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

Abstract

The present invention relates to a novel pharmaceutical composition comprising GLP-1 and a GLP-1 analog that induces 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 composition according to the present invention induces cartilage regeneration and anabolic stimulation of chondrocytes, including proliferation of chondrocytes and / or differentiation of stem cells into chondrocytes.

Description

Technology Field

[0001] The present invention relates to a novel pharmaceutical composition comprising GLP-1 and GLP-1 analogues that induce increased chondrocyte regeneration and reduced cartilage degeneration, comprising anabolic and catabolic cytokine regulation for use in the treatment of cartilage diseases. Background Technology

[0002] Osteoarthritis (OA) is the most common chronic joint disease. OA affects nearly 50% of people over the age of 65 and occurs in younger individuals following joint injury. Globally, 250 million people suffer from OA, and the disease has significant economic and social impacts on patients and healthcare systems. OA is a disease of the entire joint characterized by structural degradation of the periarticular bone, synovial joint lining, and adjacent supporting connective tissue elements. The destruction of articular cartilage is the result of chondrocytes failing to maintain a balance between the synthesis and degradation of the extracellular cartilage matrix. Pro-inflammatory cytokines, such as interleukin-1β (IL-1β), produced by macrophages, monocytes, synovial cells, and chondrocytes, play a crucial role in the development of the disease.

[0003] Glucagon-like peptide-1 (GLP-1) is a postslication product of the preproglucagon gene. The actions of GLP-1 on pancreatic β-cells include increased expression of glucose transporter 2 and insulin secretion in response to increased glucose levels. Additionally, 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 analogs are available commercially available drugs prescribed to patients for the treatment of type 2 diabetes. International patent application WO 2017 / 149070 discloses certain derivatives and analogs of glucagon-like peptide 1 (GLP-1), their manufacture, and pharmaceutical uses. This document relates to specific derivatives and analogs of GLP-1 for use in the prevention and treatment of all forms of diabetes.

[0005] Initially, osteoarthritis was considered a disease of the joint cartilage, but recent studies have shown that the condition involves the entire joint.

[0006] While the loss of articular cartilage has been considered the major change, the combination of cellular changes and biomechanical stress causes various secondary changes including subchondral bone remodeling, the formation of osteophytes, the development of bone marrow lesions, changes in the synovium, joint capsule, ligaments and periarticular muscles, and meniscal tears and protrusion.

[0007] There are two patterns in the process of cartilage growth. One is stromal growth, in which cells differentiate into chondrocytes, are surrounded by a cartilage matrix, and proliferate through cell division. Each chondrocyte secretes the matrix, and subsequently, the cartilage tissue expands. The other growth pattern is appositional growth, which occurs through the perichondrium. Cartilage tissue is covered by the perichondrium, except for the articular surface of the articular cartilage. The strong perichondrium is composed of fibroblasts, but the distinction between fibroblasts and chondrocytes is unclear because they resemble the chondrocytes in the inner layer. The cells of the inner perichondrium proliferate while gradually becoming round, and these cells grow outward by additionally secreting the cartilage matrix.

[0008] European patent EP 2 890 390 B1 relates to incretin hormones or analogs thereof for use in the treatment of osteoarthritis. More specifically, the patent discloses the use of GLP-1 and GLP-1 analogs, e.g., liraglutide, for the treatment of osteoarthritis. The peptides disclosed in patent EP 2 890 390 B1 may be administered via any known route of administration, including systemic (parenteral, intravenous, etc.), oral, rectal, topical, or subcutaneous. This patent does not disclose the role of GLP-1 in cartilage degradation nor demonstrates a relationship with chondrocytes.

[0009] The normal replacement of the cartilage matrix is ​​mediated by chondrocytes that synthesize these components and proteolytic enzymes responsible for their degradation. Chondrocytes, in turn, are influenced by various factors including polypeptide growth factors, cytokines, structural and physical stimuli, and even components of the matrix itself.

[0010] Osteoarthritis occurs when chondrocytes fail to maintain homeostasis between the anabolic and catabolic processes of these extracellular matrix components. The causes leading to this imbalance between cartilage degradation and repair are not well understood. Trauma inducing microfractures or inflammation that slightly increases enzymatic activity can lead to the formation of wear particles, which can be engulfed by resident macrophages. At some point, the generation of these wear particles overwhelms the system's ability to remove them, and they act as mediators of inflammation, stimulating chondrocytes to release degrading enzymes. Molecules derived from the degradation of collagen and proteoglycans are absorbed by synovial macrophages, triggering the release of pro-inflammatory cytokines such as TNFα, IL-1, and IL-6. There is a close relationship between cytokine expression and OA. Interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) can induce the production of interleukin-6 (IL-6) and interleukin-8 (IL-8) by synovial cells and chondrocytes. However, all studies focus on synovial tissue rather than chondrocytes.

[0011] Anabolic stimulation of chondrocytes is measured in vitro by the stimulation of proteoglycan and collagen synthesis. It has been suggested that cytokines such as GM-CSF, Granulocyte-Macrophage Colony Stimulating Factor (Quinetro et al., 2008 cytokine 44(3):366-72), and CXCL10 / IP10 (Neidlin et al. 2018, annals of biomedical engineering, volume 46, ISSUE 2 pp 345-353) stimulate the anabolic processes of chondrocytes.

[0012] It is necessary to enhance chondrocyte anabolic activity, including chondrocyte proliferation, and to reduce chondrocyte catabolic activity, including cartilage matrix degradation in OA. Such improved compositions and methods will generate significant interest in the development of novel therapeutic strategies for the treatment of osteoarthritis.

[0013] Furthermore, it is necessary to enhance chondrocyte anabolic activity, including chondrocyte differentiation, to promote cartilage regeneration and alleviate cartilage destruction, respectively. These improved compositions and methods will generate significant interest in the development of novel therapeutic strategies for the treatment of osteoarthritis.

[0014] The present invention discloses an improved pharmaceutical formulation for use in the treatment of cartilage diseases, which induces an enhancement of chondrocyte anabolic processes, including chondrocyte proliferation, and a reduction of catabolic processes, including loss of cartilage matrix and reduction of cartilage degradation.

[0015] The present invention further discloses improved pharmaceutical formulations and compositions for use in the treatment of cartilage diseases, which induce an enhancement of chondrocyte anabolic processes, including chondrocyte differentiation for cartilage regeneration, and a reduction of catabolic processes, including a reduction in cartilage matrix loss and chondrogenesis.

[0016] More specifically, this composition is useful for the treatment of osteoarthritis and for alleviating or reducing joint irritation, or for reducing the exacerbation of existing joint inflammation. The problem to be solved

[0017] The present invention relates to a pharmaceutical composition for use in treating cartilage diseases that induces anabolic stimulation of chondrocytes, including chondrocyte proliferation, and reduces catabolic activity, including loss of cartilage matrix and reduction of cartilage degeneration.

[0018] According to a specific aspect, the present invention relates to a pharmaceutical composition for use in the treatment of cartilage diseases that induces anabolic stimulation of chondrocytes, including chondrocyte proliferation for cartilage regeneration and / or differentiation of stem cells into chondrocytes, and reduces catabolic activity, including a reduction in cartilage matrix loss and cartilage degeneration.

[0019] According to a specific aspect, the present invention relates to a pharmaceutical composition for inducing anabolic stimulation of chondrocytes for use in treating cartilage diseases, comprising a glucagon-like peptide-1 analog as its active ingredient.

[0020] According to the present invention, a glucagon-like peptide-1 (GLP-1) analog is selected from the group consisting of exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, semaglutide, or liraglutide.

[0021] According to one aspect of the present invention, a pharmaceutical composition is provided for use in the treatment of cartilage disease comprising glucagon-like peptide-1 as an active ingredient, which induces anabolic stimulation of chondrocytes including chondrocyte proliferation and / or reduces catabolic activity including reduction of cartilage matrix loss.

[0022] According to another aspect of the present invention, a glucagon-like peptide-1 (GLP-1) analog is liraglutide.

[0023] According to another aspect of the present invention, the concentration of liraglutide is 1 ng / ml to 10 mg / ml.

[0024] According to another aspect of the present invention, the concentration of liraglutide is 0.1 to 10 mg / ml.

[0025] According to additional features of preferred embodiments of the present invention described below, a formulation of a pharmaceutical composition for use of the present invention provides a gel comprising a therapeutically effective amount of a GLP-1 analog and a polymer selected from the group consisting of a nonionic surfactant, cellulose, polyether, glucan, glycerophospholipid, polysaccharide, protein and combinations thereof.

[0026] According to additional features of preferred embodiments of the present invention described below, a formulation of a pharmaceutical composition for use of the present invention provides a therapeutically effective amount of a GLP-1 analog and an excipient comprising a polymer selected from the group consisting of nonionic surfactants, cellulose, polyethers, glucans, glycerophospholipids, polysaccharides, proteins, and combinations thereof.

[0027] The pharmaceutical formulation according to the present invention may also contain one or more pharmaceutically acceptable carriers / excipients.

[0028] The present invention is not limited to gel formulations, but liquid and semi-solid pharmaceutical forms suitable for topical administration, such as liquids, solutions, creams, gels, or transdermal patches, are preferred; in particular, forms suitable for intra-articular injection, such as liquids and solutions, and transdermal application and transdermal patches, such as semi-solid forms (e.g., creams or gels), are preferred. The pharmaceutical forms may also consist of some or all of the components in a dry form, possibly lyophilized and reconstituted into an aqueous solution or other suitable vehicle before use.

[0029] The above formulations may be produced by methods well known in the art using known excipients such as binders, disintegrants, fillers, stabilizers, diluents, and coloring agents. They may also include delayed-release or slow-release forms made of suitable polymers known in pharmaceutical technology.

[0030] Pharmaceutically acceptable carriers / excipients, such as solvents, preservatives and / or chelating agents and antimicrobial agents, isotonic regulators, and buffering systems, are preferred for the preparation of liquid forms suitable for injectable use.

[0031] Water is preferred as a solvent, preferably with a co-solvent such as glycol or polyalcohol (e.g., ethylene glycol).

[0032] Preservatives or chelating agents may also be used, and sodium edetate and sodium metabisulfite are preferred, as are antimicrobial agents and benzyl alcohol.

[0033] Sodium chloride or mannitol is particularly preferred as an isotonic regulator.

[0034] A desirable buffer system may be a complex of salts for phosphate and citrate buffers, preferably in the form of sodium or potassium salts.

[0035] In the preparation of liquid forms suitable for nebulization, pharmaceutically acceptable vehicles / excipients are preferred as solvents, along with preservatives such as antioxidants and / or chelating agents and antimicrobial agents, isotonic regulators, and buffering systems.

[0036] According to additional features of a preferred embodiment described in the above pharmaceutical composition, the GLP-1 analog is liraglutide and the gel contains albumin.

[0037] According to additional features of a preferred embodiment described in the above pharmaceutical composition, the GLP-1 analog is liraglutide and contains albumin.

[0038] According to additional features of a preferred embodiment described in the above pharmaceutical composition, the GLP-1 analog is liraglutide and comprises an alpha 1-acid glycoprotein (A1AGP).

[0039] According to additional features of a preferred embodiment described in a pharmaceutical composition for use according to the present invention, the albumin concentration is about 0.1% to about 10% (wt / wt) of the formulation, preferably 5% (wt / wt).

