Application of VEGFR (vascular endothelial growth factor receptor) inhibitor in preparation of medicine for treating intervertebral disc degeneration

Abstract

The invention discloses application of a VEGFR (vascular endothelial growth factor receptor) inhibitor in preparation of a medicine for treating intervertebral disc degeneration, and relates to the technical field of biological medicines. The invention evaluates the inhibition of the growth of new vessels in the nucleus pulposus tissue microenvironment and the delay of the early-stage nucleus pulposus tissue vascularization-inflammation pathological microenvironment of the medicine and / or the medicine composition of the VEGFR inhibitor through animal experiments, and discusses the effect of the medicine and / or the medicine composition on the treatment of IDD. The research finds that the medicine and / or the medicine composition of the VEGFR inhibitor can effectively inhibit the neovascularization growth of the IDD early nucleus pulposus tissue and delay the vascularization-inflammation pathological progress of the IDD early nucleus pulposus tissue, and the medicine and / or the medicine composition have important significance on early treatment and clinical intervention of IDD.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the application of a VEGFR inhibitor in the preparation of a medicine for treating intervertebral disc degeneration. Background Art

[0002] Intervertebral disc degeneration (IDD) is the main cause of low back pain. In my country, about 60-80% of people have experienced low back pain caused by IDD. Epidemiology in my country shows that IDD has a trend of becoming younger in recent years, with the majority of patients aged 25-55 years, which has an impact on my country's social productivity and economic construction. Currently, the early treatment methods for IDD used in clinical practice are mostly to control discogenic pain, which cannot change the pathological progression of IDD. Currently, the treatment methods for discogenic pain can be divided into three aspects: non-surgical treatment, surgical treatment and emerging therapies. Non-surgical treatment is the main clinical treatment for early discogenic pain, including physical therapy, analgesics and closed treatment. Although they can alleviate the pain symptoms of IDD, they cannot completely change the pathological process of IDD. The outcome of IDD patients is mostly subsequent surgical treatment. However, surgical treatment is often accompanied by the destruction of the original anatomical structure and a high recurrence rate, and can only be used for the treatment of late IDD. Therefore, the current early treatment of IDD faces the clinical problem that the main clinical treatment methods cannot meet the clinical needs of repairing early IDD lesions, and it is very necessary to develop new IDD treatment drugs. Summary of the invention

[0003] The purpose of the present invention is to provide the use of VEGFR inhibitors in the preparation of drugs for treating intervertebral disc degeneration to solve the problems existing in the above-mentioned prior art. The present invention has found that VEGFR inhibitors can effectively inhibit the growth of new blood vessels in the nucleus pulposus tissue in the early stage of IDD and delay the pathological progression of "vascularization-inflammation" in the nucleus pulposus tissue in the early stage of IDD, which is of great significance for the early treatment and clinical intervention of IDD.

[0004] Sustained abnormal stress and genetic factors are considered to be the main initiating factors of IDD. They affect the extracellular matrix (ECM) microenvironment, leading to apoptosis and functional changes of nucleus pulposus cells (NPCs), resulting in abnormal synthesis / decomposition of NPCs extracellular matrix. Vascularization of nucleus pulposus tissue is considered to be the most critical pathological change in IDD. When the intervertebral disc suffers from annular fibrosis damage, NPCs apoptosis and ECM degradation due to abnormal stress, the structural integrity of the intervertebral disc is lost. In addition, the degradation of proteoglycans in the nucleus pulposus ECM reduces the inhibitory effect of blood vessels, causing the invasion of vascular endothelium, leading to the infiltration of oxygen and inflammatory cells, destroying the previously hypoxic, avascular and immune cell-free microenvironment, and causing downstream inflammatory cascade reactions, forming a pathological microenvironment of "vascularization-inflammation". In addition, when abnormal stress causes damage to the normal intervertebral disc structure, the vascular endothelium invades the nucleus pulposus and the internal annulus fibrosus tissue due to changes in the ECM and the action of pro-angiogenic factors such as vascular endothelial growth factor; vascularization causes inflammatory cells, mainly macrophages, to invade the nucleus pulposus tissue, causing the nucleus pulposus tissue to contact with immune cells, resulting in the destruction of the blood-nucleus pulposus barrier, triggering an autoimmune cascade reaction, and further accelerating the "vascularization-inflammation" pathological microenvironment cycle of IDD. Therefore, delaying the pathological cycle of "vascularization-inflammation" in the early stage of IDD is the key to solving the above clinical problems. Therefore, the present invention applies VEGFR inhibitors to the preparation of drugs for treating intervertebral disc degeneration, and further delays the "vascularization-inflammation" pathological microenvironment of the nucleus pulposus tissue by inhibiting the growth of new blood vessels in the nucleus pulposus tissue in the early stage of IDD, and ultimately delays the early pathological process of IDD to treat IDD.

