WO2023097721A1 - Application of inhibitor and pharmaceutical composition - Google Patents

Application of inhibitor and pharmaceutical composition Download PDF

Info

Publication number
WO2023097721A1
WO2023097721A1 PCT/CN2021/136555 CN2021136555W WO2023097721A1 WO 2023097721 A1 WO2023097721 A1 WO 2023097721A1 CN 2021136555 W CN2021136555 W CN 2021136555W WO 2023097721 A1 WO2023097721 A1 WO 2023097721A1
Authority
WO
WIPO (PCT)
Prior art keywords
catenin
signaling pathway
inhibitor
icrt14
pain
Prior art date
Application number
PCT/CN2021/136555
Other languages
French (fr)
Chinese (zh)
Inventor
陈棣
陆克
Original Assignee
深圳先进技术研究院
中国科学院深圳理工大学(筹)
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 深圳先进技术研究院, 中国科学院深圳理工大学(筹) filed Critical 深圳先进技术研究院
Publication of WO2023097721A1 publication Critical patent/WO2023097721A1/en

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

Definitions

  • the invention relates to the field of treatment of geriatric diseases, in particular to the application of an inhibitor and a pharmaceutical composition.
  • Discogenic pain mainly refers to low back pain caused by disc degeneration.
  • discogenic neck pain can include all neck, shoulder and arm pain caused by intervertebral disc disease, but many pains caused by it have corresponding disease names, such as cervical disc herniation, cervical disc degeneration and stenosis, and cervical spondylosis.
  • cervical discogenic pain specifically refers to the pain caused by the internal disorder of the intervertebral disc, without radiating pain and segmental nerve dysfunction, and does not involve the adjacent spinal cord, nerve roots, and facet joints.
  • Discogenic neck pain is one of the common causes of chronic, intermittent neck and shoulder pain.
  • intervertebral discs may be a cause of low back pain. Patients with intervertebral disc herniation are often accompanied by low back pain. Another possible cause of low back pain is tearing of the fibrous annulus. Patients with such intervertebral disc herniation will induce pain after injecting normal saline or contrast medium into the intervertebral disc.
  • the main components of the intervertebral disc are collagen, proteoglycan and water, which account for 90% to 95% of the volume of the intervertebral disc.
  • the composition of these components changes with degeneration or aging, mainly manifested in the reduction of proteoglycans and water.
  • the proportion of chondroitin sulfate and keratan sulfate increased while the proportion of aggregates decreased.
  • Peripheral tearing of the annulus fibrosus can be caused by overloaded torsion during disc injury. On the basis of this tear, internal tears can appear, and finally a complete radial tear can be formed.
  • the sinus vertebral nerve innervates the structures in the spinal canal and is distributed in the posterior longitudinal ligament, ventral side of the dura sac, blood vessels and annulus fibrosus.
  • the ventral and lateral aspects of the annulus are innervated by branches originating from the ventral primary and sympathetic nerves.
  • the anterior longitudinal ligament is innervated by branches of the gray communicating sympathetic trunk.
  • the research on discogenic pain is relatively extensive. It is mainly believed that after the degenerative changes of the intervertebral disc, under the action of external force, part or all of the annulus fibrosus ruptures, protrudes alone or together with the nucleus pulposus and cartilage endplate, and stimulates or compresses the dorsal root.
  • the ganglia and nerve roots cause the adjacent spinal nerve roots to be stimulated or compressed, resulting in a series of clinical symptoms such as low back pain, numbness and pain in one or both lower limbs.
  • the inflammation of cartilage endplate and annulus fibrosus can induce abnormal hyperplasia of osteophytes to narrow the intervertebral foramen, and can also compress the spinal nerve passing through the intervertebral foramen, causing pain.
  • the chondrocytes in the endplate of the cartilage are gradually apoptotic and calcified, and the new blood vessel nerves invade into it.
  • Inflammatory factors such as TNF- ⁇ and IL-1 secreted by intervertebral disc cells and migrating inflammatory cells, as well as CCL2 secreted by endplate chondrocytes, can Stimulation of abnormally proliferating nerve endings causes a pain response.
  • the impact of the classic Wnt signaling pathway on cell metabolism is mainly through targeted regulation of the expression and localization of ⁇ -catenin protein in cells.
  • the level of ⁇ -catenin protein is often relatively stable in cells. This is mainly because the overproduced ⁇ -catenin is degraded by the protein complex composed of protein kinase GSK-3 ⁇ , Axin1/Axin2, APC (Adenomatous Polyposis Coli), Dvl (Disheveled), and CK1 (Casein Kinase I).