[0040] According to additional features of a preferred embodiment of a pharmaceutical composition for use according to the present invention, the concentration of the alpha 1-acid glycoprotein (A1AGP) is about 0.1% to about 10% (wt / wt) of the formulation, preferably 5% (wt / wt).

[0041] According to additional features of the described preferred embodiment, the pharmaceutical composition comprises 1 ng / ml and 10 mg / ml of liraglutide, and 5% (wt / wt) of albumin.

[0042] According to additional features of the described preferred embodiment, the pharmaceutical composition comprises 6 mg / ml of liraglutide and 5% (wt / wt) of albumin.

[0043] According to another aspect of the present invention, the cartilage disease is selected from the group consisting of cartilage defects caused by external injury or surgical treatment, osteochondritis dissecans, osteoarthritis, congenital cartilage disease, and cartilage damage.

[0044] According to another aspect of the present invention, a method for increasing the secretion or production of anabolic cytokines in a patient's chondrocytes is provided, said method comprising the step of administering a composition according to the present invention to the patient.

[0045] According to additional features of a preferred embodiment, the anabolic cytokine is GMCSF and / or CXCL10 / IP10.

[0046] According to another aspect of the present invention, a method for reducing the secretion or production of catabolic cytokines in a patient's chondrocytes is provided, said method comprising the step of administering a composition according to the present invention to the patient.

[0047] According to an additional feature of a preferred embodiment, the catabolic cytokine is selected from the group consisting of MMP3, MMP13, PGE2, IL7, and MCP1.

[0048] According to another aspect of the present invention, the composition is administered to an individual via intra-articular injection.

[0049] According to a further aspect of the present invention, a use of liraglutide is provided for treating cartilage disease by increasing the secretion or production of anabolic cytokines, reducing cartilage loss, and / or restoring it through the stimulation of chondrocyte proliferation.

[0050] According to a further aspect of the present invention, a use of liraglutide is provided for treating cartilage diseases by increasing the secretion or production of anabolic cytokines, reducing cartilage loss, and / or regenerating through the stimulation of chondrocyte proliferation.

[0051] According to a specific aspect of the present invention, a use of liraglutide is provided in the manufacture of a medicine for treating cartilage diseases by increasing the anabolic function of chondrocytes.

[0052] The above cartilage disease is selected from a group consisting of cartilage defects induced by external injury or surgical treatment, dissecting osteochondritis, osteoarthritis, congenital cartilage disease, and cartilage damage.

[0053] According to a further aspect of the present invention, a use of liraglutide is provided for treating cartilage diseases by reducing the secretion or production of catabolic cytokines, reducing cartilage loss, and / or restoring it through the stimulation of chondrocyte proliferation.

[0054] According to a further aspect of the present invention, a use of liraglutide is provided for treating cartilage diseases by reducing the secretion or production of catabolic cytokines, reducing cartilage loss, and / or regenerating cartilage cells through stimulation of proliferation.

[0055] The above cartilage disease is selected from a group consisting of cartilage defects induced by external injury or surgical treatment, dissecting osteochondritis, osteoarthritis, congenital cartilage disease, and cartilage damage.

[0056] According to another aspect of the present invention, a method for promoting the proliferation of chondrocytes is provided, comprising the step of contacting at least one chondrocyte with a composition according to the present invention. According to a further feature of a preferred embodiment, the cell is a mammalian cell. According to a further feature of a preferred embodiment, the cell is a human cell.

[0057] According to another aspect of the present invention, a method for treating a patient diagnosed with or at risk of developing an immunoinflammatory disorder is provided, said method comprising the step of administering a composition according to the present invention to the patient.

[0058] According to another aspect of the present invention, a method for treating an inflammatory pathology in an individual is provided, comprising the step of administering a composition according to the present invention to an individual. According to a further feature of a preferred embodiment, the inflammatory pathology is an inflammatory condition occurring in the joint and joint space, degeneration of the cartilage matrix and osteoarthritis. According to a further feature of a preferred embodiment of the method according to the present invention, the composition is administered to an individual via an intra-articular injection.

[0059] According to another aspect of the present invention, the composition is administered to the fat pad of the joint via intra-articular injection.

[0060] According to another aspect of the present invention, a method for promoting cartilage matrix repair in an individual is provided, comprising the step of administering a composition according to the present invention.

[0061] According to another aspect of the present invention, a method for promoting cartilage matrix regeneration in an individual is provided, comprising the step of administering a composition according to the present invention.

[0062] According to another aspect of the present invention, a method for improving an inflammation-promoting pathology in an individual is provided, comprising the step of administering a composition according to the present invention.

[0063] According to another aspect of the present invention, the use of liraglutide as an active ingredient in the manufacture of pharmaceutical preparations for alleviating or reducing joint irritation in mammalian individuals, or reducing the exacerbation of existing joint inflammation, is provided.

[0064] According to another aspect of the present invention, the use of liraglutide as an active ingredient for use in a method for alleviating or reducing joint irritation or reducing the exacerbation of existing joint inflammation in mammalian individuals is provided, wherein the formulation is administered via intra-articular injection into the fat pad of the joint.

[0065] According to another aspect of the present invention, 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 exacerbation of existing joint inflammation in mammalian individuals is provided, said liraglutide is formulated together with at least a second therapeutic agent, said second therapeutic agent comprises an anti-inflammatory agent, an antioxidant, a vitamin, a polyol, or a combination thereof.

[0066] According to another aspect of the present invention, a composition for use as a preparation for differentiating mesenchymal stem cells into chondrocytes is provided, comprising liraglutide as its active ingredient.

[0067] According to another aspect of the present invention, the concentration of the liraglutide is 1 ng / ml to 10 mg / ml.

[0068] According to another aspect of the present invention, a method for differentiating mesenchymal stem cells into chondrocytes is provided, comprising the following steps:

[0069] a) a step of adding the composition according to the present invention to a cell culture medium containing mesenchymal stem cells;

[0070] b) Step of differentiating mesenchymal stem cells into chondrocytes.

[0071] According to another aspect of the present invention, a method for differentiating mesenchymal stem cells into chondrocytes is provided, comprising the following steps:

[0072] c) a step of adding a GLP-1 analog to a cell culture medium containing mesenchymal stem cells;

[0073] d) Step of differentiating mesenchymal stem cells into chondrocytes.

[0074] According to another aspect of the present invention, the glucagon-like peptide-1 (GLP-1) analog is liraglutide.

[0075] According to another aspect of the present invention, the concentration of the liraglutide is 0.1 nM to 625 microM.

[0076] According to another aspect of the present invention, the cell culture medium further contains MesenPRO RS growth supplement and 1% L-glutamine.

[0077] According to another aspect of the present invention, a use of a SOX9 expression-enhancing peptide is provided in the manufacture of a medicine comprising a SOX9 expression-enhancing peptide for the treatment or prevention of arthrosis, wherein the SOX9 expression-enhancing peptide is a GLP-1 analog that selectively targets the SOX9 gene. According to another aspect of the present invention, the GLP-1 analog is liraglutide. According to another aspect of the present invention, the concentration of liraglutide is 1 ng / ml to 10 mg / ml.

[0078] According to another aspect of the present invention, a use of a SOX9 expression-enhancing peptide is provided in the manufacture of a medicine comprising a SOX9 expression-enhancing peptide for the treatment or prevention of inflammation, wherein the SOX9 expression-enhancing peptide is a GLP-1 analog that selectively targets the SOX9 gene. According to another aspect of the present invention, the GLP-1 analog is liraglutide. According to another aspect of the present invention, the concentration of liraglutide is 1 ng / ml to 10 mg / ml.

[0079] According to another aspect of the present invention, a pharmaceutical composition for use in the treatment or prevention of osteoarthritis is provided, comprising a pharmaceutically acceptable carrier and a SOX9 expression-enhancing peptide, wherein the SOX9 expression-enhancing peptide is a GLP-1 analog 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 another aspect of the present invention, a pharmaceutical composition for use in the treatment or prevention of inflammation is provided, comprising a pharmaceutically acceptable carrier and a SOX9 expression-enhancing peptide, wherein the SOX9 expression-enhancing peptide is a GLP-1 analog 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.

[0081] According to another aspect of the present invention, a pharmaceutical composition for use in the treatment or prevention of osteoarthritis comprising a pharmaceutically acceptable carrier and a SOX9 expression-enhancing peptide according to the present invention provides a gel comprising a therapeutically effective amount of liraglutide and a polymer selected from the group consisting of nonionic surfactants, cellulose, polyethers, glucans, glycerophospholipids, polysaccharides, proteins, and combinations thereof. In a preferred embodiment, the gel comprises albumin, wherein the albumin concentration is about 0.1% to about 10% (wt / wt) of the formulation, preferably 5% (wt / wt).

[0082] In another preferred embodiment, the gel comprises an alpha 1-acid glycoprotein, wherein the concentration of the alpha 1-acid glycoprotein is about 0.1% to about 10% (wt / wt) of the formulation, preferably 5% (wt / wt).

[0083] According to another aspect of the present invention, a pharmaceutical composition for use in the treatment or prevention of inflammation comprising a pharmaceutically acceptable carrier and a SOX9 expression-enhancing peptide according to the present invention provides a gel comprising a therapeutically effective amount of liraglutide and a polymer selected from the group consisting of nonionic surfactants, cellulose, polyethers, glucans, glycerophospholipids, polysaccharides, proteins, and combinations thereof. In a preferred embodiment, the gel comprises albumin, wherein the albumin concentration is about 0.1% to about 10% (wt / wt) of the formulation, preferably 5% (wt / wt).

[0084] According to another aspect of the present invention, a composition for use in cartilage regeneration comprising a glucagon-like peptide-1 analog is provided.

[0085] According to another aspect of a composition for use in cartilage regeneration, a glucagon-like peptide-1 analog is selected from the group consisting of zenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, semaglutide, or liraglutide.

[0086] According to another aspect of a composition for use in cartilage regeneration, a glucagon-like peptide-1 analog is liraglutide.

[0087] According to another aspect of a composition for use in cartilage regeneration, the concentration of the glucagon-like peptide-1 is about 0.1 nM to 625 μM.

[0088] According to another aspect, a composition for use in cartilage regeneration further comprises at least 5 weight % of a pharmaceutically acceptable formulation vehicle to be used in combination.

[0089] According to another aspect of a composition for use in cartilage regeneration, the pharmaceutically acceptable formulation vehicle is selected from the group consisting of albumin or alpha 1-acid glycoproteins.

[0090] According to another aspect of a composition for use in cartilage regeneration, the pharmaceutically acceptable concentration of the formulation vehicle is about 0.1% to about 10% (wt / wt) of the formulation, preferably 5% (wt / wt).

[0091] According to another aspect of the composition for use in cartilage regeneration, the pharmaceutically acceptable vehicle concentration of the composition is 5% (wt / wt) of the composition.

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

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

[0094] According to another aspect of the present invention, a composition for use in cartilage regeneration is intended to be administered orally, subcutaneously, intravenously, or intra-articularly.

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

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

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

[0098] Accordingly, the present invention is useful for any application where stimulation of chondrocyte proliferation or growth, including cartilage repair and / or regeneration, is considered desirable. means of solving the problem

[0099] The applicant discovered that increasing the effective concentration of liraglutide on chondrocytes of OA patients has the effect of stimulating anabolic cytokines and reducing catabolic cytokines in chondrocytes.

