Compositions and methods for modulating chondrocyte proliferation and increasing cartilage matrix production - Patents.com

Abstract

The present invention relates to novel pharmaceutical compositions comprising GLP-1 and GLP-1 analogues that induce anabolic stimulation of chondrocytes and a reduction in chondrocyte catabolism, including a reduction in cartilage matrix loss and cartilage degeneration, for use in the treatment of cartilage diseases. The compositions according to the invention induce cartilage regeneration and anabolic stimulation of chondrocytes, including chondrocyte proliferation and / or stem cell differentiation into chondrocytes.

Description

[Technical field]

[0001] The present invention relates to novel pharmaceutical compositions comprising GLP-1 and GLP-1 analogues which induce increased chondrocyte regeneration and decreased cartilage degeneration involving anabolic and catabolic cytokine regulation for use in the treatment of cartilage disorders. [Background technology]

[0002] Osteoarthritis (OA) is the most common chronic joint disease. OA affects almost 50% of people over 65 years of age, while younger people develop it following joint injury. OA affects 250 million people worldwide, and the disease has a significant economic and social impact on patients and healthcare systems. OA is a disease of the entire joint, characterized by structural degradation of periarticular bone, synovial joint lining and adjacent supporting connective tissue elements. Destruction of articular cartilage is a result of the inability of chondrocytes to maintain the balance between synthesis and degradation of the extracellular cartilage matrix. Proinflammatory cytokines such as interleukin-1β (IL-1β), produced by macrophages, monocytes, synovial cells and chondrocytes, play a key role in the development of the disease.

[0003] Glucagon-like peptide-1 (GLP-1) is the post-translational product of the preproglucagon gene. The effects of GLP-1 on pancreatic β-cells include increasing glucose transporter 2 expression and secreting insulin in response to increasing glucose levels. In addition, GLP-1 has been shown to reduce the secretion of pro-inflammatory cytokines such as interleukin-6, tumor necrosis factor-α, and interferon-c.

[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 analogues of glucagon-like peptide 1 (GLP-1), their preparation, and their pharmaceutical use. This document relates to certain derivatives and analogues of GLP-1 for use in the prevention and treatment of all forms of diabetes.

[0005] Although osteoarthritis was originally thought to be a disease of the articular cartilage, recent studies have shown that the condition may involve the entire joint.

[0006] Although loss of articular cartilage has been thought to be the primary change, a combination of cellular changes and biomechanical stresses leads to several secondary changes, including subchondral bone remodeling, osteophyte formation, development of bone marrow lesions, changes in the synovium, joint capsule, ligaments and periarticular muscles, and meniscal tears and extrusions.

[0007] There are two patterns in the process of cartilage growth. One is interstitial growth, in which cells differentiate into chondrocytes and proliferate by cell division while surrounded by the cartilage matrix. Each chondrocyte secretes matrix, and then the cartilage tissue expands. The other growth pattern is appositional growth caused by the perichondrium. Cartilage tissue is covered by perichondrium, except for the articular surface of articular cartilage. The scleroperichondrium is composed of fibroblasts, but is similar to the chondrocytes of the inner layer, and the difference between fibroblasts and chondrocytes is unclear. The perichondrium cells of the inner layer proliferate while gradually changing into a circular shape, and such cells further secrete cartilage matrix and grow outward.

[0008] US Patent No. 5,399,663 relates to incretin hormones or analogs thereof for use in the treatment of osteoarthritis. More specifically, this patent discloses the use of GLP-1 and GLP-1 analogs, such as liraglutide, for use in the treatment of osteoarthritis. The peptides disclosed in the US Patent No. 5,399,663 may be administered via any known route of administration, including systemic (parenterally, intravenously, etc.), oral, rectal, topical or subcutaneous, among others. This patent does not disclose the role of GLP-1 in cartilage degradation, nor does it demonstrate a relationship with chondrocytes.

[0009] Normal turnover of cartilage matrix is ​​mediated by chondrocytes, which synthesize these components and the proteolytic enzymes responsible for their degradation. Chondrocytes are influenced by many factors, including polypeptide growth factors and cytokines, structural and physical stimuli, and even components of the matrix itself.

[0010] Osteoarthritis results from the inability of chondrocytes to maintain homeostasis between anabolism and catabolism of these extracellular matrix components. What initiates the imbalance between cartilage degradation and repair is not well known. Trauma causing microfractures or inflammation causing a slight increase in enzyme activity can allow the formation of wear particles, which can then be phagocytosed by resident macrophages. At some point, the production of these wear particles exceeds the system's ability to eliminate them, and they become mediators of inflammation, stimulating chondrocytes to release degradative enzymes. Molecules from the degradation of collagen and proteoglycans, which are also taken up by synovial macrophages, cause the release of proinflammatory 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) can induce the production of interleukin-6 (IL-6) and interleukin-8 (IL-8) by synovial and chondrocytes. However, all studies focused on synovial tissue and not on chondrocytes.

[0011] Anabolic stimulation of chondrocytes is measured in vitro by stimulation of proteoglycan and collagen synthesis. Cytokines such as GM-CSF, granulocyte-macrophage colony-stimulating factor (Non-Patent Document 1), and CXCL10 / IP10 (Non-Patent Document 2) have been demonstrated to stimulate anabolic processes in chondrocytes.

[0012] There is a need to enhance chondrocyte anabolic activity, including chondrocyte proliferation, and reduce chondrocyte catabolic activity, including cartilage matrix degradation, in OA. Such improved compositions and methods would be of great interest in developing new therapeutic strategies in the treatment of osteoarthritis.

[0013] Also, it is necessary to enhance chondrocyte anabolic activity, including chondrocyte differentiation, to promote cartilage regeneration and reduce cartilage destruction, respectively. Such improved compositions and methods will be of great interest in the development of new therapeutic strategies in the treatment of osteoarthritis. [Prior art documents] [Patent documents]

[0014] [Patent Document 1] International Publication WO2017 / 149070 Brochure [Patent Document 2] European Patent EP2890390B1 [Non-patent literature]

[0015] [Non-Patent Document 1] Quinetro et al., 2008 Cytokines 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 discloses improved pharmaceutical formulations for use in the treatment of cartilage diseases, which induce enhanced chondrocyte anabolic processes, including chondrocyte proliferation, and reduced catabolic processes, including reduced cartilage matrix loss and cartilage degradation.

[0017] The present invention further discloses improved pharmaceutical formulations and compositions for use in the treatment of cartilage diseases, which induce enhanced chondrocyte anabolic processes, including chondrocyte differentiation for cartilage regeneration, and reduced catabolic processes, including reduced cartilage matrix loss and cartilage degradation.

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

[0019] The present invention relates to pharmaceutical compositions for use in the treatment of cartilage disorders that induce anabolic stimulation of chondrocytes, including chondrocyte proliferation, and reduce catabolic activity, including reduction in cartilage matrix loss and cartilage degeneration.

[0020] According to a particular aspect, the present invention relates to a pharmaceutical composition for use in the treatment of cartilage diseases, inducing anabolic stimulation of chondrocytes, including chondrocyte proliferation and / or stem cell differentiation into chondrocytes for cartilage regeneration, as well as reducing catabolic activity, including reducing cartilage matrix loss and cartilage degeneration.

[0021] According to a particular embodiment, the present invention relates to a pharmaceutical composition for inducing anabolic stimulation of chondrocytes, comprising as active ingredient a glucagon-like peptide-1 analogue, for use in the treatment of cartilage diseases.

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

[0023] According to one aspect of the present invention there is provided a pharmaceutical composition comprising a glucagon-like peptide-1 analogue as an active ingredient for use in the treatment of cartilage diseases, which induces anabolic stimulation of chondrocytes, including chondrocyte proliferation, and / or reduces catabolic activity, including reduced cartilage matrix loss.

[0024] According to another aspect of the invention, the glucagon-like peptide-1 (GLP-1) analogue is liraglutide.

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

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

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

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

[0029] Pharmaceutical formulations according to the invention may also contain one or more pharma- ceutically acceptable carriers / excipients.

[0030] The present invention is not limited to gel formulations, but liquid and semi-solid pharmaceutical forms suitable for topical administration are preferred, such as liquids, solutions, creams, gels or transdermal patches, and in particular forms suitable for intra-articular injection, such as liquids, solutions, and semi-solid forms such as creams or gels, and transdermal patches. Pharmaceutical forms may also consist of forms in which some or all of the ingredients are in a dry form, possibly lyophilized, to be reconstituted with an aqueous solution or other suitable vehicle before use.

[0031] The formulations may be produced by methods well known in the state of the art using known excipients such as binders, disintegrants, fillers, stabilizers, diluents and colorants, etc. They may also include delayed or sustained release forms made of suitable polymers known in the pharmaceutical art.

[0032] Pharmaceutically acceptable carriers / excipients, such as solvents, preservatives, such as antioxidants and / or chelating agents and antibacterial agents, isotonicity adjusting agents, and buffer systems, are preferred for preparing liquid forms suitable for injectable use.

