Application of P2Y12 inhibitor in medicine for treating osteoarthritis
By targeting the HNP1-P2Y12-NF-κB/IFIL44 axis and using P2Y12 inhibitors such as clopidogrel, key factors in the progression of osteoarthritis have been addressed, achieving the restoration of cartilage structure and protection of COL2, thus providing a new treatment strategy.
Patent Information
- Application Number
- CN202511665542.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-13
- Publication Date
- 2025-12-26
AI Technical Summary
Current treatment strategies for osteoarthritis mainly focus on relieving symptoms, and there are no methods to change the course of the disease. Furthermore, the potential role of endogenous host defense peptides in regulating cartilage homeostasis has not been fully studied, especially the role of α-defensins in osteoarthritis.
The application of P2Y12 inhibitors, such as clopidogrel, ticlopidine, ticagrelor, or prasugrel, targets the HNP1-P2Y12-NF-κB/IFIL44 axis, inhibiting COL2 degradation and cartilage destruction, providing a new therapeutic approach.
It effectively reduces joint damage in osteoarthritis, restores cartilage structure, reduces osteoclast activity, and inhibits COL2 degradation, filling a gap in the treatment of osteoarthritis.
Smart Images
Figure CN121197408A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the pharmaceutical technology for treating osteoarthritis. BACKGROUND
[0002] Osteoarthritis (OA) is a multifactorial joint disease, whose pathogenesis starts from the degradation of type II collagen, and then triggers the pathogenic cascade of synovial inflammation and subchondral bone remodeling, driving the progression of the disease.
[0003] Osteoarthritis poses a huge challenge to global health, with about 10% of people aged 60 and above currently affected (Hunter, D.J. and S. Bierma-Zeinstra, Osteoarthritis. Lancet, 2019. 393(10182): p. 1745-1759.). The disease involves pathological changes in multiple joint structures, including articular cartilage erosion, subchondral bone remodeling, osteophyte formation, synovial inflammation, and per-articular fibrosis. These structural changes collectively lead to the progression of joint instability, chronic pain, and dysfunction. As the disease progresses, surgical intervention is often required, with about 10% of patients with knee OA ultimately receiving joint replacement due to the poor effectiveness of conservative treatment (Losina, E., et al., Cost- effectiveness of total knee arthroplasty in the United States: patient risk and hospital volume. Arch Intern Med, 2009. 169(12): p. 1113-21; discussion 1121-2.). Current treatment strategies mainly focus on symptom relief (Bijlsma, J.W., F. Berenbaum, and F.P. Lafeber, Osteoarthritis: an update with relevance for clinical practice. Lancet, 2011. 377(9783): p. 2115-26.), with a focus on managing pain through drug therapy and lifestyle changes, as there is no treatment available to change the course of the disease.
[0004] The integrity of articular cartilage depends on the highly organized extracellular matrix (ECM) (Goldring, M.B. and S.R. Goldring, Osteoarthritis. J Cell Physiol, 2007. 213(3): p. 626-34.), which is rich in type II collagen (COL2) and aggrecan such as aggrecan (Roughley, P.J. and J.S. Mort, The role of aggrecan in normal and osteoarthritic cartilage.J Exp Orthop, 2014. 1(1): p. 8.; Hartmann, K., et al., Molecular Actions of Glucocorticoids in Cartilage and Bone During Health, Disease, and Steroid Therapy. Physiol Rev, 2016. 96(2): p. 409-47.; Loeser, R.F., Aging and osteoarthritis: the role of chondrocyte senescence and aging changes in the cartilage matrix. Osteoarthritis Cartilage, 2009. 17(8): p. 971-9.), which collectively maintain biomechanical function and load-bearing capacity. Previous studies on the pathogenesis of OA have mainly focused on the self-regulatory processes of chondrocytes, including upregulation of matrix-degrading enzymes (such as matrix metalloproteinases MMPs and ADAMTS), oxidative stress, and cell senescence (Fosang, A.J. and F. Beier, Emerging Frontiers in cartilage and chondrocyte biology. Best Pract Res Clin Rheumatol, 2011. 25(6): p. 751-66.; Loeser, R.F., et al., Osteoarthritis: a disease of the joint as an organ. Arthritis Rheum, 2012. 64(6): p. 1697-707.; van der Kraan, P.M. and W.B. van den Berg, Chondrocyte hypertrophy and osteoarthritis: role in initiation and progression of cartilage degeneration Osteoarthritis Cartilage, 2012. 20(3): p. 223-32.), and inflammatory mediators such as IL-1β and TNF-α from synovial fibroblasts and macrophages (Kapoor, M., et al., Role of proinflammatory cytokines in the pathophysiology of osteoarthritis. Nat Rev Rheumatol, 2011. 7(1): p. 33-42.; Malemud, C.J., Biologic basis of osteoarthritis: state of the evidence. Curr Opin Rheumatol, 2015. 27(3): p.289-94.). In contrast, the potential role of endogenous host defense peptides (including defensins) in regulating cartilage homeostasis has received little attention, leaving an important gap in the understanding of OA as a multifactorial disease.