[0100] The inventors have demonstrated that the use of intra-articular injection (acute or repeated) of a pharmaceutical composition and the slow release of liraglutide into the synovial fluid leads to a reduction and delay of the fibrosis process induced after cartilage damage, and actual functional and histological improvement of the articular joint after joint damage.

[0101] Furthermore, it was demonstrated that a specific dosage regimen (i.e., several injections separated by one week each) is required to induce chondrocyte proliferation without fibrosis.

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

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

[0104] According to the main aspects thereof as broadly described, the present invention provides a pharmaceutical composition comprising a glucagon-like peptide-1 analog as an active ingredient thereof for use in the treatment of a cartilage disease selected from the group consisting of cartilage defects induced by external injury or surgical treatment, dissecting osteochondritis, osteoarthritis, congenital cartilage disease and cartilage damage, said pharmaceutical composition induces enhancement of cartilage cell proliferation, cartilage repair and increase of reduced cartilage matrix.

[0105] According to the main aspects thereof as broadly described, the present invention provides a pharmaceutical composition comprising a glucagon-like peptide-1 analog as an active ingredient thereof for use in the treatment of a cartilage disease selected from the group consisting of cartilage defects induced by external injury or surgical treatment, dissecting osteochondritis, osteoarthritis, congenital cartilage disease and cartilage damage, said pharmaceutical composition induces improved cartilage cell proliferation, cartilage regeneration and reduced loss of cartilage matrix. Brief explanation of the drawing

[0106] Fig. 1: Differential expression levels of the average calculated concentration of cytokines secreted from chondrocytes of OA patients after treatment with various doses of Victoza®. Fig. 2: Release profiles for formulations 6, 8, 14, 17, 19, and 20. Fig. 3: Change in body weight-loading in percentage units (R / L) in surgery-induced OA during the study. Fig. 4: Histological findings in surgery-induced OA during the study. Fig. 5: Dose response of formulated liraglutide IA to knee measurements at termination. Fig. 6: Representative photograph of a right knee section stained with hematoxylin and eosin showing fibrous synovial chronic proliferation and tibial plate fibrosis, particularly in animals injected subcutaneously with Victoza®. Fig. 7: Representative photograph of a right knee section stained with hematoxylin and eosin showing the absence of fibrosis and chondrocyte nests in an animal injected with formulated liraglutide. Fig. 8: Long-term reduction in cartilage matrix loss in animals injected with formulated liraglutide compared to vehicle animals. Fig. 9: Long-term reduction in cartilage degeneration scores in animals injected with formulated liraglutide compared to vehicle animals. Fig. 10: Representative photograph of a right knee section stained with toluidine blue showing differences in synovial fluid thickening between animals injected with formulated liraglutide and vehicle animals. Fig. 11: Medial joint capsule repair. Fig. 12: Representative photograph of a right knee section stained with hematoxylin and eosin showing chondrocyte aggregates in group 8M. Fig. 13: Evaluation of the total number and density of chondrocyte aggregates. Fig. 14: Shows the effect of liraglutide on the sphere formation process and positive Alcian blue staining of chondrocytes formed from mesenchymal stem cells. Fig. 15: Effect of different doses of liraglutide on lactate dehydrogenase secretion by chondrocytes into culture medium. Fig. 16: SOX9 RTqPCR analysis of knee joint structures in mice injected with liraglutide-treated monoiodoacetate. Fig. 17: Total joint score of injected knees of animals from group 5M treated with A1AGP vehicle and group 6M treated with A1AGP-formulated liraglutide (histology). Fig. 18: Representative photographs of right knee sections stained with toluidine blue from animals from vehicle-treated group 5M (A) and A1AGP-formulated liraglutide-treated group 6M (B). Specific details for implementing the invention

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

[0108] Before describing the invention in detail, it should be understood that, unless otherwise indicated, the invention is not limited to specific materials or methods of manufacture and may vary. It should also be understood that the terms used herein are merely for describing specific embodiments and are not intended to be limiting.

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

[0110] As used herein, the term “about” means approximately, area, nearly, or around. When the term “about” is used in conjunction with a numerical range, it modifies the range by extending the boundaries above and below the specified numerical value. Generally, the term “about” is used herein to modify numerical values ​​above and below the specified value by a 10% change. Thus, about 50% means the 45%–55% range. Numerical ranges cited herein as endpoints include all numbers and fractions contained within that range (e.g., 1 through 5 include 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). Furthermore, it should be understood that all numbers and fractions are presumed to be modified by the term “about.”

[0111] The term "chondrocyte" refers to a cell separated from cartilage.

[0112] The terms "cartilage," "articular cartilage," or "cartilage matrix" refer to the elastic, translucent connective tissue of mammals, including humans and other species. Cartilage consists primarily of chondrocytes, type II collagen, small amounts of other collagen types, other non-collagenous proteins, proteoglycans, and water, and is generally surrounded by a perichondrium containing fibroblasts in the matrix of type I and type II collagen as well as other proteoglycans. Most cartilage becomes bone upon maturation, but some cartilage remains in its original form in locations such as the nose, ears, knees, and other joints. Cartilage lacks a 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 substances or compounds that induce desired pharmacological or physiological effects and include formulations that are therapeutically effective and prophylactically effective. The terms also include pharmaceutically acceptable, pharmacologically active derivatives and analogs of such active agents specifically mentioned herein, which include, but are not limited to, salts, esters, amides, prodrugs, active metabolites, inclusion complexes, analogs, etc.

[0114] The terms "effective amount" or "therapeutically effective amount" of a pharmacological active agent or active excipient are intended to mean an amount of formulation or excipient sufficient to provide a desired therapeutic effect without toxicity. The "effective" amount will vary from person to person. Therefore, it is not always possible to specify an exact "effective amount." However, in any individual case, an appropriate "effective" amount may be determined by a person skilled in the art using routine experiments. Furthermore, the exact "effective" amount of the active agent incorporated into the composition or formulation of the present invention is not critical as long as the concentration is within a range sufficient to allow for the immediate application of the formulation to deliver an amount of the active agent within a therapeutically effective range.

[0115] The preferred route of administration is preferably in liquid form and applied locally to a peripheral area including a hydrogel or collagen matrix or an artificial scaffold (matrix), or to a damaged cartilage area, or to a (surgical) intervention site on or near the cartilage.

[0116] The present invention also relates to a pharmaceutical composition comprising a compound of the present invention. More specifically, such compound may be formulated into a pharmaceutical composition using standard pharmaceutically acceptable carriers, fillers, solubilizers, and stabilizers known to a person skilled in the art.

[0117] The present invention comprises the preparation and use of a pharmaceutical composition comprising a compound useful for treating a disease disclosed herein as an active ingredient. Such a pharmaceutical composition may consist of the active ingredient alone in a form suitable for administration to an individual, or the pharmaceutical composition may comprise the active ingredient and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or a combination thereof.

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

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

[0120] The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredient in the pharmaceutical composition of the present invention will vary depending on the identity, size, and pathological condition of the treated subject, and additionally depending on the route of administration of the composition.

[0121] For example, the above composition may contain 0.1% to 100% (w / w) of an active ingredient. In addition to the active ingredient, the pharmaceutical composition of the present invention may further include one or more additional pharmaceutically active agents.

[0122] As used herein, “additional ingredients” include, but are not limited to, one or more of excipients; surfactants; dispersants; inert diluents; granulators and disintegrants; binders; lubricants; sweeteners; flavorings; colorings; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing agents or wetting agents; emulsifiers, demulcants; buffers; salts; thickeners; fillers; emulsifiers; antioxidants; antibiotics; antifungals; stabilizers; and pharmaceutically acceptable polymers or hydrophobic materials.

[0123] The above composition also comprises one or more substances used for the treatment of osteoarthritis, particularly preferably one or more inhibitors of dipeptidyl peptidase IV enzyme selected from the group consisting of sitagliptin, saxagliptin, vildagliptin, alogliptin, and linagliptin, or analgesics, non-steroidal anti-inflammatories, steroidal anti-inflammatories, and slow-acting anti-arthritic agents.Analgesics containing paracetamol; Acetylsalicylic acid, lysine acetylsalicylate, phenylbutazone, sulindac, diclofenac, potassium or sodium, aceclofenac, tiaprofenic acid, ibuprofen, ketoprofen, alminoprofen, fenoprofen, naproxen, flurbiprofen, indomethacin, mefenamic acid, niflumic acid, tenoxicam, meloxicam, piroxicam, and celecoxib and etoricoxib It may include selective cyclooxygenase-2 inhibitors such as etoricoxib, betamethasone, dexamethasone, prednisolone, prednisone, thixocortol, or triamcinolone; chondroitin, chondroicin sulfate (Structum, Chondrosulf), glucosamine, or glucosamine sulfate, diacerein (Art 50, Zondar), or non-saponifiable avocado and soybean extracts (piascledine).

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

[0125] The formulations of the pharmaceutical compositions described herein may be prepared by any method known in the field of pharmacology or subsequently developed. Generally, such preparation methods include the step of combining an active ingredient with a carrier or one or more other auxiliary ingredients, and then, if necessary or preferable, the step of forming or packaging the product into desired single or multiple dose units.

[0126] A person skilled in the art will understand that such pharmaceutical compositions are generally suitable for administration to all types of animals. In a preferred embodiment, the individual or patient to be treated is an animal, preferably a mammal. According to one embodiment, the individual to be treated is an animal selected from the group consisting of dogs, cats, horses, cattle, sheep, pigs, and non-human primates.

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

[0128] The composition according to the present invention may be administered via any known route of administration, particularly systemic (parenteral, intravenous, etc.), oral, rectal, local, or subcutaneous. According to one preferred embodiment, the composition may also be administered by intra-articular injection, preferably to an arthritic joint. In this case, it may be administered in combination with other locally acting substances, such as hyaluronic acid, albumin, alpha-1 glycoprotein, or analgesics.

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

[0130] The frequency of dosage will be readily apparent to a person skilled in the art, and, though not limited thereto, will vary depending on several factors such as the type and severity of the condition or disease to be treated, the type and age of the animal, etc.

[0131] The pharmaceutical composition of the present invention may be manufactured, packaged, or sold in large quantities as a single unit dose or as multiple single unit doses.

[0132] The term "unit dose" is a distinct amount of a pharmaceutical composition containing a predetermined amount of an active ingredient. The amount of the active ingredient is generally equivalent to the dose of the active ingredient to be administered to an individual, or a convenient fraction of such a dose, such as, for example, 1 / 2 or 1 / 3 of such a dose.

[0133] The present invention also relates to a method of administering a compound of the present invention to an individual. In one embodiment, the present invention provides a method of treating an individual by administering a compound identified using the method of the present invention.

[0134] As used in this literature, the terms "treatment" or "therapy" refer to any action capable of reducing, suppressing, or delaying symptoms associated with a pathological condition. This includes both curative and prophylactic treatments for a disease. Curative treatment is defined as a treatment that results in healing or treatment that alleviates, improves, and / or eliminates, reduces, and / or stabilizes the symptoms of the disease or the suffering caused by it. Prophylactic treatment includes both treatments that prevent the disease and treatments that reduce and / or delay the risk of the onset or development of the disease.

