Use of an OGG1 specific inhibitor TH5487 in the preparation of a medicament for delaying or treating intervertebral disc degeneration

Abstract

The application discloses application of an OGG1 specific inhibitor TH5487 in preparation of a medicine for delaying or treating intervertebral disc degeneration, and discloses that TH5487 is used as a core active ingredient to play a key pharmacological role, and that TH5487 is used to inhibit M1 type polarization of macrophages in intervertebral disc nucleus pulposus tissue, relieve extracellular matrix destruction of nucleus pulposus cells, and relieve fibrosis. The application discloses, for the first time, application of TH5487 in preparation of a medicine for treating or delaying intervertebral disc degeneration, and proves, through systematic research, that TH5487 has therapeutic potential for delaying or even intervening in the intervertebral disc degeneration process as an active compound, establishes a new indication of TH5487 in skeletal muscle system degenerative diseases, expands the clinical application range of the compound, and provides a solid scientific basis for subsequent clinical transformation and large-scale development of the compound.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of an OGG1 specific inhibitor TH5487 in the preparation of drugs for delaying or treating intervertebral disc degeneration. Background Technology

[0002] Lower back pain is a common musculoskeletal disorder with a high incidence and disability rate worldwide. Intervertebral disc degeneration is one of the most common organic causes, including endplate degeneration, annulus fibrosus degeneration, and nucleus pulposus degeneration. The nucleus pulposus is a crucial structure of the intervertebral disc, providing hydrostatic pressure and maintaining the biomechanical stability of the disc and the entire spine. Nucleus pulposus cells are responsible for synthesizing proteoglycans and type II collagen, which are highly hydrophilic and maintain the osmotic pressure and hydration of the nucleus pulposus, giving it excellent shock absorption capabilities. When nucleus pulposus cell function declines, proteoglycan synthesis decreases, and the activity of extracellular matrix degrading enzymes increases, leading to water loss, matrix softening, and a decrease in the disc's pressure-bearing capacity. A normal nucleus pulposus is gel-like; as degeneration progresses, the extracellular matrix is ​​gradually replaced by fibrous tissue rich in type I collagen, transforming the nucleus pulposus from a "jelly-like" state to a "fibrotic" state, significantly reducing its elasticity. Extracellular matrix degradation and fibrosis, as characteristics of intervertebral disc degeneration, are closely related to inflammation within the microenvironment.

[0003] Currently, most research on the mechanisms and treatments of intervertebral disc degeneration focuses on single cells. In addition to nucleus pulposus cells, macrophages also exist within the nucleus pulposus. Macrophages originate from monocytes. When monocytes migrate from the blood to the tissue, they differentiate into resting macrophages (M0 type) under the influence of the tissue microenvironment. Under different stimuli, M0 type macrophages polarize into M1 (pro-inflammatory) and M2 (anti-inflammatory) types. Single-cell sequencing studies have found a close relationship between M1 type macrophage polarization and the degree of intervertebral disc degeneration, suggesting that M1 type macrophage polarization may participate in the process of intervertebral disc degeneration.

[0004] TH5487 is a highly effective and selective inhibitor of 8-oxoguanine DNA glycosylase 1 (OGG1). It inhibits DNA repair by blocking the binding of OGG1 to its DNA substrate and regulates OGG1 chromatin dynamics. TH5487 antagonizes the binding of OGG1 to SMAD7, thereby reducing fibroblast differentiation and playing a therapeutic role in idiopathic pulmonary fibrosis. Furthermore, in an ovalbumin-induced mouse airway inflammation model, TH5487 effectively reduces the infiltration of inflammatory cells and the secretion of inflammatory factors in lung tissue, improving the airway inflammatory response. However, the application of TH5487 in the field of intervertebral disc degeneration is currently lacking.

[0005] In summary, current etiological interventions for early intervertebral disc degeneration are still lacking. Therefore, systematically elucidating the key molecular targets of intervertebral disc degeneration and developing novel treatment strategies with targeted and repair potential based on these targets is of significant scientific and clinical value for improving the overall prevention and treatment of low back pain and its long-term prognosis. Summary of the Invention

[0006] The present invention aims to address the above-mentioned deficiencies in the prior art by proposing the application of the OGG1 specific inhibitor TH5487 in the preparation of drugs for delaying or treating intervertebral disc degeneration. TH5487 can delay the process of intervertebral disc degeneration by inhibiting macrophage M1 polarization from the perspective of cell interaction, improving the inflammatory microenvironment, reducing the destruction of extracellular matrix and fibrosis of nucleus pulposus cells.

