Modulation of the hypertrophic signaling pathway in chondrocytes for the treatment of osteoarthritis

Abstract

The present invention relates to a pharmaceutical composition for treating osteoarthritis comprising an effective amount of a small molecule inhibitor and a pharmaceutically acceptable carrier thereof. The present invention further provides a method for treating osteoarthritis comprising administering to a subject in need thereof an effective amount of a small molecule inhibitor that modulates a signaling pathway associated with hypertrophy of chondrocytes.

Description

[0001] Cross-references to related applications

[0002] The U.S. Provisional Application No. 63 / 796,151, which claims priority, was filed on April 28, 2025. Technical Field

[0003] This invention relates to a pharmaceutical composition for treating osteoarthritis by administering an effective amount of a small molecule inhibitor to a subject in need of treatment, the inhibitor modulating signaling pathways associated with chondrocyte hypertrophy. Background Technology

[0004] Degenerative joint diseases (DJDs) are a group of progressive musculoskeletal disorders characterized by the gradual degeneration of cartilage, leading to joint pain, stiffness, and impaired mobility. Osteoarthritis is the most common of all degenerative joint diseases, affecting millions worldwide. Other common types include post-traumatic arthritis, osteochondritis, and intervertebral disc degeneration. These conditions are driven by factors such as aging, mechanical stress, inflammation, and cellular dysregulation, ultimately resulting in cartilage loss and joint dysfunction.

[0005] Articular cartilage damage and degeneration (i.e., knee osteoarthritis) has an extremely high incidence rate and has become a major global health problem. As a degenerative joint disease, osteoarthritis causes pain, stiffness, and reduced mobility. Due to the avascular and nerve-free nature of articular cartilage, its regenerative capacity is extremely limited. Despite the high incidence rate, there are currently no FDA-approved treatments that can alter the course of the disease, highlighting the urgent need for innovative therapies.

[0006] Current clinical treatments primarily focus on symptom relief and cannot address the underlying problem of cartilage degeneration—neither preventing, delaying, nor reversing the structural damage within the joint associated with osteoarthritis. Therefore, there is still a lack of effective treatments for osteoarthritis.

[0007] A key challenge in treating osteoarthritis lies in finding a cost-effective and patient-friendly alternative to joint replacement surgery. Surgical interventions, with their high medical costs, demanding postoperative care, and invasive procedures, place a heavy burden on patients. There is an urgent need for a minimally invasive therapy that can reduce these challenges while maintaining treatment effectiveness. Research has found that using small molecule inhibitors to treat osteoarthritis is more cost-effective.

[0008] For example, US patent application number 2021369714 A1 discloses an inhibitor targeting the intra-articular insulin-like growth factor-1 receptor (IGF-IR) signaling pathway, achieving the treatment or prevention of osteoarthritis by inhibiting IGF-1R phosphorylation. This is achieved by surgically implanting a small molecule tyrosine kinase inhibitor (such as NVP-AEW541) into the joint of a subject in the form of a scaffold such as nanorods or microrods. It is claimed that the small molecule inhibitor of this invention can prevent or reduce abnormal chondrocyte proliferation, abnormal angiogenesis, cartilage degradation, joint damage, and / or osteophyte formation associated with the development and / or progression of osteoarthritis. However, this invention does not disclose the reduction of any proliferative markers (such as Runx2, ALP, ColX, IHH, VEGF), which ultimately prevent the loss of proteoglycans and type II collagen, restore ICRS scores, and directly promote cartilage repair associated with osteoarthritis.

[0009] Furthermore, U.S. Patent Publication No. 9855266 B2 discloses a method for treating osteoarthritis by administering a single-dose chemotherapy to the osteoarthritis joint of a subject via intra-articular injection. The target is mouse dual-minimum protein 2 homolog (MDM2). The compound described in this invention is a small-molecule senescent cell scavenger that selectively kills senescent cells rather than non-senescent cells. The added senescent cell scavenger is 4-[[(4S,5R)-4,5-bis(4-chlorophenyl)-4,5-dihydro-2-[4-methoxy-2-(1-methylethoxy)phenyl]-1H-imidazol-1-yl]carbonyl]-2-piperazinone (PP242). This senescence scavenger treatment can inhibit the erosion of the proteoglycan layer in osteoarthritis joints, promote the production of type II collagen, thereby reducing or inhibiting the loss or erosion of the joint proteoglycan layer, and alleviating joint inflammation by promoting collagen (such as type II collagen) production. However, in osteoarthritis, inhibiting MDM2 may induce unintended apoptosis of chondrocytes, leading to accelerated cartilage loss rather than repair, as it often promotes tumor cell death by enhancing p53 activity.