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

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

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

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

[0037] In preparing a liquid form suitable for nebulization, a pharma- ceutically acceptable vehicle / excipient is preferred as the solvent, includes preservatives such as antioxidants and / or chelating agents and antibacterial agents, tonicity adjusting agents and a buffer system.

[0038] According to still further features in the described preferred embodiments of the pharmaceutical composition, the GLP-1 analog is liraglutide and the gel comprises albumin.

[0039] According to still further features in the described preferred embodiments of the pharmaceutical composition, the GLP-1 analog is liraglutide and comprises albumin.

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

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

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

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

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

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

[0046] According to yet another aspect of the present invention there is provided a method of increasing anabolic cytokine secretion or production in chondrocytes in a patient comprising administering to the patient a composition according to the present invention.

[0047] According to further features in the preferred embodiments the anabolic cytokine is GMCSF and / or CXCL10 / IP10.

[0048] According to yet another aspect of the invention there is provided a method of reducing catabolic cytokine secretion or production in chondrocytes in a patient comprising administering to the patient a composition according to the invention.

[0049] According to further features in the preferred embodiments the catabolic cytokine is selected from the group consisting of MMP3, MMP13, PGE2, IL7, MCP1.

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

[0051] According to a further aspect of the present invention there is provided a use of liraglutide for treating cartilage diseases by increasing anabolic cytokine secretion or production and reducing cartilage loss and / or repair by stimulating chondrocyte proliferation.

[0052] According to a further aspect of the present invention there is provided a use of liraglutide for treating cartilage diseases by increasing anabolic cytokine secretion or production and reducing cartilage loss and / or regeneration by stimulating chondrocyte proliferation.

[0053] According to a particular aspect of the present invention there is provided the use of liraglutide in the manufacture of a medicament for treating a cartilage disease by increasing chondrocyte anabolic function.

[0054] The cartilage disease is selected from the group consisting of cartilage defects caused by trauma or surgical procedures, osteochondritis dissecans, osteoarthritis, congenital cartilage diseases and cartilage injuries.

[0055] According to a further aspect of the present invention there is provided a use of liraglutide for treating cartilage diseases by decreasing catabolic cytokine secretion or production and reducing cartilage loss and / or repair through stimulation of chondrocyte proliferation.

[0056] According to a further aspect of the present invention there is provided a use of liraglutide for treating cartilage diseases by reducing catabolic cytokine secretion or production and reducing cartilage loss and / or regeneration through stimulation of chondrocyte proliferation.

[0057] The cartilage disease is selected from the group consisting of cartilage defects caused by trauma or surgical procedures, osteochondritis dissecans, osteoarthritis, congenital cartilage diseases and cartilage injuries.

[0058] According to yet another aspect of the present invention there is provided a method of promoting chondrocyte proliferation comprising contacting at least one chondrocyte with the composition according to the present invention. According to further features in the preferred embodiments the cell is a mammalian cell. According to further features in the preferred embodiments the cell is a human cell.

[0059] According to yet another aspect of the invention there is provided a method of treating a patient diagnosed with or at risk of developing an immunoinflammatory disorder comprising administering to the patient a composition according to the invention.

[0060] According to yet another aspect of the present invention, there is provided a method of treating an inflammatory condition in a subject, comprising administering to the subject a composition according to the present invention. According to further features of preferred embodiments, the inflammatory condition is an inflammatory condition occurring in the joints and joint cavities, as well as cartilage matrix degeneration and osteoarthritis. According to further features of preferred embodiments of the method according to the present invention, the composition is administered to the subject via intra-articular injection.

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

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

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

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

[0065] According to yet another aspect of the present invention there is provided the use of liraglutide as an active ingredient in the manufacture of a pharmaceutical formulation for alleviating or reducing joint irritation or reducing the worsening of existing joint inflammation in a mammalian subject.

[0066] According to yet another aspect of the present invention, there is provided the use of liraglutide as an active ingredient for use in a method for alleviating or reducing joint irritation or reducing the worsening of existing joint inflammation in a mammalian subject, wherein the formulation is administered via intra-articular injection into the fat pad of the joint.

[0067] According to yet another aspect of the present invention, there is provided the use of liraglutide as an active ingredient in the manufacture of an injectable pharmaceutical formulation for alleviating or reducing joint irritation or reducing the worsening of existing joint inflammation in a mammalian subject, wherein the liraglutide is formulated together with at least a second therapeutic agent, the second therapeutic agent comprising an anti-inflammatory agent, an antioxidant, a vitamin, a polyol or a combination thereof.

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

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

[0070] According to yet another aspect of the present invention, there is provided a method for differentiating mesenchymal stem cells into chondrocytes, comprising the steps of: a) adding a composition according to the present invention to a cell culture medium containing mesenchymal stem cells; and 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, comprising the steps of: 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 invention, the glucagon-like peptide-1 (GLP-1) analogue is liraglutide.

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

[0073] According to yet another embodiment of the invention, the cell culture medium further contains MesenPRO RS growth supplements and 1% L-glutamine.

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

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

[0076] According to yet another aspect of the present invention, there is provided a use of SOX9 expression enhancing peptide in the manufacture of a medicament comprising SOX9 expression enhancing peptide for treating or preventing inflammation, wherein the SOX9 expression enhancing peptide is a GLP-1 analogue that selectively targets SOX9 gene. According to another aspect of the present invention, the GLP-1 analogue is liraglutide.

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

[0078] According to yet another aspect of the present invention, there is provided a pharmaceutical composition for use in treating or preventing osteoarthritis, comprising a pharma- ceutical acceptable carrier and a SOX9 expression enhancing peptide, the SOX9 expression enhancing peptide being a GLP-1 analogue that selectively targets the SOX9 gene. In a preferred embodiment, the SOX9 expression enhancing peptide is liraglutide that selectively targets the SOX9 gene. In another preferred embodiment of the present invention, 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 treating or preventing inflammation, the pharmaceutical composition comprising a pharma- ceutically acceptable carrier and a SOX9 expression enhancing peptide, the SOX9 expression enhancing peptide being a GLP-1 analogue that selectively targets the SOX9 gene. In a preferred embodiment, the SOX9 expression enhancing peptide is liraglutide that selectively targets the SOX9 gene. In another preferred embodiment of the present invention, the concentration of liraglutide is 1 ng / ml to 10 mg / ml.

[0080] According to yet another aspect of the present invention, a pharmaceutical composition for use in treating or preventing osteoarthritis, comprising a pharma- ceutical composition comprising a pharma- ceutical acceptable carrier and a SOX9 expression enhancing peptide according to the present invention, provides a therapeutically effective amount of liraglutide and a gel comprising a polymer selected from the group consisting of non-ionic surfactants, cellulose, polyether, glucan, glycerophospholipids, polysaccharides, proteins, and combinations thereof. In a preferred embodiment, the gel comprises albumin, and the albumin concentration is about 0.1% to about 10% (wt / wt) of the formulation, preferably 5% (wt / wt).

[0081] In another preferred embodiment, the gel comprises alpha 1 acid glycoprotein, the alpha 1 acid glycoprotein concentration being from 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, a pharmaceutical composition for use in treating or preventing inflammation, comprising a pharma- ceutical acceptable carrier and a SOX9 expression enhancing peptide according to the present invention, provides a therapeutically effective amount of liraglutide and a gel comprising a polymer selected from the group consisting of non-ionic surfactants, cellulose, polyether, glucan, glycerophospholipids, polysaccharides, proteins, and combinations thereof. In a preferred embodiment, the gel comprises albumin, the albumin concentration being about 0.1% to about 10% (wt / wt) of the formulation, preferably 5% (wt / wt).

[0083] According to another aspect of the invention there is provided a composition for use in cartilage regeneration comprising a glucagon-like peptide-1 analogue.

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

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

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

[0087] According to yet another embodiment, the composition for use in cartilage regeneration further comprises at least 5% or more by weight of a pharma- ceutically acceptable formulation vehicle used in combination.

[0088] According to yet another embodiment of the composition for use in cartilage regeneration, the composition for use in cartilage regeneration, the pharma- ceutically acceptable formulation vehicle is selected from the group consisting of albumin or alpha-1 acid glycoprotein.

[0089] According to yet another embodiment of the composition for use in cartilage regeneration, the concentration of the pharma- ceutically acceptable formulation vehicle is from about 0.1% to about 10% (wt / wt) of the formulation, preferably 5% (wt / wt).

[0090] According to yet another embodiment of the composition for use in cartilage regeneration, the concentration of the pharma- ceutically acceptable formulation vehicle is 5% (wt / wt) of the formulation.

[0091]

[0092] According to yet another embodiment of the composition for use in cartilage regeneration, the pharma- ceutically acceptable formulation vehicle is albumin.

[0093] According to yet another embodiment of the composition for use in cartilage regeneration, the pharma- ceutically acceptable formulation vehicle is alpha-1 acid glycoprotein.

