Use of metformin to mitigate corticosteroid-associated osteonecrosis or osteoporosis

Abstract

Compositions and methods are provided for the prevention of osteonecrosis or osteoporosis associated with corticosteroid administration in an individual. The methods of the disclosure comprise administration of an effective dose of a metformin agent, or a derivative, mimetic or analog thereof in combination with administration of a corticosteroid. The effective dose of metformin agent decreases oxidative stress and restores osteogenic capacity of mesenchymal stem cells exposed to a glucocorticoid.

Description

ATTORNEY DOCKET NAME: STAN-2226WOCLIENT REFERENCE: S24-375USE OF METFORMIN TO MITIGATE CORTICOSTEROID-ASSOCIATED OSTEONECROSIS OR OSTEOPOROSISCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 696,263, filed September 18, 2024, which application is incorporated herein by reference in its entirety.BACKGROUND

[0002] Corticosteroids are widely used in clinical practice, such as in autoimmune disorders and chronic inflammatory diseases, for their potent anti-inflammatory and immunosuppressive effects. Despite their therapeutic benefits, corticosteroids are associated with several adverse effects that can significantly impact patient health, particularly in the musculoskeletal system. Corticosteroids inhibit osteoblast proliferation, reduce bone formation, and increase bone resorption. Additionally, corticosteroids induce oxidative stress and inflammation, which contribute to the pathology of osteonecrosis of the femoral head (ONFH) and bone and joint damage. ONFH may ultimately result in the necessity for surgical interventions, such as a hip arthroplasty. Although the development of ONFH is complex, this condition is significantly associated with the impact of corticosteroids on bone metabolism and cellular activity. Understanding the underlying mechanisms is crucial in exploring potential strategies to mitigate the adverse effects of corticosteroids on the musculoskeletal system.

[0003] Investigating potential interventions, including antioxidant therapies or agents that reduce oxidative damage, is crucial for mitigating the negative effects associated with corticosteroid use. Corticosteroids contribute to oxidative stress by increasing the production of reactive oxygen species (ROS) and impairing the body’s antioxidant defenses. For example, prednisolone has been reported to increase the level of ROS in corneal epithelial cells, while simultaneously reducing the cell viability. Subsequently, the elevated ROS damage cells and affect cell viability.

[0004] Methods of reducing the adverse effects of corticosteroids on bone are of great clinical interest, and are addressed by the present disclosure.SUMMARY

[0005] Compositions and methods are provided for the prevention of osteonecrosis or osteoporosis associated with corticosteroid administration in an individual. In some embodiments the osteonecrosis is osteonecrosis of the femoral head (ONFH). The methods of the disclosure comprise administration of an effective dose of a metformin agent, or aATTORNEY DOCKET NAME: STAN-2226WO CLIENT REFERENCE: S24-375 derivative, mimetic or analog thereof in combination with administration of a corticosteroid. In some embodiments metformin or a derivative, mimetic or analog thereof is administered concurrently with a corticosteroid. In some embodiments an effective dose of metformin or a derivative, mimetic or analog thereof is administered prior to, or shortly after administration of a corticosteroid. In some embodiments an effective dose of metformin or a derivative, mimetic or analog thereof is co-formulated with a corticosteroid. In some embodiments the drugs are separately formulated.

[0006] In some embodiments, the individual is treated with a corticosteroid for cancer, e.g. for a hematologic cancer such as leukemia, lymphoma, myeloma, etc. In some embodiments, the individual is treated with a corticosteroid for an atopic condition, e.g. asthma or severe allergies. In some embodiments, the individual is treated with a corticosteroid for an autoimmune condition, for example like systemic lupus erythematosus, rheumatoid arthritis, type I diabetes, multiple sclerosis, etc. In some embodiment the individual is treated with a corticosteroid for an inflammatory condition, e.g. chronic obstructive pulmonary disease (COPD), inflammatory bowel disease such as Crohn’s disease, ulcerative colitis; etc. In some embodiments, the individual is treated with a corticosteroid to reduce graft rejection or graft versus host disease. In some embodiments the individual is a juvenile, for example a human from birth to about 18 years of age. In some embodiment the individual is an adult. In some embodiments the individual is an aged adult.

[0007] In some embodiments the corticosteroid is a glucocorticoid. In some embodiments a glucocorticoid is, for example, one or more of beclomethasone; betamethasone; budesonide; cortisone; dexamethasone; hydrocortisone; methylprednisolone; prednisolone; prednisone; meprednisone; triamcinolone; paramethasone; and fluprednisolone. In some embodiment the corticosteroid is prednisone or prednisolone.

[0008] The administration of a metformin agent in combination with a glucocorticoid can prevent the development of corticosteroid-associated osteonecrosis during treatment. Administration of the corticosteroid prednisolone, for example, decreased cell proliferation, increased oxidative stress, decreased osteogenic differentiation and calcified matrix formation in mesenchymal stromal cells, the precursors for bone and cartilage cells. Metformin administration at concentrations above about 10 pM is shown to decrease oxidative stress and restored osteogenic capacity of MSCs exposed to prednisolone. Thus, targeting oxidative stress and increased inflammation with appropriate dosage of metformin provides a therapeutic option to alleviate impaired osteogenic differentiation in MSCs in corticosteroid- induced osteonecrosis. Metformin demonstrated significant positive effects on cell functions, including enhanced osteoblast activity and increased bone mineralization. Furthermore,ATTORNEY DOCKET NAME: STAN-2226WO CLIENT REFERENCE: S24-375 metformin effectively reduced oxidative stress, as evidenced by decreased ROS levels and increased ATP.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures.

[0010] FIGS. 1A-1 C. (A) Design and concentrations of prednisolone or metformin used in the experiments. (B, C) Mesenchymal Stem Cell (MSC) proliferation at day 3 of treatment. 3 ng / ml prednisolone significantly reduced the MSC proliferation; the addition of 10 pM metformin significantly improved cell proliferation. (*p<0.05, **p<0.01 , n=4 wells per group, PRED: prednisolone, MET: metformin)

[0011] FIGS. 2A-2B. Oxidative stress by cellular ROS. (A) Representative images of ROS expression and quantification of fluorescence intensity on day 2 of prednisolone 202 treatment. ROS signal was significantly increased in all groups of MSCs with prednisolone treatments at 3 ng / ml or above. (B) Metformin decreased ROS signal in a concentrationdependent manner. (*p<0.05, **p<0.01 , n=4 wells per group, PRED: prednisolone, MET: metformin)

[0012] FIGS. 3A-3B. Osteogenic differentiation of MSCs. Results of quantitative analysis of alkaline phosphatase (ALP)-positive area fraction. Positive ALP staining signals were significantly reduced at all concentrations of prednisolone compared to the control group. (B) The addition of metformin at 10 pM or above to 3 ng / ml of prednisolone increased ALP expression with statistical significant difference. (*p<0.05, **p<0.01 , n=4 wells per group, PRED: prednisolone, MET: metformin).

[0013] FIGS. 4A-4B. Calcified matrix formation of MSCs. (A) Quantitative analysis of the ratio of alizarin red (AR)-positive area. AR-positive area was significantly reduced at all concentrations of prednisolone compared to the control group (p < 0.01 ). (B) The addition of metformin above 10 pM to 3.0 ng / ml of prednisolone increased predominantly (p < 0.01 ). (*p<0.05, **p<0.01 , n=4 wells per group, PRED: prednisolone, MET: metformin).

[0014] FIG. 5. Assessment on inflammatory cytokines. Cytokine expression in the MSC culture supernatant on day 2 post prednisolone and / or metformin treatment. IL-1 beta was significantly higher in the PRED group (p < 0.05), while IL-6 and MCP-1 were significantly higher in the control group (p < 0.01 ). IL-10 and GM-CSF showed no significant differencesATTORNEY DOCKET NAME: STAN-2226WO CLIENT REFERENCE: S24-375 among groups. (*p<0.05, **p<0.01 , n=4 wells per group, PRED: prednisolone, MET: metformin)

[0015] FIGS. 6A-6D. Impact of different concentrations of prednisolone on MSCs viability and osteogenic potential. (A) Comparison of MSCs’ viability on day 7. (B) Representative fluorescent image of live / dead assay. Green dots represent live cells, while red dots represent dead cells. (C) Comparison of ALP level on day 7. (D) Comparison of ARS level on day 21 . (“ns” for “no significant difference”, *p < 0.05, ***p < 0.001 , ***‘p < 0.0001 ).

[0016] FIGS. 7A-7L. Metformin mitigates the osteogenic impairment by prednisolone in MSCs. (A) Statistical analysis of ROS level, (B) and the representative figures. (C) Statistical analysis of ATP level, (D) and the representative figures. (E) Representative figures of ALP level, and (F) the statistical analysis. (G) Representative figures of ARS expression, and (H) the statistical analysis. (I) qPCR comparison of osteogenic genes including RUNX2, ALPL, SPP1 , and SPARC. (L) qPCR comparison of adipogenic genes including PPARG, CEBPA, LPL, and PLIN1 . (“ns” for “no significant difference”, *p < 0.05, **p < 0.01 , ***p < 0.001 , ****p < 0.0001 ).

[0017] FIGS. 8A-8C. Impact of different concentration of prednisolone on macrophage viability and inflammation properties. (A) Histogram shows macrophages’ viability on day 7, and (B) representative fluorescent image of cells. (C) ELISA results of cytokine expressions of IL1 b, IL6, TNFa, IL10 and CCL18. (* p < 0.05, ” p < 0.01 , *** p < 0.001 , *“* p < 0.0001 .)

[0018] FIGS. 9A-9C. Metformin regulates macrophage functions. (A and B) Comparison of ROS level and ATP production in macrophages. (C) ELISA results of cytokine expressions of IL1 b, IL6, TNFa, IL10 and CCL18. (“ns” for “no significant difference”, *p < 0.05, **p < 0.01 , ***p < 0.001 , **** < 0.0001 ).

[0019] FIGS. 10A-10G The therapeutic effect of metformin on MSC-macrophage co-culture system. (A and B) Comparison of ROS expression in MSCs and macrophages in the coculture system, and (C) representative images. (D and E) Comparison of ALP and ARS on day 7 and day 21 representative. (F) qPCR comparison of osteogenic genes and adipogenic genes. (G) Cytokine secretion on day 2 and day 5. (“ns” for “no significant difference”, *p < 0.05, **p < 0.01 , ***p < 0.001 , ****p < 0.0001 ).DETAILED DESCRIPTION

[0020] Before the present methods and compositions are described, it is to be understood that this invention is not limited to particular method or composition described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.ATTORNEY DOCKET NAME: STAN-2226WOCLIENT REFERENCE: S24-375

[0021] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0022] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. It is understood that the present disclosure supercedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0023] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the peptide" includes reference to one or more peptides and equivalents thereof, e.g. polypeptides, known to those skilled in the art, and so forth.