  • This protein complex can phosphorylate specific amino acid residues at the carboxy-terminus of the ⁇ -catenin protein encoded by exon 3 (exon3) of the ⁇ -catenin gene. Therefore, when the exon 3 sequence of the ⁇ -catenin gene produces a null mutation, the ⁇ -catenin protein will accumulate abnormally in the cell, thereby abnormally activating the Wnt/ ⁇ -catenin signaling pathway. Based on this, we constructed ⁇ -catenin(ex3)Col2CreER mice (ie ⁇ -cateninAct) mice, which have abnormal accumulation of ⁇ -catenin protein in the cells expressing Col2.
  • the Wnt/ ⁇ -catenin signaling pathway is widely involved in embryonic development, regulation of biological cell proliferation, metabolism and many other aspects. A large number of Wnt/ ⁇ -catenin signaling pathway inhibitors are undergoing clinical trials. However, whether inhibition of the Wnt/ ⁇ -catenin signaling pathway has a therapeutic effect on discogenic pain, and the underlying mechanism remains unclear.
  • the inhibitor is an inhibitor of the combination of ⁇ -catenin protein and TCF7 transcription factor and/or an inhibitor of Wnt/ ⁇ -catenin signaling pathway.
  • the inhibitor is a compound iCRT14 having the following structural formula, its stereoisomer, its pharmaceutically acceptable salt, its solvate or its prodrug:
  • the effective concentration of the compound iCRT14 is 20 ⁇ M-100 ⁇ M.
  • the effective concentration of the compound iCRT14 is 50 ⁇ M.
  • a pharmaceutical composition which includes an inhibitor of the combination of ⁇ -catenin protein and TCF7 transcription factor and/or an inhibitor of Wnt/ ⁇ -catenin signaling pathway.
  • the pharmaceutical composition includes the compound iCRT14 having the following structural formula, its stereoisomer, its pharmaceutically acceptable salt, its solvate or its prodrug:
  • the effective concentration of the compound iCRT14 is 20 ⁇ M-100 ⁇ M.
  • the effective concentration of the compound iCRT14 is 50 ⁇ M.
  • the pharmaceutical composition is a therapeutic pharmaceutical composition for discogenic pain.
  • the activation of the ⁇ -catenin signaling pathway in intervertebral disc injury will cause the treatment of intervertebral disc-derived pain
  • the binding inhibitor of ⁇ -catenin protein and TCF7 transcription factor can inhibit the binding of ⁇ -catenin protein and TCF7 transcription factor Combined, thereby inhibiting the activation of ⁇ -catenin signaling pathway in intervertebral disc injury
  • Wnt/ ⁇ -catenin signaling pathway inhibitor can inhibit the activation of ⁇ -catenin signaling pathway in intervertebral disc injury.
  • the combination inhibitor of ⁇ -catenin protein and TCF7 transcription factor and/or the inhibitor of Wnt/ ⁇ -catenin signaling pathway can achieve the effect of treating discogenic pain based on the inhibition of Wnt/ ⁇ -catenin signaling pathway.
  • Figure 1A is the pain threshold detection results of ⁇ -catenin overexpression transgenic mice, where the abscissa shows ⁇ -catenin overexpression transgenic mice after 0, 4, 8, and 12 days after tamoxifen induced abnormal accumulation of ⁇ -catenin signaling pathway , 16, 20 weeks after Von Frey mechanical stress test, paw contraction response.
  • Figure 1B is the detection result of crossover frequency of ⁇ -catenin overexpression transgenic mice, where the abscissa shows that the abscissa shows that the ⁇ -catenin overexpression transgenic mice were abnormally accumulated in the ⁇ -catenin signaling pathway induced by tamoxifen 4, 8, At 12, 16, 20, and 24 weeks, LABROS system was used for behavioral testing.
  • Figure 1C is the test results of standing times of ⁇ -catenin overexpression transgenic mice, where the abscissa shows that the abscissa shows that the ⁇ -catenin overexpression transgenic mice were abnormally accumulated in the ⁇ -catenin signaling pathway induced by tamoxifen 4, 8, At 12, 16, 20, and 24 weeks, LABROS system was used for behavioral testing.
  • Figure 2A is a representative picture of the ⁇ CT scan of the vertebral body of the ⁇ -catenin overexpression transgenic mouse.
  • Fig. 2B is a representative picture of safranin O fast green staining of vertebral bodies of transgenic mice overexpressing ⁇ -catenin.
  • Figure 3A is a representative picture of immunofluorescent staining of PGP9.5 and Tuj1 in dorsal root ganglia of transgenic mice overexpressing ⁇ -catenin, in which DAPI was used for nuclear staining.
  • Figure 3B is the qPCR detection results of pain-related genes in the dorsal root ganglion of ⁇ -catenin overexpressed transgenic mice.