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

[0095] According to yet another aspect of the invention, the composition for use in cartilage regeneration is administered by intra-articular injection to the cartilage injury.

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

[0097] The present invention has utility wherever stimulation of chondrocyte proliferation or growth or chondrocyte formation from mesenchymal stem cells is deemed desirable, including cartilage repair.

[0098] Thus, the present invention has utility in any application in which stimulation of chondrocyte proliferation or growth is deemed desirable, including cartilage repair and / or regeneration.

[0099] The applicants have found that increasing the effective concentration of liraglutide on chondrocytes of OA patients has the effect of stimulating chondrocyte anabolic cytokines and decreasing catabolic cytokines.

[0100] The inventors have demonstrated that the use of intra-articular injections (acute or repeated) of a pharmaceutical composition formulation and sustained release of liraglutide into the synovial fluid reduces and slows the fibrotic process induced after cartilage damage, as well as induces actual functional and histological improvement of the joint after joint damage.

[0101] Furthermore, it was demonstrated that a specific dosing regimen (ie, several injections each spaced one week apart) was necessary to result in chondrocyte proliferation without fibrosis.

[0102] According to the present invention, the term regeneration includes chondrocyte anabolic function / proliferation in cartilage in the absence of fibrosis resulting in functional and histological improvement of the joint.

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

[0104] According to its main aspect, broadly stated, the present invention provides a pharmaceutical composition comprising a glucagon-like peptide-1 analogue as an active ingredient, for use in the treatment of cartilage diseases selected from the group consisting of cartilage defects caused by trauma or surgical procedures, osteochondritis dissecans, osteoarthritis, congenital cartilage diseases and cartilage injuries, which enhances chondrocyte proliferation, increases cartilage repair and induces loss of cartilage matrix.

[0105] According to another main aspect, broadly stated, the present invention provides a pharmaceutical composition comprising a glucagon-like peptide-1 analogue as an active ingredient, for use in treating cartilage diseases selected from the group consisting of cartilage defects caused by trauma or surgical procedures, osteochondritis dissecans, osteoarthritis, congenital cartilage diseases and cartilage injuries, which induces enhanced chondrocyte proliferation, increased cartilage regeneration and reduced cartilage matrix loss. [Brief description of the drawings]

[0106] [Figure 1] FIG. 1 shows the calculated mean concentration differential expression levels of secreted cytokines in chondrocytes of OA patients following treatment with several doses of Victoza®. [Diagram 2] FIG. 1 shows the release profiles of formulations 6, 8, 14, 17, 19, and 20. [Diagram 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. [Diagram 5] FIG. 1 shows dose response of compounded liraglutide IA on endpoint knee measurements. [Figure 6] 13 is a representative photograph of right knee sections stained with hematoxylin and eosin showing fibrous synovial chronic proliferation and tibial plate fibrosis, specifically in animals injected subcutaneously with Victoza®. [Figure 7] 13 is a representative photograph of a right knee section stained with hematoxylin and eosin showing the absence of fibrosis and chondrocyte nests in formulated 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]13 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 a medial capsule repair. [Figure 12] 13 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. 13 shows SOX9 RTqPCR analysis on knee joint structures of monoiodoacetate-injected mice treated with liraglutide. [Figure 17] FIG. 13 shows total joint scores (histology) of the injected knee of animals from Group 5M treated with A1AGP vehicle and Group 6M treated with A1AGP combined with 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 combined with A1AGP (B). Detailed Description of the Invention

[0107] One embodiment of the present invention comprises a specific composition for inducing enhanced chondrocyte differentiation and proliferation and increased cartilage matrix production for use in the treatment of cartilage diseases and osteoarthritis. Such compositions may be administered directly to the affected joint, preferably by direct injection into the closed space of the joint (intra-articular injection).

[0108] Before describing the present invention in detail, it is to be understood that, unless otherwise specified, this invention is not limited to particular materials or processes, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0109] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element, or "a protein" means more than one protein.

[0110] The term "about" as used herein means approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated numerical values. In general, the term "about" is used herein to modify numerical values ​​above and below the stated value by a variance of 10%. Thus, about 50% means within a range of 45% to 55%. Numerical ranges recited herein by endpoints include all numbers and fractions subsumed within the range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It should also be understood that all such numbers and fractions are presumed to be modified by the term "about".

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

[0112] The terms "cartilage" or "articular cartilage" or "cartilage matrix" refer to the elastic, translucent connective tissue of mammals, including humans and other species. Cartilage is composed primarily of chondrocytes, type II collagen, small amounts of other collagen types, other non-collagenous proteins, proteoglycans, and water, and is usually surrounded by a perichondrium composed of fibroblasts in a matrix of collagen types I and II and other proteoglycans. Most cartilage matures into bone, but some cartilage remains in its original form in locations such as the nose, ears, knees, and other joints. Cartilage has no blood or nerve supply, and chondrocytes are the only cell type in this tissue.

[0113] The terms "active agent," "active excipient," "active ingredient," and "pharmacologically active excipient" are used interchangeably herein to refer to chemical compounds or compounds that induce a desired pharmacological or physiological effect, and include therapeutically active and prophylactically active agents. These terms also encompass pharmacologic acceptable, pharmacologically active derivatives and analogs of the active agents specifically mentioned herein, including, but not limited to, salts, esters, amides, prodrugs, active metabolites, inclusion complexes, analogs, and the like.

[0114] The term "effective amount" or "therapeutically effective amount" of a pharmacologically active agent or active excipient is intended to mean a non-toxic but sufficient amount of the agent or excipient to provide a desired therapeutic effect. The "effective" amount varies from subject to subject. Thus, it is not always possible to specify an exact "effective amount". However, an appropriate "effective" amount in any individual case can be determined by one of ordinary skill in the art using routine experimentation. Moreover, the exact "effective" amount of the active agent incorporated into the composition or dosage form of the present invention is not critical, so long as it is within a range of concentrations sufficient to allow easy application of the formulation to deliver an amount of the active agent within the therapeutically effective range.

[0115] Preferred routes of administration include topical or local application, including locally, to the site of damaged cartilage or to a site of (surgical) intervention on or near the cartilage, preferably in fluid form, in a hydrogel or collagen matrix or artificial scaffold (matrix).

[0116] The present invention also relates to pharmaceutical compositions comprising the compounds of the present invention.More specifically, such compounds can be formulated into pharmaceutical compositions using standard pharma- ceutically acceptable carriers, fillers, solubilizers and stabilizers known to those skilled in the art.

[0117] The present invention encompasses the preparation and use of pharmaceutical compositions that contain the compounds useful for treating diseases disclosed herein as active ingredients.Such pharmaceutical compositions may consist of the active ingredient alone in a form suitable for administration to a subject, or the pharmaceutical compositions may include the active ingredient and one or more pharma- ceutically acceptable carriers, one or more additional ingredients, or any combination thereof.

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

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

[0120] The relative amounts of the active ingredient, pharma- ceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the present invention will vary depending on the identity, size, and condition of the subject being treated, as well as on the route by which the composition is administered.

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

[0122] As used herein, "additional ingredients" include, but are not limited to, one or more of the following: 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 solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifiers; antioxidants; antibiotics; antifungal agents; stabilizers; and pharma-ceutically acceptable polymeric or hydrophobic materials.

[0123] The composition may also comprise one or more substances used in the treatment of osteoarthritis, in particular one or more inhibitors of the dipeptidyl peptidase IV enzyme, preferably selected from the group consisting of sitagliptin, saxagliptin, vildagliptin, alogliptin and linagliptin, or analgesics, non-steroidal anti-inflammatory agents, steroidal anti-inflammatory agents and slow acting anti-arthritic agents, analgesics including paracetamol; acetylsalicylic acid, lysine acetylsalicylate, phenylbutazone, sulindac, diclofenac potassium or sodium, aceclofenac, tiaprofenic acid, ibuprofen, ketoprofen, alminoprofen, fenoprofen, ceclofenac ... They may also include other substances such as profen, naproxen, flurbiprofen, indomethacin, mefenamic acid, niflumic acid, tenoxicam, meloxicam, piroxicam, and selective cyclooxygenase-2 inhibitors such as celecoxib and etoricoxib, betamethasone, dexamethasone, prednisolone, prednisone, tixocortol or triamcinolone; chondroitin, chondroitin sulfate (Structum, Chondrosulf), glucosamine or glucosamine sulfate, diacerein (Art50, Zondar), or unsaponifiable extracts of avocado and soybean (piascledine).

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

[0125] The formulation of the pharmaceutical composition described herein can be prepared by any method known or hereafter developed in the field of pharmacology.In general, such preparation methods include the step of bringing the active ingredient into association with the carrier or one or more other accessory ingredients, and then, if necessary or desired, shaping or packaging the product into desired single or multiple dosage units.

