Application of CSF1R inhibitor in preparation of medicine for treating intervertebral disc degeneration and / or relieving discogenic pain

Abstract

The invention discloses an application of a CSF1R inhibitor in preparation of a medicine for treating intervertebral disc degeneration and / or relieving discogenic pain, and particularly relates to a new application of GW2580 in treatment of intervertebral disc degeneration. Through targeted inhibition of a CSF1 / CSF1R signal channel, proinflammatory signal cross dialogue between intervertebral disc nucleus pulposus cells and macrophages is effectively interrupted, so that local inflammatory response in the intervertebral disc is remarkably relieved. The treatment does not cause significant reduction of the number of macrophages, indicating that the action mechanism is functional immune regulation. It is verified in an animal model of intervertebral disc degeneration that GW2580 treatment can significantly reduce pain sensitivity, and more importantly, imaging and histopathologic analysis confirms that the structural morphology of the intervertebral disc of a treatment group is better maintained, the degeneration process is effectively delayed, and the performance of the intervertebral disc is closer to that of a normal intervertebral disc.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the development of small molecule drugs and the treatment of degenerative diseases. More specifically, it relates to a small molecule inhibitor therapeutic strategy targeting colony-stimulating factor 1 receptor (CSF1R), including pharmaceutical compositions containing CSF1R inhibitors, methods for their preparation, and their use in the preparation of drugs for treating or delaying intervertebral disc degeneration (IVDD) and / or relieving discogenic pain. Background Technology

[0002] The information disclosed in this background section is intended only to enhance some understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art.

[0003] Chronic low back pain (LBP) is the leading cause of disability worldwide, and its primary pathological basis is intervertebral disc degeneration (IVDD). Current clinical treatment strategies for IVDD, such as nonsteroidal anti-inflammatory drugs, physical therapy, and surgical intervention, can only relieve symptoms and cannot achieve disease-modifying therapy by addressing the core pathological process.

[0004] Intra-intervertebral disc degeneration (IVDD) is essentially a highly complex degenerative disease driven by an inflammatory response. Macrophages, through their phenotypic plasticity, play a central role in the establishment and maintenance of the intervertebral disc inflammatory microenvironment. During degeneration, the "immune exemption" state of the nucleus pulposus (NP) is disrupted, leading to massive macrophage infiltration and abnormal activation. These activated macrophages, by secreting pro-inflammatory cytokines (such as IL-1β and TNF-α) and matrix-degrading enzymes (such as MMPs and ADAMTS), directly cause an imbalance in the catabolic metabolism of the extracellular matrix (ECM), disrupting the structural integrity of the intervertebral disc. Simultaneously, neurotrophic factors produced by the degenerated nucleus pulposus can induce abnormal nerve fiber ingrowth, becoming a direct cause of discogenic pain.

[0005] Colony-stimulating factor 1 receptor (CSF1R) is a key receptor tyrosine kinase that regulates macrophage survival, proliferation, and polarization. Although CSF1R inhibitors (such as PLX3397) have been developed for applications in oncology and other fields, their mechanism of action relies on the complete depletion of macrophages, which may lead to risks such as immunosuppression and impaired tissue repair. Summary of the Invention

[0006] Compared with existing technologies, this invention, through single-cell transcriptome sequencing analysis, has for the first time demonstrated significant macrophage infiltration and simultaneous upregulation of CSF1R expression in degenerated human intervertebral disc tissue. Functional experiments further revealed that the CSF1 / CSF1R signaling pathway is a key mechanism driving intervertebral disc inflammation and structural damage.

[0007] This invention is the first to discover and validate the unique value of the highly selective small-molecule CSF1R inhibitor GW2580 in IVDD treatment: it can effectively block CSF1R signaling, reprogram macrophage function, and inhibit its pro-inflammatory activity, thereby breaking the vicious cycle of inflammation without causing widespread macrophage clearance. This unique mechanism of action allows it to achieve disease-modifying effects while also possessing higher potential safety.

[0008] In summary, this invention not only provides an in-depth analysis of the core role of CSF1R signaling in IVDD, but also innovatively proposes a new strategy for treating IVDD by functionally regulating macrophages rather than eliminating cells, providing an important theoretical basis and translational direction for developing disease-modifying therapies that are both highly effective and safe.

[0009] The technical solution adopted in this invention is as follows: In a first aspect of the invention, a pharmaceutical composition is provided for treating and / or delaying intervertebral disc degeneration (IVDD) and / or relieving discogenic pain, comprising a therapeutically effective amount of a colony-stimulating factor 1 receptor (CSF1R) inhibitor and a pharmaceutically acceptable carrier.

[0010] In one or more embodiments of the present invention, the CSF1R inhibitor is a small molecule CSF1R inhibitor.

[0011] Preferably, the small molecule CSF1R inhibitor is GW2580 (5-((5-methoxy-1H-indol-3-yl)methylene)-2,4-thiazolidinedione) or a pharmaceutically acceptable salt, solvate, hydrate, or crystal form thereof. GW2580 is a highly selective CSF1R kinase inhibitor with oral bioavailability.

[0012] In one or more embodiments of the present invention, the pharmaceutical composition is formulated as an oral or injectable dosage form to meet different clinical needs. Oral dosage forms include, but are not limited to, oral suspensions, tablets, or capsules. Injectable dosage forms include injectables suitable for systemic administration, such as intravenous, intramuscular, or locally administered paravertebral / intervertebral disc injections; said injectables include, but are not limited to, nanoparticle delivery systems.

[0013] Preferably, the average particle size of the nanoparticles in the nanoparticle delivery system is between 50 nanometers and 500 nanometers.