[0010] In addition to the above, WIPO publication WO 2008067427 A2 discloses the application of insulin-like growth factor receptor antagonists in weight maintenance, weight loss, and obesity treatment. The antagonist in this invention targets IGF-1R and is selected from a variety of suitable compounds, including small molecules, proteins, peptides, IGF mimics, antisense oligodeoxynucleotides, antisense RNA, small repressive RNA, triple-helix nucleic acids, dominant-negative mutants, and soluble receptor expression. However, IGF-1R antagonists may have systemic metabolic effects, potentially affecting muscle mass, glucose metabolism, and overall IGF signaling. Summary of the Invention

[0011] The present invention aims to provide a method for treating the progression of degenerative joint diseases (such as osteoarthritis) by controlling the pathological process of abnormal chondrocyte hypertrophy.

[0012] Another object of the present invention is to provide an FDA-approved treatment for controlling the progression of degenerative joint diseases such as osteoarthritis. More specifically, it aims to develop potential disease-modifying agents (DMOADs) for osteoarthritis using small molecule inhibitors of insulin-like growth factor 1 receptor (IGF-1R).

[0013] Accordingly, the above objectives can be achieved by following the teachings of this invention, which relates to a pharmaceutical composition for treating osteoarthritis, comprising an effective dose of a small molecule inhibitor and a pharmaceutically acceptable carrier thereof.

[0014] The present invention also achieves the above objectives by providing a method for treating osteoarthritis, the method comprising administering an effective dose of a small molecule inhibitor to a subject requiring treatment, the inhibitor being able to modulate signaling pathways associated with chondrocyte hypertrophy. Attached Figure Description

[0015] The features of the present invention will be more readily understood and appreciated by reading the following detailed description in conjunction with the accompanying drawings of preferred embodiments of the present invention, wherein:

[0016] Figure 1 Methods of administration of pharmaceutical compositions for preventing osteoarthritis chondrocyte hypertrophy in vitro and in vivo are shown;

[0017] Figure 2A-2B This demonstrates the ability of a specific dose of Picropodophyllin (PPP) to maintain chondrocyte proliferation and survival;

[0018] Figures 3A-3B This demonstrates the ability of a specific dose of PPP to maintain proteoglycan synthesis in in vitro microcluster culture;

[0019] Figure 4 This demonstrates the ability of a specific dose of PPP to inhibit the expression of marker genes for chondrocyte proliferation in mice under inflammatory conditions;

[0020] Figure 5A The histological staining of proteoglycans (red) in rat articular cartilage is shown by Safran O / Fast Green.

[0021] Figure 5B The quantitative results of relative integrated optical density (IOD) after saffron O staining are shown;

[0022] Figure 5C A quantitative analysis of the ICRS II score for the articular cartilage layer is shown;

[0023] Figures 6A-6D This indicates that PPP prevents osteoarthritis by maintaining collagen levels;

[0024] Figures 7A-7C This study demonstrates the effect of PPP in inhibiting articular chondrocyte hypertrophy in a mouse model of osteoarthritis (OA).

[0025] Figures 8A-8D The study showed that PPP inhibited the expression of hypertrophy markers in articular cartilage in a mouse OA model.

[0026] Figure 9 It was shown that intra-articular injection of PPP did not produce significant nephrotoxicity; and,

[0027] Figures 10A-10B It was shown that intra-articular injection of PPP does not produce significant liver toxicity. Detailed Implementation

[0028] To facilitate understanding of the principles of this invention, it is now described in conjunction with the embodiments shown in the accompanying drawings and the following written description. It should be noted that this invention covers any modifications and adjustments to the illustrated embodiments, and includes further applications of the principles of this invention that are commonly conceived by those skilled in the art.