[0024] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0025] As used herein, compounds which are "commercially available" may be obtained from commercial sources including but not limited to Acros Organics (Pittsburgh PA), Aldrich Chemical (Milwaukee Wl, including Sigma Chemical and Fluka), Apin Chemicals Ltd. (Milton Park UK), Avocado Research (Lancashire U.K.), BDH Inc. (Toronto, Canada), Bionet (Cornwall, U.K.), Chemservice Inc. (West Chester PA), Crescent Chemical Co. (Hauppauge NY), Eastman Organic Chemicals, Eastman Kodak Company (Rochester NY), Fisher Scientific Co. (Pittsburgh PA), Fisons Chemicals (Leicestershire UK), Frontier Scientific (Logan UT), ICN Biomedicals, Inc. (Costa Mesa CA), Key Organics (Cornwall U.K.), LancasterATTORNEY DOCKET NAME: STAN-2226WO CLIENT REFERENCE: S24-375 Synthesis (Windham NH), Maybridge Chemical Co. Ltd. (Cornwall U.K.), Parish Chemical Co. (Orem UT), Pfaltz & Bauer, Inc. (Waterbury CN), Polyorganix (Houston TX), Pierce Chemical Co. (Rockford IL), Riedel de Haen AG (Hannover, Germany), Spectrum Quality Product, Inc. (New Brunswick, NJ), TCI America (Portland OR), Trans World Chemicals, Inc. (Rockville MD), Wako Chemicals USA, Inc. (Richmond VA), Novabiochem and Argonaut Technology.

[0026] Compounds can also be made by methods known to one of ordinary skill in the art. As used herein, "methods known to one of ordinary skill in the art" may be identified though various reference books and databases. Suitable reference books and treatises that detail the synthesis of reactants useful in the preparation of compounds of the present invention, or provide references to articles that describe the preparation, include for example, "Synthetic Organic Chemistry", John Wiley & Sons, Inc., New York; S. R. Sandler et al., "Organic Functional Group Preparations," 2nd Ed., Academic Press, New York, 1983; H. O. House, "Modern Synthetic Reactions", 2nd Ed., W. A. Benjamin, Inc. Menlo Park, Calif. 1972; T. L. Gilchrist, “Heterocyclic Chemistry", 2nd Ed., John Wiley & Sons, New York, 1992; J. March, “Advanced Organic Chemistry: Reactions, Mechanisms and Structure”, 4th Ed., Wiley-lnterscience, New York, 1992. Specific and analogous reactants may also be identified through the indices of known chemicals prepared by the Chemical Abstract Service of the American Chemical Society, which are available in most public and university libraries, as well as through on-line databases (the American Chemical Society, Washington, D.C., may be contacted for more details). Chemicals that are known but not commercially available in catalogs may be prepared by custom chemical synthesis houses, where many of the standard chemical supply houses (e.g., those listed above) provide custom synthesis services.

[0027] "Comparable cell" shall mean a cell whose type is identical to that of another cell to which it is compared. Examples of comparable cells are cells from the same cell line.

[0028] "Inhibiting" the onset of a disorder shall mean either lessening the likelihood of the disorder's onset, or preventing the onset of the disorder entirely. In the preferred embodiment, inhibiting the onset of a disorder means preventing its onset entirely.

[0029] "Treating" a disorder shall mean slowing, stopping or reversing the disorder's progression. In the preferred embodiment, treating a disorder means reversing the disorder's progression, ideally to the point of eliminating the disorder itself. As used herein, ameliorating a disorder and treating a disorder are equivalent.

[0030] "Inhibiting" the expression of a gene in a cell shall mean either lessening the degree to which the gene is expressed, or preventing such expression entirely. "Specifically inhibit" the expression of a protein shall mean to inhibit that protein's expression (a) more than theATTORNEY DOCKET NAME: STAN-2226WO CLIENT REFERENCE: S24-375 expression of any other protein, or (b) more than the expression of all but 10 or fewer other proteins.

[0031] "Suitable conditions" shall have a meaning dependent on the context in which this term is used. When used in connection with contacting an agent to a cell, this term shall mean conditions that permit an agent capable of doing so to enter a cell and perform its intended function. In one embodiment, the term "suitable conditions" as used herein means physiological conditions.

[0032] Determining a therapeutically or prophylactically effective amount of the compositions can be done based on animal data using routine computational methods. In one embodiment, the therapeutically or prophylactically effective amount contains between about 0.01 mg and about 10 g of agent as applicable. In another embodiment, the effective amount contains between about 1 mg and about 5000 mg of agent, as applicable.

[0033] Administering the instant compositions can be effected or performed using any of the various methods and delivery systems known to those skilled in the art. The administering can be performed, for example, orally, intravenously, via implant, transmucosally, transdermally, intramuscularly, intrathecally, and subcutaneously. Oral administration may be preferred. The following delivery systems, which employ a number of routinely used pharmaceutical carriers, are only representative of the many embodiments envisioned for administering the instant compositions.

[0034] Oral delivery systems include tablets and capsules. These can contain excipients such as binders (e.g., hydroxypropylmethylcellulose, polyvinyl pyrilodone, other cellulosic materials and starch), diluents (e.g., lactose and other sugars, starch, dicalcium phosphate and cellulosic materials), disintegrating agents (e.g., starch polymers and cellulosic materials) and lubricating agents (e.g., stearates and talc).

[0035] Transmucosal delivery systems include patches, tablets, suppositories, pessaries, gels and creams, and can contain excipients such as solubilizers and enhancers (e.g., propylene glycol, bile salts and amino acids), and other vehicles (e.g., polyethylene glycol, fatty acid esters and derivatives, and hydrophilic polymers such as hydroxypropylmethylcellulose and hyaluronic acid).

[0036] Dermal delivery systems include, for example, aqueous and nonaqueous gels, creams, multiple emulsions, microemulsions, liposomes, ointments, aqueous and nonaqueous solutions, lotions, aerosols, hydrocarbon bases and powders, and can contain excipients such as solubilizers, permeation enhancers (e.g., fatty acids, fatty acid esters, fatty alcohols and amino acids), and hydrophilic polymers (e.g., polycarbophil and polyvinylpyrolidone). In one embodiment, the pharmaceutically acceptable carrier is a liposome or a transdermal enhancer.ATTORNEY DOCKET NAME: STAN-2226WOCLIENT REFERENCE: S24-375

[0037] Solutions, suspensions and powders for reconstitutable delivery systems include vehicles such as suspending agents (e.g., gums, xanthans, cellulosics and sugars), humectants (e.g., sorbitol), solubilizers (e.g., ethanol, water, PEG and propylene glycol), surfactants (e.g., sodium lauryl sulfate, Spans, Tweens, and cetyl pyridine), preservatives and Jun. 2,2005 antioxidants (e.g., parabens, vitamins E and C, and ascorbic acid), anti-caking agents, coating agents, and chelating agents (e.g., EDTA).

[0038] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a mammal being assessed for treatment and / or being treated. In an embodiment, the mammal is a human. The terms “subject,” “individual,” and “patient” thus encompass individuals having a disease of interest. Subjects may be human, but also include other mammals, particularly those mammals useful as laboratory models for human disease, e.g. mouse, rat, etc.

[0039] The definition of an appropriate patient sample encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived there from and the progeny thereof. The definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents; washed; or enrichment for certain cell populations, such as endometrial cells, etc. A sample if interest in bronchial lavage sample. The definition also includes sample that have been enriched for particular types of molecules, e.g., nucleic acids, polypeptides, etc. The term “biological sample” encompasses a clinical sample, and also includes tissue obtained by surgical resection, tissue obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow, blood, plasma, serum, and the like. A “biological sample” includes a sample obtained from a patient’s sample cell, e.g., a sample comprising polynucleotides and / or polypeptides that is obtained from a patient’s sample cell (e.g., a cell lysate or other cell extract comprising polynucleotides and / or polypeptides); and a sample comprising sample cells from a patient. A biological sample comprising a sample cell from a patient can also include normal, non-diseased cells.

[0040] Metformin agent. As used herein, the term metformin agent refers to metformin, or an analog, derivative or mimetic thereof. In an embodiment a metformin agent is metformin, which is a biguanide drug frequently used to treat Type 2 diabetes by lowering blood glucose levels. Its mechanism of action is primarily through the inhibition of hepatic gluconeogenesis, improvement in insulin sensitivity, and increased peripheral glucose uptake. Metformin is FDA approved and widely available, e.g. in oral dosage formulations.ATTORNEY DOCKET NAME: STAN-2226WO CLIENT REFERENCE: S24-375

[0041] Metformin agents may include analogs and derivatives of metformin, which have been developed to enhance its efficacy, reduce side effects, or explore its therapeutic potential beyond diabetes. Such compounds may also find use in the methods of the disclosure. For example, phenformin is chemically similar to metformin but is more lipophilic, leading to a stronger effect on glucose metabolism. Buformin is another biguanide analog of metformin. IM156 (Penta-O-galloyl-p-D-glucose) shares metformin’s inhibition of mitochondrial complex I. HL156A (HL271 ) is a structurally similar analog of metformin. Proguanil, and SYN-1 15 (Tozadenant-related derivatives) may also be included in this group. In a preferred embodiment the metformin agent is metformin.

[0042] In some embodiments metformin is administered at a dose that achieves an effectic therapeutic level on the blood of from about 0.1 to about 100 pM. The therapeutic dose is sufficient to relieve oxidative stress and restore osteogenic capacity of MSCs exposed to prednisolone. In some embodiments, a therapeutically effective dose leads to sustained serum levels of at least about 0.5 pM, of at least about 1 pM, of at least about 5 pM, of at least about 7.5 pM, of at least about 10 pM, and may be less than about 1 mM, less than about 500 pM, less than about 250 pM, less than about 100 pM, less than about 50 pM.

[0043] For example, an individual may be treated with a metformin agent at a dose of from about 100 mg / day, 200 mg / day, 300 mg / day, 500 mg / day, 750 mg / day, 1000 mg / day, 1250 mg / day, 1500 mg / day, 1750 mg / day, 2000 mg / day, 2250 mg / day to about 3000 mg / day, 2250 mg / day to about 2500 mg / day, for example in an oral formulation. Metformin may be combined in an oral formulation with an effective dose of a glucocorticoid.