  • the abscissa Ctr represents the Cre negative control group
  • Mut represents the ⁇ -catenin overexpressed transgenic mouse group
  • the ordinate represents the qPCR of the Mut group The fold change of the detected gene level relative to the gene level detected by qPCR of the Ctr group.
  • Figure 3C shows the results of qPCR detection of inflammation-related genes in the dorsal root ganglia of ⁇ -catenin overexpression transgenic mice, where the abscissa Ctr represents the Cre negative control group, Mut represents the ⁇ -catenin overexpression transgenic mouse group, and the ordinate represents the Mut group The fold change of gene levels detected by qPCR relative to the gene levels detected by qPCR in the Ctr group.
  • Figure 4A shows the changes in the expression levels of AXIN2 and DKK1 in C28/I2 cells (human chondrocyte line) treated with different concentrations of BIO, where the abscissa is the BIO concentration gradient of 0, 10, 20, and 50 ⁇ M; the ordinate is the gene level detected by qPCR in each group Fold change in gene levels detected by qPCR relative to BIO concentration 0 group.
  • Figure 4B shows the expression of ⁇ -catenin and CCL2 proteins after BIO treatment of C28/I2 cells (human chondrocyte line), wherein the BIO treatment concentration was 1 ⁇ M and the treatment time was 12 hours.
  • Figure 4C is the effect of BIO and IL-1 ⁇ on the level of ⁇ -catenin protein in rat primary annulus fibrosus (AF) cells, wherein the BIO treatment concentration was 1 ⁇ M, the treatment time was 12 hours, the IL-1 ⁇ treatment concentration was 10 nM, and the treatment The time is 12 hours.
  • Figure 4D is the effect of BIO and IL-1 ⁇ on the level of ⁇ -catenin protein in rat nucleus pulposus (NP) cells, wherein the BIO treatment concentration is 1 ⁇ M, and the treatment time is 12 hours, and the IL-1 ⁇ treatment concentration is 10nM, and the treatment time is 12 hours.
  • Figure 4E shows the changes in the expression levels of CCL2 and CCR2 in C28/I2 cells (human chondrocyte line) treated with different concentrations of BIO, where the abscissa is the BIO concentration gradient of 0, 10, 20, and 50 ⁇ M; the ordinate is the gene level detected by qPCR in each group Fold change in gene levels detected by qPCR relative to BIO concentration 0 group.
  • Figure 4F is a representative image of immunofluorescence of CCL2 after BIO treatment of C28/I2 cells (human chondrocyte cell line), in which DAPI was used for nuclei staining.
  • Figure 4G is the average optical density statistics of CCL2 immunofluorescence after BIO treatment of C28/I2 cells (human chondrocyte line), where the ordinate is the average optical density of CCL2 immunofluorescence, the abscissa is the different treatment methods of C28/I2 cells, and DMSO is Control treatment, BIO treatment for the experimental group.
  • Fig. 5A is the expression level change of TCF7 in C28/I2 cells (human chondrocyte cell line) transfected with TCF7.
  • Figure 5B shows the changes in the expression levels of AXIN2 and DKK1 in C28/I2 cells (human chondrocyte line) transfected with TCF7.
  • Figure 5C shows the expression of TCF7 and CCL2 proteins in C28/I2 cells (human chondrocyte line) transfected with TCF7, wherein the transfection time of pc-DNA3-TCF7 plasmid and empty plasmid pc-DNA3 was 24 hours, and the concentration was 1 ⁇ g/ ⁇ L.
  • Figure 5D shows the changes in the expression levels of CCL2 and CCR2 in C28/I2 cells (human chondrocyte line) transfected with TCF7.
  • FIG. 5E is a representative image of immunofluorescence of CCL2 after TCF7 transfection of C28/I2 cells (human chondrocyte cell line), in which DAPI was used for nuclei staining.
  • Figure 5F is the average optical density statistics of CCL2 immunofluorescence after TCF7 transfection of C28/I2 cells (human chondrocyte line), where the ordinate is the average optical density of CCL2 immunofluorescence, and the abscissa is the different treatment methods of C28/I2 cells, DMSO was the control treatment, and BIO was the treatment of the experimental group.
  • Figure 6A is the pain threshold detection results of mice with intervertebral disc degeneration after different treatments.
  • the abscissa represents the different treatments for mice with intervertebral disc degeneration, in which LSI is the surgical model for intervertebral disc instability, Celebrex is the positive control, and the intraperitoneal injection concentrations of Celebrex and iCRT14 are both 50 mg/kg, both every 3 days One injection, starting one month after the operation, and a total of 6 weeks of injection, 1 week before the operation, 4, 7, 10, and 13 weeks after the operation, the Von Frey mechanical stress test was performed to detect the contraction response of the hind paw of the mouse.