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

[0136] As used herein, the term "therapeutically effective dose" refers to the amount necessary to observe therapeutic or preventive activity against osteoarthritis, in particular the amount necessary to observe inhibition or deceleration of arthritic cartilage destruction. The amount of peptide to be administered and the duration of treatment are evaluated by a person skilled in the art based on the physiological pathology of the subject to be treated and the nature of the arthritic joint(s) to be treated.

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

[0138] The present invention also relates to the use of liraglutide as an active ingredient in the manufacture of pharmaceutical preparations for the alleviation or reduction of joint irritation in mammalian individuals, or for the reduction of the exacerbation of existing joint inflammation.

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

[0140] According to one embodiment, a method for treating an inflammatory pathology in an individual requiring such treatment is provided. The method comprises the step of administering a pharmaceutical composition comprising at least one compound of the present invention to an individual requiring such treatment. The compound identified by the method of the present invention may also be administered together with a known compound or other drug.

[0141] All references mentioned in the description of the invention are incorporated into this application by reference. Other features and advantages of the invention will become more apparent when reading the following examples given as non-limiting examples.

[0142] Example 1: Effect of VICTOZA® on Cytokine Release in Chondrocytes of OA Patients

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

[0144] Materials and Methods

[0145] Test item: Victoza® (Novo Nordisk).

[0146] Reference Item: Water for Injection.

[0147] Cell culture materials: DMEM, fetal bovine serum, penicillin streptomycin, phosphate-buffered saline, Liberase Blendzyme 3, IL-1β.

[0148] Test systems: MMP3 ELISA kit, MMP13 ELISA kit, PGE2 ELISA kit, cytokine 30-Plex panel analysis. ELISA and multiplex analyses were performed according to the manufacturer's instructions.

[0149] Preparation of test and reference item formulations: Victoza stock solution is 6 mg / ml. The molecular weight is 3751.202 g / mol.

[0150] For each patient, 4 ml of medium containing 5, 25, 50, 125, and 625 nM of Victoza® was prepared. To this end, 5, 25, 50, 125, and 625 μM solutions were prepared as follows.

[0151] 5 μM (18.756 μg / ml): 6 mg / ml qsp 1.56 μl stock solution in 500 μl sterile water

[0152] 25 μM (93.78 μg / ml): 6 mg / ml qsp 1.56 μl stock solution in 100 μl sterile water

[0153] 50 μM (187.56 μg / ml): 6 mg / ml qsp 1.56 μl stock solution in 50 μl sterile water

[0154] 125 μM (468.2 μg / ml): 6 mg / ml qsp 1.56 μl stock solution in 20 μl sterile water

[0155] 625 μM (2344.5 μg / ml): 6 mg / ml qsp 1.95 μl of stock solution in 5 μl of sterile water.

[0156] These solutions were prepared for each patient and diluted 1:1000 in culture medium to reach the final concentration (4 μl in 4 ml). The vehicle consisted of 4 μl of sterile water in 4 ml of culture medium.

[0157] Preparation of the liberase solution: A 26 U / ml liberase stock solution was prepared in DMEM containing 1% P / S and 2% glutamine and stored at -20°C. For the two first digestion steps, a 0.52 U / ml solution was prepared fresh in DMEM. For the final digestion step, the 0.52 U / ml solution was diluted 1:4 to prepare a 0.13 U / ml solution.

[0158] Preparation of cell culture medium: 15% FBS, 2% L-glutamine, 1% penicillin / streptomycin in DMEM with 4.5 g / L glucose.

[0159] Experimental Design and Conditions

[0160] Cartilage was isolated from four osteoarthritis patients undergoing knee surgery with prosthetic insertion at Saint Antoine Hospital. The isolation, seeding, culture, and activation of chondrocytes and sample preparation were performed. ELISA and multiplex analysis were performed later.

[0161] The day the human joint cartilage was isolated was considered "Day 1," and the end of the study was considered "Day 14."

[0162] Isolation of chondrocytes from human cartilage: Cartilage was isolated from osteoarthritis patients undergoing knee surgery with implants. The cartilage from one patient was processed at a time. The newly 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 minutes. After 45 minutes, the Liberase was removed, and a fresh Liberase solution at 0.52 U / ml was added for 45 minutes. Then, the solution was removed, and the cartilage pieces were incubated overnight in 40 ml of Liberase (0.13 U / ml).

[0163] Chondrocyte Seeding: 16 hours after chondrocyte isolation, the cells were homogenized by pipetting the solution up and down. Subsequently, the solution was filtered through a 100 µm cell strainer. The filtered solution was centrifuged at 1600 rpm for 6 minutes at room temperature. The pellet was resuspended in 15 ml of complete medium (DMEM + 15% FBS + 2% glutamine + 1% P / S). Cells were counted using a hemocytometer and seeded into 12-well culture plates at a density of 200,000–250,000 cells per well. The cultures were incubated under sterile conditions (37 ℃, 5% CO2).

[0164] Chondrocyte culture: 48 hours after seeding, the medium was replaced with fresh medium. Thereafter, the medium was updated every 2 days until confluence occurred (12-13 days). At confluence, 24 hours prior to treatment, the medium was replaced with a medium free of FBS and containing 0.1% BSA.

[0165] The next day, chondrocytes were pre-incubated for 2 hours with 5 doses of Victoza® (5 nM, 25 nM, 50 nM, 125 nM, 625 nM) or vehicle, and then stimulated with IL-1β (5 ng / ml) for 24 hours according to the study design (Table 1) and schedule (Table 2).

[0166]

[0167]

[0168] Testing and Evaluation: ELISA and Multiplex Analysis

[0169] Sample preparation: At the end of each study, culture media were collected, centrifuged, and the supernatant was frozen. Samples were shipped from the testing facility on dry ice and stored at -80°C immediately upon receipt until analysis.

[0170] Detection analysis.

[0171] PGE2 Analysis: The analysis is based on competition between free PGE2 and the PGE2-acetylcholinesterase conjugate (PGE2 tracer) against a limited amount of PGE2 monoclonal antibody. While the concentration of the PGE2 tracer was kept constant, the free PGE2 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. Since the enzymatic reaction involves an acetylcholinesterase substrate, the color was barely intense in samples with high PGE2 concentrations and very intense in samples with low PGE2 concentrations.

[0172] The concentration was calculated by determining % B / B0 according to the manufacturer's instructions, where B0 represents the absorbance obtained by reading the well combined with the maximum amount of PGE2 tracer (in the absence of free PGE2) and B represents the absorbance obtained for each standard or sample well. To obtain accurate results, the supernatant from the IL-1β-treated wells was diluted 1:1000, and the remainder was diluted 1:500 before proceeding with the test. The measurement range for PGE2 is 7.8 to 1000 pg / ml.

[0173] MMP3 Analysis: This analysis is based on a classic sandwich ELISA combined with a colorimetric peroxidase system detection. To obtain accurate results, the supernatant from IL-1β-treated wells was diluted 1:1000, and the remainder was diluted 1:500 before proceeding with the test. The measurement range for MMP3 is 0.156 to 10 ng / ml.

[0174] MMP13 Analysis: The analysis was based on a classic sandwich ELISA combined with a colorimetric biotin-streptavidin system detection. To obtain accurate results, the supernatant from IL-1β-treated wells was diluted 1:100, and the remainder was diluted 1:10 before proceeding with the test. The measurement range for MMP13 is 8.23 ​​to 6000 pg / ml.

[0175] Multiplex Analysis: Luminex technology is based on the combination of ELISA sandwich technology and a mixture of fluorescent polystyrene beads representing a solid phase for detection. Each bead is bound to a specific antibody and a different fluorescent dye (fluorochrome). This analysis can quantify 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 / p70), IL-13, IL-15, IL-17, IP-10, MCP1, MIG, MIP1, MIP1β, RANTES, TNF-α, and VEGF.

[0176] The samples were analyzed without dilution in accordance with the manufacturer's instructions.

[0177] After the incubation was finished, the culture medium was collected and centrifuged, and then ELISA analysis (MMP3 ELISA kit, MMP13 ELISA kit, PGE2 ELISA kit, and cytokine 30-Plex panel analysis) was performed on the supernatant.

[0178] result

[0179] The concentration of each cytokine was calculated according to the manufacturer's guidelines.

[0180] The baseline inflammation profile of each patient was evaluated by accumulating the average concentrations calculated for each detected cytokine.

[0181] The results indicated that the underlying inflammation profiles were highly variable, including 2 patients with high concentrations of inflammatory cytokines and 2 patients with low concentrations of cytokines.

[0182] The inflammatory profile of the Victoza® response for each patient was evaluated by comparing the ratio of the mean concentrations of each cytokine calculated before and after Victoza® treatment. The general results showed varying responses among patients. However, a similar response to Victoza® was observed for four cytokines in chondrocytes of 3 out of 4 patients treated with Victoza® in the context of IL-1β. The mean calculated concentrations of GMCSF and CXCL10 / IP-10 (anabolic cytokines) increased, while IL7 and MCP1 (catabolic cytokines) decreased (Figure 1).

[0183] conclusion

[0184] The inventors demonstrated different response profiles in each of the four patients studied. Liraglutide increased the secretion of some anabolic cytokines and decreased the secretion of some catabolic cytokines in chondrocytes from OA patients.

[0185] Example 2: Preparation of an Intra-articular Formulation of Liraglutide with Extended Release - Determination of Release Profile

[0186] The inventors tested 20 different formulations of viscous hydrogels containing intra-articular injectable liraglutide to select three best formulations for additional in vivo preclinical studies and determined the release profile of each formulation into artificial synovial fluid.

[0187] Materials and Methods

[0188] Test material: Liraglutide.

[0189] Materials for hydrogel formulations

[0190] Dextran 70 EP (70 kDa),

[0191] Poloxamer 407: Kolliphor® P 407, oxyethylene 71,5-74,9%,

[0192] Polyethylene Glycol (PEG) 3350

[0193] Sodium alginate,

[0194] (Hydroxypropyl)methylcellulose (HPMC), viscosity 2,600-5,600 cP, 2% in H2O (20 ℃) ​​(lit.),

[0195] Albumin bovine fraction V, pH 7.0, Mr 67.000.00,

[0196] Polysorbate 80: Tween® 80

[0197] Lecithin from soybeans,

[0198] Polyethylene Glycol (PEG) 400,

[0199] Chitosan 95 / 500, high viscosity,

[0200] Sodium hyaluronate 1.9 MDa,

[0201] PBS pH 7.4

[0202] Materials for artificial synovial fluid preparations

[0203] Sodium Hyaluronate 1.9 MDa, Wellcos - Markus Grauel

[0204] Albumin bovine fraction V, pH 7.0, Mr 67.000.00, SERVA

[0205] γ-Globulin Sodium, Mr 150.000,00, SERVA

[0206] PBS pH 7.4, Panreac AppliChem

[0207] Test system for determining emission profile

[0208] Semipermeable membrane used: Dialysis tube Visking, cellulose, thickness 0.023 mm, MWCO 12-14 kDa

[0209] ELISA kit for determining liraglutide concentration

[0210] Artificial synovial fluid preparation: To prepare the artificial synovial fluid preparation, 3.5 mg of sodium hyaluronate, 9 mg of albumin, and 3.5 mg of γ-globulin were resuspended in PBS pH 7.4 every 1 ml. The volume of the prepared artificial synovial fluid was 10 ml for each experiment.