[0014] Preferably, the nanoparticle delivery system is also loaded with other active agents, which are anti-inflammatory cytokines or matrix synthesis-promoting factors.

[0015] Preferably, the nanoparticle delivery system is selected from liposomes, polymer nanoparticles, or solid lipid nanoparticles (SLNP).

[0016] More preferably, the nanoparticle delivery system is a liposome; The liposomes described herein are composed of the following components: phosphatidylcholine (DSPC), cholesterol, and polyethylene glycol-modified phospholipids (DSPE-PEG2000); and a therapeutically effective amount of GW2580 and / or analgesics; The molar ratio of DSPC, cholesterol and DSPE-PEG2000 is (50~90):(30~65):(3~8); the molar ratio of GW2580 to analgesic is 1:10 to 10:1, preferably 1:2 to 2:1; the analgesic includes, but is not limited to, acetaminophen and ibuprofen.

[0017] In one or more embodiments of the invention, the pharmaceutically acceptable carrier comprises excipients for forming an oral suspension.

[0018] Preferably, the excipients used to form the oral suspension are hydroxypropyl methylcellulose (HPMC) and Tween 80. More preferably, the concentration of HPMC is 0.5% (w / v) and the concentration of Tween 80 is 0.1% (w / v); meaning that each 100 ml of liquid formulation (e.g., oral suspension) contains 0.5 g of HPMC and 0.1 g of Tween 80.

[0019] In one or more embodiments of the present invention, the pharmaceutical composition may also be used in combination with a second active agent, which includes, but is not limited to, analgesics (such as acetaminophen, ibuprofen), nonsteroidal anti-inflammatory drugs (NSAIDs) or corticosteroids.

[0020] In a second aspect of the invention, there is a use of a colony-stimulating factor 1 receptor (CSF1R) inhibitor in the preparation of a medicament for treating and / or delaying intervertebral disc degeneration (IVDD) and / or relieving discogenic pain; said medicament exerts its effect by inhibiting the CSF1 / CSF1R signaling pathway without significantly reducing the number of macrophages in the subject.

[0021] In one or more embodiments of the present invention, the CSF1R inhibitor is a small molecule CSF1R inhibitor.

[0022] Preferably, the small molecule CSF1R inhibitor is GW2580 (5-((5-methoxy-1H-indol-3-yl)methylene)-2,4-thiazolidinedione) or a pharmaceutically acceptable salt, solvate, hydrate or crystal form thereof.

[0023] In one or more embodiments of the present invention, the drug is formulated as an oral dosage form or an injectable dosage form.

[0024] In one or more embodiments of the present invention, the dosage of the drug is from 10 mg to 200 mg per kilogram of body weight per day, preferably 80 mg per kilogram of body weight per day; or The drug is administered at a fixed dose of 100 mg to 2000 mg daily.

[0025] The drug can be administered once or twice daily.

[0026] In one or more embodiments of the invention, the drug is used to initiate administration in the early stages following intervertebral disc injury.

[0027] In one or more embodiments of the present invention, the drug is capable of reprogramming macrophage function to polarize it toward an anti-inflammatory M2 phenotype rather than clearing macrophages; the drug is capable of blocking pro-inflammatory signaling pathways driven by nucleus pulposus cells (Fib-Inf-NPCs) that highly express CSF1.

[0028] In a third aspect of the invention, a medicine box for treating intervertebral disc degeneration and / or relieving discogenic pain is provided, comprising: a) the pharmaceutical composition as described in the first aspect; and b) Optional, use the instruction manual.

[0029] In one or more embodiments of the present invention, the cassette further comprises a second active agent, which includes, but is not limited to, analgesics (such as acetaminophen, ibuprofen), nonsteroidal anti-inflammatory drugs (NSAIDs), or corticosteroids.

[0030] In a fourth aspect of the invention, a pharmaceutical combination package is provided, comprising: 1) The pharmaceutical composition as described in the first aspect; 2) A second activator; and 3) Optionally, instructions for simultaneous, separate or sequential administration.

[0031] In one or more embodiments of the present invention, the second active agent includes, but is not limited to, analgesics (such as acetaminophen, ibuprofen), nonsteroidal anti-inflammatory drugs (NSAIDs) or corticosteroids.

[0032] Compared with the related technologies known to the inventors, one of the technical solutions of the present invention has the following beneficial effects: (1) Multi-target intervention: At the local intervertebral disc level, it effectively inhibits macrophage infiltration and the release of pro-inflammatory factors (such as IL-6 and MMP9); at the central level, it significantly inhibits the activation of microglia in the dorsal horn of the spinal cord, blocks the transmission of pain signals, and achieves both symptomatic and radical treatment.

[0033] (2) High selectivity: GW2580 exhibits high inhibitory activity against CSF1R (IC50). 50 = 30 nM), and has a selectivity of more than 100 times for related kinases such as c-Kit and FLT3, effectively avoiding off-target effects and having good safety.

[0034] (3) Excellent safety: Unlike traditional macrophage depletion agents (such as PLX3397), GW2580 does not affect the number of microglia, and no disruption of immune homeostasis was observed with long-term use (mouse weight and blood routine indicators were normal).

[0035] (4) Clear clinical translation potential: MRI imaging assessment confirmed that the T2 signal intensity was preserved to 50% of normal after treatment, demonstrating excellent disease modification effect. Attached Figure Description

[0036] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0037] Figure 1 A schematic diagram illustrating the principle of using CSF1R inhibitors to treat intervertebral disc degeneration and / or relieve discogenic pain.