  • Figure 6B and Figure 6C are the changes in the expression levels of AXIN2 (6B) and DKK1 (6C) in C28/I2 cells (human chondrocyte line) treated with different concentrations of iCRT14, where the abscissa is the iCRT14 concentration gradient 0, 25, 50 ⁇ M, treatment time is 12 hours; the vertical axis is the fold change of the gene level detected by qPCR in each group relative to the level of the gene detected by qPCR in group 0 with iCRT14 concentration.
  • the * sign indicates the comparison with the iCRT4 concentration of 0 and the IL1 ⁇ concentration of 0 group
  • the # sign indicates the comparison with the iCRT14 concentration of 0 and the IL1 ⁇ concentration of 20nM.
  • the concentration of IL1 ⁇ pretreatment of C28/I2 cells was 20nM, and the treatment time was 4 hours.
  • Figure 6D and Figure 6E show the changes in the expression levels of CCL2 (6D) and CCR2 (6E) in C28/I2 cells (human chondrocyte line) treated with different concentrations of iCRT14, where the abscissa is the iCRT14 concentration gradient 0, 25, 50 ⁇ M, treatment time is 12 hours; the vertical axis is the fold change of the gene level detected by qPCR in each group relative to the level of the gene detected by qPCR in group 0 with iCRT14 concentration.
  • the * sign indicates the comparison with the iCRT4 concentration of 0 and the IL1 ⁇ concentration of 0 group
  • the # sign indicates the comparison with the iCRT14 concentration of 0 and the IL1 ⁇ concentration of 20nM.
  • the concentration of IL1 ⁇ pretreatment of C28/I2 cells was 20nM, and the treatment time was 4 hours.
  • Figure 6F is a representative image of immunofluorescence of CCL2 after iCRT14 treatment of C28/I2 cells (human chondrocyte cell line), in which DAPI was used for nuclei staining.
  • Figure 6G is the average optical density statistics of CCL2 immunofluorescence after iCRT14 treatment of C28/I2 cells (human chondrocyte line), where the ordinate is the average optical density of CCL2 immunofluorescence, the abscissa is the different treatment methods of C28/I2 cells, and DMSO is Control treatment, iCRT14 treatment for the experimental group.
  • the present invention discloses the application of an inhibitor of one embodiment in the field of preparation of therapeutic drugs or therapeutic equipment.
  • the inhibitor is an inhibitor of the combination of ⁇ -catenin protein and TCF7 transcription factor and/or an inhibitor of Wnt/ ⁇ -catenin signaling pathway.
  • the activation of the ⁇ -catenin signaling pathway in intervertebral disc injury will cause the treatment of intervertebral disc-derived pain
  • the binding inhibitor of ⁇ -catenin protein and TCF7 transcription factor can inhibit the binding of ⁇ -catenin protein and TCF7 transcription factor Combined, thereby inhibiting the activation of ⁇ -catenin signaling pathway in intervertebral disc injury
  • Wnt/ ⁇ -catenin signaling pathway inhibitor can inhibit the activation of ⁇ -catenin signaling pathway in intervertebral disc injury.
  • discogenic pain refers to low back pain caused by intervertebral disc degeneration.
  • the activation of the ⁇ -catenin signaling pathway in intervertebral disc injury will cause the treatment of intervertebral disc-derived pain
  • the binding inhibitor of ⁇ -catenin protein and TCF7 transcription factor can inhibit the binding of ⁇ -catenin protein and TCF7 transcription factor Combined, thereby inhibiting the activation of ⁇ -catenin signaling pathway in intervertebral disc injury
  • Wnt/ ⁇ -catenin signaling pathway inhibitor can inhibit the activation of ⁇ -catenin signaling pathway in intervertebral disc injury.
  • the combination inhibitor of ⁇ -catenin protein and TCF7 transcription factor and/or the inhibitor of Wnt/ ⁇ -catenin signaling pathway can achieve the effect of treating discogenic pain based on the inhibition of Wnt/ ⁇ -catenin signaling pathway.
  • the inhibitor is a compound iCRT14 having the following structural formula, its stereoisomer, its pharmaceutically acceptable salt, its solvate or its prodrug:
  • iCRT14 can effectively reduce the expression level of Dv1 and inhibit the binding of TCF and DNA downstream of the Wnt/ ⁇ -catenin signaling pathway. And studies have shown that administering iCRT14 to mice transplanted with colon cancer can effectively inhibit tumor cell proliferation. However, whether iCRT14 has a therapeutic effect on discogenic pain and the underlying mechanism is still unclear.