[0044] In some embodiments, the dose of a metformin agent is a dose that provides for a plasma concentration of greater than about 1 pM and less than about 100 pM, for example achieving a circulating level of from about 5 pM to about 75 pM, from about 10 pM to about 60 pM, from about 10 pM to about 40 pM. For example, see He (2020) Metformin and Systemic Metabolism. Trends Pharmacol Sci. Nov;41 (1 1 ):868-881 , herein specifically incorporated by reference, which found that a flat dose of 2.5 g / day of metformin resulted in circulating metformin concentrations of around 10-40 pM in humans.

[0045] A metformin agent is administered in combination with a dose of a glucocorticoid agent, delivered in a dose effective to treat a condition of interest. In some embodiments a glucocorticoid is, for example, one or more of beclomethasone; betamethasone; budesonide; cortisone; dexamethasone; hydrocortisone; methylprednisolone; prednisolone; prednisone; meprednisone; triamcinolone; paramethasone; and fluprednisolone. In some embodiment the corticosteroid is prednisone, methylprednisolone, or prednisolone.ATTORNEY DOCKET NAME: STAN-2226WO CLIENT REFERENCE: S24-375

[0046] The dose of glucocorticoid may be selected based on the intended treatment. The dosage may be conventional as is known in the art. The dose may be equivalent to the following prednisone dosages. For example, a low dose of prednisone for maintenance, treatment of chronic conditions such as autoimmune disease, etc. may be a flat dose equal to or less than about 7.5 mg / day, e.g. from about 2.5 to about 7.5 mg / day. A moderate dose for treatment of conditions such as asthma exacerbations, rheumatoid arthritis, or moderate inflammation may range from about 7.5 mg / day to about 30 mg / day. A high dose of prednisone for short-term treatment of severe inflammation, autoimmune flares, cancer, etc. may range from about 30 mg / day to about 100 mg / day.

[0047] Osteonecrosis (ON) is a disabling clinical disease characterised by death of the osteocytes and the bone marrow, followed by resorption of the necrotic tissues and formation of new but weaker osseous tissue which leads to a progressive destruction of bone architecture, subchondral fracture, and collapse of joints, mostly occurring at the femoral head, and finally loss of joint function. ON occurs mostly in large weight-bearing joints, such as the hip that may subsequently develop to joint collapse and end up with joint replacement surgery.

[0048] An individual selected for treatment with the methods disclosed herein may be diagnosed with osteonecrosis, or at risk of osteonecrosis. For example, steroid-associated ON (SAON) is a common nontraumatic ON caused by use of steroids, which are initially prescribed for many nonorthopaedic medical conditions, including systemic lupus erythematosus (SLE), organ transplantation, asthma, rheumatoid arthritis, and severe acute respiratory syndrome (SARS). ON in large joints caused by corticosteroid administration is usually associated with the worst prognosis due to the degeneration of the bone around the prosthesis. ON development is associated with dose and duration of administrating corticosteroids. Frequently the chronic administration of high-dose steroids will result in ON. The interval between steroid administration and the ON onset can be from 6 months to > 3 years. Using MRI to detect early ON of the femoral head has shown that the initial changes of necrosis were found at about 3 months after administration of steroids.

[0049] Avascular necrosis of the femoral head is a type of osteonecrosis due to disruption of blood supply to the proximal femur. Chronic steroid use and excessive alcohol consumption represent the bulk of non-traumatic etiologies, contributing to more than 80% of them. Steroid- associated osteonecrosis represents the second most common cause of osteonecrosis overall, after trauma. Despite evidence demonstrating the correlation between steroid use and osteonecrosis, the exact pathophysiology is not clear and probably multifactorial. The cause is most likely an aggregate of factors such as fat emboli, fat cell hypertrophy leading to increased intraosseous pressure, endothelial dysfunction, hyperlipidemia, and abnormality ofATTORNEY DOCKET NAME: STAN-2226WO CLIENT REFERENCE: S24-375 the stem cell pool of the bone marrow; all of which contribute to ischemia and subsequent necrosis. Autoimmune and chronic inflammatory disorders, e.g., systemic lupus erythematosus (SLE), are well-known to be associated with osteonecrosis of the femoral head. The risk of developing the condition in these patients is usually attributed to long-term steroid treatment.

[0050] The exact pathophysiologic mechanisms behind avascular necrosis of the femoral head are not always clear and generally regarded as being multifactorial. Regardless of the precipitating factor, the outcome is essentially the death of osteocytes and bone marrow that results from insufficient blood flow to the subchondral bone of the proximal femur. This cell death will inevitably lead to a collapse of the femoral head and subsequent osteoarthritis, if not treated effectively in the early stages.

[0051] Patients may be asymptomatic early on in the disease process. When they become symptomatic, however, the stated history is usually of hip pain that may radiate to the groin and / or thigh. The pain is typically aggravated by activities such as walking and climbing stairs and alleviated by rest. The pain will often still be present, even in the absence of movement. Some examples of physical exam findings indicative of osteonecrosis of the femoral head are restricted range of motion, pain upon abduction and internal rotation, and tenderness to palpation of the hip region. Treatments are best implemented at the pre-collapse stage and include both operatives as well as non-operatives options. If left untreated, femoral head necrosis may lead to subchondral fractures within only 2 to 3 years.

[0052] Osteoporosis is defined as low bone mineral density caused by altered bone microstructure, ultimately predisposing patients to low-impact, fragility fractures. Osteoporotic fractures lead to a significant decrease in quality of life, increasing morbidity, mortality, and disability. Over 50% of postmenopausal white women will have an osteoporotic-related fracture. Only 33% of senior women who have a hip fracture will be able to return to independence.

[0053] Primary osteoporosis is related to the aging process in conjunction with decreasing sex hormones. The bones demonstrate deterioration in microarchitecture, leading to loss of bone mineral density and increased risk of a fracture. Importantly, medications that can lead to secondary osteoporosis include glucocorticoids.

[0054] Osteoporosis is caused by an imbalance of bone resorption and bone remodeling, leading to decreased skeletal mass. In most individuals, bone mass peaks in the third decade, after which bone resorption exceeds bone formation. Failure to reach a normal peak bone mass or acceleration of bone loss can lead to osteoporosis.ATTORNEY DOCKET NAME: STAN-2226WO CLIENT REFERENCE: S24-375

[0055] Dual X-ray absorptiometry tests scans of the central skeleton are the best test for assessing bone mineral density. Dual X-ray absorptiometry scans measure all calcified tissue in the path of the scan, and specificity is better than sensitivity. A dual X-ray absorptiometry scan reports the t-score and a z-score. A t-score reflects the difference between the measured bone mineral density and the mean value of bone mineral density in young adults. It is measured in standard deviations. The WHO has defined normal bone mineral density for women as a t-score within one standard deviation of the young adult mean. Scores between negative 1 and negative 2.5 reflect a diagnosis of osteopenia. Scores below negative 2.5 reflect a diagnosis of osteoporosis.

[0056] An individual selected for treatment with the methods disclosed herein may be diagnosed with osteoporosis, or at risk of osteoporosis.

[0057] The methods of the disclosure may be combined with conventional treatment of osteonecrosis or osteoporosis. For example, pharmacotherapy agents may be prescribed, which work through either anti-resorptive or anabolic means. Bisphosphonates are the most commonly prescribed medication class. These drugs are divided into non-nitrogen and nitrogen-containing compounds. The latter are considered first-line therapy. The nitrogencontaining compounds inhibit farnesyl pyrophosphate synthase and ultimately inhibit osteoclast resorption and induce osteocyte apoptosis. Common agents include: Alendronate may reduce the rate of hip, spine, and wrist fractures by 50%; Risedronate may reduce vertebral and nonvertebral fractures by 40% over three years; IV zoledronic acid reduces the rate of spine fractures by 70% and hip fractures by 40% over three years.

[0058] As used herein, the terms “treatment,” “treating,” and the like, refer to administering an agent, or carrying out a procedure for the purposes of obtaining an effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of effecting a partial or complete cure for a disease and / or symptoms of the disease. “Treatment,” as used herein, covers any treatment of a mammal, particularly in a human, and includes: (a) preventing the development of osteonecrosis or osteoporosis; (b) inhibiting ongoing osteonecrosis or osteoporosis, i.e., arresting its development; and (c) relieving osteonecrosis or osteoporosis, i.e., causing regression of disease.

[0059] Treating may refer to any indicia of success in the treatment or amelioration or prevention of disease, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degenerationATTORNEY DOCKET NAME: STAN-2226WO CLIENT REFERENCE: S24-375 less debilitating. The treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of an examination by a physician. Accordingly, the term "treating" includes the administration of the compounds or agents of the present invention to prevent or delay, to alleviate, or to arrest or inhibit development of the symptoms or conditions associated with osteonecrosis or osteoporosis. The term "therapeutic effect" refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in the subject.

[0060] "In combination with", "combination therapy" and "combination products" refer, in certain embodiments, to the concurrent administration to a patient of a first therapeutic (i.e., first therapeutic agent) and a second therapeutic agent. When administered in combination, each component can be administered at the same time or sequentially in any order at different points in time. Thus, each component can be administered separately but sufficiently closely in time so as to provide the desired therapeutic effect.

[0061] "Concomitant administration" of a known therapeutic agent with a pharmaceutical composition of the present invention means administration of two therapeutic agents at such time that both the agents will have a therapeutic effect. Such concomitant administration may involve concurrent (i.e. at the same time), prior, or subsequent administration of a drug with respect to the administration of a compound according to the present disclosure. A person of ordinary skill in the art would have no difficulty determining the appropriate timing, sequence and dosages of administration for particular drugs and compositions of the present invention.

[0062] As used herein, the term “correlates,” or “correlates with,” and like terms, refers to a statistical association between instances of two events, where events include numbers, data sets, and the like. For example, when the events involve numbers, a positive correlation (also referred to herein as a “direct correlation”) means that as one increases, the other increases as well. A negative correlation (also referred to herein as an “inverse correlation”) means that as one increases, the other decreases.

[0063] "Dosage unit" refers to physically discrete units suited as unitary dosages for the particular individual to be treated. Each unit can contain a predetermined quantity of active compound(s) calculated to produce the desired therapeutic effect(s) in association with the required pharmaceutical carrier. The specification for the dosage unit forms can be dictated by (a) the unique characteristics of the active compound(s) and the particular therapeutic effect(s) to be achieved, and (b) the limitations inherent in the art of compounding such active compound(s).