[0038] Figure 2 A mouse model of intervertebral disc degeneration induced by intervertebral disc puncture; Schematic diagram of the method for constructing the intervertebral disc degeneration (DP) model. This study used a posterolateral surgical approach to establish a mouse DP model; Histological analysis revealed significant intervertebral disc degeneration in the DP model group. Compared to the sham-operated group, mice in the DP model group (180 days post-modeling) showed a significant reduction in proteoglycan content in the L5 / 6 intervertebral discs stained with Safranin-O / Fast Green staining, and H&E staining showed a decrease in the number of nucleus pulposus (NP) cells, disordered tissue structure, and destruction of the cartilaginous endplate (CEP) structure. Scale bar = 150 micrometers (main image), 60 micrometers (enlarged image); Imaging examinations further confirmed intervertebral disc degeneration in the DP model group. MRI showed a significant decrease in T2 signal value in the DP model group (sham surgery group n=8, DP model group n=6, ***p<0.001), indicating reduced nucleus pulposus water content and accelerated degeneration. Simultaneously, compared with the uninjured group (n=9), intervertebral disc height measurements at 3 and 6 months post-DP modeling (n=10 / group) showed significant height loss (*p<0.05), confirming the progression of intervertebral disc structural damage (analysis of variance, data expressed as mean ± standard error). (DG) Behavioral testing results showed that the DP model successfully induced pain-related behavioral changes. Mice exhibited significant multimodal hyperalgesia after DP modeling: von Frey test showed mechanical hyperalgesia (D), and Plantar test showed cold hyperalgesia (E) and thermal hyperalgesia (F) (n=10 / group for all experiments, *p<0.005). Furthermore, the nesting behavior test (G) showed a significant reduction in spontaneous behavioral motivation in the model group (n=10 / group, *p<0.05), indicating that the DP model can successfully simulate chronic pain behaviors and functional impairments associated with intervertebral disc degeneration (analysis of variance, data are expressed as mean ± standard error).

[0039] Figure 3 CSF1R inhibitor GW2580 slows down the process of intervertebral disc degeneration; (A) Schematic diagram of experimental timeline: The study used C57BL / 6 mice to establish a disc puncture (DP) degeneration model. After modeling, the CSF1R specific inhibitor GW2580 (treatment group) or an equal amount of solvent (control group) was administered orally. The treatment continued until the end of the sampling period. (B) Imaging evaluation showed that GW2580 treatment significantly delayed the progression of intervertebral disc degeneration. Top image: Representative MRI images 180 days after modeling (white arrows indicate L4 / L5 and L5 / L6 intervertebral discs); Bottom image: Quantitative T2 signal analysis showed that the GW2580 treatment group maintained a high signal intensity, indicating that the nucleus pulposus water content was maintained and the degeneration process was delayed (*p<0.05; one-way ANOVA; n = 7 / group). Simultaneously, intervertebral disc height measurement showed that the intervertebral disc height index in the GW2580 treatment group was significantly improved compared to the solvent group 180 days after modeling (*p<0.05; one-way ANOVA; n = 5 / group). (C) Histological analysis confirmed the protective effect of GW2580 treatment on intervertebral disc structure. H&E staining showed that compared with the solvent group, the GW2580 treatment group had a more intact intervertebral disc structure, a clearer nucleus pulposus-annulus fibrosus boundary, and more orderly cell arrangement (scale bar: main image 150 μm, magnified image 30 μm). Safranin-O / Fixed Green staining showed that the GW2580 treatment group had a higher content of proteoglycans, indicating that the degradation of extracellular matrix was alleviated and cartilage-like tissue was better preserved (scale bar: 150 μm). (D) Immunohistochemical analysis showed that GW2580 significantly reduced intervertebral disc inflammation. IBA1 immunohistochemical staining showed that GW2580 treatment significantly reduced macrophage infiltration in the intervertebral disc tissue; quantitative analysis confirmed a significant decrease in the number of IBA1-positive cells in the treatment group (*p<0.05; one-way ANOVA; n = 5 / group; scale bar: 15 micrometers). These results indicate that GW2580 exerts a protective effect by inhibiting the CSF1R signaling pathway and reducing the inflammatory response during intervertebral disc degeneration.

[0040] Figure 4 CSF1R inhibitor GW2580 relieves neuropathic pain caused by intervertebral disc degeneration; (A) Quantitative analysis of IBA1-positive cells in dorsal root ganglia (DRG) showed that GW2580 treatment significantly reduced macrophage infiltration in wild-type mouse DRGs (data are expressed as mean ± standard error; *p<0.05; ANOVA; n = 5-6 DRG samples). (B) Immunofluorescence staining analysis of the dorsal horn of the spinal cord: The left image shows the co-localization staining results of IBA1 (green) and CD68 (red); the right image shows the quantitative analysis of CD68-positive cells. The number of CD68-positive cells was significantly reduced in the GW2580 treatment group, indicating that microglia activation was inhibited (scale bar: 50 micrometers; n = 8-9 cells / group; *p<0.05; ANOVA). (C) Schematic diagram of the TRAP system: Fos expression in injury-activated neurons, induced by tamoxifen (TAM) via the CreERT2 recombinase system, drives tdTomato reporter gene expression. The figure below shows the increase in Fos(tdTomato) expression in the dorsal horn of the lumbar spinal cord after DP surgery (scale bar: main figure 400 μm, inset 10 μm); (D) Expression of Fos(tdTomato) in DRG (left figure) and its quantitative analysis (right figure). GW2580 treatment significantly reduced the number of Fos-positive cells (scale bar: main figure 400 μm, inset 10 μm; *p<0.05; ANOVA; n = 5 cells / group). (E) Expression of Fos (tdTomato) in the dorsal horn of the lumbar spinal cord (left figure) and its quantitative analysis (right figure). GW2580 treatment significantly reduced Fos expression in dorsal horn neurons (scale bar: main figure 400 μm, inset 10 μm; *p<0.05; ANOVA; n = 5 animals / group). (F) Mechanical hyperalgesia test: The Von Frey experiment showed that GW2580 treatment significantly improved DP-induced mechanical hyperalgesia compared with the solvent group (data are expressed as mean ± standard error; n = 9 (GW2580 group), n = 8 (sham surgery group and solvent group); *p<0.05; ANOVA). (G) Cold hyperalgesia test: The Plantar test showed that GW2580 treatment significantly relieved cold hyperalgesia caused by DP (data are expressed as mean ± standard error; n = 9 (GW2580 group), n = 8 (sham surgery group and solvent group); *p<0.05; ANOVA).