[0211] The composition of the artificial synovial fluid was selected based on many information sources (e.g., Biological Performance of Materials: Fundamentals of Biocompatibility. Fourth Edition, Jonathan Black, CRC Press, 20 gru 2005; 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).

[0212] Liraglutide preparation for stability study: For the stability study, 1 mg of liraglutide in 1.02 ml of PBS was mixed with 10 ml of artificial synovial fluid.

[0213] Test Procedure

[0214] Determination of Release Profile: The release profile was determined for each prepared formulation. On day 0, liraglutide hydrogel formulations (1 mg / ml - 1.02 ml) were placed in a semipermeable membrane test system immersed in 10 ml of artificial synovial fluid. Released molecules passed through the membrane into the artificial synovial fluid at 37 °C. This membrane was intended to allow the free diffusion of liraglutide monomers but served as a physical barrier for liraglutide oligomers and hydrogel formulations. Artificial synovial fluid samples (0.2 ml each) were collected after days 1, 2, 4, 7, 10, and 14. The collected samples were replaced with fresh artificial synovial fluid to maintain an equal volume of fluid outside the membrane. The artificial synovial fluid samples were stored at 2–8 °C until the end of the study for further ELISA analysis to determine liraglutide concentrations.

[0215] Stability study: To determine the stability of liraglutide under study conditions, a liraglutide solution was prepared at an appropriate concentration in artificial synovial fluid and treated in the same manner as the liraglutide hydrogel formulation.

[0216] ELISA Procedure: Quantification of liraglutide concentrations was performed using an ELISA kit, and the ELISA procedure was carried out according to the kit provider's recommendations with one exception. The standards for the calibration curves used had concentrations higher than suggested, ranging from 0.977 to 1000 ng / ml. Samples were diluted with EIA buffer. Standards and controls were prepared from a single stock solution of liraglutide. Analysis was performed on multiple EIA plates. The same standard and control solutions were used in all plates. The expected liraglutide concentrations in the samples after dilution ranged from 5 to 227 ng / ml. Three quality controls were used at concentrations covering the expected concentration ranges of the samples (15, 100, and 200 ng / ml). Each time point was measured in duplicate. Absorbance was measured at 450 nm using a 96-well plate reader.

[0217] Experimental Design and Conditions

[0218] Experiment Period: The day the formulation was prepared and membrane loading was performed was considered "Day 0," and the end of the study was considered "Day 14."

[0219] Group Design and Study Schedule: Twenty different hydrogel formulations containing 1 mg / ml of liraglutide were tested according to the study design (Table 3) and study schedule (Table 4). Three types of formulations were included.

[0220] Standard formulation (1,2,3,4,5,7,8,10,11,12,13,14,15,16),

[0221] Formulations having potential active excipients (17,18,19,20)

[0222] and albumin-based preparations (6,9).

[0223]

[0224]

[0225] result

[0226] The analysis of liraglutide was performed accurately and provided reliable results. Raw ELISA data, including calibration curves, are presented in Appendix II. In all plates, two of the three quality controls met the acceptance criteria, i.e., fell within ± 20% of the theoretical concentration. The acceptance criteria were accepted in accordance with the EMA guidelines for the validation of bioanalytical methods (EMEA / CHMP / EWP / 192217 / 2009 Rev. 1 Corr. 2**).

[0227] Liraglutide concentration: Artificial synovial fluid samples were diluted 1:400 before proceeding with ELISA staining. All samples collected at the first five time points were analyzed. Additionally, eight samples from the last time point were analyzed based on the consistency with the formulation at the start of the study.

[0228] Determination of release profile: The results were calculated as a percentage of the maximum expected liraglutide concentration for all hydrogel formulations. The amount of liraglutide taken from experiments using analytical samples was not included in the calculation. The expected liraglutide concentration in artificial synovial fluid (when all liraglutide is released from the semipermeable membrane) is 90.74 μg / ml (representing 100%).

[0229] The calculated percentages of the best formulations for the six best formulations are presented below (Table 5).

[0230]

[0231] Experiments conducted to determine the release profiles of 20 different formulations containing liraglutide showed variability among the tested formulations. Of the original 20 tested formulations, 6 provided liraglutide concentrations greater than 1% after 10 days. These are formulations 6, 8, 14, 17, 19, and 20, which have the release profiles shown in Figure 2.

[0232] Example 3: Efficacy Study of Three Liraglutide-Based Formulations Using a Surgically Induced Osteoarthritis Model in Rats

[0233] The purpose of this study is to evaluate the efficacy of three liraglutide-based preparations using a surgically induced osteoarthritis model in rats.

[0234] Materials and Methods

[0235] Test item: Liraglutide

[0236] Vehicle: PBS

[0237] Liraglutide formulations: Three viscous hydrogel formulations were tested:

[0238] Formulation 6: High-release

[0239] Formulation 8: Intermediate release

[0240] Formulation 20: Low release

[0241] Unformulated liraglutide corresponds to liraglutide dissolved in PBS.

[0242] For each of the three cycles, the formulation was prepared on the day of treatment. For each formulation, 4 mg of liraglutide was dissolved in 2 ml of the formulation solution or PBS (in the case of unformulated liraglutide) to reach a dose level of 0.18 mg / kg in 25 μl for intra-articular injection. Assuming an average body weight of 280 g, the dose administered to each rat was 50 μg. Liraglutide was used for animal administration within 1 hour of preparation.

[0243] experimental model

[0244] Animal Species / Strain: Rat / Sprague Dawley (SD)

[0245] Average Gender / Quantity / Weight: Male / 60 / 6-8 weeks at the start of the study

[0246] Diet: The animals were freely fed a commercial rodent diet.

[0247] Experimental Design and Conditions

[0248] Rats were assigned to one of five stratified study groups based on body weight.

[0249] Medial meniscectomy (MMx) after OA induction by medial ligamentectomy (MLT) procedure.

[0250] Anesthesia was induced in each rat using chamber induction techniques with inhalation anesthesia (4.0% isoflurane). During surgery, the animals were maintained at 1.5 to 2.5% isoflurane levels at an oxygen flow rate of 1–2 L / min. Ophthalmic ointment was applied to the eyes to prevent tissue dehydration during anesthesia. After anesthesia induction, hair was removed from the skin surface of the right leg using an electric animal clipper. After shaving the knee joint, the skin was disinfected with iodine, and the skin around the patella was incised on the medial side of the joint. The medial side of the joint space was incised. The medial ligament was incised, and the medial meniscus was resected using a microsurgical knife. The wounds were 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 schedule is shown in Table 7.

[0251]

[0252]

[0253] Testing and Evaluation

[0254] Changes in body weight bearing in rats with OA were measured using an incapacitance tester. Postural imbalance, known to indicate changes in pain threshold and limb weight distribution, was reduced. Each rat was positioned so that each hind leg rested on a separate force plate of the body weight bearing device, and the weight supported by each hind leg was measured for 5 seconds. The ratio of the weight supported by the right hind leg to the left hind leg was calculated. The average of 5 consecutive measurements was recorded for each rat. Body weight bearing function (body weight bearing test) was performed a total of 4 times at baseline (-1 day), 14 days, 28 days, and 35 days. The experimenter(s) were blinded to the group.

[0255] The rats were sacrificed by CO2 asphyxiation on day 36. The knee joint structures were fixed in a 4% buffered formalin solution for further histological analysis. The contralateral (undamaged) knee was also fixed in a 4% buffered formalin solution.

[0256] Numerical results were presented as mean ± SD. Outlier data points (indicated by asterisks) were identified after Grubbs’ test analysis with alpha = 5% and were not included in the calculation of group means. Where applicable, statistical analysis was performed using two-way (followed by Bonferroni post-hoc test) or one-way ANOVA (followed by Dunnett’s multiple comparison post-hoc test). A probability of 5% (p ≤ 0.05) was considered significant. In the plot, the degree of statistically significant difference between groups was indicated as *p≤0.05, **p<0.01, and ***p<0.001.

[0257] result

[0258] weight-bearing test

[0259] Changes in body weight bearing in rats with OA were evaluated using a weight bearing scale that independently measures the weight distributed by the animals to each hind paw. Prior to OA induction, all animals distributed their body weight equally to both hind paws. On day 14, a significant increase in the difference in body weight bearing (R / L weight %) between the control group and group 3M was observed with the prolonged release of liraglutide (Fig. 3).

[0260] Histological evaluation

[0261] A pathologist blinded the slides of the treatment group examined them. The knee cross-section was evaluated for the following parameters.

[0262] Cartilage matrix loss range (0% cartilage damage, 100% high range (tidemark), 50% mid range (Midzone))

[0263] Chondrogenic score (score 0-5, see Section 9.1).

[0264] Total cartilage degeneration range (0% cartilage is no damage, 100% is the highest range, 50% is the middle range). This includes all possible degenerative changes.

[0265] Significant cartilage degeneration width. A measurement exceeding 50% of the thickness is considered severely compromised (+ / -).

[0266] Ratio of zone depth of the lesion (microns).

[0267] Osteophytes (score 0-4, see Section 9.2).

[0268] Calcified cartilage and subchondral bone damage score (score 0-5, see Section 9.3).

[0269] Synovial response (score 0-4, see Section 9.4).

[0270] Medial joint capsule repair (unit: μm)

[0271] 10. Increase in plate thickness (Unit: μm)

[0272] The width of cartilage matrix loss (%) was lowest in Group 3M (35.8%) compared to the vehicle-treated control group 1M (43.6%). The width of total cartilage degeneration (%) was also lowest in Group 3M (37.5%) compared to the vehicle-treated control group 1M (43.2%). Bonferroni post-hoc comparisons following a two-way ANOVA revealed a statistically significant difference in cartilage degeneration between the control group and the liraglutide-treated animal group 3M, as shown in Figure 4 (*P<0.05). Mean + / - SD, n = 11-12.

[0273] conclusion

[0274] The objective of this study was to evaluate the efficacy of three liraglutide-based preparations using a surgically induced osteoarthritis model in rats.

[0275] The results of this study indicated that liraglutide in Formulation 6, corresponding to high release (Group 3M) of liraglutide, induced statistically significantly reduced OA damage on weight-bearing measurements using a weight-bearing meter compared to vehicle-treated animal controls 14 days after OA induction. The Frey test did not show statistically significant differences among all animal groups. Chondrodysplasia was also reduced in liraglutide treated with Formulation 6 compared to the control group and other formulations. Based on weight-bearing tests and histological evaluations, the OA rat model was clearly sensitive to the testing of novel therapeutic treatments.

[0276] Liraglutide formulated as formulation 6 has an in vivo chondroprotective effect compared to other formulations or unformulated liraglutide.

[0277] Example 4 - Dose-response study using an albumin-based formulation of liraglutide in a surgically induced osteoarthritis model in rats

[0278] The principle of the study is based on the evaluation of albumin-formulated liraglutide for the measurement of disease parameters in a rat OA model.

[0279] Materials and Methods

[0280] The test item is liraglutide and the positive control is dexamethasone (and unformulated liraglutide Victoza® (6 mg / ml injectable solution) from Novo Nordisk).

[0281] preparation

[0282] The vehicle (preparation excipient) consisted of albumin resuspended in phosphate-buffered saline for intra-articular injection (25 μl) in groups 1M and 7M.

[0283] Albumin-based formulations of liraglutide (high, medium, and low doses) were prepared as follows.