[0007] The technical concept of this invention can be realized through the following implementation scheme:

[0008] The first objective of this invention is to propose the application of the OGG1-specific inhibitor TH5487 in the preparation of drugs for delaying or treating intervertebral disc degeneration, with TH5487 as the core active ingredient to exert key pharmacological effects. The molecular structure of TH5487 is as follows:

[0009] .

[0010] Furthermore, the intervertebral disc degeneration is described as intervertebral disc nucleus pulposus degeneration.

[0011] Furthermore, TH5487 inhibits M1 polarization of macrophages in the nucleus pulposus tissue of the intervertebral disc, alleviating extracellular matrix damage and fibrosis of nucleus pulposus cells.

[0012] A second objective of this invention is to provide a drug for delaying or treating intervertebral disc degeneration, said drug comprising the OGG1-specific inhibitor TH5487.

[0013] Furthermore, the concentration at which the drug exerts its therapeutic effect, calculated as TH5487, is 1~50 μM.

[0014] Furthermore, the concentration at which the drug exerts its therapeutic effect, calculated as TH5487, is 10 μM.

[0015] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0016] Furthermore, the pharmaceutically acceptable excipients include one or more of solvents, carriers, and excipients.

[0017] Furthermore, the pharmaceutically acceptable excipients include one of nanomaterials, hydrogels, exosomes, etc.

[0018] Thirdly, the present invention provides a method for delaying intervertebral disc degeneration in vitro for non-therapeutic purposes, by treating cells in the nucleus pulposus tissue of the intervertebral disc with the OGG1-specific inhibitor TH5487.

[0019] Furthermore, the cells within the intervertebral disc nucleus pulposus tissue include nucleus pulposus cells, macrophages, T lymphocytes, and neutrophils.

[0020] Compared with the prior art, the present invention has the following outstanding advantages:

[0021] (1) This invention discloses for the first time the application of TH5487 in the preparation of drugs for treating or delaying intervertebral disc degeneration. A targeted intervention strategy is constructed using TH5487 as the active ingredient. By inhibiting macrophage M1 polarization, the inhibitory effect of the inflammatory microenvironment on the activity of nucleus pulposus cells is effectively alleviated, the imbalance between extracellular matrix synthesis and degradation is improved, and the fibrosis of nucleus pulposus tissue is inhibited, thereby achieving the delay and intervention of intervertebral disc degeneration.

[0022] (2) This invention is the first to extend the application of TH5487 to the prevention and treatment of intervertebral disc degeneration. Systematic research has confirmed that TH5487, as an active compound, has the therapeutic potential to delay and even intervene in the process of intervertebral disc degeneration. In vitro experiments show that TH5487 can effectively inhibit the polarization of M0 macrophages to the pro-inflammatory M1 type, thereby alleviating nucleus pulposus cell degeneration. In vivo experiments further verify that local injection of TH5487 into the coccygeal intervertebral disc can significantly alleviate acupuncture-induced polarization of M1 macrophages within the intervertebral disc and the destruction of the intervertebral disc structure. This establishes a new indication for TH5487 in degenerative diseases of the musculoskeletal system, expands the clinical application scope of this compound, and provides a solid scientific basis for its subsequent clinical translation and large-scale development. Attached Figure Description

[0023] Figure 1 The effect of TH5487 on macrophage M1 polarization in Example 1;

[0024] Figure 2 The effect of TH5487 on nucleus pulposus cell activity in Example 2;

[0025] Figure 3 This illustrates the effect of TH5487 on the expression levels of extracellular matrix-related proteins in nucleus pulposus cells, as shown in Example 2.

[0026] Figure 4 This illustrates the effect of TH5487 on the levels of fibrosis markers in nucleus pulposus cells in Example 2.

[0027] Figure 5 This refers to the preventive and therapeutic effect of TH5487 on intervertebral disc degeneration in rats in Example 3;

[0028] Figure 6 This refers to the inhibition of M1 type polarization of rat intervertebral disc macrophages by TH5487 in Example 3. Detailed Implementation

[0029] The present invention will be further explained below with reference to the accompanying drawings. The presented embodiments are mainly used to help those skilled in the art to better grasp the technical essence and do not constitute a limitation on the scope of the claims of the present invention. In addition, without departing from the basic principles of the present invention, those skilled in the art can make adaptive adjustments, functional extensions or performance improvements to the details of the solution according to actual application scenarios; all improvements and optimizations made based on the same technical concept are within the protection scope covered by the present invention.

[0030] Unless otherwise specified, all reagents or materials used in this embodiment can be obtained through commercial channels.

[0031] Example 1: In vitro experimental verification of the inhibitory effect of the OGG1-specific inhibitor TH5487 on macrophage M1 polarization

[0032] Human monocytic leukemia THP-1 cell line was cultured in RPMI-1640 basal medium, supplemented with 10% (v / v) fetal bovine serum and 1% penicillin-streptomycin solution, and routinely passaged in a 37℃, 5% CO2 constant temperature and humidity incubator. Fresh culture medium was changed daily, and cell adhesion, morphology and proliferation activity were evaluated under a microscope.