[0126] Such pharmaceutical compositions are generally suitable for administration to any kind of animal, as will be understood by those skilled in the art. In a preferred embodiment, the subject or patient to be treated is an animal, preferably a mammal. According to one embodiment, the subject to be treated is an animal selected from the group consisting of dogs, cats, horses, cows, sheep, pigs and non-human primates.

[0127] According to one preferred embodiment, the subject to be treated is a human being, preferably an adult, particularly preferably an adult over 50 years of age.

[0128] The composition according to the present invention can be administered via any known administration route, including in particular systemically (parenterally, intravenously, etc.), orally, rectally, topically or subcutaneously.According to one preferred embodiment, the composition can also be administered by intra-articular injection, preferably into arthritic joints.In this case, it can be administered in combination with other local agents such as hyaluronic acid, albumin, alpha-1 glycoprotein, analgesic substances, etc.

[0129] The compound may be administered to the animal as frequently as several times per day, or less frequently, for example, once per day, once per week, once every two weeks, once per month, or even less frequently, for example, once every few months, or even less than once per year.

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

[0131] A pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses.

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

[0133] The present invention also relates to methods of administering the compounds of the present invention to a subject, hi one embodiment, the present invention provides a method of treating a subject by administering a compound identified using the methods of the present invention.

[0134] As used herein, the term "treatment" or "therapy" refers to any action that can reduce, inhibit or delay symptoms associated with a pathological condition. It includes both curative and preventive treatment of a disease. Curative treatment is defined by treatment that brings about a cure or that relieves, improves and / or eliminates, reduces and / or stabilizes the symptoms of a disease or the burden it causes. Preventive treatment includes both treatment that brings about the prevention of a disease and treatment that reduces and / or delays the incidence of a disease or the risk of it occurring.

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

[0136] The term "therapeutically effective dose" as used herein refers to the amount required to observe therapeutic or preventive activity against osteoarthritis, particularly the amount required to observe inhibition or suppression of arthritis cartilage destruction.The amount of peptide administered and the duration of treatment are evaluated by those skilled in the art according to the physiological condition of the subject to be treated, the nature of the joint to be treated.

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

[0138] The present invention also relates to the use of liraglutide as an active ingredient in the manufacture of a pharmaceutical formulation for alleviating or reducing joint irritation or reducing the worsening of existing joint inflammation in a mammalian subject.

[0139] The present invention also relates to a method for increasing chondrocyte proliferation in a patient, comprising administering to the patient a therapeutically effective dose of a composition consisting of 1 ng / ml to 10 mg / ml of liraglutide and 0.1% to 10% albumin.

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

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

[0142] Example 1: Effect of VICTOZA® on cytokine release in chondrocytes from OA patients

[0143] This study aimed to evaluate the effect of Victoza®, a GLP-1 analogue, on the release of inflammatory regulators from IL-1β-stimulated human chondrocytes isolated from cartilage from osteoarthritis patients.

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

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

[0146] These solutions were prepared for each patient and diluted 1:1000 in culture medium (4 μl in 4 ml) to final concentrations. The vehicle consisted of 4 μl of sterile water in 4 ml of culture medium.

[0147] Liberase solution formulation: A stock solution of 26 U / ml of Liberase was prepared in DMEM containing 1% P / S and 2% glutamine and stored at -20°C. For the two first steps of the digestion, a 0.52 U / ml solution was extemporaneously prepared in DMEM. For the last step of the digestion, a 0.13 U / ml solution was prepared by diluting the 0.52 U / ml solution 1:4.

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

[0149] Experimental design and conditions Cartilage was isolated from four patients with osteoarthritis undergoing prosthetic knee surgery at Saint Anthony Hospital. Chondrocyte isolation, seeding, culture and activation as well as sample preparation were performed. 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: Cartilage was isolated from osteoarthritis patients undergoing total knee replacement surgery. Cartilage from one patient was processed at a time. Freshly isolated cartilage was cut into pieces 2-3 mm in diameter, placed in a 50 ml tube and rinsed with PBS. The cartilage pieces were incubated in 40 ml of Liberase at 0.52 U / ml in DMEM for 45 min. After 45 min, Liberase was removed and fresh Liberase solution at 0.52 U / ml was added for 45 min. The solution was then removed and the cartilage pieces were incubated overnight in 40 ml of Liberase (0.13 U / ml).

[0152] Chondrocyte seeding: 16 hours after chondrocyte isolation, the solution was moved up and down with a pipette to homogenize the cells. The solution was then filtered through a 100 μm cell strainer. The filtered solution was centrifuged at 1600 rpm for 6 min at room temperature. The pellet was resuspended in 15 ml of complete medium (DMEM + 15% FBS + 2% glutamine + 1% P / S). The cells were counted by hemocytometer and seeded at a density of 200000–250000 cells / well in 12-well culture plates. The cultures were incubated under sterile conditions (37 °C, 5% CO 2 ).

[0153] Chondrocyte culture: 48 hours after seeding, the medium was replaced with fresh medium. Thereafter, the medium was renewed every 2 days until confluence (days 12-13). At confluence, 24 hours prior to treatment, the medium was replaced with medium containing 0.1% BSA without FBS.

[0154] The next day, chondrocytes were preincubated with five doses of Victoza® (5 nM, 25 nM, 50 nM, 125 nM, 625 nM) or vehicle for 2 hours according to the study design (Table 1) and schedule (Table 2) and then stimulated with IL-1β (5 ng / ml) 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 study, the medium was collected, centrifuged, and the supernatant was frozen. Samples were shipped on dry ice within the testing facility and stored at -80°C upon receipt until analysis.

[0159] Detection assays PGE2 assay: The assay was based on the competition between free PGE2 and a PGE2-acetylcholinesterase conjugate (PGE2 tracer) for a limited amount of PGE2 monoclonal antibody. The concentration of the PGE2 tracer was kept constant, while the free PGE2 was varied in each sample. The amount of PGE2 tracer bound to the monoclonal antibody was inversely proportional to the amount of free PGE2 in the sample. Because the enzymatic reaction involved an acetylcholinesterase substrate, the color was less intense in the PGE2-high sample and very intense in the PGE2-low sample.

[0160] Concentrations were calculated after determination of %B / B0 according to the manufacturer's instructions, where B0 represents the absorbance obtained from reading the well with the maximum amount of PGE2 tracer bound (in the absence of free PGE2) and B represents the absorbance obtained for each standard or sample well. To obtain accurate results, supernatants from IL-1β-treated wells were diluted 1:1000 and others 1:500 before proceeding with the test. The measurement range of PGE2 was 7.8-1000 pg / ml.

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

[0162] MMP13 assay: The assay was based on a classical sandwich ELISA combined with colorimetric biotin-streptavidin-based detection. To obtain accurate results, supernatants from IL-1β-treated wells were diluted 1:100 and others 1:10 before proceeding with the test. The measurement range for MMP13 was 8.23-6000 pg / ml.

[0163] Multiplex assay: Luminex technology is based on the combination of an ELISA sandwich technique with a mixture of fluorescent polystyrene beads that represent the solid phase for detection. Each bead is coupled with a specific antibody and a different fluorescent dye. This assay allows the quantification of various cytokines in the same sample with minimal volume utilization. The Human Cytokine Magnetic 30-plex from Life Technologies was used to quantify EGF, Eotaxin, FGF basic, GCSF, GMCSF, HGF, IFN-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.

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

[0165] At the end of incubation, the culture medium was collected and centrifuged, and then the supernatant was subjected to ELISA (MMP3 ELISA kit, MMP13 ELISA kit, PGE2 ELISA kit and cytokine 30-plex panel assay) assay.

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

[0167] The basal inflammatory profile of each patient was assessed by accumulating the concentration means calculated for each cytokine detected.

[0168] The results show that the basal inflammatory profile was highly variable, with two patients having high levels of inflammatory cytokines and two patients having low levels of cytokines.

[0169] The Victoza® response inflammatory profile of each patient was evaluated by comparing the ratio of the mean calculated concentrations of each cytokine before and after Victoza® treatment. The general results show different responses from patient to patient. However, a comparable response to Victoza® emerged for four cytokines in three of four patients' chondrocytes treated with Victoza®, with IL-1β increasing in the mean calculated concentrations of GMCSF and CXCL10 / IP-10 (anabolic cytokines), whereas IL7, MCP1 (catabolic cytokines) decreased (Figure 1).

[0170] conclusion We demonstrated a different response profile in each of the four patients tested: liraglutide increased some anabolic cytokine secretion and decreased some catabolic cytokine secretion in chondrocytes from OA patients.

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

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

[0173] material and method Test material: Liraglutide.