[0005] Based on this, the present invention provides the following solutions:

[0006] The present invention provides application of a VEGFR inhibitor in preparing a medicine for treating intervertebral disc degeneration.

[0007] Furthermore, the VEGFR inhibitor is cabozantinib or a derivative thereof.

[0008] Furthermore, the derivative is cabozantinib malate, an N-substituted cabozantinib derivative, a ring structure-modified cabozantinib derivative, a fluorine-substituted cabozantinib derivative, an amide bond-modified cabozantinib derivative, a pyrrolo[3,2-d]pyrimidinyl cabozantinib derivative or an antibody-drug conjugate of cabozantinib.

[0009] Furthermore, the VEGFR inhibitor is sorafenib, sunitinib, apatinib, bevacizumab, rilapanib, basotinib, olalimumab, ramucirumab, axitinib, regorafenib, vandetanib, pazopanib, lenvatinib, ponatinib, aflibercept or fruquintinib.

[0010] The present invention also provides a medicine for treating intervertebral disc degeneration, wherein the active ingredient comprises a VEGFR inhibitor.

[0011] Furthermore, the medicine also includes pharmaceutically acceptable excipients.

[0012] Furthermore, the auxiliary material includes a carrier.

[0013] Furthermore, the carrier is at least one of a gel, a nanocarrier, a liposome, a bioceramic, a bioglass, a scaffold carrier and a liquid solvent.

[0014] Furthermore, the gel is HAMA hydrogel, hyaluronic acid hydrogel, type I collagen hydrogel, sodium alginate hydrogel or gelatin hydrogel, but the gel is not limited to the above-mentioned types of gels. Those skilled in the art believe that feasible and acceptable gels are within the scope of protection of the present invention.

[0015] Furthermore, the nanocarrier is preferably a polymer nanoparticle, and may also be a nanobody, a cell membrane, a DNA structure, an inorganic nanoparticle or a liposome. Those skilled in the art believe that feasible nanocarriers are within the scope of protection of the present invention.

[0016] Furthermore, the scaffold carrier is preferably a hydrogel scaffold, and can also be an inorganic material scaffold, an organic material scaffold, a composite material scaffold, a microsphere scaffold or a 3D printed scaffold. Those skilled in the art believe that feasible and acceptable scaffold carriers are within the scope of protection of the present invention.

[0017] Furthermore, the liquid solvent is physiological saline, phosphate buffer, physiological saline, dimethyl sulfoxide or glycerol. Those skilled in the art consider that feasible and acceptable liquid solvents are within the protection scope of the present invention.

[0018] Furthermore, the dosage form of the drug is oral or injection.

[0019] Furthermore, the VEGFR inhibitor is cabozantinib; the drug also includes hyaluronic acid methacrylate, photoinitiator 2959, DMEM culture medium, type I collagen and bone marrow mesenchymal stem cells.

[0020] The present invention discloses the following technical effects:

[0021] The present invention uses animal experiments to evaluate the effect of VEGFR inhibitor drugs and / or drug compositions on inhibiting the growth of new blood vessels in the microenvironment of nucleus pulposus tissue and delaying the pathological microenvironment of "vascularization-inflammation" of early nucleus pulposus tissue, and explores the effect of the drugs and / or drug compositions on the treatment of IDD. Studies have found that VEGFR inhibitor drugs and / or drug compositions can effectively inhibit the growth of new blood vessels in the early nucleus pulposus tissue of IDD and delay the pathological progression of "vascularization-inflammation" of the early nucleus pulposus tissue of IDD, which is of great significance for the early treatment and clinical intervention of IDD. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 The Micro-CT images of the effect of the cabozantinib composition on the intervertebral disc of IDD rats; wherein A is a sagittal cross-sectional image; B is a three-dimensional reconstruction stereogram; and C is a statistical analysis graph of the relative intervertebral disc volume percentage of Micro-CT;

[0024] Figure 2 MRI images of the effects of the cabozantinib composition on the intervertebral disc of IDD rats; wherein A is a sagittal cross-sectional image of MRI, and the nucleus pulposus is a high signal shadow; B is a Pfirrnman score image of the MRI image;

[0025] Figure 3 The figure is a histological staining result diagram of the effect of the cabozantinib composition on the intervertebral disc of IDD rats; wherein A and B are HE staining and Safranin-fix O-green staining (OS staining) diagrams of each group, respectively;

[0026] Figure 4 It is a pathological evaluation chart obtained according to the results of HE staining; AE represents the comprehensive pathological score of the intervertebral disc, the pathological score of the nucleus pulposus, the pathological score of the end plate, the pathological score of the annulus fibrosus and the pathological score of the junction area;