[0284] Liraglutide (supplied as a powder) was dissolved in a vehicle of an appropriate volume to reach dose levels of 0.18 mg / kg (for groups 2M and 8M), 0.06 mg / kg (for group 3M), or 0.02 mg / kg (for group 4M) in 25 μl for intra-articular injection. The formulation was prepared on the day of treatment for each of the three cycles.

[0285] The positive control used in Group 5M was dexamethasone. The human clinical dose for knee treatment is 4 mg / injection, which corresponds to 0.4 mg / kg in rats. Dexamethasone injectable solution was supplied "ready-to-use" at a concentration of 4 mg / ml. The volume administered to rats was 25–50 μl, depending on the rat mean BW on each treatment day.

[0286] Unformulated liraglutide (Victoza®) was administered as a 6 mg / ml stock solution. The human clinical "starting" dose for diabetic patients was 0.6 mg / day (administered via repeated SC injection), which corresponds to 0.06 mg / kg in rats. The Victoza® stock solution was diluted 1000-fold with injectable saline to reach a final SC injection concentration of 0.006 mg / ml for Group 6M (10 ml / kg).

[0287] experimental model

[0288] Animal: Rat / Strain: SD

[0289] Gender: Male / Number: 72 / Age: 6-8 weeks at study start

[0290] Source: Janvier Labs, France.

[0291] Initial body weight: The average body weight was 260 g at the start of the study (-1 day). The minimum and maximum body weights recorded in each group were within ± 20% of the group mean.

[0292] Diet: The animals were freely fed commercial rodent diet (Safe ref # A04). The animals were allowed to drink filtered osmotic water freely.

[0293] Contaminants: There were no contaminants in the feed and water supply that could affect the results of this test.

[0294] Experimental Design and Conditions

[0295] Definition of study start and end: The day OA was induced was defined as "Day 1". For the main study, the end of the study was "Day 36". For the study using satellite groups, the end of the study was "Day 57".

[0296] Treatment group assignment: Rats were randomly assigned to one of eight groups based on body weight.

[0297] Research Design and Schedule: The research was conducted in three cycles according to Table 1 for the research plan and Table 2 for the research schedule.

[0298] Resection of the medial meniscus after inducing OA via MLT (medial collateral ligament incision) procedure

[0299] Anesthesia was induced using chamber induction techniques with inhalation anesthesia (5.0% isoflurane). During surgery, the animals were maintained under isoflurane at a concentration of 1.5 to 3.5% at an airflow rate of 1–2 liters / min. Ophthalmic ointment was applied to the eyes to prevent tissue dehydration during anesthesia. After anesthesia induction, hair was removed from the skin surface of the right leg using an electric animal clipper. After shaving the knee joint, the skin was disinfected with iodine, and an incision was made in the peripapillary skin on the medial side of the joint. The exposed joint was accessed through a medial incision of the patellar tendon; the medial ligament was incised, and the medial meniscus was resected using a microsurgical knife. The wound was closed with Vicryl 5-0 sutures. All surgical procedures were performed using a surgical microscope. Group assignments are shown in Table 8, and the study schedule in Table 9.

[0300]

[0301]

[0302] Testing and Evaluation

[0303] Joint Edema: Knee diameter measurements were performed to infer joint edema as an indicator of inflammation. The diameters of both knees were measured using digital calipers during anesthesia in rats. Measurements were performed on the day before surgery (baseline), the day after surgery, and once a week until the end of the study. The experimenter(s) were blinded to the group.

[0304] Weight-bearing test: Changes in weight-bearing in rats after OA induction were monitored using a weight-bearing measurement system. Postural imbalances, known to indicate changes in limb pain thresholds and weight distribution, were tracked. Each rat was positioned so that each hind leg rested on a separate force plate on the weight-bearing device, and the weight supported by each hind leg was measured for 5 seconds. The ratio of the weight supported by the right hind leg to the left was calculated. The average of three consecutive measurements was recorded for each rat. The weight-bearing function (weight-bearing test) was performed on the animals a total of four times at baseline (-1 day), 14 days, 28 days, and 35 days. The experimenter(s) were blinded to the groups.

[0305] Animal Sacrifice and Tissue Fixation: Bleeding was performed on all animals on day 36 (end day for groups 1M–6M). Rats were euthanized with a lethal dose of Euthasol vet. The 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. Rats were euthanized on day 57 (end day for satellite groups 7M–8M). The 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.

[0306] Histological analysis: Histological analysis was performed. Knee joint sections were stained with hematoxylin-eosin or toluidine blue to assess the extent of pathological lesions. Slides were scored as in N. Gerwin et al., Osteoarthritis and Cartilage 18 (2010) S24-S34.

[0307] Histological analysis was performed:

[0308] Right knee (diseased): All groups harvested on day 36, 10 animals / group. Sample size: n = 60

[0309] Left knee (healthy group): Vehicle group 1M, 5 animals / group. Sample size: n = 5

[0310] Right knee (diseased): All animals in groups 7M–8M harvested at 57 days, 6 animals / group. Sample size: n = 12

[0311] Total number of samples: n = 77

[0312] Statistical Analysis: Numerical results were presented as mean ± standard deviation (SD). Outliers or excluded data points ($) were not included in the calculation of group means. Where applicable, statistical analysis was performed using two-way or one-way ANOVA (followed by Dunnett's multiple comparison post-hoc test). A probability of 5% (p ≤ 0.05) was considered significant. Results were presented in the figures as mean ± SEM, and the degree of statistically significant difference between groups was indicated as *p≤0.05, **p<0.01, and ***p<0.001.

[0313] result

[0314] For this study, six groups (1M-6M, n = 9-10 per group) were followed from -1 day to 36 days. Animal #2 of group 2M was excluded from the entire study because there were no lesions in the right knee joint in histological analysis.

[0315] Knee measurements (KM): Knee measurements were recorded before surgery, the day after surgery, and once a week thereafter. For each group, the average of the left and right knee diameters was calculated in two dimensions.

[0316] At the end, dose-response was observed in rats of Group 2M treated with IA high dose (width and thickness) of liraglutide and rats of Group 3M treated with IA medium dose (thickness) of liraglutide. As shown in Figure 5, rats of Group 6M treated SC with Victoza® (thickness) showed a significant decrease in knee measurements compared to vehicle-treated Group 1M.

[0317] Body Weight Loading Test: Changes in body weight load in rats with OA were evaluated using a body weight load meter that measures the weight distributed by the animal to each hind leg. Body weight load tests were performed on -1 day (baseline) and on days 14, 28, and 35. On day 14, a significant increase in the R / L ratio, expressed as %, was observed in Group 6M treated with Victoza®. Although not significant, this result was also observed at other measurement periods (days 28 and 35). A slight, insignificant trend in body weight differences was observed between the control group and animals treated with a medium dose (days 14 and 35 after OA induction) or a high dose (day 35).

[0318] Histological analysis

[0319] The left hind leg (animals 1M1, 1M9, 1M26, 1M31, and 1M57) was provided as a control. As expected, no lesions were observed in the knee joint.

[0320] The lesions observed in all groups ranged from significant to severe. The typically observed pattern was an intensive and extensive tearing of the cartilage, generally involving the entire medial tibial plate.

[0321] The margins of the ulcers were generally characterized by necrosis accompanied by chondrofibrillation and / or complete loss of the proteoglycan matrix. The subchondral bone was typically entangled with large fibroma bundles (fibrosis). At the osteochondral junction (highest extent) containing the friction zone, the subchondral bone exhibited necrosis (cells showing hyperacidophilia, caryorrexis, and pycnosis along with surrounding fibrin).

[0322] The dexamethasone IA treatment group (Group 5M) and the Victoza® SC treatment group (Group 6M) showed a tendency for lower cartilage loss. However, in some individuals, the tibial plate was partially replaced by fibrous tissue, forming synechia between the medial meniscus, femoral cartilage, and synovium as shown in Figure 6.

[0323] The observed repair changes were characterized by fibroma bundles within the synovial bursa in the dexamethasone IA treatment group (5M) and the Victoza® SC treatment group (6M), which sometimes exhibited large fibrous papillary hypervascular processes forming adhesions in all joints and other structures (medial meniscus remnants, articular cartilage, ligament remnants). In the liraglutide IA treatment group, no fibrosis was observed and no lesion differences were observed: chondrocyte aggregates were observed within the cartilage of the liraglutide IA treatment group with a dose-response pattern (Fig. 7): 3 animals / 9 in the high-dose group, 2 animals / 8 in the medium-dose group, and 1 animal / 9 in the low-dose group. In the Victoza® SC treatment group, only 1 in 7 animals showed chondrocyte aggregate proliferation, whereas none were found in the vehicle treatment group (0 animals / 10 animals).

[0324] This observed chondrocyte proliferation suggests an attempt at cartilage regeneration.

[0325] Satellite group research

[0326] For the study using satellite groups, two groups (7M-8M, n = 6 per group) were followed from -1 to 57 days. Histological parameters were measured as in the previous primary study. The recovery rate 3 weeks after discontinuation of treatment was evaluated and analyzed by comparing the high-dose liraglutide IA treatment group with the vehicle group.

[0327] Table 10. Satellite Group Study.

[0328]

[0329] The lesions observed overall for both groups were indicated. As observed in the primary study, the typically observed pattern was intensive and extensive tearing of the cartilage, including a large portion of the medial tibial plate. The margins of the ulcers were characterized by necrosis accompanied by cartilaginous fibrillation and / or complete loss of the proteoglycan matrix.

[0330] Animals treated with high-dose liraglutide IA (Group 8M) not only had lower substrate loss compared to vehicle-treated animals (Group 7M), but also showed a tendency to exhibit reduced loss throughout the study process in the liraglutide IA-treated group (Fig. 8).

[0331] No difference was observed in regression scores and total regression width. However, throughout the study process, the regression scores after treatment discontinuation were lower in the liraglutide IA treatment groups among Z1 and Z3 (Fig. 9).

[0332] There was no significant difference in osteophyte formation between the two groups. However, throughout the study, the percentage of osteophytes tended to be lower in the liraglutide IA-treated group after treatment discontinuation.

[0333] The high-dose treatment group showed more significant synovial repair, observed as membrane thickening, compared to the control group (Figs. 10 and 11).

[0334] An example of an aggregate diagram is presented in Fig. 12. As evaluated in Fig. 13, significantly more chondrocyte aggregates were observed in the high-dose test treatment group (5 animals / 6 animals; 30.6 ± 7.0 aggregates / μm²) compared to the vehicle group (2 animals / 5 animals; 8.1 ± 4.1 aggregates / μm²).

[0335] conclusion

[0336] As previously observed in major studies, significant histological changes were observed in each group regarding cartilage, subchondral bone, and synovium.

[0337] Changes in synovial repair were stronger in the IA treatment group using high-dose liraglutide compared to the vehicle group. Furthermore, changes in cartilage degeneration were less pronounced in the liraglutide treatment group compared to the vehicle group, but were not statistically different.

[0338] In particular, more chondrocyte aggregates were observed in the liraglutide IA-treated group, demonstrating attempts at regeneration within the cartilage.

[0339] Under the study conditions, dexamethasone IA and Victoza® SC treatments were able to alleviate several OA-related defects (histological findings related to knee edema, weight-bearing, and cartilage loss), but repairs were accompanied by fibrosis not observed in the IA treatment group.

[0340] Interestingly, histological analysis revealed the presence of chondrocyte aggregates within the cartilage of the group treated with liraglutide.