[0174] Materials for Hydrogel Formulation Dextran 70EP (70kDa), 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, H 22% (literature value) in O (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 compound Sodium Hyaluronate 1.9MDa, 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 system for determining release profiles Semipermeable membrane used: dialysis tubing Visking, cellulose, thickness 0.023 mm, MWCO 12-14 kDa Liraglutide concentration measurement ELISA kit

[0177] Artificial synovial fluid formulation: To formulate the artificial synovial fluid, 3.5 mg of sodium hyaluronate, 9 mg of albumin and 3.5 mg of γ-globulin were resuspended in PBS pH 7.4 per ml. The volume of artificial synovial fluid prepared was 10 ml for each experiment.

[0178] The composition of the artificial synovial fluid was selected based on many sources, e.g. Biological Performance of Materials: Fundamentals of Biocompatibility. 4th ed., Jonathan Black, CRC Press, 20gru2005; Synovial Fluid Composition and Functions. Dr Arun Pal Singh, http: / / boneandspine.com / synovial-fluid / ; Concentration of Hyaluronic Acid in Synovial Fluid. Barry Decker et al., Clinical Chemistry 1959, 5(5):465-469.

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

[0180] Testing Procedure Determination of release profile: For each formulation prepared, the release profile was determined. On day 0, liraglutide hydrogel formulations (1mg / ml-1.02ml) were placed in a semipermeable membrane test system immersed in 10ml of artificial synovial fluid. The released molecules passed through the membrane into the artificial synovial fluid at 37°C. This membrane should allow free diffusion of liraglutide monomers, but is a physical barrier for liraglutide oligomers and hydrogel formulations. Samples of artificial synovial fluid (0.2ml each) were collected after 1, 2, 4, 7, 10 and 14 days. The collected samples were replaced with fresh artificial synovial fluid to maintain the same volume of fluid outside the membrane. The samples of artificial synovial fluid were stored at 2-8°C until the end of the study for further ELISA analysis to determine liraglutide concentrations.

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

[0182] ELISA procedure: Quantification of liraglutide concentrations was performed with an ELISA kit. The ELISA procedure was performed according to the kit provider's recommendations with one exception. The calibration standards used had higher concentrations than suggested and were in the range of 0.977-1000ng / ml. Samples were diluted in EIA buffer. Standards and controls were prepared from one stock solution of liraglutide. The analysis was performed on several EIA plates. In all plates, the same standard and control solutions were used. The expected concentrations of liraglutide in the samples after dilution were in the range of 5-227ng / ml. Three quality controls were used, with concentrations covering the expected concentration range of the samples: 15, 100 and 200ng / ml. Each point was measured in duplicate. The absorbance was measured at 450nM by 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 study was considered as "day 14".

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

[0185] standard formulations (1, 2, 3, 4, 5, 7, 8, 10, 11, 12, 13, 14, 15, 16), potential active excipient formulations (17, 18, 19, 20) and albumin-based formulations 6, 9).

[0186] [Table 3]

[0187] [Table 4]

[0188] result

[0189] The analysis of liraglutide was performed accurately and with reliable results. The ELISA raw data including the calibration curve are shown in Appendix II. In all plates, two out of three quality controls met the acceptance criteria, i.e., fell within ±20% of the theoretical concentration. The acceptance criteria were based on the EMA's Guideline on bioanalytical method validation (EMEA / CHMP / EWP / 192217 / 2009Rev.1Corr.2 ** ) was accepted.

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

[0191] Release profile determination: Results were calculated as a percentage of the maximum expected liraglutide concentration for all hydrogel formulations. The amount of liraglutide obtained from the analytical sample experiments was not included in the calculation. The expected liraglutide concentration in the artificial synovial fluid (if all liraglutide was released from the semipermeable membrane) 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] Experiments carried out with the aim of determining the release profiles of 20 different formulations containing liraglutide showed variability between the formulations tested. From the original 20 test formulations, six resulted in a liraglutide concentration of greater than 1% after 10 days. These are formulations 6, 8, 14, 17, 19 and 20, with the release profile shown in Figure 2.

[0195] Example 3: Efficacy study of three liraglutide-based formulations using a surgically induced model of osteoarthritis in rats

[0196] The objective of this study was to evaluate the efficacy of three liraglutide-based formulations utilizing a surgically induced model of osteoarthritis in rats.

[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] Formulations were prepared on the day of treatment for each of the three cycles. For each formulation, 4 mg of liraglutide was dissolved in 2 ml of formulation solution or PBS (for unformulated liraglutide) to a dose level of 0.18 mg / kg in 25 μl for intra-articular injection. Assuming an average BW of 280 g, the dose administered to each rat was 50 μg. Liraglutide was used to dose the animals within 1 hour of preparation.

[0201] Experimental model Animal species / strain: Rat / Sprague Dawley (SD) Sex / number / weight average: Male / 60 / 6-8 weeks old at start of 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] Induction of OA by medial ligament transection (MLT) procedure followed by resection of the medial meniscus (MMx) Anesthesia was induced in each rat by a chamber induction technique using inhalation anesthesia (isoflurane 4.0%). During surgery, the animals were maintained with isoflurane at a level of 1.5-2.5% with an oxygen flow rate of 1-2 liters / min. Ophthalmic ointment was applied to the eyes to prevent tissue drying during the anesthesia period. After induction of anesthesia, the hair on the right leg skin surface was clipped using electric animal clippers. After shaving the knee joint, the skin was disinfected with iodine and a parapatellar skin incision was made medial to the joint. An incision was made medial to the joint cavity. 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 using 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 Weight-bearing changes in OA rats were measured using an incapacity tester. Postural imbalance, which has been reported to indicate changes in limb pain threshold and weight distribution, is reduced. Each rat was placed so that each hind paw rested on a separate force plate on the incapacity apparatus, and the weight supported by each hind paw was measured for 5 seconds. The ratio of the weight of the right hind paw to the left hind paw was calculated. The average value of five consecutive measurements for each rat was recorded. Weight-bearing function (incapacity test) was performed a total of four times at baseline (day 1), days 14, 28, and 35. The experimenter was blinded to the groups.

[0207] On day 36, rats were subjected to CO 2The animals were sacrificed by asphyxiation. The knee joint structures were fixed in 4% buffered formalin solution for further histological analysis. The contralateral (uninjured) knee was also fixed in 4% buffered formalin solution.

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

[0209] result Weight Load Test Weight bearing changes in rats with OA were assessed using an incapacity meter, which measures the weight that the animal distributes to each hind paw independently. All animals prior to OA induction distributed weight equally to both hind paws. On day 14, a significant increase in weight bearing difference (R / L weight percent) was observed between the control group and group 3M, accompanied by a prolonged release of liraglutide (Figure 3).

[0210] Histological evaluation Slides were reviewed by a single pathologist blinded to the treatment groups. Knee cross sections were evaluated for the following parameters: Cartilage matrix loss range (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 reached, 50% is the intermediate region). This 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). Synovial response (score 0-4, see section 9.4). Inner Capsular Repair (measured in μm) 10. Increase in plate thickness (measured in μm)

[0211] Cartilage matrix loss width (%) was lowest in Group 3M (35.8%) compared to vehicle-treated control Group 1M (43.6%). Total cartilage degeneration width (%): This was also lowest in Group 3M (37.5%) compared to vehicle-treated control Group 1M (43.2%). Two-way ANOVA followed by Bonferroni post-hoc comparison revealed a statistically significant difference in cartilage degeneration between control and liraglutide-treated animal groups, as shown in Figure 4 ( * P<0.05). Mean + / -SD, n=11~12.

[0212] conclusion The study objective was to evaluate the efficacy of three liraglutide-based formulations using a surgically induced model of osteoarthritis in rats.

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

[0214] Liraglutide formulated in Formulation 6 has chondroprotective effects in vivo compared to other formulations or unformulated liraglutide.

[0215] Example 4: Dose-response study using an albumin-based formulation of liraglutide utilizing a surgically induced model of osteoarthritis in rats

[0216] The principle of the test was based on the evaluation of liraglutide formulated with albumin on disease parameter measurements in a rat OA model.

[0217] material and method

[0218] The test substance was liraglutide and the positive control was dexamethasone (and unformulated liraglutide Victoza® (6 mg / ml injection) from Novo Nordisk).

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

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

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

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

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

[0224] Experimental model Animal: Rat / Strain: SD Gender: Male / Number: 72 / Age: 6-8 weeks old at the start of the study Supplier: Janvier Labs, France Initial Body Weight: The average body weight at the start of the study (day 1) was 260 g. The minimum and maximum weights recorded in each group 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: There were no contaminants in the food and water supplies that could have affected the results of this test.

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

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

[0227] Study design and timeline: The study was conducted in three cycles according to Table 1 for study design and Table 2 for study timeline.

[0228] Induction of OA by medial ligament cut (MLT) procedure followed by resection of the medial meniscus Anesthesia was induced by a chamber induction technique using inhalation anesthesia (isoflurane 5.0%). During surgery, animals were maintained under isoflurane at a level of 1.5-3.5% with an air flow rate of 1-2 liters / min. Ophthalmic ointment was applied to the eyes to prevent tissue drying during the anesthesia period. After induction of anesthesia, the hair on the right leg skin surface was clipped using electric animal clippers. After shaving the knee joint, the skin was disinfected with iodine and a parapatellar skin incision was made medial to the joint. An incision medial to the patellar tendon provides access to the exposed joint. The medial ligament was cut and the medial meniscus was excised using a microsurgical knife. The wound was closed with vicryl 5-0 sutures. All surgical procedures were performed using a surgical microscope. Group allocation is shown in Table 8 and the study timeline is shown in Table 9.