[0064] "Pharmaceutically acceptable excipient "means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use.ATTORNEY DOCKET NAME: STAN-2226WOCLIENT REFERENCE: S24-375Such excipients can be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.

[0065] The terms "pharmaceutically acceptable", "physiologically tolerable" and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a human without the production of undesirable physiological effects to a degree that would prohibit administration of the composition.

[0066] A "therapeutically effective amount" means the amount that, when administered to a subject for treating a disease, is sufficient to effect treatment for that disease.

[0067] The phrase “determining the treatment efficacy” and variants thereof can include any methods for determining that a treatment is providing a benefit to a subject. The term “treatment efficacy” and variants thereof are generally indicated by alleviation of one or more signs or symptoms associated with the disease and can be readily determined by one skilled in the art. “Treatment efficacy” may also refer to the prevention or amelioration of signs and symptoms of toxicities typically associated with standard or non-standard treatments of a disease. Determination of treatment efficacy is usually indication and disease specific and can include any methods known or available in the art for determining that a treatment is providing a beneficial effect to a patient. For example, evidence of treatment efficacy can include but is not limited to remission of the disease or indication. Further, treatment efficacy can also include general improvements in the overall health of the subject, such as but not limited to enhancement of patient life quality, increase in predicted subject survival rate, decrease in depression or decrease in rate of recurrence of the indication (increase in remission time). (See, e.g., Physicians' Desk Reference (2010).)Methods

[0068] Methods are provided for treating a subject with a therapeutic dose of a metformin agent in combination with a glucocorticoid treatment to prevent or reduce incidence of osteonecrosis or osteoporosis. The subject methods include a step of administering the glucocorticoid, in combination with, or followed by a step of administering a therapeutically effective dose of the metformin agent to the subject. In an embodiment the agents are coformulated. In an embodiment, the agents are separately formulated. In some embodiments the agents are administered concomitantly, e.g. simultaneously. In some embodiments the metformin agent is administered shortly after administration of the glucocorticoid.

[0069] In some embodiments, the step of administering the metformin agent is performed within at least about 3 days, e.g., within at least about 4 days, within at least about 5 days, within at least about 6 days, within at least about 7 days, within at least about 8 days, withinATTORNEY DOCKET NAME: STAN-2226WO CLIENT REFERENCE: S24-375 at least about 9 days, within at least about 10 days, within at least about 2 weeks, within at least 3 weeks after administration of the glucocorticoid. This period of time is, for example, sufficient to provide for decreased oxidative stress and restored osteogenic capacity of mesenchymal stem cells by the individual.

[0070] Dosage and frequency may vary depending on the half-life of the agent in the patient. It will be understood by one of skill in the art that such guidelines will be adjusted for the molecular weight of the active agent. The dosage may also be varied for localized administration, e.g. oral, intranasal, inhalation, etc., or for systemic administration, e.g. i.m., i.p. , i.v., and the like. The effective dose may range from about 100 mg / day, 200 mg / day, 300 mg / day, 500 mg / day, 750 mg / day, 1000 mg / day, 1250 mg / day, 1500 mg / day. 1750 mg / day. 2000 mg / day, 2250 mg / day to about 2500 mg / day, for example in an oral formulation. Metformin may be combined in an oral formulation with an effective dose of a glucocorticoid.

[0071] The metformin agent may be administered one or a plurality of days, and in some embodiments is administered daily, every two days, semi-weekly, weekly, etc. for a period of from about 1 , about 2, about 3, about 4, about 5, about 6, about 7 or more weeks, up to a chronic maintenance level of dosing. Alternatively, therapeutic entities of the present invention can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the polypeptide in the patient.

[0072] In some embodiments, for prophylactic applications, pharmaceutical compositions or medicaments are administered to a patient susceptible to, or otherwise at risk of osteonecrosis in an amount sufficient to eliminate or reduce the risk, lessen the severity, or delay the outset of the disease, including biochemical, histologic and / or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease.

[0073] According to the present invention, compositions can be administered by oral, parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intracranial, intraperitoneal, intranasal, aerosol, or intramuscular means. The most typical route of administration is intravenous although other routes can be equally effective.

[0074] For parenteral administration, compositions of the invention can be administered as injectable dosages of a solution or suspension of the substance in a physiologically acceptable diluent with a pharmaceutical carrier that can be a sterile liquid such as water, oils, saline, glycerol, or ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, surfactants, pH buffering substances and the like can be present in compositions. Other components of pharmaceutical compositions are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, and mineral oil. In general, glycols suchATTORNEY DOCKET NAME: STAN-2226WO CLIENT REFERENCE: S24-375 as propylene glycol or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions. Antibodies and / or polypeptides can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained release of the active ingredient.

[0075] The pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration. Preferably, a therapeutically effective dose will provide therapeutic benefit without causing substantial toxicity.

[0076] Toxicity of the agent described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD50 (the dose lethal to 50% of the population) or the LD100 (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index. The data obtained from these cell culture assays and animal studies can be used in formulating a dosage range that is not toxic for use in human. The dosage of the agents described herein lies preferably within a range of circulating concentrations that include the effective dose with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See, e.g., Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, Ch. 1 ).

[0077] Also within the scope of the invention are kits comprising the compositions of the invention and instructions for use. The kit can further contain a least one additional reagent. Kits typically include a label indicating the intended use of the contents of the kit. The term label includes any writing, or recorded material supplied on or with the kit, or which otherwise accompanies the kit.

[0078] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible. It is also understood that the terminology used herein is for the purposes of describing particular embodiments

[0079] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changesATTORNEY DOCKET NAME: STAN-2226WOCLIENT REFERENCE: S24-375 and modifications may be made thereto without departing from the spirit or only and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[0080] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the appended claims.EXPERIMENTAL

[0081] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.Example 1Metformin Modulates Oxidative Stress in Murine Mesenchymal Stem Cells In Vitro and Alleviates Corticosteroid-Induced Inflammation and Impairment of Bone Formation

[0082] Corticosteroid use is a known risk factor for developing osteonecrosis of the femoral head (ONFH). Corticosteroids disrupt the balance between oxidative and glycolytic energy metabolism, increases oxidative stress and reactive oxygen species (ROS); associated with prolongation of inflammation, cell apoptosis, deficits in mesenchymal stem cell (MSC) and osteoclast differentiation. Metformin, a drug for diabetes, has antioxidant properties by inhibiting NADPH oxidase (NOX), which promotes the production of ROS. The purpose of this study was to evaluate the effects of corticosteroid and metformin administration on MSCs in vitro.

[0083] Primary mouse bone marrow MSCs were used to evaluate effects of prednisolone on cell proliferation, oxidative stress, osteogenic differentiation, and mineralization; then, the effect of metformin on corticosteroid-induced inhibition of bone formation. Three different concentrations (1 pM, 10pM, 100pM) of metformin were added to 3 ng / ml prednisolone, followed by assessments above. Cytokines in the supernatant were assessed using the Luminex assay during the acute phase. One-way analysis of variance with Tukey's multipleATTORNEY DOCKET NAME: STAN-2226WO CLIENT REFERENCE: S24-375 comparison was used for comparisons between multiple groups at p<0.05 considered statistically significant.

[0084] Prednisolone at 3 ng / ml significantly reduced cell proliferation; the addition of 10 pM metformin significantly restored cell proliferation (p < 0.05). Oxidative stress evaluated by ROS production was significantly increased with prednisolone (p < 0.05); metformin decreased ROS in a concentration-dependent manner (p < 0.01 ), with the greatest decrease at concentration of 100 pM. Osteogenic differentiation and mineralization were significantly reduced with prednisolone administration above 3 ng / ml (p < 0.01 ), while negative effects were alleviated with the addition of 10 pM and 100 pM metformin to prednisolone administration at 3 ng / ml (p < 0.01 ). Interleukin 1 -beta, a proinflammatory cytokine, was increased with prednisolone administration, however it was significantly decreased with metformin administration.

[0085] Prednisolone administration decreased cell proliferation, increased oxidative stress, decreased osteogenic differentiation and calcified matrix formation in MSCs. In contrast, metformin administration at concentrations above 10 pM decreased oxidative stress and restored osteogenic capacity of MSCs exposed to prednisolone. Targeting oxidative stress and increased inflammation with appropriate dosage of metformin may be a promising therapeutic option to alleviate impaired osteogenic differentiation in MSCs in corticosteroid- induced ONFH.

[0086] Osteonecrosis of the femoral head (ONFH) is a progressive bone disease that typically results in femoral head collapse and hip dysfunction. Corticosteroid use is a known risk factor for developing ONFH, however the exact biological mechanisms are unknown and potential preventive interventional strategies are very limited. Corticosteroids disrupt the physiological balance between oxidative and glycolytic energy metabolism leading to increases in oxidative stress and production of reactive oxygen species (ROS); these events are associated with prolongation of inflammation, cell apoptosis, and osteoclast differentiation. ROS are present in the physiological environment and are involved in signal transduction, immune regulation, and aging by inducing apoptosis. Once the redox balance is disrupted, the generation of ROS adversely affects oxidative stress and triggers tissue damage and various pathological conditions.

[0087] The pathophysiological feature of ONFH is apoptosis and deficits in mesenchymal stem cell (MSC) differentiation, which ultimately leads to structural changes in the femoral head and secondary degenerative arthritis of the hip joint. High doses of corticosteroids not only cause apoptosis in MSCs, but also result in an imbalance in osteogenesis and adipogenesis. Therefore, controlling ROS production, inhibiting apoptosis of MSCs, andATTORNEY DOCKET NAME: STAN-2226WO CLIENT REFERENCE: S24-375 maintaining a balance in MSC-directed bone formation may be a promising early-stage interventional strategy during the onset of steroid-induced ONFH. Metformin, a therapeutic agent for type 2 diabetes, has recently attracted attention for its ability to regulate oxidative stress and control the production of ROS by modulation of AMP activated protein kinase (AMPK), mitochondrial complex I inhibition, and increasing antioxidant enzyme activity.