  • the present invention clarified that the abnormal activation of the Wnt/ ⁇ -catenin signaling pathway promoted the structural and functional damage of the intervertebral disc by constructing a transgenic mouse overexpressing ⁇ -catenin, activated the dorsal root ganglion of the mouse, and resulted in small Increased pain in mice.
  • iCRT14 treats disc-derived pain mainly by inhibiting the interaction of ⁇ -catenin/TCF7 function, thereby inhibiting the expression of the downstream chemokine CCL2, inhibiting the abnormal activation of pain sensory nerves in the dorsal root ganglion, and finally relieving discogenic pain.
  • iCRT14 inhibits the binding of ⁇ -catenin protein and TCF7 transcription factor, thereby inhibiting the activation of ⁇ -catenin signaling pathway in intervertebral disc injury, and then inhibiting the expression of downstream chemokine CCL2, so as to achieve the effect of treating discogenic pain.
  • the effective concentration of the compound iCRT14 is 20 ⁇ M-100 ⁇ M.
  • the effective concentration of the compound iCRT14 is 50 ⁇ M.
  • the therapeutic drug is a therapeutic drug for intervertebral disc-derived pain
  • the therapeutic device is a therapeutic device for intervertebral disc-derived pain
  • the invention also discloses a pharmaceutical composition according to one embodiment.
  • the pharmaceutical composition is an inhibitor of the combination of ⁇ -catenin protein and TCF7 transcription factor and/or an inhibitor of Wnt/ ⁇ -catenin signaling pathway.
  • the activation of the ⁇ -catenin signaling pathway in intervertebral disc injury will cause the treatment of intervertebral disc-derived pain
  • the binding inhibitor of ⁇ -catenin protein and TCF7 transcription factor can inhibit the binding of ⁇ -catenin protein and TCF7 transcription factor Combined, thereby inhibiting the activation of ⁇ -catenin signaling pathway in intervertebral disc injury
  • Wnt/ ⁇ -catenin signaling pathway inhibitor can inhibit the activation of ⁇ -catenin signaling pathway in intervertebral disc injury.
  • discogenic pain refers to low back pain caused by intervertebral disc degeneration.
  • the combination inhibitor of ⁇ -catenin protein and TCF7 transcription factor and/or the inhibitor of Wnt/ ⁇ -catenin signaling pathway can achieve the effect of treating discogenic pain based on the inhibition of Wnt/ ⁇ -catenin signaling pathway.
  • the pharmaceutical composition includes the compound iCRT14 having the following structural formula, its stereoisomer, its pharmaceutically acceptable salt, its solvate or its prodrug:
  • iCRT14 can effectively reduce the expression level of Dv1 and inhibit the binding of TCF and DNA downstream of the Wnt/ ⁇ -catenin signaling pathway. And studies have shown that administering iCRT14 to mice transplanted with colon cancer can effectively inhibit tumor cell proliferation. However, whether iCRT14 has a therapeutic effect on discogenic pain and the underlying mechanism is still unclear.
  • the present invention clarified that the abnormal activation of the Wnt/ ⁇ -catenin signaling pathway promoted the structural and functional damage of the intervertebral disc by constructing a transgenic mouse overexpressing ⁇ -catenin, activated the dorsal root ganglion of the mouse, and resulted in small Increased pain in mice.
  • iCRT14 treats disc-derived pain mainly by inhibiting the interaction of ⁇ -catenin/TCF7 function, thereby inhibiting the expression of the downstream chemokine CCL2, inhibiting the abnormal activation of pain sensory nerves in the dorsal root ganglion, and finally relieving discogenic pain.
  • iCRT14 inhibits the binding of ⁇ -catenin protein and TCF7 transcription factor, thereby inhibiting the activation of ⁇ -catenin signaling pathway in intervertebral disc injury, and then inhibiting the expression of downstream chemokine CCL2, so as to achieve the effect of treating discogenic pain.
  • the effective concentration of the compound iCRT14 can be 20 ⁇ M-100 ⁇ M.
  • the effective concentration of the compound iCRT14 is 50 ⁇ M.
  • the pharmaceutical composition is a therapeutic pharmaceutical composition for discogenic pain.
  • C28/I2 cell line was purchased from Shanghai Jinyuan Biotechnology, and was preserved and cultivated by our laboratory; DMEM medium, fetal bovine serum, double antibodies (penicillin and streptomycin), and trypsin were purchased from Gibico; IL-1 ⁇ , BIO, iCRT14 were purchased from Selleckchem; ⁇ -catenin, CCL2 antibodies were purchased from CST; Actin, ⁇ -Tub ⁇ Lin, Tuj1 antibodies were purchased from Santa Cruz; PGP9.5, antibodies were purchased from Abcam; The TCF7 plasmid was purchased from Addgene; the primers used in qPCR were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.