[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 of both knee diameters were taken with a digital caliper while the rats were anesthetized. Measurements were taken the day before surgery (for baseline), the day after surgery, and then once a week until the end of the study. The experimenter was blinded to the groups.

[0232] Weight-bearing test: Weight-bearing changes of rats after OA induction were monitored using an incapacity test system. Postural imbalance, which has been reported to indicate changes in limb pain threshold and weight distribution, was tracked. Each rat was placed so that each hind paw rested on a separate force plate on the incapacity apparatus, and the weight supported by each hind paw 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 three consecutive measurements for each rat was recorded. The animals were subjected to weight-bearing function (incapacity test) a total of four times at baseline (day 1), day 14, day 28, and day 35. The experimenter was blinded to the groups.

[0233] Animal Sacrifice and Tissue Fixation: On day 36 (the termination day of groups 1M-6M), all animals were bled. Rats were euthanized by a lethal dose of Euthasol vet. Knee joint structures were harvested and fixed in 4% buffered formalin solution for further histological analysis. The contralateral (non-injured) knee was also fixed in 4% buffered formalin solution. On day 57 (the termination day of satellite groups 7M-8M), rats were euthanized. Knee joint structures were harvested and fixed in 4% buffered formalin solution for further histological analysis. The contralateral (non-injured) knee was also fixed in 4% buffered formalin solution.

[0234] Histological analysis: Histological analysis was performed. Knee joint sections were stained with hematoxylin-eosin or toluidine blue stain to evaluate the degree of pathological lesions. Slides were scored as in N. Gerwin et al., Osteoarthritis and Cartilage 18 (2010) 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. Number of samples: n=5 Right knee (affected): all animals in groups 7M-8M harvested on day 57, 6 animals / group. Sample size: n=12 Total sample size: n=77 Statistical analysis: Numerical results were presented as mean ± standard deviation (SD). Outliers or excluded data points (marked with $) were not included in the group mean calculation. Statistical analysis was performed using two-way or one-way ANOVA (followed by Dunnett's multiple comparison post-hoc test) where applicable. A probability of 5% (p ≤ 0.05) was considered significant. In the figures, results are presented as mean ± SEM, and the degree of statistically significant differences between groups was * p ≤ 0.05, ** p<0.01 and *** Presented as p<0.001.

[0236] result In this study, six groups (1M–6M, n = 9–10 per group) were followed from days 1 to 36.

[0237] Animal 2 from group 2M was excluded from the entire study as histological analysis revealed no lesions in the right knee joint.

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

[0239] At termination, a dose response is observed in rats from Group 2M treated IA with high dose liraglutide (width and thickness), and in rats from Group 3M treated IA with medium dose liraglutide (thickness). As shown in Figure 5, rats from Group 6M treated SC with Victoza® (thickness) also had significantly decreased knee measurements compared to vehicle-treated Group 1M.

[0240] Weight bearing test: Weight bearing changes in rats with OA were assessed using an incapacity meter that measures the weight that the animal distributes to each hind paw. Incapacity tests were performed on day 1 (baseline) and on days 14, 28 and 35. A significant increase in the R / L ratio in % was observed for group 6M treated with Victoza® on day 14. This result could also be observed during other measurement periods (days 28 and 35), although not significant. A slight, non-significant trend for weight bearing differences was observed between control animals and liraglutide IA-treated animals with the medium dose (days 14 and 35 after OA induction) or the high dose (day 35).

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

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

[0243] The edges of the ulcerations were generally characterized by necrosis with fibrillation of the cartilage and / or complete loss of the proteoglycan matrix. The subchondral bone was generally intertwined with large fibrous collagenous bundles (fibrosis). At the osteochondral interface (highest point), including the area beyond the friction zone, the subchondral bone showed necrosis (cells showing eosinophilia, karyorrhexis, and pyknotic nuclei with surrounding fibrin).

[0244] The dexamethasone IA (5M) and Victoza® SC (6M) treatment groups tended to show less cartilage loss, however, some individuals showed near complete replacement of the tibial plate with fibrous tissue forming adhesions between the medial meniscal remnant, femoral cartilage, and synovium, as shown in Figure 6.

[0245] The observed repair changes were characterized by fibrous collagen bundles within the synovial capsule, which in the dexamethasone IA (5M) and Victoza® SC (6M) treated groups showed large fibrous papillary-like highly vascularized projections and could form adhesions within all joints with other structures (medial meniscus remnants, articular cartilage, ligament remnants). In the liraglutide IA treated groups, no fibrosis was observed and no pathological differences were observed. In the liraglutide IA treated groups, chondrocyte nests were observed within the cartilage with a dose-response pattern (Figure 7): 3 / 9 in the high dose group, 2 / 8 in the mid dose group and 1 / 9 in the low dose group. In the Victoza® SC treated group, only 1 out of 7 animals showed proliferation of chondrocyte nests, whereas none in the vehicle treated group (0 / 10).

[0246] The observed chondrocyte proliferation is suggestive of an attempt at cartilage regeneration.

[0247] Satellite group study In the satellite study, two groups (7M-8M, n=6 per group) were followed from day 1 to day 57. Histological parameters were measured as previously in the main study. Evaluation of the percentage recovery 3 weeks after cessation of treatment was analyzed comparing the liraglutide high-dose IA treated group and the vehicle group.

[0248] [Table 10]

[0249] Overall, the lesions observed were remarkable for both groups. As observed during this study, the typical pattern observed was a focally extensive tear of the cartilage involving most of the medial tibial plate. The margins of the ulceration were characterized by fibrillation of the cartilage and / or necrosis with complete loss of the proteoglycan matrix.

[0250] Animals treated IA with high doses of liraglutide (Group 8M) showed lower matrix loss compared to vehicle-treated animals (Group 7M), as well as a trend towards reduced loss over the course of the study in the liraglutide IA-treated group (Figure 8).

[0251] No differences were observed in degeneration scores and total degeneration width, however, throughout the course of the study after cessation of treatment, degeneration scores were lower in the liraglutide IA-treated groups in Z1 and Z3 (Figure 9).

[0252] There was no significant difference in osteophyte formation between the two groups, however, throughout the course of the study after treatment was stopped, 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 (FIGS. 10 and 11).

[0254] Examples of nest plots are shown in Figure 12. As assessed in Figure 13, the vehicle group (2 / 5 mice; 8.1±4.1 nests / μm 2 ), compared with the high-dose test substance treatment group (5 / 6; 30.6 ± 7.0 foci / μm 2 ) significantly more chondrocyte nests were observed.

[0255] conclusion As previously observed during this study, significant histological changes in each group regarding cartilage, subchondral bone and synovium were observed.

[0256] Synovial repair changes were more pronounced in the IA-treated group with high-dose liraglutide compared to the vehicle group. Furthermore, cartilage degeneration changes were less pronounced in the liraglutide-treated group compared to the vehicle group, but were not statistically different.

[0257] Notably, more chondrocyte nests were observed in the liraglutide IA-treated group, demonstrating attempts at endochondral regeneration.

[0258] Under the test conditions, dexamethasone IA and Victoza® SC treatment were able to reduce some OA-associated defects (knee joint swelling, incapacity, histological findings related to cartilage loss), but the repair was accompanied by fibrosis that was not observed in the IA-treated group.

[0259] Interestingly, histological analysis showed intrachondrocyte nests for the liraglutide-treated group.

[0260] The figures show chondrocyte proliferation demonstrating attempts at cartilage repair. In the main study (day 36), the presence of nests was not associated with changes in synovial repair in the IA liraglutide-treated group. However, in the satellite study (day 57), chondrocyte nests seen in animals treated IA with high doses of liraglutide were associated with more pronounced synovial repair changes (hypertrophy).

[0261] Overall the study results indicate that liraglutide targets relevant mechanisms involved in the inflammatory and regenerative processes associated with OA.

[0262] Example 5: Effect of liraglutide on chondrogenesis in an in vitro differentiation model of human mesenchymal stem cells

[0263] We tested the effect of liraglutide on chondrogenesis in an in vitro differentiation model of human mesenchymal stem cells (hMSCs) to evaluate whether liraglutide promotes chondrogenesis.

[0264] material and method Test material: Liraglutide. Test system: human mesenchymal stem cells (StemPro BM, Cat A15652, ThermoFisher Scientific). Basal medium: MesenPRO RS basal medium (ThermoFisher Scientific) supplemented with MesenPRO RS growth supplement, L-glutamine (1%) and gentamicin (10 mg / ml, 50 μl for 100 ml of medium). Differentiation medium (used as positive control): StemPRO Chondrogenic Differentiation Medium (ThermoFisher Scientific) supplemented with StemPRO Chondrogenic Differentiation Supplement and Gentamicin (10 mg / ml, 50 μl per 100 ml of medium).