[0088] Published reports together suggest a biphasic effect of metformin on oxidative stress and thus an appropriate metformin dosage capable of controlling ROS production is important. There have been no previous studies of metformin on the targeting elevated oxidative stress in ONFH. In this study, we hypothesized that controlling ROS with an appropriate dose of metformin administration would alleviate osteogenic deficits in MSCs caused by corticosteroid-induced increase in oxidative stress. There are two purposes of this study. The first aim is to evaluate the effect of prednisolone at various concentrations on the production of ROS, osteogenic differentiation, and mineralization of MSCs in vitro. The second aim is to evaluate the effect of metformin on the pharmacokinetics of ROS production by MSCs during prednisolone administration.Methods

[0089] Isolation of mice MSCs Isolated MSCs from male 10- to 12-week-old BALB / c mice (Jackson Laboratory, Bar Harbor, ME, USA) were used for primary cell culture. MSCs were isolated from mice as described by Lin et al. A total of 20 mice were sacrificed to collect sufficient cells. Mice were euthanized according to protocol, using carbon dioxide at a flow rate of 100% displacement / min for 10 minutes. Mice were then removed from the box, and cervical dislocation was per- formed as a secondary method of euthanasia. Bone marrow was collected from the femurs and tibias, and MSCs were isolated and cultured in a minimal essential medium (MEM). The suspension was then filtered through a 70-Dm cell strainer, spun down, and resuspended in D-MEM supplemented with 10% certified fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, MA, USA) and 1 % antibiotic / antifungal solution (A / A, Thermo Fisher Scientific) and resuspended in a-MEM. To remove unattached cells, the medium was replaced the next day. Flow cytometry (Sca1 + / CD105+ / CD44+ / CD45- / CD34- / CD1 1 b-) was used to identify MSCs, and cells from passages 4 to 6 were used in this study. The experimental design of this study was approved by the Institutional Committee on Laboratory Animal Care and followed institutional guidelines for the care and use of laboratory animals.

[0090] Cell cultures and groups. Prednisolone (Sigma-Aldrich, St. Louis, MO, USA) was used for corticosteroid administration at 3 different concentrations (3, 30, and 300 ng / mL)ATTORNEY DOCKET NAME: STAN-2226WO CLIENT REFERENCE: S24-375 based on previous reports. MSCs were cultured overnight in MEM and then treated with each concentration of prednisolone. The effect of prednisolone on MSCs was evaluated for the following 4 groups: (1 ) control group (no prednisolone); (2) 3PRED (MSCs + 3 ng / mL prednisolone group); (3) 30PRED (MSCs + 30 ng / mL prednisolone group); and (4) 300PRED (MSCs + 300 ng / mL prednisolone group). Based on the prednisolone results, the optimal prednisolone concentration (3 ng / mL) was determined, and the effect of metformin (EMD Millipore Corp) administration was then evaluated. The concentration of metformin was determined based on previous reports (1 , 10, 100 pM). In addition to the control group without prednisolone, a total of 5 groups were investigated (Table 1 ).

[0091] The effects of prednisolone were evaluated by cell proliferation rate, oxidative stress expression, alkaline phosphatase (ALP) staining for bone differentiation, and alizarin red (AR) staining for matrix calcification. In addition, protein expression in the supernatant was also evaluated to determine the effect of metformin administration.Table 1 Grouping in metformin administration.PRED; prednisolone, MET; metformin

[0092] Cell proliferation assay AlamarBlue Cell Viability Reagent (Thermo Fisher Scientific) was used to evaluate cell proliferation in each group according to the manufacturer’s protocol. After seeding 1 .0 x 104cells / 100 ,uL / group into 96-well plates and incubating for 3 days, 10 pL of alamarBlue reagent was added to each well and incubated at 37°C for 1 hour [3]. After incu- bation, absorbance was measured using a SpectraMax iD3 (Molecular Devices, San Jose, CA, USA); absorbance at 570 and 600 nm was measured and cell proliferations were calculated as in previous reports [24,33]. Briefly, the average absorbance value at 600 nm of the cell culture medium only was subtracted from the absorbance value at 570 nm of the experimental wells and evaluated relative to the time course. Cell proliferation results are expressed as normalized absorbance versus the control group as the baseline.

[0093] Oxidative stress by cellular ROS. Oxidative stress was evaluated using the DCFDA / H2DCFDA Cellular ROS Assay Kit (Abeam, Burlingame, CA, USA). Cells were seeded in dark clear bottom 96-well microplates at 1 .0 x 104cells / 100 pL / well and cultured in each group of mediaATTORNEY DOCKET NAME: STAN-2226WO CLIENT REFERENCE: S24-375 for 2 days. After incubation, all groups of cells were washed with 1 x buffer, and DCFDA solution was added and kept at 37°C for 45 minutes in the dark. The cells were then observed using a fluorescence microscope (BZ-X810, Keyence), and the fluorescence intensity (485 / 535 nm) of ROS was measured using a SpectraMax iD3 (Molecular Devices, Osaka, Japan). ROS expression results were expressed as normalized fluorescence intensity relative to the control group as baseline.

[0094] Osteogenic differentiation assay For the assay of osteogenic differentiation, 4.0 x 104 / 500 iL cells were seeded into 24-well plates and cultured overnight in osteogenic medium (a-MEM, Thermo Fisher Scientific), 10% FBS (Thermo Fisher Scientific), 1 % A / A solution (Thermo Fisher Scientific), 10 mM [3-glycerol phosphate (Sigma-Aldrich), and 50 pM 1 -ascorbic acid (Sigma-Aldrich) for 7 days

[0032] , ALP staining was evaluated using 1 Step NBT / BCIP substrate solution (Thermo Fisher Scientific) on day 7 of culture. Images were taken using a BZ-X810 (Keyence), and the positive ratio of ALP staining was quanti- tied using QuPath (vO.5.1 ).

[0095] Mineralization by calcified matrix formation assay. Mineralization was assessed using AR staining on day 21 . 2.0 x 104 / 500 pL cells were seeded in 24-well plates and cultured in an osteogenic medium. The osteo- genic medium was changed twice a week. Calcified matrix formation was assessed on day 21 using AR staining (pH 4.1 , Sigma-Aldrich). Images were taken using a BZ-X810, and the ratio of positive staining was quantified using QuPath.

[0096] Cytokine measurement Cytokines were evaluated by multiplex quantification using Luminex xMAP technology. Cytokine quantification was compared in four groups (control, PRED, 10MET, 100MET) based on previous results. After culturing 1 x105cells / ml in 12-well plates for 2 days, the supernatant was collected. Multiplex analysis was performed using a Luminex™ 200 system (Luminex, Austin, TX, USA) from Eve Technologies Corp. (Calgary, Alberta). Cytokine expression in the acute phase of inflammation included growth factor of granulocyte macrophage colony-stimulating factor (GM-CSF); pro-inflammatory cytokines of interleukin- 1 beta (IL-1 beta), IL-6, and monocyte chemotactic protein-1 (MCP-1 ); and antiinflammatory marker of IL-10. The concentrations in the supernatant were determined according to the manufacturer's protocol.

[0097] Statistical analysis Statistical analysis was performed using Prism 9 (GraphPad Software, San Diego, CA, USA). One-way analysis of variance with Tukey's multiple comparison test was used for comparisons between multiple groups. p<0.05 results were considered statistically significant.ResultsATTORNEY DOCKET NAME: STAN-2226WO CLIENT REFERENCE: S24-375

[0098] After 3 days of administration, prednisolone decreased MSC proliferation in a dosedependent manner. Prednisolone at concentrations above 3 ng / mL significantly reduced cell pro- liferation compared to the control group (P< .01 ). In the metformin treatment experiment, cell proliferation was significantly restored by 10 pM of metformin in the PRED + 10MET group versus the PRED group (FIG. 1 , P < .05). The results of metformin treatment at the other 2 concentrations (1 .0 and 100 pM) to MSCs showed no significant enhancement in cell proliferation compared to the PRED group.

[0099] Prednisolone treatment significantly increased oxidative stress of the MSCs at each of the 3 concentrations of 3, 30, and 300 ng / mL, as shown by the cellular ROS staining, in which the highest result at the 300PRED group (Figure 2a). In contrast, the addition of metformin to 3 ng / mL prednisolone decreased ROS production in a concentration-depen- dent manner (P < .01 ), with the greatest reduction in the PRED + 100MET group (Figure 2b). ROS production was significantly lower in the PRED + 100MET group than in the control group. Representative images of cellular ROS assay and a comparison of fluorescence intensity are shown in FIG. 2.

[0100] Osteogenic differentiation evaluated by ratio of ALP- positive staining was significantly decreased by the treatment of prednisolone at each of the 3 doses, compared to the control group (P < .01 ); significant lower signal was also detected in the 300PRED group than the 3PRED group (Figure 3a, P < .05, control at 0.57 ± 0.03, 3PRED at 0.38 ± 0.02, 30PRED at 0.29 ± 0.01 , and 300PRED at 0.26 ± 0.02). The addition of metformin at 1 .0 pM showed no significant improvement to the ALP signal compared to the PRED group. When metformin concentration was increased further, this resulted in a significantly lower positive signal for ALP staining in the PRED and PRED + 1.0MET groups and a predominant increase in the PRED + 10MET and PRED + 100MET groups (Figure 3b, P < .01 , control; 0.53 ± 0.02, PRED; 0.28 ± 0.03, PRED + 1.0MET; 0.30 ± 0.02, PRED + 10MET; 0.49 ± 0.02, PRED + 100MET; 0.45 ± 0.03). ALP staining positivity ratio was higher in the 10MET group than in the 100MET group.