  • transgenic mice ⁇ -catenin(ex3)flox/flox mice were crossed with Col2-CreER mice to construct chondrocyte ⁇ -cateninAct mice targeting endplate and annulus fibrosus, and genotyping the mice genotype identification.
  • the cells expressing Col2 in this mouse will have abnormal accumulation of ⁇ -catenin protein and activate the Wnt/ ⁇ -catenin signaling pathway.
  • This mouse model mimics pathological conditions in which disc cells overexpress ⁇ -catenin.
  • a mouse model of intervertebral disc degeneration was constructed by resection of the supraspinous and medial spinous ligaments.
  • the C28/I2 human chondrocyte cell line was used as a cell model, in which transfection of TCF7 plasmid can increase the expression level of TCF7 in C28/I2 chondrocytes; To activate Wnt/ ⁇ -catenin signaling pathway.
  • the effective concentration of BIO was 1 ⁇ M, and the treatment time was 12 hours; the effective concentration of iCRT14 was 50 ⁇ M, and the treatment time was 12 hours; the transfection concentration of TCF7 plasmid was 1 ⁇ g/ml, the transfection time was 48 hours, and the effective concentration of IL-1 ⁇ It is 10ng/ml, and the treatment time is 4 hours.
  • Crossings Frequency reflects the exercise ability of mice, and Rearing numbers also reflects the changes of mouse movement and pain threshold from the side.
  • Nerve compression caused by intervertebral foraminal stenosis is an important cause of numbness and low back pain caused by intervertebral disc degeneration.
  • ⁇ CT nerve compression caused by intervertebral foraminal stenosis
  • DRG dorsal root ganglion
  • BIO an agonist of ⁇ -catenin signaling pathway
  • C28/I2 human chondrocyte cell line
  • NP nucleus pulposus
  • BIO can inhibit the degradation of ⁇ -catenin, which leads to abnormal accumulation of ⁇ -catenin signaling pathway in cells. It can be seen from Figure 4A that as the BIO concentration increased, the expression levels of AXIN2 and DKK1 downstream of the TCF7 gene increased after 4 hours. This suggests that BIO can promote TCF7 activation in C28/I2 human chondrocytes.
  • BIO can inhibit the degradation of ⁇ -catenin, resulting in the abnormal accumulation of ⁇ -catenin signaling pathway in cells. It can be seen from Figure 4E that with the increase of BIO concentration, the expression levels of CCL2 and CCR2 in C28/I2 cells after 4 hours It rises accordingly. This indicates that the abnormal activation of ⁇ -catenin signaling pathway induced by BIO can promote the expression of CCL2 and CCR2 in C28/I2 human chondrocytes.
  • BIO activates the ⁇ -catenin signaling pathway to promote CCL2/CCR2 signaling pathway expression.
  • the expression level of the TCF7 gene was significantly up-regulated 24 hours after transfection with the pc-DNA3-TCF7 plasmid compared with the empty plasmid transfection group (pcDNA3 group). This indicated that transfection of pc-DNA3-TCF7 plasmid could effectively up-regulate the expression of TCF7 gene in chondrocytes.
  • FIG. 5B it can be seen that the expression levels of AXIN2 and DKK1 genes were significantly up-regulated 24 hours after transfection with the pc-DNA3-TCF7 plasmid compared with the empty plasmid transfection group (pcDNA3 group). This indicated that transfection of pc-DNA3-TCF7 plasmid could effectively up-regulate the expression of AXIN2 and DKK1 genes downstream of TCF7 gene in chondrocytes.
  • iCRT14 can block the interaction between ⁇ -catenin and TCF to inhibit the abnormal activation of ⁇ -catenin signaling pathway.
  • solvent corn oil was used as the negative control group, and the classic anti-inflammatory drug Celebrex was used as the positive control group.
  • iCRT14 can inhibit the combination of ⁇ -catenin and downstream TCF7, thereby inhibiting the transduction of ⁇ -catenin signaling pathway.
  • Figure 6B and Figure 6C that, with the increase of iCRT14 concentration, after 12 hours, the inflammatory factor IL1 ⁇ pretreated C28/ The expression levels of AXIN2 and DKK1 in I2 cells decreased accordingly.
  • iCRT4 can inhibit the abnormally activated ⁇ -catenin signaling pathway due to inflammation, thereby inhibiting the increased expression of AXIN2 and DKK1 downstream of TCF7.