[0265] procedure Mesenchymal stem cells between 60-80% confluence were used. The cells were detached from their supports and a cell suspension was prepared at 1.6 x 107 cells / milliliter in basal medium (MesenPRO RS basal medium + supplements).

[0266] 5 μl of this suspension was placed in the center of a well of a 24-well plate. The plate was incubated for 2 hours in a humid incubator at 37°C. After 2 hours, 1 ml of basal medium was added to each well without (negative control) or with test substance according to Table 11 for the study design and Table 12 for the study timeline. As a positive control, 1 ml of differentiation medium (StemPRO Chondrocyte Differentiation Basal Medium + Supplements) was used. The plate was incubated at 37°C + 5% CO 2 The cells were then returned to the incubator for 7, 14 or 21 days. During the differentiation phase, medium was changed every 3-4 days.

[0267] [Table 11]

[0268] [Table 12]

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

[0270] The 4% formaldehyde was then removed and the fixed cells were gently rinsed twice with 1 ml of distilled water. The distilled water was removed and 1 ml of Alcian Blue 1% (prepared in 0.1N HCl) was added for 2 hours at room temperature and protected from light. The staining solution was removed and the cells were washed two or three times with 1 ml of 0.1N HCl. The hydrochloric acid 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. In addition, 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) and confirms the formation of chondrocyte spheroids.

[0272] [Table 13]

[0273] As shown in Table 13, no sphere formation was observed for vehicle-treated cells in basal medium during the study. Sphere formation was observed with a dose response for the two test doses of liraglutide. Indeed, for cells treated with 10 nM and 100 nM liraglutide, sphere formation was observed in 67% and 100% of treated wells on day 22, respectively. Differentiation medium contains all the reagents necessary to induce hMSCs to commit to the chondrogenic pathway and generate chondrocytes. As expected, sphere formation was observed for vehicle-treated cells in this medium (83% of treated wells on day 22). Alcian blue staining confirmed that the liraglutide-induced spheres were chondrocyte spheroids. This result indicates that liraglutide alone can induce the differentiation of hMSCs into chondrocytes. An example of the sphere formation process and positive Alcian blue staining is shown in Figure 14.

[0274] conclusion In this study, an in vitro assay for chondrogenesis was used to examine the effects of liraglutide on this process.

[0275] We demonstrated that in the presence of basal medium, liraglutide induced sphere formation with a dose-response, whereas no spheres were observed in vehicle-treated cells. Chondrocyte sphere formation was confirmed by positive Alcian blue staining, a marker of cartilage matrix synthesis.

[0276] Under the test conditions, our data show that liraglutide alone induces hMSCs to commit to the chondrogenic pathway and generate chondrocytes. This anabolic function of liraglutide allows targeting resident stem cell populations in the joint area to stimulate cartilage repair via chondrocyte differentiation, which is considered a promising approach for the treatment of OA.

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

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

[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), and 1% penicillin / streptomycin was used for cell culture from day 1 to day 7. On day 7, DMEM containing 2 mM L-glutamine, 0.1% bovine serum albumin (BSA), and 1% penicillin / streptomycin (P / S) was used to act in FBS-free conditions.

[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 study was considered "day 9."

[0281] procedure Isolation of mouse articular cartilage Immature mouse chondrocytes were derived from newborn mice (5–6-day-old C57Bl / 6). The procedure was performed in a sterile flow hood. After euthanasia of the mice by decapitating the head with scissors, the animals were fixed in a prone position and the front legs were fixed with needles. The skin of the hind limbs was removed using scissors and pliers. The hind limbs were cut along the spine. The skin and muscle remnants were removed from the limbs. The foot was flattened with curved forceps to free a small translucent, hard sphere corresponding to the femoral head. When the sphere was separated, the muscle and other tissues were removed from the remainder of the foot which was placed in 30 ml of 1x PBS. The bone appeared reddish brown and the cartilage appeared white. The bone was cut on both sides of the white part, which is the joint (forming two spheres). After removing the surrounding tissue from the joint with a scalpel, it 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 1× PBS.

[0282] Isolation of immature mouse chondrocytes Cartilage pieces were placed in a 100 mm Petri dish in 10 ml of digestion solution (DMEM, 2 mM L-glutamine + 1% P / S + collagenase 3 mg / ml) at 37°C and 5% CO 2 The cartilage pieces were incubated twice for 45 min in a 37°C incubator. Between the two digestions, the cartilage pieces were collected using a 25 ml pipette and placed in a new Petri dish. After the two digestions, a dispersion of the aggregates was made using a 25 ml pipette. The cartilage pieces were incubated in 10 ml of DMEM, 2 mM L-glutamine + 1% P / S, and 0.5 mg / ml (diluted 1 / 6) collagenase D solution at 37°C, 5% CO 2 The mixture was incubated overnight in a 300-°C incubator.

[0283] Chondrocyte seeding After overnight digestion, 10 ml of DMEM, 2 mM L-glutamine + 10% FBS was added to each Petri dish to stop the action of collagenase D. The medium and residual cartilage were collected and placed in a 50 ml Falcon tube. Dispersion of aggregates was performed using a reduced size pipette to obtain a suspension of isolated cells, which was filtered through a sterile 70 μm cell strainer. The cells were then centrifuged at 400 g for 10 min at 20 °C. The medium was removed and the pellet was resuspended in 5 ml of PBS to wash the cells. The cells were centrifuged at 400 g for 10 min at 20 °C, the PBS was removed and 15 ml of DMEM 2 mM L-glutamine + 10% FBS + 1% P / S was added. The chondrocytes were counted with a Neubauer hemocytometer and observed to assess the viability of the extracted cells. Chondrocytes were cultured at 40 × 10 in 2 ml of DMEM 2 mM L-glutamine + 10% FBS + 1% P / S / well in a 12-well plate. 3 The cells were seeded at a density of 100x100 / mL. The cultures were incubated at 37°C and 5% CO 2 The cells were maintained under inorganic conditions in a 100-mL incubator.

[0284] Chondrocyte culture Immature mouse articular chondrocytes became confluent after 6-7 days. The medium was changed after 3 days of culture. On day 7, the DMEM medium containing 10% FBS was removed, the wells were rinsed twice with 1 ml PBS and 1 ml DMEM, and 2 mM L-glutamine + 1% P / S + 0.1% BSA was added. On day 8, the medium was removed and treatments with 12 different concentrations of liraglutide in 500 μl DMEM, 2 mM L-glutamine + 1% P / S + 0.1% BSA per well were performed (Table 14). The plates were incubated at 37°C + 5% CO 2 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 study (day 9), the culture medium from each well (±500 μl) was collected into 1.5 ml tubes (one tube per well), centrifuged at 4000 rpm for 10 min at room temperature, and the supernatant was placed into 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 the injured cells. LDH assays were performed according to the procedure detailed in the instructions for the specific LDH assay kit and analyzed by a Plate Reader (96 well) (Multiskan FC, Thermo Fisher). The wavelength for measuring absorbance was 450 nM. The average optical density (OD) of the reading blank wells was subtracted from each reading.

[0289] result Lactate dehydrogenase is a stable enzyme present in all cell types and rapidly released into cell culture medium upon plasma membrane damage. LDH enzyme was detected using an enzyme-coupled reaction and measured by SkanIt software for microplate reader, Thermo Fisher. LDH oxidizes lactate to generate NADH, which then reacts with WST substrate to generate a yellow color. The intensity of the coloration directly correlates with the number of lysed cells. LDH activity was quantified at OD450nM by spectrophotometer. LDH activity was measured after 24 hours of incubation with 12 doses of liraglutide (1.7nM to 300μM). A positive control was used, where 5μl of LDH enzyme was directly placed in the wells. The % of 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 doses of liraglutide (up to 11.1 μM) induced the release of small amounts of lactate dehydrogenase in the medium. However, there was no significant difference compared to vehicle-treated cells. In the presence of the highest tested doses of liraglutide (>30 μM), the levels of lactate dehydrogenase enzyme detected were significantly increased compared to vehicle, with a dose response. Indeed, the calculated mortality % was as follows: vehicle: 0.0% ± 0.008 and liraglutide 33.3 μM: 8.5% ± 0.006; liraglutide 100 μM: 11.1% ± 0.051 and liraglutide 300 μM: 11.5% ± 0.069, p<0.001). The use of a positive control (marked in yellow) confirmed that all reagents of the kit were functioning properly.

[0290] conclusion This study shows that depending on the liraglutide dose tested, mortality can be observed after 24 hours of incubation on chondrocytes.