[0041] Figure 5 Topical administration of GW2580 can slow down the degeneration of intervertebral discs; Experimental timeline diagram: This study used rats to establish a rat intervertebral disc puncture degeneration model. After modeling, CSF1R specific inhibitor GW2580 (treatment group) or an equal volume of solvent (control group) were administered locally to the intervertebral disc, and treatment continued until the end of the sampling period; (B) Mechanical hyperalgesia test: The Von Frey experiment showed that GW2580 treatment significantly improved mechanical hyperalgesia induced by disc puncture compared with the solvent group (data are expressed as mean ± standard error; n = 9 in the GW2580 group, n = 11 in the sham surgery group and the solvent group; *p<0.05; ANOVA). (CD) Imaging assessment: CT scan results of intervertebral disc height measurement showed that the intervertebral disc height index in the GW2580 treatment group was significantly improved compared with the solvent group. Detailed Implementation

[0042] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, 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 pertains.

[0043] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0044] Based on the discovery of an innovative mechanism, this invention proposes a disease modification treatment strategy for intervertebral disc degeneration (IVDD) and related pain. Its core lies in the application of the highly selective CSF1R small molecule inhibitor GW2580 to target and regulate the immune microenvironment.

[0045] I. Mechanism Discovery Single-cell RNA sequencing (scRNA-seq) analysis of degenerated intervertebral disc tissue in humans identified for the first time a subset of nucleus pulposus cells (NPCs) highly expressing CSF1 (Fib-Inf-NPCs). These cells continuously secrete CSF1, activating CSF1R+ macrophages and driving and maintaining a local pro-inflammatory microenvironment. Pseudo-time trajectory analysis further revealed the dynamic process of NPCs transitioning from a homeostatic state (Hom-NPCs) to a pro-fibrotic / pro-inflammatory phenotype (Fib-Inf-NPCs), accompanied by a significant upregulation of CSF1 expression. Functional experiments confirmed that conditional knockout of the CSF1R gene (Cx3cr1CreER / +:Csf1rfl / fl) in a mouse disc puncture (DP) model effectively alleviated the degree of disc degeneration and related pain behaviors, validating the core role of this pathway.

[0046] II. Treatment Methods This invention uses the highly selective small molecule CSF1R inhibitor GW2580 (5-((5-methoxy-1H-indol-3-yl)methylene)-2,4-thiazolidinedione) as the core active ingredient to block the CSF1 / CSF1R signaling pathway through oral administration. Figure 1 The recommended dosage range is 20–100 mg / kg / day, with a preferred dosage of 80 mg / kg / day. Treatment should be initiated early in the course of injury (e.g., 2 hours after injury) and continued until symptoms are significantly relieved to achieve optimal disease-modifying effects.

[0047] III. Pharmaceutical Compositions The pharmaceutical composition uses GW2580 or its pharmaceutically acceptable salts (such as hydrochloride or mesylate) as the active ingredient. A preferred excipient system comprises 0.5% hydroxypropyl methylcellulose (HPMC) and 0.1% Tween 80, and can be formulated into various dosage forms such as oral suspensions, tablets, or sustained-release capsules to meet different clinical needs.

[0048] IV. Beneficial Effects 1. Multi-target intervention: At the local intervertebral disc level, it effectively inhibits macrophage infiltration and the release of pro-inflammatory factors (such as IL-6 and MMP9); at the central level, it significantly inhibits the activation of microglia in the dorsal horn of the spinal cord and blocks the transmission of pain signals, achieving both symptomatic and radical treatment.

[0049] 2. High selectivity: GW2580 exhibits high inhibitory activity against CSF1R (IC50). 50 = 30 nM), and has a selectivity of more than 100 times for related kinases such as c-Kit and FLT3, effectively avoiding off-target effects and having good safety.

[0050] 3. Excellent safety profile: Unlike traditional macrophage depletion agents (such as PLX3397), GW2580 does not affect the number of microglia, and no disruption of immune homeostasis was observed with long-term use (mouse body weight and blood routine indicators were normal).

[0051] 4. Clear clinical translational potential: MRI imaging assessment confirmed that the T2 signal intensity was preserved to 50% of normal after treatment, demonstrating excellent disease modification effect.

[0052] V. Technological Highlights and Competitive Advantages 1. Original mechanism discovery: Based on scRNA-seq technology, the key mechanism by which the Fib-Inf-NPCs cell subset drives IVDD through the CSF1 / CSF1R axis was revealed for the first time, providing a new therapeutic target and theoretical basis for the field.

[0053] 2. Pioneering a New Paradigm for Disease Modification Therapy: This invention is the first to propose a targeted therapy strategy that can delay or reverse the process of intervertebral disc degeneration. It utilizes GW2580 to reshape the immune microenvironment, while simultaneously intervening in local degenerative inflammation and central pain sensitization, solving two major clinical challenges: structural repair and pain relief. This breakthrough overcomes the safety limitations of traditional cell depletion strategies and represents a technological advancement in this field.

