that SOX9 expression was decreased after MIA injection.

[0305] Therefore, this study demonstrated that topically administered albumin-combined liraglutide has the potential to improve the Targeting the relevant mechanisms related to anabolism in MIA-induced OA and inflammatory pain models in This indicates that

[0306] Example 7: Relaxation using type II collagenase-induced osteoarthritis model in rats Efficacy study of Lutide alpha 1 acid glycoprotein-based formulation

[0307] The purpose of this study was to utilize a collagenase-induced model of osteoarthritis in rats, To conduct an efficacy study using an alpha-1 acid glycoprotein-based formulation of liraglutide It was.

[0308] material and method compound Type II collagenase: Type II collagenase was dissolved in PBS at a concentration of 20,000 U / ml, and 50 μL of collagenase was added. 0 U was delivered. Treatment formulation: Alpha 1-acid glycoprotein (A1AGP) vehicle was PBS (25%) for intra-articular injection. The suspension consisted of 5% alpha 1-acid glycoprotein resuspended in 10 μl of PBS. -Alpha-1 acid glycoprotein-based formulation of liraglutide: Liraglutide (supplied as a powder) in an appropriate volume of alpha-1 acid glycoprotein vehicle It was dissolved to a dose level of 0.18 mg / kg in 25 μl for intra-articular injection.

[0309] Experimental model species / strain Rat / SD Gender / Number / Age Male / 20 / 6-7 weeks old at the start of the test supplier Janvier Labs, France

[0310] Animal Control Housing Animal care was carried out in accordance with the regulations of the Federation of European Laboratories. ory Animal Science Associations (FELASA) The animals were fed pelleted food and plastic bottles. Plastic cages with stainless steel top grills to facilitate drinking water (cage per Bedding: Steam-cleaned rice husks (Safe) were used. The scrubs were replaced at least once a week along with the scrubs.

[0311] diet Animals were fed commercial rodent chow (Safe ref#A04) ad libitum. had free access to filtered drinking percolate water.

[0312] Experimental design and conditions Definition of test start In this study, the day of OA induction was defined as "day 1" and the end of the study was defined as "day 43." .

[0313] OA induction by intra-articular (IA) injection of type II collagenase Animals were anesthetized by chamber induction technique using inhalation anesthesia (isoflurane 5%). During the procedure, the animals were kept under isoflurane at a level of 1.5-3% and an air flow rate of 1-2 liters. Type II collagenase was administered intra-articularly (IA) in 25 μl containing 500 U. ) were injected. Animals were injected twice: one injection on day 1 and a second injection on day 4.

[0314] Study Design and Timeline The study was conducted according to Table 18 for the study design and Table 19 for the study timeline. Ta.

[0315] [Table 18]

[0316] [Table 19]

[0317] Testing and Evaluation body weight Body weights were recorded on arrival, before the start of the study, and weekly thereafter.

[0318] Animal sacrifice and tissue fixation On day 43 (end of study), all animals were bled. Rats were euthanized. Knee joint structure The contralateral side was harvested and fixed in 4% buffered formalin solution for further histological analysis. The (uninjured) knee was also fixed in 4% buffered formalin solution.

[0319] Histological analysis Rat knees, immersed in buffered 3.7% formalin, were delivered to a subcontractor for histological analysis. Histological analysis was performed in animals blinded to group treatment and protocol during the entire analysis procedure. The knee joint sections were then cut into osteoarthritic sections by a surgeon (DVM, DESV Anatomic Pathology). thritis Cartilage.2010Oct;18Suppl3:S24-3 Scoring was done according to 4.

[0320] statistical analysis Numerical results were presented as mean ± standard deviation (SD). or one-way ANOVA (followed by Dunnett's multiple comparison post-hoc test) or t-test Statistical analysis was performed using the . A probability of 5% (p≦0.05) was considered significant. Results are presented as mean ± SEM, and the degree of statistical significance between groups is * p ≤ 0.05, ** p<0.01 and *** Shown as p<0.001.

[0321] result The results showed that Group 6M (intra-articular administration of liraglutide in A1AGP vehicle) was significantly superior to Group 5M (A1 There was a clear trend towards less cartilage lesions than with AGP vehicle. Joint scores were calculated using the following subsections (de Visser et al., PLoS One. 201 8Apr23;13(4):e0196308) were calculated based on the sum of: cartilage matrix Loss of bone (0-2), cartilage degeneration (0-5), cartilage degeneration (0-4), osteophyte (0-4) , calcified cartilage and subchondral bone damage (0-5), and synovial inflammation (0-4). There was a significant decrease in total joint score in Group 6M compared to Vehicle Group 5M.

[0322] Representative photographs of right knee sections from animals in Groups 5M and 6M are shown in FIG.

[0323] conclusion Histological findings showed that liraglutide IA induced less cartilage loss compared with vehicle. showed a tendency to significantly reduce the total joint score, and local administration of liraglutide This suggests that cartilage protection may be achieved.