[0005] Cationic host defense peptides are evolutionarily conserved components of the innate immune system, playing important roles in antimicrobial defense and immunomodulation (Hancock, R.E. and H.G. Sahl, Antimicrobial and host- defense peptides as new anti-infective therapeutic strategies.NatBiotechnol, 2006. 24(12): p. 1551-7.). In humans, the major family of defensive peptides includes the antimicrobial peptide LL-37 and defensins. LL-37 was first identified by Gudmundsson et al. from bone marrow and testes, and is formed by an α-helix (Gudmundsson, GH, et al., The human gene FALL39 and processing of the cathelin precursor to the antibacterial peptide LL-37 in granulocytes. Eur J Biochem, 1996. 238(2): p. 325-32.). Defensins are formed by β-sheets via disulfide bonds and are classified into α, β, and θ subclasses according to their linkage mode (Ganz, T., Defensins: antimicrobial peptides of innate immunity. alpha-Defensins in human innate immunity. Nat Rev Immunol, 2003. 3(9): p. 710-20.). Among them, α-defensins—also known as human neutrophil peptides (HNPs)—were first isolated from neutrophils and macrophages by Lehrer and colleagues (Lehrer, RI and W. Lu, Multispecific myeloid defensins. Immunol Rev, 2012.245(1): p. 84-112.; Lehrer, RI, Figure 1 Current Opinion in Hematology, 2007. 14(1): p. 16-21.). Although HNPs have been well-established as microbial killers that disrupt microbial membranes and regulate immune cell activity, their role in aseptic chronic inflammatory diseases such as osteoarthritis (OA)—especially at pathophysiologically relevant concentrations—remains unclear. Interestingly, some β-defensins are expressed in OA synovial membranes and chondrocytes and may influence the inflammatory response, but opinions on their role in COL2 are inconsistent, while the role of α-defensins like HNP1 in OA has not been systematically studied. Summary of the Invention
[0006] The purpose of this invention is to provide a new drug for treating osteoarthritis.
[0007] This invention relates to the application of P2Y12 inhibitors in drugs for treating osteoarthritis, and P2Y12 inhibitors can be used to treat osteoarthritis.
[0008] The benefits of this invention lie in its enhanced understanding of the mechanism by which HNP1 promotes OA progression and in opening up possibilities for novel intervention strategies for OA. Targeting the HNP1-P2Y12-NF-κB / IFIL44 axis may provide a new approach to inhibiting COL2 degradation and cartilage destruction, filling a gap in OA treatment. Attached Figure Description
[0009] Figure 2 is a diagram of key amino acid residues of HNP1 interacting with P2Y12, Figure 3 is a graph of surface plasmon resonance (SPR) analysis of P2Y12 binding kinetics to immobilized HNP1, Figure 4 is a graph for determining the binding affinity between HNP1 and P2Y12, Figure 5 is a graph of experimental time course, Figure 6 is a representative micro-CT image of the sagittal plane of the knee joint of each experimental group, Figure 7 is a representative micro-CT image of the coronal plane of the knee joint of each experimental group, Figure 8 is a graph showing the structure of cartilage by SO-FG staining, Figure 9 is a graph showing the activity of osteoclasts by tartrate-resistant acid phosphatase (TRAP) staining, Figure 10 is a graph showing the preservation of cartilage matrix by COL2 immunohistochemistry, Figure 11 is a graph of bone volume / total bone volume analysis in the modeling region, Figure 12 is a graph of trabecular bone thickness in the modeling region, Figure 13 is a graph of trabecular bone space in the modeling region, Figure 14 is a graph of ICRS score for gross observation of cartilage in the modeling region, Figure 15 、 Figure 1 is the mean ± SD for 5 random fields per group. DETAILED DESCRIPTION
[0010] The present application is the use of P2Y12 inhibitors in the treatment of osteoarthritis drugs, P2Y12 inhibitors can be used to treat osteoarthritis.
[0011] The above P2Y12 inhibitor, the inhibitor is clopidogrel, or is ticlopidine, or is ticagrelor, or is prasugrel, or is cangrelor.