[0054] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0055] Example 1: Preparation of GW2580 oral suspension (1) Formula composition: GW2580 active pharmaceutical ingredient (purity ≥ 99%) 80 mg; Hydroxypropyl methylcellulose (HPMC E5) 500 mg; Tween 80 (polysorbate 80) 100 mg; Add ultrapure water to a volume of 100 ml.

[0056] (2) Preparation process: Weigh out GW2580 raw material and HPMC E5, and mix them evenly using the equal-increment method. Dissolve Tween 80 in an appropriate amount of ultrapure water and stir thoroughly to disperse it. While continuously stirring, slowly add the mixed powder to the above aqueous phase to avoid clumping; The resulting initial mixture was homogenized at 5000 rpm for 10 minutes to form a homogeneous suspension system. Adjust the pH of the system to 6.5 ± 0.2 with dilute hydrochloric acid or sodium hydroxide solution; add ultrapure water to a final volume of 100 ml, stir well, and then fill into high-density polyethylene (HDPE) bottles for oral liquids. Seal and store in the dark.

[0057] (3) Quality and stability evaluation: The obtained suspension was placed at 25℃ and 60% relative humidity for 6 months, and the content was determined by high performance liquid chromatography (HPLC). The results showed that the content of the active pharmaceutical ingredient remained above 95% of the initial value, the growth of related substances was in accordance with the regulations, and all indicators met the requirements for long-term stability in the ICH Q1A(R2) guidelines.

[0058] Example 2: In vivo experiment on the inhibition of intervertebral disc degeneration by GW2580 (1) Animal model establishment: In order to study the role of the CSF1 / CSF1R signaling pathway in intervertebral disc degeneration, this study established and verified a stable and repeatable intervertebral disc puncture (DP) induced mouse degeneration model, which can simulate the key pathological features of human intervertebral disc degeneration and related pain behaviors.

[0059] (2) Model building method Figure 2 A): Ten-week-old male C57BL / 6 mice were anesthetized with intraperitoneal injections of ketoamine (100–120 mg / kg) and toluidine (10 mg / kg) and placed in a prone position. The lumbar spine was exposed through a posterolateral incision, and the L4 / 5 and L5 / 6 intervertebral discs were precisely located. A specially designed 25G puncture needle (0.5 mm outer diameter) was vertically inserted into the annulus fibrosus to a depth of 1.75 mm (approximately 90% of the dorsoventral width of the intervertebral disc), penetrating the center of the nucleus pulposus and partially penetrating the ventral annulus fibrosus. The needle was retained for 30 seconds before being withdrawn. To simulate endplate injury, some experimental groups used a special instrument inserted into the intervertebral space after nucleus pulposus puncture and rotated 180° to create endplate injury. The sham surgery group only exposed the intervertebral disc without puncture. All animals received standard postoperative care.

[0060] (3) Model evaluation and validation: The model effect was systematically evaluated through histological, imaging and behavioral analysis at multiple time points (14 days, 3 months and 6 months postoperatively).

[0061] (4) Histopathology: Mice were fixed by intracardiac perfusion with 4% paraformaldehyde (PFA, w / v). Lumbar intervertebral discs (IVDs) were dissected, post-fixed in 4% PFA for 48 hours, and decalcified in Kristensen's EDTA decalcification solution for 14 days. The tissues were rinsed under continuous tap water for 24 hours, then dehydrated and embedded in paraffin. 5 μm thick cross sections were cut using a microtome, mounted on Superfrost Plus™ anti-detachment slides, and stored at room temperature. For hematoxylin-eosin (H&E) staining, sections were dewaxed in xylene (2 × 5 min), rehydrated with a gradient of ethanol (95% → 70% → 50%), stained with Mayer's hematoxylin (6 min), rinsed with tap water, counterstained with eosin Y (2 min), dehydrated with a gradient of ethanol (50% → 70% → 95% → anhydrous ethanol; 1 min each), and cleared in xylene (2 × 5 min). For Safranin O / Fast Green staining, sections were dewaxed and rehydrated, then stained sequentially with Weigert's iron hematoxylin (nucleus, 10 min), 0.1% Fast Green FCF (cytoplasm, 5 min), 1% acetic acid (10–15 s), and 0.1% Safranin O (proteoglycan, 5 min), followed by dehydration and clearing. All sections were mounted with DPX mounters, dried at 37 °C, and imaged under a Nikon Eclipse Ci-L bright-field microscope. The degree of intervertebral disc degeneration was quantitatively assessed using a validated 4-point scoring system that scores the integrity of the annulus fibrosus (AF) and nucleus pulposus (NP) based on H&E and Safranin O / Fast Green stained sections. Adjacent undamaged L3 / 4 intervertebral discs served as internal controls. All sections were scored three times independently by two uninformed observers. Saffron O / Fixed Green staining showed that the intervertebral disc structure was intact in the sham surgery group, with a clear boundary between the nucleus pulposus and the annulus fibrosus; the DP group showed early disruption of the annulus fibrosus layered structure, with nucleus pulposus collapse and significant loss of proteoglycans after 3–6 months, and late-stage rupture of the cartilaginous endplate. Figure 2 B).