[0027] Figure 5 The immunohistochemical staining results of the effect of the cabozantinib composition on the "vascularization-inflammation" microenvironment of the nucleus pulposus are shown in Figure 1; wherein A is an immunohistochemical staining picture of proteoglycan (Aggrecan, ANCA), and brown is a positive area; and B is a quantitative result picture of the immunohistochemical staining picture of ANCA;

[0028] Figure 6The figures are the immunofluorescence staining results of the effect of the cabozantinib composition on the "vascularization-inflammation" microenvironment of the nucleus pulposus; wherein A is a staining image of type I collagen (Collagen I, green fluorescent area) and type II collagen (Collagen II, red fluorescent area); B is a staining image of CD68 (green fluorescent area) and CD31 (red fluorescent area). DETAILED DESCRIPTION

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0031] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0032] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0033] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0034] Terminology Note:

[0035] The cabozantinib referred to in the present invention has a molecular formula of C 28 H 24 FN 3 O 5 , molecular weight is 501.51, CAS number is 849217-68-1, structural formula is:

[0036]

[0037] Example 1

[0038] Preparation method of cabozantinib composition:

[0039] 10 μL 1% (w / v) methacrylated hyaluronic acid (HAMA), 500 μL 10% (w / v) photoinitiator 2959, 10 μL 10× DMEM, 5000 μL type I collagen, 200 μL bone marrow mesenchymal stem cell (MSC) suspension (a total of 1×10 2 MSC) and 10 μL 0.01 mM cabozantinib were mixed evenly and placed on ice. 405 nm ultraviolet light was used for cross-linking for 10 minutes, and then placed in a 37° C. constant temperature incubator for 10 minutes to obtain a cabozantinib composition. The above operations were all performed under a sterile environment.

[0040] Example 2

[0041] Preparation method of cabozantinib composition:

[0042] 500 μL 10% (w / v) HAMA, 300 μL 5% (w / v) photoinitiator 2959, 250 μL 10× DMEM, 3000 μL type I collagen, and 200 μL MSC cell suspension (a total of 1×10 5 MSC) and 5 μL 5 mM cabozantinib were mixed evenly and placed on ice. 405 nm ultraviolet light was used for cross-linking for 8 minutes, and then placed in a 37° C. constant temperature incubator for 20 minutes to obtain a cabozantinib composition. The above operations were all performed under a sterile environment.

[0043] Example 3

[0044] Preparation method of cabozantinib composition:

[0045] 5 mL 50% (w / v) HAMA, 10 μL 0.1% (w / v) photoinitiator 2959, 500 μL 10× DMEM, 100 μL type I collagen, 200 μL MSC cell suspension (a total of 1×10 7 MSC) and 0.01 μL 10 mM cabozantinib were mixed evenly and placed on ice. 405 nm ultraviolet light was used for cross-linking for 3 minutes, and then placed in a 37° C. constant temperature incubator for 30 minutes to obtain a cabozantinib composition. The above operations were all performed under a sterile environment.

[0046] Comparative Example 1

[0047] Preparation method of drug carrier:

[0048] 500 μL 10% (w / v) HAMA, 300 μL 5% (w / v) photoinitiator 2959, 250 μL 10× DMEM, 3000 μL type I collagen and 200 μL MSC cell suspension (a total of 1×10 5 MSCs) were mixed evenly and placed on ice for standing. The mixture was cross-linked with 405 nm ultraviolet light for 8 minutes, and then placed in a 37° C. constant temperature incubator for 20 minutes to obtain a drug carrier. The above operations were all performed under a sterile environment.

[0049] Example 4 Restorative effect of cabozantinib composition on IDD rat model

[0050] 1. Materials and Methods

[0051] Eight-week-old Sprague-Dawley (SD) rats with an average weight of 280±10 g were ordered from Viton Lever Company for animal experiments, with 10 rats in each group. The animal experiment groups were as follows:

[0052] (1) Sham operation group, only anesthesia and incision treatment, corresponding to the coccyx (Co) 4-5 intervertebral disc;

[0053] (2) surgical modeling group, who underwent surgical treatment of IDD, corresponding to Co 4-5 disc;

[0054] (3) carrier group, IDD postoperative injection of the drug carrier prepared in Comparative Example 1, corresponding to Co 4-5 intervertebral disc;

[0055] (4) The cabozantinib composition group: the cabozantinib composition prepared in Example 2 was injected after IDD surgery, corresponding to Co 4-5 intervertebral disc.

[0056] The ethics of this animal study was approved by the Institutional Review Board and Animal Care Committee of Guangdong Huawei Testing Co., Ltd. (approval number: HWT-BG-117(20210606)), and the animal samples were further analyzed at the First Affiliated Hospital of Sun Yat-sen University.