[0341] These figures show chondrocyte proliferation demonstrating an attempt at cartilage repair. In the main study (36 days), the presence of aggregates was not associated with synovial repair changes in the IA liraglutide-treated group. However, in the satellite group study (57 days), chondrocyte aggregates found in the animal group treated with IA at high doses of liraglutide were associated with more pronounced synovial repair changes (thickening).

[0342] The overall research results indicate that liraglutide targets associated mechanisms related to inflammation and regeneration processes associated with OA.

[0343] Example 5: Effect of liraglutide on chondrogenesis in an in vitro differentiation model of human mesenchymal stem cells

[0344] The inventors tested the effect of liraglutide on chondrogenesis in an in vitro differentiation model of human mesenchymal stem cells (hMSC) and evaluated whether liraglutide promotes chondrogenesis.

[0345] Materials and Methods

[0346] Test materials: Liraglutide.

[0347] Test System: Human mesenchymal stem cells (StemPro BM, Cat A15652, ThermoFisher Scientific).

[0348] Basic Badge: MesenPRO RS basal medium (ThermoFisher Scientific) supplemented with MesenPRO RS growth supplement, L-glutamine (1%), and gentamicin (10 mg / ml, 50 μl per 100 ml of medium).

[0349] Differentiation medium (Used as a positive control): StemPRO chondrogenic differentiation medium supplemented with StemPRO chondrogenic differentiation supplement and gentamicin (10 mg / ml per 100 ml of medium, 50 μl) (ThermoFisher Scientific).

[0350] procedure

[0351] Mesenchymal stem cells with a fusion rate of 60–80% were used. Cells were detached from the scaffold and 1.6 x 10⁶ cells per milliliter were placed in basal medium (MesenPRO RS basal medium + supplement). 7 A cell suspension of cells was prepared.

[0352] 5 μl of this suspension was placed in the center of each well of a 24-well plate. The plates were incubated in a 37°C incubator with high humidity for 2 hours. After 2 hours, 1 ml of basal medium with or without the test item (negative control) was added to each well according to Table 11 for the study design and Table 12 for the study schedule. 1 ml of differentiation medium (StemPRO Chondrocyte Differentiation Basal Medium + Supplement) was used as a positive control. The plates were placed back into an incubator at 37°C + 5% CO2 for 7, 14, or 21 days. During the differentiation phase, the medium was replaced every 3–4 days.

[0353]

[0354]

[0355] At the end of each study time (8, 15, or 22), plates were retrieved for Alcian blue staining and microscopic analysis. The medium was removed, and the cells were gently rinsed with 1 ml of PBS. The PBS was removed, and 1 ml of 4% formaldehyde was added at room temperature for 30 minutes.

[0356] The 4% formaldehyde was 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 0.1 N HCl) was added and the cells were protected from light at room temperature for 2 hours. The staining solution was removed, and the cells were washed 2 or 3 times with 1 ml of 0.1 N 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 photographs were taken.

[0357] result

[0358] The effect of liraglutide on sphere formation was evaluated by microscopic observation at 5-day intervals. In addition, Alcian blue staining was performed at three time points (e.g., 7, 14, and 21 days after treatment). This dye incorporation reflects the presence of sulfated glycosaminoglycans (GAGs) and confirms the formation of chondrocyte spheroids.

[0359]

[0360] As shown in Table 13, no sphere formation was observed in vehicle-treated cells in the basal medium during the study. Sphere formation was observed in a dose-response manner for 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 the wells treated on day 22, respectively. The differentiation medium contains all the reagents necessary to induce hMSCs to enter the chondrogenic pathway and generate chondrocytes. As expected, sphere formation was observed in vehicle-treated cells in this medium (83% of the wells treated on day 22). Alcian blue staining confirmed that the liraglutide-induced spheres were chondrocyte spheroids. These results indicate that hMSCs can be induced to differentiate into chondrocytes using liraglutide alone. An example of the sphere formation process and positive Alcian blue staining is presented in Figure 14.

[0361] conclusion

[0362] In this study, we tested the effect of liraglutide on this process using an in vitro analysis of cartilage formation.

[0363] The inventors demonstrated that liraglutide induced dose-response sphere formation in the presence of a basic medium, but no spheres were observed in vehicle-treated cells. Chondrocyte sphere formation was confirmed by Alcian blue positive staining (a cartilage matrix synthesis marker).

[0364] Under the study conditions, the inventors' data indicate that liraglutide alone induces hMSCs to enter the chondrogenic pathway and generate chondrocytes. This anabolic characteristic of liraglutide targets the resident stem cell population in the joint region to stimulate cartilage repair through chondrocyte differentiation, which is considered a promising approach for the treatment of OA.

[0365] Example 6: Effect of liraglutide on the viability of murine primary chondrocytes

[0366] The purpose of this study is to evaluate the effect of liraglutide on cell viability using murine primary chondrocytes.

[0367] Materials and Methods

[0368] Test materials: liraglutide

[0369] Test System: Murine primary chondrocytes

[0370] Cell culture medium preparation

[0371] 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, the experiment was conducted under FBS-free conditions using 2 mM L-glutamine, 0.1% bovine serum albumin (BSA), and 1% penicillin / streptomycin (P / S).

[0372] Experimental Design and Conditions

[0373] Definition of research initiation

[0374] The day the cells were plated in the well was considered "Day 1," and the end of the study was considered "Day 9."

[0375] procedure

[0376] Separation of murine articular cartilage

[0377] Immature murine chondrocytes were derived from neonatal mice (5-6 day old C57Bl / 6). This procedure was performed in a sterile flow hood. After euthanizing the mice by decapitating them with scissors, the animals were secured in a prone position, and the forelimbs were fixed with needles. The skin of the hind limbs was removed using scissors and forceps. The hind limbs were amputated along the spine. Remnants of skin and muscle were removed from the limbs. The feet were flattened with curved forceps to dismantle the small, translucent, and hard spheres corresponding to the femoral heads. Once the spheres were separated, they were placed in 30 ml of 1X PBS. The remaining foot contained no muscle or other tissues. The bone appeared reddish-brown, and the cartilage white. The bone was cut on each side of the white portion, forming the joints (forming two spheres). The joints were cleaned of surrounding tissue using a scalpel, then cut in half to separate the two spheres, and then cut in half again. This allows for easier digestion. The femoral condyles and tibial plateau were also placed in 30 ml of 1X PBS.

[0378] Isolation of immature murine chondrocytes

[0379] Cartilage pieces were incubated twice in a 100 mm Petri dish with 10 ml of digestion solution (DMEM, 2 mM L-glutamine + 1% P / S + 3 mg / ml collagenase) at 37°C with 5% CO2 for 45 minutes. Between the two digestions, the cartilage pieces were retrieved using a 25 ml pipette and placed in a new Petri dish. After the two digestions, a dispersion of aggregates was prepared using a 25 ml pipette. The cartilage pieces were incubated overnight in a 5% CO2 incubator at 37°C with 10 ml of 0.5 mg / ml collagenase D solution (diluted 1 / 6) in 2 mM L-glutamine + 1% P / S.

[0380] cartilage cell seeding

[0381] After overnight digestion, 10 ml of DMEM, 2 mM L-glutamine, and 10% FBS were added to each Petri dish to stop collagenase D activity. The medium and residual cartilage were collected and placed in 50 ml Falcon tubes. Aggregates were dispersed using a small pipette to obtain a separated cell suspension, which was filtered through a sterile 70 μm cell strainer. Then, the cells were centrifuged at 400 g at 20 °C for 10 minutes. 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 at 20 °C for 10 minutes, the PBS was removed, and 15 ml of DMEM, 2 mM L-glutamine, 10% FBS, and 1% P / S were added. Chondrocytes were counted using a Neubauer hemocytometer, and the extracted cells were observed to evaluate viability. Chondrocytes were cultured in 40x10 wells of 2 ml DMEM per well in 2 mM L-glutamine + 10% FBS + 1% P / S in a 12-well plate. 3 Seeding was performed at cell density. The culture was maintained under sterile conditions in an incubator at 37°C with 5% CO2.

[0382] Cartilage cell culture

[0383] Immature murine articular chondrocytes were confluent after 6–7 days. The culture medium was replaced after 3 days of culture. On day 7, the DMEM medium containing 10% FBS was removed, the wells were rinsed twice with 1 ml of PBS, and 1 ml of DMEM, 2 mM L-glutamine + 1% P / S + 0.1% BSA was added. On day 8, the medium was removed, and treatment with 12 different concentrations of liraglutide was performed in 500 μl of DMEM, 2 mM L-glutamine + 1% P / S + 0.1% BSA per well (Table 14). The plates were incubated at 37°C + 5% CO2 for 24 hours. The study schedule is presented in Table 15.

[0384]

[0385] Each condition processing was executed in triples.

[0386]

[0387] Testing and Evaluation

[0388] At the end of the study (day 9), culture medium (± 500 μl) from each well was collected in 1.5 ml tubes (one tube per well), centrifuged at 4000 rpm for 10 minutes at room temperature, and the supernatant was transferred to new 1.5 ml tubes. The samples were frozen at -70 ℃ until the dose was administered.

[0389] LDH analysis

[0390] Lactate dehydrogenase secretion into the culture medium was measured by LDH analysis (Abcam). 100 μl of the supernatant was used to measure the levels of lactate dehydrogenase secreted from damaged cells. LDH analysis was performed according to the procedure detailed in the instructions for the specific LDH analysis kit and analyzed using a plate reader (96-well) (Multiskan FC, Thermo Fisher). The wavelength for measuring absorbance was 450 nm. The average optical density (OD) of the read blank wells was subtracted from each reading.

[0391] result

[0392] Lactate dehydrogenase is a stable enzyme present in all cell types and is rapidly released into the cell culture medium upon plasma membrane damage. LDH enzyme was detected using enzyme coupling reactions and measured using SkanIt software on a Thermo Fisher microplate reader. LDH oxidizes lactate to produce NADH, which then reacts with the WST substrate to produce a yellow color. The intensity of the color is directly correlated with the number of eluted cells. LDH activity is OD 450nm It was quantified using a spectrophotometer. LDH activity was measured after 24 hours of incubation with 12 doses of liraglutide (1.7 nM–300 μM). A positive control was used by directly adding 5 μl of LDH enzyme to the wells. The % cytotoxicity was calculated by the following formula: ((Test Sample – Low Control) / (High Control – Low Control)) x 100.

[0393] As shown in Figure 15, the presence of the minimum test dose of liraglutide (max. 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 maximum dose of liraglutide (> 30 μM), the detected levels of lactate dehydrogenase increased significantly compared to the vehicle with a dose-response. Indeed, the calculated mortality percentages are 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). A positive control (indicated in yellow) was used to confirm that all reagents in the kit were functioning correctly.

[0394] conclusion

[0395] This study indicates that mortality may be observed in chondrocytes after 24 hours of incubation depending on the liraglutide test dose.

[0396] Example 6: SOX9 expression in the knee joint of monoiodoacetate-injected mice following intra-articular administration of an albumin-based liraglutide formulation

[0397] SOX9 is a pivotal transcription factor in developing and adult cartilage. The gene is expressed during the pluripotent skeletal progenitor stage and is activated throughout chondrocyte differentiation. While it is suppressed in hypertrophic chondrocytes of the chondroblast, it is expressed throughout life in permanent chondrocytes of healthy articular cartilage. SOX9 is necessary for chondrogenesis: it secures the commitment of chondrocyte lineages, promotes cell survival, and transcriptionally activates genes for many cartilage-specific structural components and regulatory factors.