[0012] 1. In vitro verification of HNP1-P2Y12 interaction As shown in Figure 2 , the key amino acid residues of HNP1 interacting with P2Y12 were predicted by structure analysis. The distance between the interaction residues has been marked. As shown in Figure 3 , the binding kinetics of P2Y12 to immobilized HNP1 was analyzed by surface plasmon resonance (SPR). The data are presented as response units (RU) of P2Y12 at different concentrations over time, and the dissociation constant (Kd) is calculated. As shown in Figure 1 , the polarization experiment was used to determine the binding affinity between HNP1 and P2Y12. The dissociation constant (Kd) and the correlation coefficient (R) are shown, and the data are represented as mean ± standard deviation. The results represent 3 independent experiments.
[0013] To verify the direct interaction between HNP1 and P2Y12, molecular docking, surface plasmon resonance and fluorescence polarization experiments were performed.
[0014] First, molecular docking analysis visualized the binding conformation of HNP1 to P2Y12 (as shown in Figure 2 By analysis, the key amino acid residues mediating the interaction were determined: Arg15 and Arg24 of HNP1 formed tight contacts with Thr379 (distance: 2.7 Å), Glu197 (2.6 Å) and Leu193 (3.7 Å) of P2Y12, suggesting a stable molecular interaction interface.
[0015] Second, surface plasmon resonance technique was used to quantitatively analyze the affinity of P2Y12 protein binding to immobilized HNP1 protein at a fixed concentration. The experiment showed that only P2Y12 protein had no specific binding to the control buffer solution, while the binding to immobilized HNP1 was dose-dependent, with a dissociation constant of 26.5 nM (as shown in Figure 3 .
[0016] In addition, fluorescence polarization experiment was also performed to further confirm this interaction. When the concentration of P2Y12 increased, the polarization value of HNP1 increased in a concentration-dependent manner, and the dissociation constant (Kd) obtained was 29.75 nM, and the correlation coefficient (R) was 0.93 (as shown in Figure 4 . The consistency of the results of SPR and fluorescence polarization experiments strongly supports the specific and high-affinity binding of HNP1 to P2Y12 in vitro.
[0017] 2. P2Y12 activation is a key factor for HNP1-mediated exacerbation of OA As shown in Figure 5 , SD rats received DMM surgery and / or HNP1 injection on day 0, followed by local P2Y12 inhibitor treatment from day 14, and tissue collection started on day 28 (n=5 for each group).
[0018] As shown in Figure 6 , Figure 7 , representative micro-CT images of knee joints in each experimental group.
[0019] As shown in Figure 8 , SO-FG staining showed cartilage structure (scale: 200 μm; local magnification scale 50 μm).
[0020] As shown in Figure 9 , TRAP staining showed osteoclast activity (scale: 50 μm).
[0021] AsFigure 10~Figure 15 COL2 immunohistochemistry showed the expression of COL2 in the cartilage matrix, scale bar: 100 pm; magnified view scale bar 50 pm).
[0022] Figure 10 For quantitative analysis: Figure 11 Bone volume / total bone volume (BV / TV, %) in the modeling region, Figure 12 Trabecular thickness (Tb.Th, pm) in the modeling region, Figure 13 Trabecular space (Tb.Sp, pm) in the modeling region, Figure 14 Macroscopic ICRS score in the modeling region (B, D-H n=5 per group), Figure 15 , Figure 4 Mean ± SD of 5 random fields per group.
[0023] P2Y12 activation is a key factor in HNP1-mediated exacerbation of OA To verify the causal relationship between HNP1-induced P2Y12 activation and the exacerbation of OA progression, the effect of P2Y12 intervention on joint damage was studied in an in vivo rat OA model.
[0024] First, an OA model was established in 6- to 8-week-old male SD rats by medial meniscus destabilization (DMM) surgery (as shown in Figure 5 ). The rats were then divided into five groups: Sham group (sham operation group), OA group (DMM operation only), HNP1 group (HNP1 treatment only), OA+HNP1 group (DMM + HNP1), and OA+HNP1+P2Y12 inhibitor group (DMM + HNP1 + local injection of P2Y12 inhibitor).
[0025] Micro-CT analysis showed that the knee joint structure of the rats in the Sham group was intact, while the OA group showed obvious cartilage and subchondral bone damage. HNP1 treatment alone also caused joint abnormalities, especially the OA+HNP1 group showed more severe joint destruction than the OA group or the HNP1 group. Notably, P2Y12 inhibitor treatment significantly alleviated the joint damage in OA+HNP1 rats, and the joint morphology was restored in the sagittal and coronal planes of micro-CT (as shown in Figure 6 , Figure 7 ).