[0101] Prednisolone treatment significantly reduced the AR-positive area in each concentration group compared to the control group (Figure 3c, P < .01 , control; 0.49 ± 0.02, 3PRED; 0.36 ± 0.03, 30PRED; 0.29 ± 0.01 , 300PRED; 0.30 ± 0.04). The positive signal of AR staining with the administration of metformin was significantly lower in the PRED group and the PRED + 1.0MET group, while it was increased in the PRED + 10MET and PRED + 100MET groups (Figure 3d, P < .01 , control; 0.51 ± 0.02, PRED; 0.34 ± 0.05, 1 .0MET; 0.36 ± 0.02, 10MET; 0.55 ± 0.04, 100MET ; 0.48 ± 0.07). AR-positive was also higher in the PRED + 10MET group than in the PRED + 100MET group. Cytokine expression in the supernatant onATTORNEY DOCKET NAME: STAN-2226WO CLIENT REFERENCE: S24-375 day 2 after treatment is shown in Figure 4. The expression of the proinflammatory cytokine IL- 1 was significantly higher in the PRED group than the control group, and subsequently reduced by the addition of metformin at either concentration (P < .05, control: 26.5 ± 2.2 pg / mL, PRED:32.5 ± 2.3 pg / mL, PRED + 10MET: 26.3 ± 1 .9 pg / mL, PRED + 100MET: 27.3 ± 2.5 pg / mL). In contrast, IL-6 and MCP-1 expression was significantly lower in the 3 prednisolone- containing groups than in the control group and was not changed by the administration of metformin (IL-6; P < .01 , control: 2167.5 ± 133.2 pg / mL, PRED: 406.5 ±15.9 pg / mL, PRED + 10MET: 380.5 ± 15.2 pg / mL, PRED + 100MET: 326.8 ± 19.2 pg / mL, MCP-1 ; P < .01 , control: 1748.3 ± 133.2 pg / mL, PRED: 851 .3 ± 46.7 pg / mL, PRED + 10MET: 741 .3 ± 34.5 pg / mL, PRED + 100MET: 450 ± 15.7 pg / mL). The expression of the anti- inflammatory cytokine IL-10 and the growth factor GM-CSF did not differ significantly among the groups (IL- 10; control: 47.5 ± 1 .7 pg / mL, PRED: 41.8 ± 4.0 pg / mL, PRED + 10MET: 39.5 ± 1 .8 pg / mL, PRED + 100MET: 37.5 ± 1 .8 pg / mL, MCP1 ; P < .01 , control: 17.3 ± 3.3 pg / mL, PRED; 19.0 ± 0.7 pg / mL, PRED + 10MET: 18.8 ± 1 .9 pg / mL, PRED + 100MET: 18.8 ± 1 .3 pg / mL).

[0102] This study evaluated the effects of prednisolone treatment at various concentrations on the oxidative stress, osteogenic differentiation, and mineralization of murine MSCs in vitro. We expanded research into the use of metformin as a treatment that specifically targets oxidative stress during the development of corticosteroid-induced ON and bone injury. Briefly, we found that the administration of prednisolone to MSCs increased oxidative stress and ROS production at a concentration of 3 ng / mL, along with inhibiting osteogenic differentiation and mineralization. In contrast, metformin in the presence of prednisolone suppressed the increase of oxidative stress and ROS production, increased osteogenic differentiation, and mineralization, accompanied by suppression of IL-1 p protein expression. As for the concentration of in vitro metformin treatment, a concentration of 1 pM was not sufficient, a concentration of 10 pM produced notable effects with statistical significance in most readouts, and a concen- tration of 100 pM attenuated the effects, possibly due to over- suppression of the oxidative pathways. The expression of IL-6 and MCP-1 was decreased by prednisolone treatment, and the expression did not change with varying concentra- tions of additional metformin. The expression of the anti- inflammatory cytokine IL-10 and the growth factor GM- CSF did not differ among the groups on day 2, regardless of whether prednisolone or metformin was used or not.

[0103] Although there are many aspects of the pathogenesis of ON and other steroid-induced bone injury that remain to be elucidated, impaired differentiation of MSCs into osteoblasts and pathologies related to oxidative stress have been noted. ROS exist in the physiological envi-ATTORNEY DOCKET NAME: STAN-2226WO CLIENT REFERENCE: S24-375 ronment and are involved in signal transduction, immune response, and programmed cell death. However, once the redox balance is disrupted by non-physiological factors (smoking, aging, obesity, diabetes mellitus, corticosteroid use, etc.), excessive ROS causes tissue damage due to increased oxidative stress. Prolonged corticosteroid administration leads to increased ROS, which is a risk for producing oxidative stress and leads to cell apoptosis, prolonged inflammation, and impaired differentiation of MSCs, for example in ON of the femoral head. Since oxidative stress due to ROS affects pathophysiology immediately after corticosteroid administration, the timing of evaluation after corticosteroid administration was determined with reference to previous reports. In the present results, prednisolone at concentrations of 3 ng / mL or higher also decreased cell proliferation rate, increased cellular ROS, and inhibited osteogenic differentiation and mineralization. Previous studies on prednisolone concentrations have shown that 0.01 pM prednisolone inhibits spheroid formation in MSCs. In addition, the clinical pharmacokinetics of 20 mg of prednisolone taken internally per day resulted in a prednisolone concentration of 4.5 ng / mL in peripheral blood. Considering these previous reports, the prednisolone concentration used in this study and the results are reasonable.

[0104] The function of metformin on oxidative stress is still being investigated. Metformin reduces mutagenesis by activating the ataxia telangiectasia mutated (ATM) pathway, which plays a central role in the DNA damage response and inhibits NOX, the source of ROS production. For example, there are reports showing that high concentrations of metformin increase the production of ROS in MSCs, induce cell apoptosis, and increase adipose tissue. In the current study, 10 pM metformin was shown to promote cell proliferation, osteogenic differentiation, and mineralization, whereas 100 pM treatment resulted in lower ROS than the control group, inferior cell proliferation, and osteogenic differentiation and mineralization than 10 pM metformin treatment. As in the controls in this study, ROS are present in the physiological environment and are involved in immune regulation and signal transduction. The results of this study show that controlling oxidative stress and ROS at a biologically suitable level with an appropriate concentration (dosage) of metformin provides promising interventional direction in the treatment of corticosteroid-induced bone differentiation disorder, particularly when administered at the start of corticosteroid treatment.

[0105] During the inflammatory phase of bone formation, the introduction of inflammatory cytokines and the immune response of macrophages and other immune cells are critical. MSCs secrete MCP-1 and IL-6 as part of an immune response to maintain tissue homeostasis. MCP-1 enhances macrophage migration and is involved in immune modulation. During inflammation, MSCs genetically modified to overexpress MCP-1 enhanced macrophage migration and promoted bone differentiation and mineralization. In this study, prednisoloneATTORNEY DOCKET NAME: STAN-2226WO CLIENT REFERENCE: S24-375 treatment dramatically reduced IL-6 and MCP-1 expression. The reduction of this immune response by corticosteroids may be involved in the inhibition of bone formation. Conversely, IL-1 p expression was increased by prednisolone administration and decreased by metformin treatment at a concentration of 10 pM or above. Mature IL-1 is generated during the nuclear factor-KB pathway, which is associated with increased ROS expression. This is consistent with the reduction in ROS levels by metformin, suppressing NOX. Modulating ROS and inflammation with appropriate doses of metformin may alleviate the oxidative stress caused by corticosteroids and provide a treatment option for bone diseases such as ON and osteoporosis.

[0106] In conclusion, this study examined the effects of corticosteroids on oxidative stress and subsequent bone formation capacity in murine MSCs and the efficacy of metformin in alleviating corticosteroid-induced oxidative stress. Prednisolone administration decreased cell proliferation, increased oxidative stress, and decreased calcified matrix formation in MSCs. In contrast, metformin treatment at concentrations above 10 ,u.M alleviated the increase in oxidative stress secondary to prednisolone and promoted osteogenic differentiation and calcified matrix formation. Increasing concentrations of metformin, however, tended to excessively suppress ROS than controls and inhibited the formation of calcified matrices. Moderate ROS and inflammatory cytokine expression are also important in the acute phase, and control of ROS and inflammation with appropriate doses of metformin may be an important treatment strategy in the prevention of corticosteroid-induced bone injury, such as ON.EXAMPLE 2Metformin modulates cell oxidative stress to mitigate corticosteroid-induced suppression of osteogenesis in a 3D model

[0107] Corticosteroids provide well-established therapeutic benefits, however they are also accompanied by adverse effects on bone. Metformin is a widely used medication for managing type 2 diabetes mellitus.

[0108] This research investigates the effects of prednisolone on cellular metabolic functions and bone formation using a 3D in vitro model. Then, we demonstrate the potential therapeutic effects of metformin on oxidative stress and the formation of calcified matrix due to corticosteroids. Human mesenchymal stem cells (MSCs) and macrophages were cultured in 3D GelMA scaffold and subjected to stimulation with prednisolone, with and without the presence of metformin. The adverse effect of prednisolone and therapeutic effect of metformin were assessed by analyzing cell viability, osteogenesis markers, bone mineralization, andATTORNEY DOCKET NAME: STAN-2226WO CLIENT REFERENCE: S24-375 inflammatory markers. Oxidative stress was measured by evaluating reactive oxygen species (ROS) levels and ATP production.

[0109] Prednisolone exhibited cytotoxic effects, resulting in reduced viability of MSCs and macrophages. Lower osteogenesis potential was also detected in the MSC group. Metformin demonstrated significant positive effects on cell functions, including enhanced osteoblast activity and increased bone mineralization. Furthermore, metformin effectively reduced oxidative stress, as evidenced by decreased ROS levels and increased ATP. These findings indicate that metformin shows protective effects against oxidative damage, thus supporting osteogenesis. Metformin exhibits promising therapeutic potential beyond its role in diabetes management. The capacity to alleviate oxidative stress highlights the potential of metformin in supporting bone formation in inflammatory environments.

[0110] Advanced 3D cell culture models have become essential tools for studying complex cellular interactions and simulating in vivo systems. The 3D culture system provides a more physiologically relevant environment for exploring cellular interactions and effects of a drug. Mesenchymal stem cells (MSCs) have significant advantages when investigating bone formation due to their potential for differentiating into various cell types, including osteoblasts, chondrocytes, and adipocytes. Macrophages contribute to the regulation of bone remodeling via secreting inflammatory cytokines that license MSCs, and by forming osteoclasts. By incorporating both MSCs and macrophages within a 3D scaffold, our goal was to elucidate how prednisolone influences cellular interactions, and how metformin might alleviate any adverse effects induced by prednisolone.

[0111] Here, we applied a 3D culture system that encapsulates MSCs and macrophages to mimic the bone microenvironment and investigated the effects of prednisolone on MSCs and their interactions with macrophages. We focused on oxidative stress, osteogenic potential, and inflammatory regulation. Our results indicate that metformin promotes osteogenesis potential and enhances bone mineralization by MSCs. Additionally, metformin successfully mitigated oxidative stress, as indicated by lower levels of ROS and elevated ATP production. This comprehensive research provides valuable insights into the mechanisms underlying corticosteroid effects on MSCs and the benefits of combining metformin with corticosteroids, contributing to the development of more effective treatment strategies in regenerative medicine.