[0398] The purpose of this study was to investigate SOX9 expression in the knee joints of monoiodoacetate (MIA)-injected mice after intra-articular administration of an albumin-based liraglutide formulation.

[0399] Materials and Methods

[0400] preparation

[0401] Monoiodoacetate (MIA):

[0402] MIA as a powder was resuspended in injectable saline, and 0.75 mg of 5 μl per mouse was injected into the knee joint for the 2M, 3M, 4M, and 5M groups.

[0403] Item preparations for treatment:

[0404] - The vehicle (preparation excipient) consisted of 5% human albumin resuspended in phosphate-buffered saline (PBS) for intra-articular injection (5 μl) in the 1M and 2M groups.

[0405] - Albumin-based formulations of liraglutide:

[0406] 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 per mouse in 5 μl for the 3M, 4M, and 5M groups, respectively.

[0407] experimental model

[0408] Animal species / strain

[0409] Mouse / C57Bl / 6

[0410] Gender / Age

[0411] Male / 12 weeks on Day 1

[0412] source

[0413] Janvier Labs, France.

[0414] Diet

[0415] The animals were freely fed a commercial rodent diet (Safe ref # A04). The animals had free access to filtered osmotic water.

[0416] Experimental Design and Conditions

[0417] Definition of research initiation

[0418] In this study, the day MIA was induced was defined as "Day 1," and the end of the study was defined as "Day 11."

[0419] Induction of OA by intra-articular (IA) injection in MIA

[0420] Animals were anesthetized via chamber induction techniques using inhalation anesthesia (isoflurane at 5%). During the procedure, animals were maintained under isoflurane at 1.5 to 3% levels at an airflow rate of 1–2 liters / min. The area around the knee joint was wiped with alcohol. 5 μl of MIA containing 0.75 mg was injected intra-articularly (IA) through the patellar tendon. A 30-gauge, 0.5-inch needle fitted with a duct insertion tube was used to ensure that only 2 to 3 mm of the needle penetrated the joint. After injection, the knee was massaged to ensure even distribution of the solution. Animals were injected once on Day 1 (Groups 2M, 3M, 4M, and 5M). For Group 1M (sham control), 5 μl of injectable saline was injected into the knee joint.

[0421] Research Design and Schedule

[0422] The study was conducted in three cycles according to Table 16 for the study design and Table 17 for the study schedule.

[0423]

[0424] Identical treatment mice were randomly assigned to group 1M on day 1. For MIA-injected mice, group assignment was performed on day 3 based on mouse BW.

[0425]

[0426] Testing and Evaluation

[0427] Animal sacrifice and tissue collection

[0428] Mice were euthanized on the 11th (end date). Knee joint structures (including synovium) were harvested and flash-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 was identified as the first transcription factor essential for chondrocyte differentiation and chondrogenesis.

[0429] result

[0430] SOX9 RTqPCR analysis of knee joint structure

[0431] As shown in Figure 16, vehicle mice (Group 2M) administered 0.75 mg of MIA to the knee joint on day 1 showed a 40% decrease in relative expression of SOX9 on day 11 compared to the vehicle same-treatment control group (Group 1M). When albumin-prepared liraglutide was injected intra-articularly into mice injected with MIA on day 3, SOX9 expression was restored and was similar to the same-treatment control group 1M for Groups 3M (10 μg liraglutide) and 4M (20 μg liraglutide). In the case of the 5M group injected with 30 μg liraglutide, the relative expression of SOX9 increased by 55% compared to the same-treatment control group 1M.

[0432] conclusion

[0433] The objective of the study was to perform RTqPCR for SOX9 on the knee joints of mice injected with monoiodoacetate after intra-articular administration of the formulated liraglutide.

[0434] 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 delivered to the knee joint disrupts chondrocyte glycolysis by inhibiting glyceraldehyde-3-phosphatase dehydrogenase, thereby inducing chondrocyte apoptosis in particular. Chondrocytes, which differentiate following the 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, the inventors demonstrated that while SOX9 expression decreases after MIA injection, intra-articular injection of the formulated liraglutide restored or increased the relative expression of SOX9 compared to healthy controls under the same treatment.

[0435] Therefore, this study indicated that locally administered albumin-formulated liraglutide targets the relevant mechanisms associated with anabolic action in MIA-induced OA and inflammatory pain models in mice.

[0436] Example 7: Efficacy Study of Liraglutide Alpha 1-Acid Glycoprotein-Based Formulation Using a Collagenase Type II-Induced Osteoarthritis Model in Rats

[0437] The purpose of this study is to conduct an efficacy study using an alpha 1-acid glycoprotein-based liraglutide preparation using a collagenase induction model in rats.

[0438] Materials and Methods

[0439] preparation

[0440] Collagenase type II:

[0441] Collagenase type II was dissolved in PBS at a concentration of 20,000 U / ml to deliver 500 U in 25 μl.

[0442] Item formulations for treatment:

[0443] The alpha 1-acid glycoprotein (A1AGP) vehicle consists of alpha 1-acid glycoprotein (25 μl) resuspended in 5% PBS for intra-articular injection.

[0444] - Alpha 1-acid glycoprotein-based formulations of liraglutide:

[0445] Liraglutide (supplied as an acid) was dissolved in an appropriate volume of alpha 1-acid glycoprotein vehicle to reach a dose level of 0.18 mg / kg in 25 μl for intra-articular injection.

[0446] experimental model

[0447] Species / Strain

[0448] Rat / SD

[0449] Gender / Number / Age

[0450] Male / 20 / 6-7 weeks at the start of research

[0451] source

[0452] Janvier Labs, France.

[0453] Animal care housing

[0454] Animal handling was performed in accordance with the guidelines of the Federation of European Laboratory Animal Science Associations (FELASA). Animals lived in plastic cages (2-3 animals per cage) and were provided with pellet food and drinking water in plastic bottles using a stainless steel top grill; bedding: steam-cleaned rice husks (Safe) were used and the bedding was replaced with the cage at least once a week.

[0455] Diet

[0456] The animals were freely fed a commercial rodent diet (Safe ref # A04). The animals had free access to filtered osmotic water.

[0457] Experimental Design and Conditions

[0458] Definition of research initiation

[0459] In this study, the day OA was induced was defined as "Day 1," and the end of the study was defined as "Day 43."

[0460] Induction of OA by intra-articular (IA) injection of collagenase type II

[0461] Animals were anesthetized via chamber induction techniques using inhalation anesthesia (isoflurane at 5%). During the procedure, animals were maintained under isoflurane at 1.5 to 3% levels at an airflow rate of 1–2 liters / min. Collagenase type II was injected intra-articularly (IA) at a volume of 25 μl containing 500 U. The animals were injected twice: the first injection on day 1 and the second injection on day 4.

[0462] Research Design and Schedule

[0463] Regarding the research design Table 18 and regarding the research schedule Table 19 It was performed according to.

[0464]

[0465]

[0466] Testing and Evaluation

[0467] weight

[0468] Body weight was recorded upon arrival, before the start of the study, and once a week thereafter.

[0469] Animal sacrifice and tissue fixation

[0470] Hemorrhages were performed on all animals on day 43 (end date). The rats were euthanized. The knee joint structures were harvested and fixed in 4% buffered formalin solution for further histological analysis. The contralateral (undamaged) knee was also fixed in 4% buffered formalin solution.

[0471] Histological analysis

[0472] Rat knees soaked in buffered 3.7% formalin were delivered to a subcontractor for histological analysis. Histological analysis was performed by a veterinarian (DVM, DESV-anatomical pathology) who was blinded to the group's handling and protocol throughout the entire analysis procedure. Knee joint sections were scored according to Osteoarthritis Cartilage. 2010 Oct;18 Suppl 3:S24-34.

[0473] Statistical analysis

[0474] Numerical results were presented as mean ± standard deviation (SD). Where applicable, statistical analysis was performed using two-way or one-way ANOVA (followed by Dunnett's multiple comparison post-hoc test) or t-tests. A probability of 5% (p≤0.05) was considered significant. In the figures, results were presented as mean ± SEM, and the degree of statistically significant difference between groups was indicated as * p≤0.05, ** p<0.01, and *** p<0.001.

[0475] result

[0476] The results showed a clear trend in which Group 6M (intra-articular administration of liraglutide in A1AGP vehicle) exhibited fewer cartilage lesions than Group 5M (A1AGP vehicle). To support 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): width of cartilage matrix loss (0-2), cartilage degeneration (0-5), width of cartilage degeneration (0-4), osteophytes (0-4), calcified cartilage and subchondral bone damage (0-5), and synovial inflammation (0-4). Figure 17 shows a significant decrease in the total joint score for Group 6M compared to Vehicle Group 5M.

[0477] Representative photographs of the right knee sections of the 5M and 6M group animals are shown in Fig. 18.

[0478] conclusion

[0479] Histological results suggest that local administration of liraglutide protects cartilage, as liraglutide IA induces less cartilage loss and a significant reduction in the total joint score compared to the vehicle.

[0480] The overall results of the study indicate that when administered topically, liraglutide exhibits excellent tolerance in a rat collagenase-induced OA model and targets associated mechanisms related to chondroprotection.

Claims

Claim 1 A pharmaceutical composition for use in cartilage regeneration comprising a glucagon-like peptide-1 analog, wherein the glucagon-like peptide-1 analog is selected from the group consisting of exenatide, lixisenatide, albiglutide, dulaglutide, semaglutide, and liraglutide. Claim 2 A pharmaceutical composition according to claim 1, wherein the glucagon-like peptide-1 analog is liraglutide. Claim 3 A pharmaceutical composition according to claim 2, wherein the concentration of the glucagon-like peptide-1 analog is 0.1 nM to 625 μM. Claim 4 A pharmaceutical composition according to claim 3, further comprising at least 5 weight % of a pharmaceutically acceptable formulation vehicle used in combination. Claim 5 A pharmaceutical composition according to claim 4, wherein the pharmaceutically acceptable formulation vehicle is selected from the group consisting of albumin and alpha 1-acid glycoproteins. Claim 6 A pharmaceutical composition for use in cartilage regeneration according to claim 5, wherein the pharmaceutically acceptable formulation vehicle concentration is 0.1% to 10% (wt / wt) of the formulation. Claim 7 A pharmaceutical composition according to claim 6, wherein the pharmaceutically acceptable formulation vehicle concentration is 5% (wt / wt) of the formulation. Claim 8 A pharmaceutical composition according to claim 7, wherein the pharmaceutically acceptable formulation vehicle is albumin. Claim 9 A pharmaceutical composition according to claim 8, wherein the pharmaceutically acceptable formulation vehicle is an alpha1-acid glycoprotein. Claim 10 A pharmaceutical composition according to any one of claims 1 to 9, wherein the composition is administered by intra-articular injection to a cartilage damage lesion. Claim 11 delete Claim 12 delete Claim 13 delete Claim 14 delete Claim 15 delete Claim 16 delete Claim 17 delete Claim 18 delete Claim 19 delete Claim 20 delete Claim 21 delete Claim 22 delete Claim 23 delete Claim 24 delete Claim 25 delete Claim 26 delete Claim 27 delete Claim 28 delete

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