[0029] This invention provides a pharmaceutical composition for treating osteoarthritis, comprising an effective dose of a small molecule inhibitor and a pharmaceutically acceptable carrier thereof, such as... Figure 1 As shown.

[0030] In one embodiment of the present invention, the small molecule inhibitor is an insulin-like growth factor 1 receptor (IGF-1R) inhibitor, which can inhibit the IGF-1R signaling pathway.

[0031] In one embodiment of the present invention, the effective dose range of the small molecule inhibitor is 50 nM to 100 nM.

[0032] The present invention further provides a method for treating osteoarthritis, comprising administering an effective dose of a small molecule inhibitor to a subject requiring treatment, the inhibitor modulating signaling pathways associated with chondrocyte hypertrophy.

[0033] In one embodiment of the invention, an effective dose of the small molecule inhibitor is administered to the affected joint of the subject requiring treatment via intra-articular administration.

[0034] In one embodiment of the invention, the method further includes co-administering an effective dose of a small molecule inhibitor with an anti-inflammatory agent.

[0035] The present invention further provides a method for preventing chondrocyte hypertrophy in the joints of subjects with osteoarthritis, comprising administering an effective dose of a small molecule inhibitor into the joint cavity, which reduces hypertrophic differentiation of chondrocytes and alleviates cartilage degeneration associated with osteoarthritis.

[0036] In one embodiment of the present invention, the reduction in chondrocyte hypertrophy leads to a decrease in the expression of hypertrophy markers, including but not limited to: Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), type X collagen (Col X), Indian hedgehog protein (IHH), vascular endothelial growth factor (VEGF), and matrix metalloproteinase 13 (MMP13).

[0037] In one embodiment of the invention, administration of small molecule inhibitors maintained the viability, proliferation, and proteoglycan synthesis of chondrocytes.

[0038] The present invention further provides a method for manufacturing drugs using small molecule inhibitors to treat osteoarthritis, thereby achieving therapeutic effects by preventing or reducing chondrocyte hypertrophy.

[0039] This invention provides a method for repairing osteoarthritis-related lesions by using IGF-1R small molecule inhibitors to inhibit chondrocyte hypertrophy and maintain chondrocyte phenotype. The aim is to control abnormal chondrocyte hypertrophy, a pathological process that is a key feature of osteoarthritis progression, characterized by abnormal enlargement and terminal differentiation of chondrocytes, ultimately leading to hyaline cartilage remodeling and mineralization.

[0040] More specifically, by administering doses of potent small molecule IGF-1R inhibitors (including but not limited to PPP) to reduce chondrocyte hypertrophy, cartilage degeneration in osteoarthritis can be alleviated.

[0041] Current management methods for osteoarthritis primarily rely on conservative treatment. Therefore, this approach bridges the gap in treatment for both early and late-stage osteoarthritis, providing patients with an alternative treatment option. More specifically, one drug delivery technique—intra-articular injection of small-molecule drugs—requires minimally invasive surgery, making it more patient-friendly than joint replacement surgery. This method can reduce the medical costs associated with surgery and postoperative care.

[0042] More specifically, this invention treats osteoarthritis by controlling chondrocyte hypertrophy by applying an IGF-1R inhibitor (PPP) to regulate IGF-1R signaling in chondrocytes.

[0043] Example

[0044] To facilitate understanding of the present invention, the specific details will be further described below in conjunction with embodiments. However, the present invention is not limited to the following embodiments, and those skilled in the art can make various modifications or alterations to the present invention, and these equivalent forms are also within the scope of the claims listed in this application.

[0045] This study investigated the effect of IGF-1R inhibition on controlling chondrocyte hypertrophy under conditions of osteoarthritis.