  • Figure 6D and Figure 6E the expression levels of CCL2 and CCR2 genes were also down-regulated after iCRT14 treatment.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Epidemiology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Engineering & Computer Science (AREA)
  • Neurology (AREA)
  • Biomedical Technology (AREA)
  • Rheumatology (AREA)
  • Neurosurgery (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pain & Pain Management (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

An application of an inhibitor and a pharmaceutical composition. The pharmaceutical composition is a β-catenin protein and TCF7 transcription factor binding inhibitor and/or a Wnt/β-catenin signaling pathway inhibitor. The β-catenin protein and TCF7 transcription factor binding inhibitor and/or the Wnt/β-catenin signaling pathway inhibitor can achieve the effect of treating discogenic pain on the basis of inhibition of a Wnt/β-catenin signaling pathway.

Description

抑制剂的应用以及药物组合物Use of inhibitors and pharmaceutical compositions 技术领域technical field
本发明涉及老年病治疗领域，尤其是涉及一种抑制剂的应用以及药物组合物药物。The invention relates to the field of treatment of geriatric diseases, in particular to the application of an inhibitor and a pharmaceutical composition.
背景技术Background technique
椎间盘源性疼痛主要指由椎间盘退变引起的腰背部疼痛。广义上椎间盘源性颈痛可包括所有因椎间盘病变导致的颈肩臂疼痛，但许多因此引起的疼痛已有相应的病名，如颈椎间盘突出症、颈椎间盘退变狭窄、颈椎病等。近年来提出的椎间盘源性颈痛(cervical discogenic pain)特指局限在椎间盘内部紊乱引起的疼痛，无放射痛及节段性神经功能障碍，不涉及其相邻的脊髓、神经根、小关节。椎间盘源性颈痛是慢性的、间歇性颈肩痛的常见原因之一。Discogenic pain mainly refers to low back pain caused by disc degeneration. In a broad sense, discogenic neck pain can include all neck, shoulder and arm pain caused by intervertebral disc disease, but many pains caused by it have corresponding disease names, such as cervical disc herniation, cervical disc degeneration and stenosis, and cervical spondylosis. In recent years, cervical discogenic pain (cervical discogenic pain) specifically refers to the pain caused by the internal disorder of the intervertebral disc, without radiating pain and segmental nerve dysfunction, and does not involve the adjacent spinal cord, nerve roots, and facet joints. Discogenic neck pain is one of the common causes of chronic, intermittent neck and shoulder pain.
一些临床研究认为椎间盘可能是腰背疼痛的一个原因。椎间盘突出的病人常伴有腰背疼痛，另一个腰背疼痛的可能原因是纤维环撕裂，像此类椎间盘突出的患者的椎间盘内注入生理盐水或造影剂后会诱发疼痛。Some clinical studies suggest that intervertebral discs may be a cause of low back pain. Patients with intervertebral disc herniation are often accompanied by low back pain. Another possible cause of low back pain is tearing of the fibrous annulus. Patients with such intervertebral disc herniation will induce pain after injecting normal saline or contrast medium into the intervertebral disc.
椎间盘的主要组成为胶原、蛋白多糖和水，其占椎间盘容积的90％～95％。这些成分组成随退变或衰老而变化，主要表现为蛋白多糖和水的减少。另外，硫酸软骨素和硫酸角质素比例增加而聚合体比例减少。在椎间盘损伤的过程中，纤维环周边撕裂可由超载扭转所造成。在此撕裂的基础上可出现内部的撕裂，并最终形成完全的放射状撕裂。The main components of the intervertebral disc are collagen, proteoglycan and water, which account for 90% to 95% of the volume of the intervertebral disc. The composition of these components changes with degeneration or aging, mainly manifested in the reduction of proteoglycans and water. In addition, the proportion of chondroitin sulfate and keratan sulfate increased while the proportion of aggregates decreased. Peripheral tearing of the annulus fibrosus can be caused by overloaded torsion during disc injury. On the basis of this tear, internal tears can appear, and finally a complete radial tear can be formed.
窦椎神经(SVN)支配椎管内结构，分布于后纵韧带、硬膜囊的腹侧、血管和纤维环的后方。纤维环的腹侧和侧方由起自腹侧原发支和交感神经发出的分支支配。前纵韧带由灰交通支交感神经干的分支支配。这些神经中含有痛觉纤维，被认为感受椎间盘源性腰背痛，并在椎间盘损伤时变的敏感。The sinus vertebral nerve (SVN) innervates the structures in the spinal canal and is distributed in the posterior longitudinal ligament, ventral side of the dura sac, blood vessels and annulus fibrosus. The ventral and lateral aspects of the annulus are innervated by branches originating from the ventral primary and sympathetic nerves. The anterior longitudinal ligament is innervated by branches of the gray communicating sympathetic trunk. These nerves contain pain-sensing fibers that are thought to sense discogenic low back pain and become sensitized during disc injury.