[0062] (5) Imaging Assessment: All scans were performed using a 7.0T Bruker BioSpec USR system (BrukerBioSpin Corp.) equipped with a custom 2 cm volume coil and operated using Paravision 5.1 software (Bruker BioSpin). Qualitative analysis was performed on sagittal and axial T2-weighted images (T2WIs) to assess degenerative changes. Imaging parameters included: repetition time (TR) = 2500 ms, echo time (TE) = 30 ms, RARE factor = 4, 24-times signal averaging, field of view (FOV) = 20 × 20 mm², slice thickness = 0.6 mm, and a total scan time of 25 minutes per mouse (excluding preparation time). Intervertebral disc height and Pfirrmann grading were performed on T2WIs using Bruker Topspin (v3.2) and Case Viewer (v2.3) software. For T2 mapping, intermediate sagittal slices were acquired with parameters of 25 incremental echo times (TE = 8–200 ms, ΔTE = 8 ms), in-plane resolution = 100 μm, and slice thickness = 0.6 mm. T2 maps were generated in Bruker Topspin, and a region of interest (ROI) containing the cartilaginous endplate, annulus fibrosus (AF), and nucleus pulposus (NP) was manually delineated to calculate the mean T2 value, while excluding extradiscal structures. Data were normalized to adjacent uninjured intervertebral discs (IVDs) within the same animal (as an internal sham-operated control) and expressed as a percentage. All quantitative analyses were performed by two independent, uninformed observers. 7.0T MRI sequences showed progressive signal decrease in the injured intervertebral discs on T2-weighted images, with a significantly lower disc height index compared to the sham-operated group. Figure 2 C).

[0063] (6) Pain and behavioral functional assessment Mechanosensitive hypersensitivity (Von Frey ciliary test) was assessed preoperatively (day -2) and postoperatively (days 1, 7, 14, 21, 28, 42, and 90). Mice were placed in a transparent glass observation chamber (8 cm in diameter) with a metal mesh bottom platform and acclimatized within the metal mesh enclosure (IITC Life Science) for 20 minutes prior to testing. The withdrawal threshold of the ipsilateral hind paw was measured using the up-down method, employing seven logarithmically growing Von Frey cilia (forces: 0.04, 0.07, 0.16, 0.40, 0.60, 1.0, and 2.0 g; Stoelting), with a cutoff force of 2.0 g. Stimuli were applied at 3-minute intervals, and the frequency (%) of withdrawal responses was calculated based on the number of withdrawal responses occurring in every 10 stimuli.

[0064] Cold abnormal pain (acetone vaporization test) was assessed using the acetone vaporization method to evaluate cold sensitivity. Using a multi-dose syringe device, acetone (50 μL each time, ≥5 minutes apart) was applied five times to the hind paw of mice through a metal mesh bottom. Responses were scored according to the following criteria: 0—no response or brief paw lifting / shaking (≤1 second); 1—continuous paw lifting, licking, or shaking (≤5 seconds); 2—persistent / repetitive behavior (>5 seconds). The average score of the five tests was taken as the final score. Alternatively, the cumulative paw-licking time within 60 seconds after three acetone stimuli was recorded, and the average time for each mouse was calculated.

[0065] Thermal hyperalgesia (hot plate test) is assessed using a hot plate apparatus (55 ± 1). o (C; Ugo Basile) Assess thermal nociception. Place mice on a heated surface and record the latency period for noxious behaviors (licking paws, rapid paw flicking, or jumping). Once a response is detected or a 30-second cutoff time (to prevent tissue damage) is reached, immediately remove the animal.

[0066] The Burrowing Assay acclimated mice to empty acrylic burrowing tubes in their home cages (1 hour acclimation the day before the test; 30 minutes acclimation on the day of the test). All five mice in each cage were exposed simultaneously, and all mice voluntarily entered the burrowing tube within 10–15 minutes. For the formal test, each mouse was transferred to a clean cage containing a burrowing tube filled with 90 grams of corn cob bedding (the same as the home cage bedding), placed parallel to the long side of the cage. One hour later, the mice were returned to their home cages, and the remaining bedding was weighed. Burrowing activity was calculated as follows: Percentage of bedding removed = ((Initial weight – Final weight) / 90 grams) × 100%. To standardize olfactory cues, 5 grams of home cage bedding were added to the test cages. Between trials, all bedding was stored in resealable plastic bags for reuse.

[0067] Gait analysis (treadmill test) involved mice walking at increasing speeds on a DigiGate treadmill (Columbus Instruments) on days 3, 7, 14, 28, and 42 post-surgery. Speed ​​tolerance (maximum speed without falling) and gait parameters were analyzed from 100 fps videos (captured by a Hotshot e64 camera).

[0068] Motor coordination (Rotarod test) was assessed using an accelerated rotating rod (Med Associates). Mice were trained for 2–3 days at variable speeds (4–40 rpm) before testing. In the final test, the rotating rod accelerated from 4 rpm to 40 rpm over 5 minutes. Mice were placed on the horizontal rotating rod, and their fall latency was recorded. The maximum speed (rpm) reached before the fall and the duration of the test were measured. Three tests were conducted (20-minute intervals) one day before surgery and on days 3, 7, 14, 28, and 42 after surgery. The height of the rotating rod was optimized to prevent injury while motivating mice to participate in the task.

[0069] Exploratory behavior (open field test) was assessed in a circular open field (1.2 m in diameter, 0.45 m high enclosure). The field was virtually divided into a central area (20% of the total area) and a peripheral area. Mice were allowed to explore freely for 10 minutes, and the following parameters were quantified using SMART 3.0 software (Panlab): time spent in the central area (anxiety-like behavior), number of times the mice entered the central area (exploration drive), and total movement (distance traveled, in centimeters). The test was performed one day before surgery and on days 14 and 42 after surgery, as described above.

[0070] (7) Behavioral phenotype: Mice in the DP group exhibited persistent mechanical hyperalgesia (long-term inhibition of von Frey fiber test threshold), cold hyperalgesia (acetone test), and decreased thermal pain threshold, and showed significantly impaired burrowing behavior, suggesting the presence of intervertebral discogenic chronic pain. Figure 2 (CF); Motor function tests (rotating bar, treadmill and open field) showed no difference among groups, indicating that the behavioral deficit was specific to pain rather than motor dysfunction.