[0112] Fabrication of GelMA. GelMA scaffold was synthesized according to a previously described procedure. Briefly, a solution of 15 grams of gelatin was prepared by dissolving it completely in 500 mL water. Subsequently, 15 mL of methacrylic anhydride was introduced into the solution. The mixture was then incubated in a shaker at 150 rpm and 37°C for 24 hours. The methacrylated gelatin (mGL) solution was dialyzed for 4 days against water atATTORNEY DOCKET NAME: STAN-2226WO CLIENT REFERENCE: S24-375 room temperature. The mGL was subsequently dissolved in Hank's balanced salt solution (HBSS) at a concentration of 10% (w / v), with the incorporation of 0.15% (w / v) of the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) and 1 % of Antibiotic- Antimycotic.[001 13] Culture of MSC and Macrophage and 3D system setup. Human MSCs were cultured in DMEM medium supplied with 10% FBS, 1 % antibiotic-antimycotic, and 1 ng / mL fibroblast growth factor (FGF). Cells were passaged upon reaching 70-80% confluency. Human buffy coats were ordered from Stanford Blood center. Monocytes were isolated using EasySep™ Human Monocyte Isolation Kit (STEMCELL Technogies). Monocytes were cultured in macrophage induction medium (RPMI supplemented with 10% FBS, 1% antibiotic- antimycotic, and 100 ng / mL M-CSF) for 5 days.[001 14] MSCs or macrophages were loaded to GelMA scaffold at a concentration of 10 million / mL. 3D MSC scaffolds were cultured in osteogenic medium (DMEM, 10% FBS, 1% antibiotic-antimycotic, 50 pM L-ascorbic acid, 10 mM p-glycerophosphate, 100 mM Vitamin D3, and 100 ng / mL bone morphogenetic proteins 7 (BMP-7)). 3D macrophage scaffolds were cultured in macrophage induction medium. In the coculture system, scaffolds were cultured in a medium that consisted of half osteogenic medium and half macrophage induction medium.[001 15] Live / dead validation. Cell viabilities were measured using LIVE / DEAD™ Viability / Cytotoxicity Kit (ThermoFisher SCIENTIFIC, L3224). The working solution contains 0.1 pM calcein AM solution and 8 pM ethidium homodimer-1 . Scaffolds were washed using PBS, and then incubated in working solution for 30 min in an incubator. After that, scaffolds were washed using PBS again and imaged using Keyence microscope. Three images were taken per scaffold, and three scaffolds were measured in each group.[001 16] Cellular Oxidative Stress Assay (ROS and ATP staining). DCFDA - Cellular ROS Assay Kit (Abeam, ab113851 ) was applied to assess ROS production. Scaffolds were stained with DCFDA for 45 min in a 37°C incubator. Then, the fluorescent images were captured using confocal microscope (Leica STELLARIS 5) with an excitation / emission at 485 nm / 535 nm.[001 17] To measure the ATP production, scaffolds were stained with 5 pM BioTracker ATP- Red Live Cell Dye (Millipore Sigma, SCT045) for 15 min. After washing scaffolds with PBS buffer, the fluorescent images were captured with emission 570 nm. For each scaffold, three images were recorded, and within each group, three scaffolds were evaluated.[001 18] Quantification of Inflammatory Cytokine by ELISA. ELISA was conducted according to the manufacturer’s introduction. ELISA kit for measuring IL10 (88-7106-22), TNFa (88-7346- 22), and IL6 (88-7066-86) were ordered from ThermoFisher SCIENTIFIC. ELISA kit for measuring CCL18 (DY394) and IL1 p (DY201 -05) were ordered from R&D Systems. Briefly,ATTORNEY DOCKET NAME: STAN-2226WO CLIENT REFERENCE: S24-375 we first coat the plate with corresponding antibody overnight at 4°C. After washing, block with a blocking buffer for 1 hour at room temperature. Then, samples or standards were added to the wells and incubated for 2 hours. Subsequently, wash the plate again and add the secondary antibody to each well. After adding substrate solution for 15 min, the reactions were stopped with a stop solution and the absorbance was measured using a microplate reader.[001 19] Gene Expression by real-time PCR. mRNA was extracted by crushing scaffolds in TRIzol to release nucleic acids. After a 5-minute incubation, chloroform was added to separate the mixture. After centrifuge, the upper aqueous phase contains RNA was transferred to a new tube. Then the RNA was precipitated by adding isopropanol and incubating for 10 min. After centrifugation, the RNA pellet was washed with 75% ethanol, air dry, and dissolved in RNase-free water. Finally, measure RNA concentration using nanodrop. RNA was converted to cDNA using iScript™ cDNA Synthesis Kit. Primers for qPCR were ordered from ThermoFisher SCIENTIFIC, including CCL18 primer (Hs002681 13_m1 ), IL6 primer(Hs00174131_m1 ), IL10 primer (Hs00961622_m1 ), IL1 b primer (Hs01555410_m1 ), RUNX2 primer (Hs01047973_m1 ), ALPL primer (Hs01029144_m1 ), PPARG primer(Hs01115513_m1), PLIN1 primer (Hs00160173_m1 ), CEBPA primer (Hs00269972_s1 ), LPL primer (Hs00173425_m1 ), and GAPDH primer (Hs02786624_g1 ).

[0120] Osteoblast Differentiation Assay by ALP staining and APS staining. For ALP quantification, scaffolds were cultured for 7 days and fixed using 4% paraformaldehyde. Then, scaffolds were stained with 1 -Step™ NBT / BCIP Substrate Solution (ThermoFisher SCIENTIFIC, 34042) for 4 hours. For Alizarin Red S staining (ARS) quantification, scaffolds were cultured for 21 days. Then, the fixed scaffolds were stained with ARS solution for 30 seconds. The scaffolds were dehydrated by 15% sucrose and 30% sucrose. After that, the scaffolds were embedded in optimal cutting temperature (OCT) and stored in -80°C. Microtome was applied to section frozen OCT-embedded scaffolds. Keyence microscope was used to capture images.

[0121] Statistical analysis. Three independent experiments were performed for all assays. Statistical analysis of data was performed with Prime Graphpad software using ANOVA. Results with a p-value of less than 0.05 was considered to be statistically significant. All results in the graphs are presented as mean ± SD.Results

[0122] Dose effect of prednisolone on the cell viabilities of MSCs. \Ne investigated the effect of prednisolone on MSC viability, and whether there was a concentration-dependence using a 3D GelMA system. Previous research has suggested that the effective concentration of prednisolone typically peaks at a several hundred nanogram per milliliter. Therefore, we testedATTORNEY DOCKET NAME: STAN-2226WO CLIENT REFERENCE: S24-375 the MSCs viability treated with 3, 30, 150, and 300 ng / ml prednisolone. As shown in Figure 6, a reduced cell viability was observed at concentration of 3 ng / ml on day 7. This suggests that even relatively low concentrations of prednisolone can have a measurable impact on cells.

[0123] We further explored the osteogenic effect of prednisolone on MSCs. MSC scaffolds were cultured in osteogenic medium for 3 weeks. We stained our scaffolds with alkaline phosphatase (ALP) on day 7 to assess MSC osteogenic potential. We found that an increase in staining area was observed at the 150 ng / mL concentration compared to the 3 ng / mL group, at which no significant change was observed in other groups. We stained our scaffolds with Alizarin Red S (ARS) on day 21 to quantify areas of calcium deposition. A decrease in staining area was observed in the prednisolone groups, which correlated with the concentration of prednisolone. Higher concentrations of prednisolone reduced the staining area. Our analysis revealed that prednisolone significantly impacts the osteogenic differentiation of MSCs.

[0124] Protective Effect of Metformin on Prednisolone-Suppressed Osteogenesis by MSCs. \Ne assessed the impact of prednisolone and metformin on reactive oxygen species (ROS) and ATP production in MSCs on day 1 , which provided valuable insights into the drug’s early effects on cellular stress and energy metabolism. Cellular ROS production was quantified by cell-permeant reagent 2',7'-dichlorofluorescin diacetate (DCFDA), which is oxidized to a fluorescent compound by ROS. 10 pM metformin was used in our experiments. The levels of ROS increased by 50% in the 3 ng / mL prednisolone group, while the levels of ROS did not change in the metformin group. The treatment of the MSC scaffold with prednisolone and metformin reduced ROS levels, which were lower than those observed in the control group. ATP level is another critical energy marker to indicate metabolic features. A significantly reduced ATP level was observed in the prednisolone group, which suggested that prednisolone impairs cellular energy metabolism. Adding 10 pM metformin resulted in a recovery of ATP levels; however, these levels remained lower than those observed in the control group. The results demonstrated that metformin contributes to the recovery of normal ROS and ATP expression when stimulated with prednisolone, although it does not achieve complete normalization.

[0125] We further investigated the protective effect of metformin on the osteogenic potential of MSCs in the context of prednisolone and metformin treatment together. MSCs were cultured with osteogenic medium with or without prednisolone for 3 weeks. Separate groups of MSCs were co-treated with metformin and prednisolone, or metformin only. Runt-related transcription factor 2 (RUNX2) and ALPL are essential markers for MSC osteogenesis. As shown in FOG. 7, qPCR results indicated that the decreased expression of RUNX2 caused by prednisolone is completely reversed by metformin. In addition, a higher expression of the ALPL gene was detected in the prednisolone group, and metformin enhanced ALPL levels.ATTORNEY DOCKET NAME: STAN-2226WO CLIENT REFERENCE: S24-375 We further checked calcium matrix formation by ARS staining. Metformin increased ARS area observed in the group treated with both prednisolone and metformin. Thus, we found that metformin significantly mitigated the adverse impacts of prednisolone on osteogenesis.

[0126] The adipogenic potential of MSCs was also investigated by comparing the gene expression of peroxisome proliferator-activated receptor gamma (PPARG), CCAAT / enhancer-binding protein-alpha (CEBPA), lipoprotein lipase (LPL), and perilipin 1 (PLIN1). \Ne found that 3 ng / mL prednisolone increased the expression of CEBPA, LPL, and PLIN1 indicating an effect on adipogenesis. Metformin did not change the adipogenic potential of MSCs. However, the combination of prednisolone and metformin exhibited the most significant effect on adipogenesis.

[0017] The findings demonstrate that metformin can effectively protect against the impairment in osteogenesis caused by prednisolone-exposed MSCs. By normalizing ROS levels and supporting cellular energy metabolism, metformin helps preserve the osteogenic potential of MSCs.

[0128] Dose Effect of Prednisolone on the Macrophage Function. Macrophages serve as key regulators in both inflammatory responses and bone homeostasis. To assess the impact of prednisolone on macrophage viability at different concentrations, we also used 0 (control group), 3 ng / mL, 30 ng / mL, 150 ng / mL, and 300 ng / mL to cover a range of physiological and potentially toxic levels. Compared to the control group, significant decreases in viability were detected beginning at a dose of 3 ng / mL group, as shown in FIG. 8A.