[0057] Steps for constructing rat IDD model: On the day of surgical modeling, all animals were anesthetized with isoflurane using a small animal anesthesia machine. The rat IDD model was established by percutaneous needle puncture. Three days after surgery, rats were given meloxicam (2 mg / kg), streptomycin (0.125 mg), and penicillin (0.1 mg) once a day for analgesia and anti-infection. One week after modeling, 20 μL of hydrogel from each group was injected into different caudal intervertebral discs (Co 4-9) of the same rat using a 26G needle. The operation process of percutaneous needle puncture was as follows: first, we used a marking pen to mark the Co 4-9 intervertebral disc segment; then, we used a 20G needle to puncture from the top to the bottom, and repeatedly rotated the needle by hand; finally, we pressed the puncture point with iodine cotton balls to stop bleeding and disinfect it. Magnetic resonance imaging (MRI) scans were performed one week before, one week after surgery, and nine weeks after surgery. Two people simultaneously evaluated and statistically analyzed the images using the Pfirrnman score. Micro-CT scanning and pathological staining were performed in the first week after surgery.

[0058] Immunohistochemical staining: All specimens were decalcified and sent to Google Bio for paraffin embedding and sectioning. Google Bio performed immunofluorescence and immunohistochemical staining on the sections.

[0059] 2. Results

[0060] The experimental results of the repair effect of the cabozantinib composition on IDD rats are as follows Figure 1-Figure 4 shown. Figure 1 It can be seen that the cabozantinib composition can effectively alleviate the damage caused by IDD surgery and can restore the volume of the intervertebral disc to an effect similar to that of a normal intervertebral disc. Figure 2 It was shown that the cabozantinib combination can effectively delay the damage of nucleus pulposus tissue. Figure 3 and Figure 4 The results showed that the cabozantinib composition plays a role in inhibiting vascular invasion, and after intradiscal injection of the cabozantinib composition, it can restore the nucleus pulposus tissue, endplate and annulus fibrosus structure of the IDD model to a level similar to that of normal intervertebral disc tissue.

[0061] The immunohistochemical staining results of the effect of the cabozantinib combination on the "vascularization-inflammation" microenvironment of the nucleus pulposus are shown in Figure 5 . Figure 5 It was shown that the cabozantinib combination could alleviate the degeneration of IDD nucleus pulposus tissue to a certain extent and promote ANCA expression.

[0062] The results of immunofluorescence staining of the effect of the cabozantinib combination on the "vascularization-inflammation" microenvironment of the nucleus pulposus are shown in Figure 6 . Figure 6The results showed that the cabozantinib combination can alleviate the degeneration of nucleus pulposus tissue of IDD to a certain extent, and can reduce the early vascular growth and inflammatory infiltration of IDD to a certain extent.

[0063] The results of the experiment of injecting cabozantinib combination into IDD rats showed that cabozantinib combination showed good effects in slowing down vascular invasion, preventing inflammatory cascade reaction, regenerating and repairing nucleus pulposus tissue and breaking the "vascularization-inflammation" pathological microenvironment cycle.

[0064] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of VEGFR inhibitors in the preparation of drugs for treating intervertebral disc degeneration.

2. The use according to claim 1, characterized in that: The VEGFR inhibitor is cabozantinib or a derivative thereof.

3. The use according to claim 2, characterized in that The derivative is cabozantinib malate, an N-substituted cabozantinib derivative, a ring structure-modified cabozantinib derivative, a fluorine-substituted cabozantinib derivative, an amide bond-modified cabozantinib derivative, a pyrrolo[3,2-d]pyrimidinyl cabozantinib derivative or an antibody-drug conjugate of cabozantinib.

4. The use according to claim 1, characterized in that: The VEGFR inhibitor is sorafenib, sunitinib, apatinib, bevacizumab, rilaparib, basotinib, olalimumab, ramucirumab, axitinib, regorafenib, vandetanib, pazopanib, lenvatinib, ponatinib, aflibercept or fruquintinib.

5. A drug for treating intervertebral disc degeneration, characterized in that: The active ingredients include VEGFR inhibitors.

6. The drug according to claim 5, characterized in that The drug also includes pharmaceutically acceptable excipients.

7. The drug according to claim 6, characterized in that The auxiliary materials include carriers.

8. The drug according to claim 7, characterized in that The carrier is at least one of a gel, a nanocarrier, a liposome, a bioceramic, a bioglass, a scaffold carrier and a liquid solvent.

9. The drug according to claim 6, characterized in that The dosage form of the drug is oral preparation or injection.

10. The drug according to claim 5, characterized in that The VEGFR inhibitor is cabozantinib; the drug also includes hyaluronic acid methacrylate, photoinitiator 2959, DMEM culture medium, type I collagen and bone marrow mesenchymal stem cells.

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