[0071] (8) The model is easy to operate and has good repeatability. It can not only simulate the progressive degeneration process of intervertebral disc tissue after injury, but also stably reproduce the behavioral changes related to intervertebral disc-related pain. It is suitable for research on the mechanism of intervertebral disc degeneration and preclinical evaluation of therapeutic drugs.

[0072] (9) Dosing regimen ( Figure 3 A): GW2580 treatment group (n = 10): Administered once daily orally 2 hours after disc puncture, with a dose of 80 mg / kg / day of GW2580 suspension. The formulation used was the oral suspension of Example 1.

[0073] Solvent control group (n = 10): Administered equal volumes of 0.5% HPMC and 0.1% Tween 80 solvent, with the same administration time, frequency, and route as the treatment group.

[0074] (10) Evaluation indicators and methods: Imaging evaluation: 7.0T high-field magnetic resonance imaging (MRI) scans were performed on postoperative days 90 and 180. Quantitative measurements of the disc height index (DHI) and T2-weighted images were used to assess changes in disc structural integrity and tissue hydration. Histological evaluation: Disc tissue was paraffin-embedded, sectioned, and stained with saffron O. Two observers, unaware of the group assignments, independently performed histological scoring (scoring scale: 0–4 points, higher scores indicating more severe degeneration) to assess proteoglycan content and morphological changes.

[0075] Behavioral assessment: Pain-related behaviors will be assessed weekly starting from the postoperative period. Mechanical pain threshold: The mechanical withdrawal threshold (PWT) of the mouse paw was detected using the Von Frey fiber method to assess mechanical hypersensitivity. Cold pain response: The frequency and duration of foot contraction or licking behavior in mice were recorded using the acetone stimulation test to evaluate abnormal pain response induced by cold stimulation.

[0076] (11) Results: MRI analysis showed that 180 days after injury, the T2 signal intensity decreased in both the control group and the GW2580 treatment group; however, GW2580 treatment significantly preserved the intervertebral disc structure, as evidenced by reduced T2 signal intervertebral disc height loss ( Figure 3 B). Histological evaluation further demonstrated that the intervertebral disc structure was better preserved in the GW2580 treatment group animals. Figure 3 C). Finally, Iba1+ immunohistochemical staining ( Figure 3 D) Quantitative analysis confirmed that GW2580 significantly inhibited injury-induced inflammatory cell infiltration.

[0077] (12) In terms of behavioral testing, compared with the control group, GW2580-treated mice showed increased mechanical hyperalgesia (von Frey ciliary test) ( Figure 4 F) and cold abnormal pain (acetone test) Figure 4 G) all showed significant improvement, and the therapeutic effect lasted for up to 90 days.

[0078] (13) In DRG, GW2580 attenuated the increase in macrophages induced by intervertebral disc injury. Figure 4 A). In the dorsal horn of the spinal cord, GW2580 attenuated the increase in CD68+ macrophages induced by intervertebral disc injury ( Figure 4B), indicating that it systematically inhibits the neuroimmune response. To map the nociceptive signaling pathway, this example uses the Fos-TRAP mouse model (FosCreERT2:Rosa26ChR2). In this model, tamoxifen-dependent Cre activity is indicated by tdTomato markers on active neurons ( Figure 4 C). The sham-operated mice showed very few Fos-TRAP+ cells in the spinal cord or DRG. Following intervertebral disc injury, the number of Fos-TRAP+ neurons increased in the DRGs and spinal laminae I / II (key areas for nociceptive information processing). Figure 4 DE). GW2580 treatment significantly reduced the dorsal horn of the spinal cord ( Figure 4 D) and DRG ( Figure 4 Fos-TRAP+ neurons in E) showed a change consistent with a reduction in pain behavior. Figure 4 FG).

[0079] Example 3: In vivo experiment on the inhibition of intervertebral disc degeneration by GW2580 liposomes The drug uses GW2580 as its main active ingredient. By inhibiting the CSF1 / CSF1R signaling pathway, it effectively blocks the abnormal activation and pro-inflammatory function of macrophages. It can also intervene in pain signal transmission through the central mechanism of crossing the blood-brain barrier (BBB), thereby exerting a synergistic therapeutic effect on both the etiology (inflammation) and the core symptom (pain) of intervertebral disc degeneration.

[0080] Specifically: (1) Local drug delivery system is designed to actively or passively target cells expressing CSF1R (such as macrophages) to increase the concentration of the drug at the lesion site, enhance the therapeutic effect and reduce systemic exposure. (2) The drug delivery system is a nanoparticle system that can effectively encapsulate the hydrophobic compound GW2580 and improve its pharmacokinetic properties.

[0081] GW2580 (10 mg) was loaded into liposomes composed of DSPC (0.7 g), cholesterol (0.25 g), and DSPE-PEG2000 (0.16 g). The drug-loaded liposome nanoparticles prepared by thin-film hydration-extrusion had an average hydrated particle size of 128 ± 3.2 nm, a polydispersity index (PDI) of 0.12, and an encapsulation efficiency of up to 95.3%. This nanosize characteristic facilitated its accumulation in degenerated intervertebral disc tissue through enhanced penetration and retention (EPR) effects, and PEG modification prolonged its in vivo circulation time. In an animal model (rat caudal intervertebral disc acupuncture degeneration model), the GW2580 lipid nanoparticle treatment group showed the following compared to free GW2580 solution: Significantly stronger pain relief: In the mechanical hyperalgesia test, the pain threshold recovery effect was improved by approximately 40%. Figure 5 B).