[0129] Macrophages regulate immune responses by polarization to different phenotypes and secreting various cytokines. We collected the culture supernatants on days 2 and 5 and measured the pro-inflammatory cytokines (IL1 p, IL6, and TNFa) and anti-inflammatory cytokines (IL10 and CCL18) by ELISA. As shown in FIG. 8, a significant increase in the levels of CCL18 was observed in the group treated with 3 ng / mL prednisolone. The expression of TNFa and IL6 was found to be reduced in the 3 ng / mL group. The level of IL10 and IL1 b did not change at low concentrations of prednisolone.

[0130] Protective Effects of Metformin on Prednisolone-Treated Macrophages. Prednisolone impacts ROS production, which plays a crucial role in inflammation. We measured the ROS and ATP production in macrophages after 1 day of incubation treated with or without prednisolone. As shown in FIG. 9A and B, prednisolone did not change ROS and ATP expression in macrophages. In contrast, metformin was found to enhance the expression of both ROS and ALP.

[0131] We expanded our analysis of inflammation-related cytokine levels by ELISA. As shown in FIG. 4G, pro-inflammatory markers (IL-1 and IL6) and anti-inflammatory markers (CCL18 and IL-10) were measured. Within the group treated with prednisolone and metformin, theATTORNEY DOCKET NAME: STAN-2226WO CLIENT REFERENCE: S24-375 concentrations of CCL18 tend to return to their control levels. We also checked the gene expression on day 5, but most of the genes were not detectable, indicating low cytokine expression after day 5.

[0132] Effect of Metformin on the Co-Culture of MSCs and Macrophages. We co-cultured MSCs with macrophages in a 3D scaffold to provide a more realistic system. The 3D co-culture model was applied to test the effects of prednisolone and metformin on the interactions between MSCs and macrophages. Before co-culturing, we stained the MSCs with a Deep-red dye to facilitate tracking, which allows for a clear distinction between MSCs and macrophages. FIG. 10A - C shows the ROS expression level in MSCs and macrophages. The results indicated that prednisolone led to an increase in ROS levels in MSCs, but this effect was not seen in macrophages. In contrast, treatment with metformin resulted in a reduction of ROS levels, which were lower than those observed in the control group.

[0133] We evaluated the osteogenic and adipogenic markers, and the results are shown in FIG. 10D and E . ALP staining on day 7 shows no significant difference among different groups. ARS staining on day 21 indicated that prednisolone reduced calcium deposition, while metformin contributed to the preservation of this effect. Gene expressions on day 21 were tested by qPCR as shown in FIG. 10F. Cells subjected to prednisolone and metformin showed high expression of SPARC, contrasted by a decrease in CEBPA level. These results show that metformin contributes to the recovery of osteogenic capabilities in MSCs that have been adversely affected by prednisolone. We also measured cytokine levels and other secreted factors to assess the impact of co-culture conditions on inflammation. The results are shown in FIG. 10G. Metformin recovered IL1 b and TNFa reduced by prednisolone. However, metformin did not affect other cytokine levels.

[0134]

[0135] Prednisolone is widely used to manage inflammation and reduce pain in arthritic conditions, asthma, and many other diseases. While prednisolone demonstrates considerable effectiveness, it is primarily utilized for short-term management due to the risk of adverse effects from long-term use. Prolonged prednisolone treatment can lead to osteoporosis due to decreased bone formation and increased bone resorption. This effect has been documented in both animal models and human studies. Osteoporosis is induced through several mechanisms. For example, prednisolone promotes Notum expression and inhibits the PI3K / AKT / GSK3|3 / |3- catenin pathway which will induce osteocyte apoptosis. Prednisolone also modulates the Wnt / - catenin signaling pathway and ERK signaling pathway. In addition, prednisolone directly regulates the production of intracellular ROS. The increased ROS production occurs mainly through disruption of the oxidative and glycolytic balance in cells. This leads to long-term inflammation, increased cell apoptosis, and alternated cell activities. The administration ofATTORNEY DOCKET NAME: STAN-2226WO CLIENT REFERENCE: S24-375 prednisolone led to a reduction in the generation of ROS at the cellular level, as assessed by the DCFH-DA assay. The activation of the NOX / ROS / NF-KB signaling pathway leads to MSC apoptosis and interferes with MSC differentiation, which plays a pivotal role in the development of ONFH. The effects of prednisolone on the production of intracellular ROS were also reported in human platelets, which may contribute to the anti-inflammatory actions of these agents. The possible mechanisms underlying the effects of prednisolone on immune cells also include heightened oxidative stress and reduced energy production, which can account for alterations in cell functionality and phenotype. For example, prednisolone suppressed the M1 macrophage markers, including chemokine ligand 2, C-X-C chemokine motif 10, tumor necrosis factor- a and CD80. The results indicated that prednisolone inhibited the polarization of monocytes / macrophages towards the M1 phenotype. ROS recruits and polarizes macrophages to the M2 phenotype. Evaluating ROS production and ATP levels provides a comprehensive view of how prednisolone impacts MSCs and macrophages at the biochemical level. Considerable progress has been made in the development of therapeutic strategies that target ROS in stem cells for bone therapy.

[0136] Previous research has suggested that the effective concentration of prednisolone typically peaks at several hundred nanograms per milliliter. We tested the effects of different concentrations of prednisolone (ranging from 3 ng / mL to 300 ng / mL) on cell viability and function. We found that 3 ng / mL of prednisone significantly affected the viability of MSCs and macrophages. We chose this concentration for subsequent studies. The values of plasma metformin concentrations range from 0.129 to 90 mg / L. However, the therapeutic serum concentration of metformin for bone protection is poorly investigated as most research focuses on glucose modulation. Future research is needed to determine the therapeutic range of metformin in the context of corticosteroid-induced bone diseases.

[0137] One strategy to mitigate the adverse effects of prednisolone is combining corticosteroids with drugs that promote bone formation and minimize bone destruction. Metformin’s potential to mitigate oxidative stress through its antioxidant properties is of significant interest. Metformin enhances cellular antioxidant defenses and improves mitochondrial function through the activation of the AMP-activated protein kinase (AMPK) pathway, resulting in a reduction of oxidative damage. Metformin enhances the proliferative capacity and survival of multipotent stromal stem cells, suggesting an anti-apoptotic effect that is associated with reduced ROS activity. Metformin promotes cell proliferation and osteogenesis under high glucose conditions by regulating the ROS-AKT-mTOR axis. Others have suggested that the mechanism by which metformin treatment decreases oxidative stress is associated with the metabolic pathway involving Sirtuin 3 (SIRT3). Activation of the SIRT3 pathway by metformin contributed to the reduction of oxidative stress in chondrocytes. BesidesATTORNEY DOCKET NAME: STAN-2226WO CLIENT REFERENCE: S24-375 MSCs, metformin demonstrated an inhibitory effect on the production of ROS by human M2 macrophages through the activation of AMPK.

[0138] 3D culture more closely replicates in vivo tissue architecture than 2D culture and is a more authentic methodology for the assessment of biological mechanisms and drug effects on tissue regeneration. 3D culture of MSCs mimics the cellular behavior and differentiation pattern closely resembling those found in vivo. This enhanced physiological relevance provides a more authentic and accurate prediction of prednisolone and metformin affecting cellular processes and interactions within a complex tissue context. Our study evaluated the effects of metformin administration following corticosteroid exposure on cellular behavior, oxidative stress mechanism, and matrix mineralization in in-vitro 3D cell cultures encapsulating MSCs ± macrophages. Our findings reveal that prednisolone significantly impairs osteogenic differentiation, as evidenced by reduced MSC viability, mineralization, and osteogenic markers compared to controls. We also detected a high expression of anti- inflammatory cytokines in the macrophage culture group. We subsequently administered metformin to the prednisolone- treated MSCs, demonstrating the protective effect of metformin against the adverse effects induced by prednisolone. We further conducted a 3D co-culture of MSCs with macrophages and treated the cells with prednisolone to study the interactions between these cells and the effects of corticosteroid treatment and metformin administration. Our results show that metformin preserves MSC osteogenic potential in the co-culture environment. These interactions highlight the complexity of corticosteroid effects on stem cell behavior in an inflammatory context. Understanding the mechanisms can facilitate the development of more focused therapeutic approaches for mitigating corticosteroid-related adverse effects and improving regenerative treatment outcomes.

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[0140] The preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied by the appended claims.

Claims

ATTORNEY DOCKET NAME: STAN-2226WOCLIENT REFERENCE: S24-375What is Claimed is:1 . A method of reducing corticosteroid associated osteonecrosis or osteoporosis in an individual, the method comprising: administering a dose of a metformin agent effective to decrease oxidative stress and / or restore osteogenic capacity of mesenchymal stem cells exposed to corticosteroids.

2. The method of claim 1 , wherein the osteonecrosis is osteonecrosis of the femoral head (ONFH).

3. The method of claim 1 or claim 2, wherein the metformin agent is metformin or a metformin prodrug.

4. The method of any of claims 1 -3, wherein the metformin agent and the corticosteroid are co-formulated.

5. The method of any of claims 1 -3, wherein the metformin agent and the corticosteroid are separately formulated.

6. The method of any of claims 1 -5, wherein the metformin agent is formulated for oral administration.

7. The method of any of claims 1 -6, wherein the individual is treated with the corticosteroid for cancer, autoimmune disease, atopic disease, inflammatory disease or graft rejection.

8. The method of any of claims 1 -7 wherein the individual is a juvenile human.

9. The method of any of claims 1 -7 wherein the individual is an adult human.

10. The method of any of claims 1 -9, wherein the corticosteroid is a glucocorticoid.1 1 . The method of claim 10, wherein the glucocorticoid is one or more of beclomethasone; betamethasone; budesonide; cortisone; dexamethasone; hydrocortisone; methylprednisolone; prednisolone; prednisone; meprednisone; triamcinolone; paramethasone; and fluprednisolone.ATTORNEY DOCKET NAME: STAN-2226WOCLIENT REFERENCE: S24-37512. The method of claim 10, wherein the glucocorticoid is prednisone or prednisolone.

13. The method of any of claims 1 -12, wherein metformin is administered at a dose that achieves a sustained serum levels of from about 0.1 to about 100 y.M.

14. The method of any of claims 1 -12, wherein metformin is administered at a dose that achieves a sustained serum levels of from about 10 p.M to about 50 |j.M.

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