[0026] Second, histological evaluation was performed using SO-FG staining to assess the integrity of the cartilage. The Sham group showed strong Safranin O staining (indicating intact proteoglycan content), while the OA group showed reduced staining and cartilage degradation (as shown in Figure 8OA+HNP1 rats exhibited severe proteoglycan depletion and structural disruption. In contrast, P2Y12 inhibitor treatment largely preserved Safranin O staining and cartilage structure in OA+HNP1 rats, with significantly reduced cartilage degradation. Tartrate-resistant acid phosphatase (TRAP) staining, which was used to assess osteoclast activity, showed that the Sham group had very few stained cells, while the OA and HNP1 groups exhibited more TRAP-positive cells; OA+HNP1 rats exhibited a significant accumulation of osteoclasts, which was markedly reduced following P2Y12 inhibitor treatment (as shown in FIG. 6). Figure 9
[0027] In addition, quantitative analysis of micro-CT data showed that the Sham group had the highest bone volume (BV / TV) in the modeled region, while the OA and HNP1 groups had decreased BV / TV, and the OA+HNP1 group had further decreased BV / TV (as shown in FIG. 7). Figure 10 Figure 11 Figure 11
[0028] In addition, the Osteoarthritis Research Society International (OARSI) score was used to quantify cartilage degradation, and the Sham group had the lowest OARSI score, while the OA and HNP1 groups had increased OARSI scores, and the OA+HNP1 group had a significantly higher OARSI score (as shown in FIG. 8). Figure 8 Figure 9 Figure 14 Figure 15
[0029] Overall, these in vivo experimental results indicate that P2Y12 activation mediates the progression of HNP1-induced osteoarthritis, and inhibiting P2Y12 can effectively alleviate joint damage in osteoarthritis.
[0030] Studies related to this invention have demonstrated that HNP1 promotes the progression of osteoarthritis (OA) by acting on its receptor P2Y12; this interaction leads to the degradation of COL2 (an important component of articular cartilage) through activation of the NF-κB / IFIL44 pathway. Evidence supporting the biological plausibility of this mechanism is the significant elevation of HNP1 in OA samples, providing a physiological basis for its pathogenic role in osteoarthritis progression. This elevation is consistent with the view that HNP1 accumulates in the OA microenvironment and exerts a pro-degenerative effect, similar to how the presence of HD5 in colonic tissue supports its role in Shigella invasion.
[0031] This invention has identified P2Y12 as the receptor for HNP1-mediated COL2 loss. Human α-defensins (including HNP1) play an "antibacterial and anti-inflammatory" role in inflammation and degenerative processes, but the findings of this experiment completely overturn previous conventional understanding of HNP1.
[0032] Although this invention characterizes P2Y12 as a specific receptor for HNP1, its classification as a G protein-coupled receptor (GPCR) is consistent with existing trends: almost all known receptors that interact with human defensins belong to the GPCR family, reflecting the conservation of defensin-mediated cellular responses within the signal transduction framework. As a well-known purinergic receptor, P2Y12 differs from its family members in several ways. P2Y12 is classically thought to regulate platelet activation and hemostasis; however, this invention expands its functional scope to include the pathogenesis of OA, where it transduces pro-degenerative signals from HNP1. Notably, the NF-κB / IFIL44 pathway activated by the HNP1-P2Y12 interaction is a recognized driver of chondrocyte degradation in OA, further validating the relevance of this signaling pathway—previous studies have linked NF-κB activation to COL2 degradation and chondrocyte apoptosis, key evidence for OA progression.
[0033] Furthermore, in vitro validation of the HNP1-P2Y12 interaction confirmed the direct and specific binding between the ligand and receptor, ruling out the interference of non-specific off-target effects. More importantly, this invention also demonstrated the role of P2Y12 activation in HNP1-mediated OA invasion, directly linking the HNP1-P2Y12 axis to the progressive destruction of articular cartilage and joint structures in OA. However, whether HNP1 regulates the spatial distribution of P2Y12 in articular chondrocytes requires further investigation to fully elucidate the fine-tuning mechanisms of this pathway in OA.
[0034] In summary, these results further deepen the understanding of the mechanism of HNP1 promoting OA progression, and open up the possibility of new intervention strategies for OA. Targeting the HNP1-P2Y12-NF-κB / IFIL44 axis may provide a new approach to inhibit COL2 degradation and cartilage destruction, filling the gap in OA treatment.
[0035] Animal experiments have been approved by the Experimental Animal Ethics Committee of Fudan University School of Basic Medical Sciences (Ethical Approval Number: 20240229-035). The study on the mechanism of human alpha-defensin HNP1 promoting osteoarthritis involves relevant biomedical aspects and has been approved by the Ethics Committee of Fudan University (Ethical Approval Number: FE24138R).
Claims
1. Application of P2Y12 inhibitors in the treatment of osteoarthritis: P2Y12 inhibitors can be used to treat osteoarthritis.
2. The P2Y12 inhibitor according to claim 1, characterized in that: The inhibitor is clopidogrel, or ticlopidine, or ticagrelor, or prasugrel, or canagrelor.