[0291] Example 6: SOX9 expression in knee joints of monoiodoacetate-injected mice following intra-articular administration of an albumin-based formulation of liraglutide

[0292] SOX9 is a pivotal transcription factor in developing and adult cartilage. The gene is expressed from the multipotent skeletal progenitor cell stage and is active throughout chondrocyte differentiation. It is repressed in hypertrophic chondrocytes of the cartilage growth plate but remains expressed throughout life in permanent chondrocytes of healthy articular cartilage. SOX9 is required for chondrogenesis: it ensures chondrocyte lineage commitment, promotes cell survival and transcriptionally activates genes for many cartilage-specific structural components and regulators.

[0293] The aim of this study was to examine SOX9 expression in the knee joints of monoiodoacetate (MIA)-injected mice following intra-articular administration of an albumin-based formulation of liraglutide.

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

[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 a commercial rodent diet (Safe ref#A04) ad libitum. Animals had free access to filtered drinking osmotic 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.”

[0298] Induction of OA by intra-articular (IA) injection of MIA Animals were anesthetized by a chamber induction technique using inhalation anesthesia (isoflurane 5%). During the procedure, animals were maintained under isoflurane at a level of 1.5-3% with an air flow rate of 1-2 liters / min. The area around the knee joint was swabbed with alcohol. MIA was injected intra-articularly (IA) through the patellar tendon in 5 μl containing 0.75 mg. A 30-gauge 0.5-inch needle fitted with a cannulation tube was used to allow only 2-3 mm of the needle to puncture the joint. After injection, the knee was massaged to ensure uniform distribution of the solution. Animals were injected once on day 1 (groups 2M, 3M, 4M, 5M). For group 1M (sham control), 5 μl of injectable saline was injected into the knee joint.

[0299] Study Design and Timeline The study was conducted in three cycles according to Table 16 for study design and Table 17 for study timeline. [Table 16]

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

[0301] Testing and Evaluation Animal sacrifice and tissue collection On day 11 (end date), mice were euthanized. Knee joint structures (including synovium) were harvested and snap frozen in liquid nitrogen. RNA extraction was performed using the SV Total RNA Isolation System Kit (Promega) according to the manufacturer's recommendations. RT-q-PCR analysis was performed for the SOX9 marker. SOX9 has been identified as the first transcription factor essential for chondrocyte differentiation and chondrogenesis.

[0302] result SOX9 RTqPCR analysis of knee joint structures As shown in Figure 16, vehicle mice (group 2M) administered 0.75 mg MIA into the knee joint on day 1 showed a 40% decrease in SOX9 relative expression on day 11 compared to the vehicle sham control (group 1M). When MIA-injected mice received an intra-articular injection of liraglutide formulated with albumin on day 3, SOX9 expression was restored and is similar for groups 3M (10 μg liraglutide) and 4M (20 μg liraglutide) to the sham control group 1M. Group 5M, which received 30 μg liraglutide, shows a 55% increase in SOX9 relative expression compared to the sham control group 1M.

[0303] conclusion The study objective was to perform RTqPCR of SOX9 in the knee joints of mice injected with monoiodoacetate following intra-articular administration of formulated liraglutide.

[0304] The monoiodoacetate (MIA) model has become the standard for modeling joint destruction in osteoarthritis (OA) in both rats and mice. In this model, a single injection of MIA is delivered to the knee joint to disrupt chondrocyte glycolysis by inhibiting glyceraldehyde-3-phosphatase dehydrogenase, specifically inducing chondrocyte death. Chondrocytes, which differentiate after condensation of mesenchymal stem cells, are responsible for the secretion of extracellular matrix molecules such as collagen and proteoglycans. The transcription factor SOX9 is important for chondrocyte differentiation and function. Using this animal model of OA, we showed that SOX9 expression was decreased after MIA injection, while intra-articular injection of formulated liraglutide restored or increased SOX9 relative expression, compared to sham healthy controls.

[0305] Thus, this study shows that locally administered albumin-compounded liraglutide targets relevant mechanisms linked to anabolism in MIA-induced OA and inflammatory pain models in mice.

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

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

[0308] material and method compound Type II collagenase: Type II collagenase was dissolved in PBS at a concentration of 20000 U / ml to deliver 500 U in 25 μl. Treatment formulation: -Alpha 1-acid glycoprotein (A1AGP) vehicle consisted of 5% alpha 1-acid glycoprotein resuspended in PBS (25 μl) for intra-articular injection. -Alpha-1 acid glycoprotein-based formulations of liraglutide: Liraglutide (supplied as a powder) was dissolved in an appropriate volume of alpha-1 acid glycoprotein vehicle to give 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 study supplier Janvier Labs, France

[0310] Animal Control Housing Animal handling was performed in accordance with the guidelines of the Federation of European Laboratory Animal Science Associations (FELASA). Animals were housed in plastic cages (2–3 per cage) with stainless steel top grills facilitating pelleted food and drinking water in plastic bottles. Bedding: steam-cleaned rice husk (Safe) was used, and bedding material was changed along with the cages at least once a week.

[0311] Diet Animals were fed a commercial rodent diet (Safe ref#A04) ad libitum. Animals had free access to filtered drinking osmotic 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] Induction of OA by intra-articular (IA) injection of type II collagenase Animals were anesthetized by a chamber induction technique using inhalation anesthesia (isoflurane 5%). During the procedure, animals were maintained under isoflurane at a level of 1.5-3% with an air flow rate of 1-2 liters / min. Type II collagenase was injected intra-articularly (IA) in 25 μl containing 500 U. 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 study design and Table 19 for study timeline.

[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 (termination day), all animals were bled. Rats were euthanized. Knee joint structures were harvested and fixed in 4% buffered formalin solution for further histological analysis. The contralateral (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 by a veterinarian (DVM, DESV Anatomic Pathology) who was blinded to group treatments and protocols during the entire analysis procedure. Knee joint sections were scored according to Osteoarthritis Cartilage. 2010 Oct;18 Suppl3:S24-34.

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

[0321] result The results showed a clear trend for group 6M (intra-articular administration of liraglutide in A1AGP vehicle) to show less cartilage lesions than group 5M (A1AGP vehicle). In support of this, the total joint score was calculated based on the sum of the following subsections (de Visser et al., PLoS One. 2018 Apr 23; 13(4): e0196308): cartilage matrix loss width (0-2), cartilage degeneration (0-5), cartilage degeneration width (0-4), osteophytes (0-4), calcified cartilage and subchondral bone damage (0-5) and synovial inflammation (0-4). Figure 17 showed a significant reduction in the total joint score for group 6M versus 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 and tended to significantly reduce total joint scores, suggesting chondroprotection with local administration of liraglutide.

[0324] Overall study results indicate that liraglutide was well tolerated when administered locally and targeted relevant mechanisms associated with chondroprotection in this collagenase-induced OA model in rats.

Claims

1. A pharmaceutical composition for use in cartilage regeneration comprising a glucagon-like peptide-1 analogue selected from the group consisting of exenatide, lixisenatide, albiglutide, dulaglutide, semaglutide and liraglutide.

2. The pharmaceutical composition for use according to claim 1, wherein the glucagon-like peptide-1 analogue is liraglutide.

3. 3. The pharmaceutical composition for use according to claim 2, wherein the concentration of said glucagon-like peptide-1 analog is from about 0.1 nM to 625 μM.

4. 4. The pharmaceutical composition for use according to claim 3, further comprising at least 5% by weight or more of a pharma- ceutically acceptable formulation vehicle used in combination.

5. 5. The pharmaceutical composition for use according to claim 4, wherein said pharma- ceutically acceptable formulation vehicle is selected from the group consisting of albumin and alpha-1 acid glycoprotein.

6. 6. The pharmaceutical composition for use according to claim 5, wherein the concentration of said pharma- ceutically acceptable formulation vehicle is from about 0.1% to about 10% (wt / wt) of said formulation.

7. 7. The pharmaceutical composition for use according to claim 6, wherein the concentration of said pharma- ceutically acceptable formulation vehicle is 5% (wt / wt) of said formulation.

8. 8. The pharmaceutical composition for use according to claim 7, wherein said pharma- ceutically acceptable formulation vehicle is albumin.

9. 8. The pharmaceutical composition for use according to claim 7, wherein said pharma- ceutically acceptable formulation vehicle is alpha 1 acid glycoprotein.

10. 10. The pharmaceutical composition for use according to any one of claims 1 to 9, which is administered by intra-articular injection to cartilage damage lesions.

11. 11. A pharmaceutical composition for use according to any one of claims 1 to 10, for inducing anabolic stimulation of chondrocytes, including chondrocyte proliferation and / or stem cell differentiation into chondrocytes.

12. 12. A pharmaceutical composition for use according to any one of claims 1 to 11, comprising a therapeutically effective amount of a glucagon-like peptide-1 analogue and an excipient comprising a polymer selected from the group consisting of non-ionic surfactants, cellulose, polyethers, glucans, glycerophospholipids, polysaccharides, proteins, and combinations thereof.

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