[0033] The following three experimental groups were set up:

[0034] (1) M0 group: THP-1 cells were induced into M0 macrophages, i.e. resting macrophages, by treating them with 50 nM Phorbol 12-myristate 13-acetate (PMA) for 24 hours.

[0035] (2) M1 group: After PMA treatment, M0 cells were induced into M1 macrophages, i.e. inflammatory macrophages, by treatment with 100 ng / ml Lipopolysaccharide (LPS) and 20 ng / ml Interferon gamma (IFNγ) for 48 hours.

[0036] (3) M1+TH5487 group: After PMA treatment, LPS+IFNγ was added for 24 hours, and then 10μMTH5487 was added for co-culture for 24 hours.

[0037] Immunofluorescence was used to detect the fluorescence intensity of interleukin-1β (IL1β) and CD86, markers of M1 macrophages, in the cells of the three experimental groups. Stronger fluorescence intensity indicated higher protein expression levels. Results are as follows: Figure 1 As shown, the fluorescence intensity of IL1β and CD86 in M1 macrophages of group M1 was significantly higher than that of group M0. After treatment with TH5487, the fluorescence intensity of IL1β and CD86 in M1+TH5487 group decreased, indicating that M1 polarization of macrophages was successfully induced and that TH5487 could inhibit M1 polarization of macrophages.

[0038] Example 2: In vitro experimental verification of the alleviating effect of the OGG1 specific inhibitor TH5487 on nucleus pulposus cell degeneration

[0039] In this embodiment, the three groups of cells treated in Example 1 were replaced with fresh RPMI-1640 medium and cultured in a cell culture incubator for 24 hours. The inner culture medium was then aspirated from the culture dishes and centrifuged at 3000 rpm for 10 minutes to obtain cell supernatants. These three groups of cell supernatants were added to culture dishes of human nucleus pulposus cells. The human nucleus pulposus cells corresponding to the M0, M1, and M1+TH5487 macrophage supernatants were designated as M0@HNPC, M1@HNPC, and M1+TH5487@HNPC, respectively. To ensure stable biological activity, primary human nucleus pulposus cells were all obtained from the 2nd to 3rd generation logarithmic growth phase. After culturing for 24-48 hours, nucleus pulposus cells were harvested for CCK8 assay, Western blot experiments, and immunofluorescence.

[0040] The CCK8 kit can be used to detect cell viability, and the results are as follows: Figure 2 As shown, M1@HNPC cells had the lowest cell viability, followed by TH5487@HNPC cells, while M0@HNPC cells had the highest cell viability.

[0041] Western blot experiments are used to detect the expression levels of proteins within cells, such as... Figure 3As shown, in the M1@HNPC group, the expression of extracellular matrix synergistic metabolic markers aggrecan and collagen II in nucleus pulposus cells treated with M1 macrophage supernatant decreased, while the expression of extracellular matrix catabolism markers matrix metalloproteinases 3 and 13 (MMP3 and 13) and human metallopeptidase containing thromboretin type 1 motif 4 and 5 (ADAMTS4 and 5) increased. Compared with the M1@HNPC group, the expression of extracellular matrix synergistic metabolic markers in nucleus pulposus cells of the M1+TH5487@HNPC group increased, while the expression of catabolism-related markers decreased.

[0042] Figure 4 Immunofluorescence results showed that the fluorescence intensity of connective tissue growth factor (CTGF) and transforming growth factor β1 (TGFβ1), indicators of nucleus pulposus fibrosis, increased after stimulation with M1 macrophage supernatant, but decreased in the M1+TH5487@HNPC group.

[0043] This embodiment demonstrates that TH5487 can alleviate nucleus pulposus cell degeneration by inhibiting macrophage M1 polarization, improving cell activity of nucleus pulposus cells, and inhibiting extracellular matrix degradation and fibrosis.

[0044] Example 3: In vivo experimental verification of the relief or treatment of intervertebral disc degeneration by the OGG1 specific inhibitor TH5487.

[0045] Thirty adult SD rats were randomly divided into the following three groups:

[0046] (1) SHAM group: sham surgery group, only fine needle puncture of the skin without affecting the intervertebral disc.

[0047] (2) IVDD group: A degeneration model was constructed by puncturing the intervertebral disc of the rat caudal vertebrae with a fine needle.

[0048] (3) IVDD+ TH5487 group: After intervertebral disc puncture and modeling, 10μM TH5487 was injected into the intervertebral disc of the same segment.