[0324] Overall study results showed that liraglutide was well tolerated when administered topically and that this We demonstrated that we targeted relevant mechanisms involved in chondroprotection in a collagenase-induced OA model. is doing.

Claims

1. Use in the treatment of cartilage disorders comprising a glucagon-like peptide-1 analogue as an active ingredient Induce anabolic stimulation of chondrocytes, including chondrocyte proliferation, and / or promote cartilage growth. Pharmaceutical compositions for reducing catabolic activity, including reducing ribosomal loss.

2. The glucagon-like peptide-1 (GLP-1) analog is liraglutide.

2. A pharmaceutical composition for use as described in 1.

3. The use according to claim 2, wherein the concentration of liraglutide is 1 ng / ml to 10 mg / ml. A pharmaceutical composition for

4. The formulation comprises a therapeutically effective amount of a GLP-1 analogue, a non-ionic surfactant, cellulose , polyethers, glucans, glycerophospholipids, polysaccharides, proteins, and combinations thereof and an excipient comprising a polymer selected from the group consisting of a combination of A pharmaceutical composition for use in

5. The use of claim 4, wherein the GLP-1 analog is liraglutide and contains albumin. A pharmaceutical composition for use.

6. The albumin concentration is from about 0.1% to about 10% (wt / wt) of the formulation, preferably The pharmaceutical composition for use according to claim 5, wherein the amount of the active ingredient is 5% (wt / wt).

7. The cartilage disease is a cartilage defect caused by trauma or surgical treatment, osteochondritis dissecans, osteitis, osteoarthritis, congenital cartilage disease and cartilage damage.

7. A pharmaceutical composition for use according to any one of claims 1 to 6.

8. 1. A method for increasing anabolic cytokine secretion or production in chondrocytes in a patient, comprising: A method comprising administering to said patient a composition according to claims 1 to 7.

9. the anabolic cytokine is GMCSF and / or CXCL10 / IP10; The method of claim 8.

10. 1. A method for reducing catabolic cytokine secretion or production in chondrocytes in a patient, comprising: A method comprising administering to said patient a composition according to claims 1 to 7.

11. The catabolic cytokines are selected from the group consisting of MMP3, MMP13, PGE2, IL7, and MCP1.

11. The method of claim 10, wherein the compound is selected from the group consisting of:

12. 12. The method of claim 8, wherein the composition is administered to the subject via intra-articular injection. method.

13. Increases anabolic cytokine secretion or production, and reduces cartilage loss and inflammation by stimulating chondrocyte proliferation. In the manufacture of a medicament for treating cartilage disorders by reducing cartilage damage and / or repair. Use of liraglutide.

14. Cartilage disorders include cartilage defects caused by trauma or surgical procedures, such as osteochondritis dissecans.

13. The method according to claim 12, wherein the cartilage is selected from the group consisting of osteoarthritis, congenital cartilage disease, and cartilage damage. Use as described in.

15. A composition for use in cartilage regeneration comprising a glucagon-like peptide-1 analogue.

16. The glucagon-like peptide-1 analog is selected from the group consisting of exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, semaglutide, or liraglutide.

16. The composition for use in cartilage regeneration according to claim 15,

17. The cartilage regeneration agent according to claim 16, wherein the glucagon-like peptide-1 analog is liraglutide. Composition for use in livestock.

18. 10. The method of claim 1, wherein the concentration of glucagon-like peptide-1 is about 0.1 nM to 625 μM.

8. A composition for use in cartilage regeneration according to claim 7.

19. At least 5% by weight of a pharmaceutically acceptable vehicle used in combination 19. The composition for use in cartilage regeneration according to claim 18, further comprising:

20. The pharmaceutically acceptable formulation vehicle is albumin or alpha-1 acid glycoprotein.

20. The composition for use in cartilage regeneration according to claim 19, selected from the group consisting of: 。

21. The concentration of the pharmaceutically acceptable formulation vehicle is from about 0.1% to about 10% of the formulation. (wt / wt), preferably 5% (wt / wt). A composition for use in

22. The concentration of the pharmaceutically acceptable formulation vehicle is 5% (wt / wt) of the formulation.

22. A composition for use in cartilage regeneration according to claim 21 .

23. 23. The softgel of claim 22, wherein the pharmaceutically acceptable formulation vehicle is albumin. A composition for use in bone regeneration.

24. 2. The method of claim 1, wherein the pharmaceutically acceptable formulation vehicle is alpha-1 acid glycoprotein.

24. A composition for use in cartilage regeneration according to 23.

25. 25. Any one of claims 1 to 24, administered by intra-articular injection to cartilage damage lesions.

2. A composition for use in cartilage regeneration according to claim 1.

26. induces anabolic stimulation of chondrocytes, including chondrocyte proliferation and / or stem cell differentiation into chondrocytes; 26. A composition for use in cartilage regeneration according to any one of claims 1 to 25, 。

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