[0046] In in vitro experiments, primary chondrocytes were isolated from newborn mice, and the dosage of the IGF-1R inhibitor (PPP) was optimized by cell viability assay. Chondrocytes were treated with a specific dose of PPP, and gene expression and proteoglycan synthesis were analyzed. In in vivo experiments, medial meniscus instability surgery was performed on adult C57BL / 6 mice to induce osteoarthritis. Mice received intra-articular PPP injections for 6 weeks, followed by knee joint samples for processing, paraffin embedding, and sectioning. Knee joint histological sections were stained with H&E, safran O stain, and immunohistochemically stained for articular cartilage markers (Protocol 1). The small molecule IGF-1R inhibitor maintained chondrocyte proliferation, survival rate, and proteoglycan synthesis levels at specific doses in in vitro chondrocyte microcluster culture. This molecule can reduce the expression of chondrocyte hypertrophy marker genes under inflammatory conditions (IL-1β treatment), prevent the loss of proteoglycans and type II collagen (COL II), restore the ICRS II score of articular cartilage, and downregulate chondrocyte hypertrophy markers (such as MMP13 and COL X) in a mouse model of osteoarthritis induced by medial meniscus instability.

[0047] Preliminary experimental results

[0048] like Figure 2A-2B As shown, primary mouse chondrocytes were cultured in different concentrations of IGF-1R inhibitor (PPP) for 3 and 7 days, respectively. The optimal concentration of IGF-1R inhibitor for mouse chondrocytes was determined by cell proliferation assay. Data were analyzed using one-way ANOVA, *** P < 0.001 (compared to the control group), n=4.

[0049] Figure 3A Mouse chondrocytes treated with 20 nM insulin-like growth factor-1 (IGF-1) or different doses of phosphate (PPP) for 7 days were shown, and the proteoglycans synthesized by the chondrocytes were visualized by Alsin blue staining. Figure 3B The relative integrated optical density (IOD) of Alsin blue staining in each group was quantified. Data were analyzed using one-way ANOVA, * P < 0.05 (compared to the control group), n = 3.

[0050] Figure 4Mouse chondrocytes were seeded in 6-well plates, pretreated with 10 ng / mL IL-1β for 24 hours, and then cultured in 100 nM PPP for 30 minutes. Cells were subsequently harvested for gene expression analysis. mRNA expression levels were expressed as the ratio of target gene to GAPDH. Data were analyzed using a Student's t-test; *P < 0.05 (compared to the control group), n=3.

[0051] Figure 5A The distribution of proteoglycans (red) in mouse articular cartilage after intra-articular injection of saline (control group) or PPP (treatment group) at 6 weeks was visualized using saffron O / fast green histological staining. Figure 5B Further, the quantitative results of the relative IOD value of saffron O staining are presented. Figure 5C The ICRS II score quantification results of the articular cartilage layer are presented. L: low dose (50 nM); H: high dose (100 nM); AC: articular cartilage; SB: subchondral bone. Scale bar = 100 μm. * P < 0.05, **** P ≤ 0.0001 (compared with the sham surgery group). Data were processed by one-way ANOVA, # P < 0.05, ### P < 0.001, #### P ≤ 0.0001 (compared with the DMM control group), n=5.

[0052] Figures 6A-6D This demonstrates that PPP prevents arthritis and joint degeneration by maintaining collagen. Specifically, Figure 6A and Figure 6B Immunohistochemical (IHC) staining results of SOX9 and type II collagen in mouse articular cartilage after intra-articular injection of saline (control group) or PPP (treatment group) for 6 weeks are presented. Figure 6C and Figure 6D Immunohistochemical quantitative results of SOX9 and type II collagen in articular cartilage are shown. L: low dose (50 nM); H: high dose (100 nM); AC: articular cartilage; SB: subchondral bone. Scale bar = 100 μm. ** P < 0.01, *** P < 0.001 (compared with the sham-operated control group). Data were analyzed by one-way ANOVA, ## P < 0.01 (compared with the DMM control group), n=5.

[0053] Figures 7A-7C As shown, in a mouse model of osteoarthritis, PPP can prevent the hypertrophy of articular chondrocytes. For example, Figure 7A The results of hematoxylin-eosin (H&E) histological staining of chondrocytes in the articular cartilage of mice after intra-articular injection of saline (control group) or PPP (treatment group) for 6 weeks are shown. Figure 7BQuantitative analysis of the mean size of chondrocytes in articular cartilage is shown. L represents low dose (50 nM); H represents high dose (100 nM); SZ is the superficial zone; HZ is the hypertrophic zone; SB is the subchondral bone. Scale bar = 100 μm. * P < 0.05, ** P < 0.01, *** P < 0.001. Compared with the sham-operated control group. Data were processed by one-way ANOVA, # P < 0.05, ## P < 0.01. Compared with the DMM control group, n=5. Figure 7C Quantitative analysis of the percentage of hypertrophic chondrocytes in articular cartilage is shown. L, low dose (50 nM); H, high dose (100 nM). ** P < 0.01, compared with the sham-operated control group. # P < 0.05, compared with the DMM control group, n = 5.