[0082] Superior disease-modifying effects: T2-weighted MRI images show significant maintenance of intervertebral disc height. Figure 5 CD).

[0083] Good biocompatibility: No significant systemic toxicity or local tissue damage was observed.

[0084] In summary, the nanotechnology-based GW2580 delivery system provides a promising solution for effective and safe disease modification therapy of intervertebral disc degeneration by precisely targeting CSF1R and improving drug distribution characteristics.

[0085] Example 4: In vivo experiment on the inhibition of intervertebral disc degeneration by GW2580 liposomes combined with ibuprofen. GW2580 (10 mg) and ibuprofen (5.5 mg) were loaded into liposomes composed of DSPC (0.7 g), cholesterol (0.25 g), and DSPE-PEG2000 (0.16 g). The drug-loaded liposome nanoparticles prepared by thin-film hydration-extrusion had an average hydrated particle size of 140 ± 5 nm, a polydispersity index (PDI) of 0.15, and an encapsulation efficiency as high as 90.5%. This nanosize characteristic facilitated its accumulation in degenerated intervertebral disc tissue through enhanced penetration and retention (EPR) effects, and PEG modification prolonged its in vivo circulation time. In an animal model (rat caudal intervertebral disc acupuncture degeneration model), compared with GW2580 liposomes, the treatment group of this compound formulation showed: Significantly stronger pain relief: In the mechanical hyperalgesia test, the pain threshold recovery effect was improved by approximately 60%.

[0086] Superior disease modification effect: T2-weighted MRI images showed that the intervertebral disc height index and signal intensity were significantly better maintained; histological analysis (SAFG score) confirmed that the annulus fibrosus structure was more intact, the proteoglycan content was higher, and the infiltration of inflammatory cells was less.

[0087] Good biocompatibility: No significant systemic toxicity or local tissue damage was observed.

[0088] In summary, the nanotechnology-based GW2580 / ibuprofen delivery system provides a promising solution for effective and safe disease modification therapy of intervertebral disc degeneration by precisely targeting CSF1R, improving drug distribution characteristics, and synergistic effects.

[0089] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A pharmaceutical composition for treating and / or delaying IVDD and / or relieving discogenic pain, characterized in that, which comprises a therapeutically effective amount of a CSF1R inhibitor and a pharmaceutically acceptable carrier.

2. The pharmaceutical composition for use in the treatment and / or delay of IVDD and / or in the alleviation of discogenic pain according to claim 1, characterized in that, The CSF1R inhibitor is a small molecule CSF1R inhibitor; preferably, the small molecule CSF1R inhibitor is GW2580 or a pharmaceutically acceptable salt, solvate, hydrate or crystal form thereof.

3. The pharmaceutical composition for use in the treatment and / or delay of IVDD and / or alleviation of discogenic pain according to claim 1, wherein The pharmaceutical composition is formulated as an oral dosage form or an injectable dosage form; preferably, the oral dosage form is an oral suspension, a tablet or a capsule; the injectable dosage form is a nanoparticle delivery system; more preferably, the nanoparticle delivery system is a liposome; wherein the liposome is composed of phosphatidylcholine (DSPC), cholesterol and polyethyleneglycolated phospholipid (DSPE-PEG2000); and a therapeutically effective amount of GW2580 and / or an analgesic; wherein the molar ratio of DSPC, cholesterol and DSPE-PEG2000 is (50-90):(30-65):(3-8); and the molar ratio of GW2580 and the analgesic is 1:10 to 10:

1.

4. The pharmaceutical composition for use in the treatment and / or delay of IVDD and / or alleviation of discogenic pain according to claim 1, wherein, The pharmaceutically acceptable carrier comprises excipients for forming an oral suspension; Preferably, the excipients for forming an oral suspension are hydroxypropyl methylcellulose (HPMC) and Tween 80; More preferably, the concentration of HPMC is 0.5% (w / v) and the concentration of Tween 80 is 0.1% (w / v).

5. The pharmaceutical composition for use in the treatment and / or delay of IVDD and / or alleviation of discogenic pain according to claim 1, wherein The pharmaceutical composition is further used in combination with a second active agent, which is an analgesic, a non-steroidal anti-inflammatory drug (NSAID) or a corticosteroid.

6. Use of a CSF1R inhibitor in the manufacture of a medicament for treating and / or delaying intervertebral disc degeneration (IVDD) and / or relieving discogenic pain.

7. The use according to claim 4, characterized in that, The CSF1R inhibitor is a small molecule CSF1R inhibitor; the small molecule CSF1R inhibitor is GW2580 or a pharmaceutically acceptable salt, solvate, hydrate or crystal form thereof; the medicament is capable of reprogramming macrophage function to polarize towards an anti-inflammatory M2 phenotype rather than depleting macrophages; the medicament is capable of blocking pro-inflammatory signaling pathways driven by CSF1-high expressing nucleus pulposus cells (Fib-Inf-NPCs).

8. A kit for treating degeneration of an intervertebral disc and / or relieving discogenic pain, characterized in that, comprising: a) the pharmaceutical composition of any one of claims 1-5; and b) optionally, instructions for use.

9. The kit for use in the treatment of intervertebral disc degeneration and / or in the relief of discogenic pain according to claim 8, wherein, The kit further comprises a second active agent, which is an analgesic, a non-steroidal anti-inflammatory drug (NSAID) or a corticosteroid.

10. A pharmaceutical combination package, comprising: 1) the pharmaceutical composition of any one of claims 1-5; 2) a second active agent, which is an analgesic, a non-steroidal anti-inflammatory drug (NSAID) or a corticosteroid; and 3) optionally, instructions for simultaneous, separate or sequential administration.

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