[0049] The specific experiments for the above-mentioned animal experiments are as follows:

[0050] SD rats were anesthetized and placed in a prone position. Using X-ray image guidance combined with fine-needle localization, the intervertebral disc space between the 8th and 9th vertebrae of the coccyx was precisely located, and the puncture point was marked on the body surface with a marker pen. Subsequently, the localized area was disinfected with povidone-iodine. A 27G syringe needle was inserted vertically into the intervertebral space, rotated 180° at the needle tip, held for 60 seconds, and then removed. The needle was then disinfected with povidone-iodine. Two weeks later, the IVDD model group received an injection of 2 μL of physiological saline into the intervertebral space, while the IVDD + TH5487 group received an injection of 2 μL of 10 μM TH5487 solution. The injections were disinfected with povidone-iodine afterward. Four weeks later, all rats were euthanized, and the coccygeal intervertebral disc tissue was harvested, fixed with paraformaldehyde, decalcified, embedded, and sectioned. The sections were stained with safranin and fast green for histological scoring and immunofluorescence staining.

[0051] Safranin-Fix-Green staining can be used for the histological assessment of intervertebral disc degeneration; the higher the score, the greater the degree of degeneration. For example... Figure 5 As shown, the IVDD group exhibited the most severe nucleus pulposus tissue damage, with an indistinct boundary between the annulus fibrosus and the nucleus pulposus, and a lighter red staining of the nucleus pulposus, resulting in the highest histological score. However, after administration of TH5487, the volume of preserved nucleus pulposus tissue increased, the boundary between the nucleus pulposus and the annulus fibrosus became clearer, and the intensity of red staining of the nucleus pulposus improved, leading to a decrease in histological score compared to the IVDD group.

[0052] Regarding macrophage polarization in inflammatory vivo, this embodiment uses green fluorescence and red fluorescence to label the universal macrophage marker CD68 and the M1 macrophage marker IL1β, respectively. Figure 6 As shown, immunofluorescence of tissues revealed that after acupuncture, the fluorescence intensity of green cells in the intervertebral discs of the IVDD group increased, as did the fluorescence intensity of red cells. After TH5487 injection, the green fluorescence intensity in the intervertebral discs of the IVDD+TH5487 group decreased, and the ratio of red fluorescence intensity to green fluorescence intensity also decreased.

[0053] This embodiment demonstrates that intradiscal injection of TH5487 into the intervertebral disc of degenerated rats can reduce macrophage infiltration and M1 polarization within the intervertebral disc, and reduce damage to the intervertebral disc structure, thus playing a role in delaying or treating intervertebral disc degeneration.

[0054] Although the present invention has been illustrated and described in detail through several specific embodiments, those skilled in the art should understand that as long as the technical principles and core ideas on which the present invention is based are not departed from, the above embodiments can be appropriately optimized, specifically improved, or have equivalent technical features replaced in terms of appearance, internal structure, etc., or can be reasonably evolved based on the same functional objectives. The legal protection boundary of the present invention shall be determined by the technical features recorded in the claims and the technical solutions extended and covered by the principle of equivalence.

Claims

1. The use of TH5487, an OGG1-specific inhibitor, in the preparation of drugs for delaying or treating intervertebral disc degeneration.

2. Use according to claim 1, characterized in that: TH5487 is used as the core active ingredient to exert key pharmacological effects. The molecular structure of TH5487 is as follows: 。 3. Use according to claim 1, characterized in that: The intervertebral disc degeneration is described as intervertebral disc nucleus pulposus degeneration.

4. The use according to claim 1, characterized in that: The TH5487 inhibits M1 polarization of macrophages in the nucleus pulposus tissue of the intervertebral disc, and alleviates the destruction of the extracellular matrix and fibrosis of nucleus pulposus cells.

5. A medicament for delaying or treating intervertebral disc degeneration, characterized by: The drug mentioned includes the OGG1-specific inhibitor TH5487.

6. The medicament according to claim 5, wherein: The concentration at which the drug exerts its effect, calculated as TH5487, is 1~50 μM.

7. The medicament according to claim 5 or 6, characterized in that: The concentration at which the drug exerts its effect, expressed as TH5487, is 10 μM.

8. The drug as described in claim 5, characterized in that: The drug also includes pharmaceutically acceptable excipients.

9. The medicament according to claim 8, characterized in that: Pharmaceutically acceptable excipients include one or more of solvents, carriers, excipients, nanomaterials, hydrogels, and exosomes.

10. A method of delaying intervertebral disc degeneration for non-therapeutic purposes in vitro, characterized in that: Cells within the nucleus pulposus tissue of the intervertebral disc were treated with the OGG1-specific inhibitor TH5487.

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