[0054] Figures 8A-8D This study demonstrated that PPP can suppress the expression of hypertrophy markers in articular cartilage of a mouse OA model. Specifically, Figure 8A -B shows the immunohistochemical (IHC) staining results of collagen X and MMP13 in mouse articular cartilage after intra-articular injection with saline (control group) or PPP (treatment group) for 6 weeks. Figure 8C -D shows the IOD quantification results of collagen X and MMP13 in articular cartilage, respectively. L: low dose (50 nM); H: high dose (100 nM); AC: articular cartilage; SB: subchondral bone. Scale bar = 100 μm. Data were processed by one-way ANOVA, * P < 0.05, *** P < 0.001 (compared with the sham-operated control group); ## P < 0.05 (compared with the DMM control group), n=5.

[0055] Figure 9 This study demonstrates that intra-articular injection of PPP does not produce significant nephrotoxicity. Specifically, kidney structure was observed by H&E staining after 6 weeks of intra-articular administration of saline (control group) or PPP (treatment group). L: low dose (50 nM); H: high dose (100 nM). Scale bar = 200 μm.

[0056] Figures 10A-10B This study demonstrates that intra-articular injection of PPP did not produce significant liver toxicity. Specifically, liver structures in different treatment groups were observed 7 weeks post-surgery using H&E staining. The tissue structures are presented at high and low magnification in the figures. L: Low-dose group (50 nM); H: High-dose group (100 nM). Scale bars in Figures A and B are 100 μm and 50 μm, respectively.

[0057] The present invention described above is not limited to the foregoing embodiments and figures. It will be apparent to those skilled in the art that various substitutions, modifications and alterations can be made without departing from the scope of the invention.

Claims

1. A pharmaceutical composition for treating osteoarthritis, characterized in that, This includes effective doses of small molecule inhibitors and their pharmaceutically acceptable carriers.

2. The pharmaceutical composition according to claim 1, characterized in that, The small molecule inhibitors mentioned above are insulin-like growth factor 1 receptor (IGF-1R) inhibitors, which can inhibit the IGF-1R signaling pathway.

3. The pharmaceutical composition according to claim 1, characterized in that, The effective dose of small molecule inhibitors is 50 nM to 100 nM.

4. A method for treating osteoarthritis, characterized in that, This includes administering an effective amount of a small molecule inhibitor to subjects in need of treatment, which modulates signaling pathways associated with chondrocyte hypertrophy.

5. The method according to claim 4, characterized in that, The effective dose of small molecule inhibitors is administered intra-articularly to the affected joint of the subject requiring treatment.

6. The method according to claim 4, characterized in that, The method further includes co-administering an effective dose of a small molecule inhibitor with an anti-inflammatory agent.

7. A method for preventing articular chondrocyte hypertrophy in subjects with osteoarthritis, characterized in that, This includes administering an effective dose of a small molecule inhibitor intra-articularly into the joint cavity, wherein the administration reduces hypertrophic differentiation of chondrocytes and alleviates cartilage degeneration associated with osteoarthritis.

8. The method according to claim 7, characterized in that, The reduction in chondrocyte hypertrophy leads to a decrease in the expression of hypertrophy markers, including but not limited to Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), type X collagen (Col X), Indian hedgehog protein (IHH), vascular endothelial growth factor (VEGF), and matrix metalloproteinase 13 (MMP13).

9. The method according to claim 7, characterized in that, Small molecule inhibitors maintain the survival, proliferation, and proteoglycan synthesis capabilities of chondrocytes.

10. Use of a small molecule inhibitor in the manufacture of a medicament for the treatment of osteoarthritis, by preventing or reducing chondrocyte hypertrophy.

Images