目前关于椎间盘源性疼痛的研究较为广泛，主要认为椎间盘发生退行性改变以后，在外力作用下，纤维环部分或全部破裂，单独或者连同髓核、软骨终板向外突出，刺激或压迫背根神经节和神经根，导致相邻脊神经根遭受刺激或 压迫，从而产生腰部疼痛，一侧下肢或双下肢麻木、疼痛等一系列临床症状。除此以外，在椎间盘退变病程中，软骨终板与纤维环的炎症可以诱导异常增生的骨赘造成椎间孔的狭窄，同样可以对从椎间孔通过的脊神经进行压迫，从而引起疼痛。软骨终板中的软骨细胞逐渐凋亡钙化，新生血管神经侵入其中由椎间盘细胞以及迁徙而来的炎症细胞分泌的炎症因子如TNF-α与IL-1，以及终板软骨细胞分泌的CCL2，可以对异常增生的神经末梢进行刺激，引起疼痛反应。At present, the research on discogenic pain is relatively extensive. It is mainly believed that after the degenerative changes of the intervertebral disc, under the action of external force, part or all of the annulus fibrosus ruptures, protrudes alone or together with the nucleus pulposus and cartilage endplate, and stimulates or compresses the dorsal root. The ganglia and nerve roots cause the adjacent spinal nerve roots to be stimulated or compressed, resulting in a series of clinical symptoms such as low back pain, numbness and pain in one or both lower limbs. In addition, during the course of intervertebral disc degeneration, the inflammation of cartilage endplate and annulus fibrosus can induce abnormal hyperplasia of osteophytes to narrow the intervertebral foramen, and can also compress the spinal nerve passing through the intervertebral foramen, causing pain. The chondrocytes in the endplate of the cartilage are gradually apoptotic and calcified, and the new blood vessel nerves invade into it. Inflammatory factors such as TNF-α and IL-1 secreted by intervertebral disc cells and migrating inflammatory cells, as well as CCL2 secreted by endplate chondrocytes, can Stimulation of abnormally proliferating nerve endings causes a pain response.
经典的Wnt信号通路对细胞代谢的影响主要通过靶向调控细胞内的β-catenin蛋白的表达以及定位。生理状态下，由于缺少上游Wnt蛋白的刺激，β-catenin蛋白水平在细胞内往往是相对稳定的。这主要是由于过量产生的β-catenin被蛋白激酶GSK-3β，Axin1/Axin2，APC(Adenomatous Polyposis Coli)，Dvl(Disheveled)，以及CK1(Casein Kinase I)共同构成了蛋白复合物所降解。这种蛋白质复合物可磷酸化β-catenin基因外显子3(exon3)编码的β-catenin蛋白羧基末端的特定氨基酸残基。因此，当β-catenin基因外显子3序列产生无效突变时，β-catenin蛋白便会在细胞内异常积累，从而异常激活Wnt/β-catenin信号通路。据此我们构建了β-catenin(ex3)Col2CreER小鼠(即β-cateninAct)小鼠，该小鼠的表达Col2的细胞中会存在β-catenin蛋白的异常积累。The impact of the classic Wnt signaling pathway on cell metabolism is mainly through targeted regulation of the expression and localization of β-catenin protein in cells. Under physiological conditions, due to the lack of upstream Wnt protein stimulation, the level of β-catenin protein is often relatively stable in cells. This is mainly because the overproduced β-catenin is degraded by the protein complex composed of protein kinase GSK-3β, Axin1/Axin2, APC (Adenomatous Polyposis Coli), Dvl (Disheveled), and CK1 (Casein Kinase I). This protein complex can phosphorylate specific amino acid residues at the carboxy-terminus of the β-catenin protein encoded by exon 3 (exon3) of the β-catenin gene. Therefore, when the exon 3 sequence of the β-catenin gene produces a null mutation, the β-catenin protein will accumulate abnormally in the cell, thereby abnormally activating the Wnt/β-catenin signaling pathway. Based on this, we constructed β-catenin(ex3)Col2CreER mice (ie β-cateninAct) mice, which have abnormal accumulation of β-catenin protein in the cells expressing Col2.
由于Wnt/β-catenin信号通路广泛参与机体胚胎发育、调控生物体细胞增殖、代谢等多个方面。大量Wnt/β-catenin信号通路抑制剂正在进行临床实验。然而，Wnt/β-catenin信号通路的抑制对于椎间盘源性疼痛是否有治疗效果，以及潜在的作用机制却并不清楚。The Wnt/β-catenin signaling pathway is widely involved in embryonic development, regulation of biological cell proliferation, metabolism and many other aspects. A large number of Wnt/β-catenin signaling pathway inhibitors are undergoing clinical trials. However, whether inhibition of the Wnt/β-catenin signaling pathway has a therapeutic effect on discogenic pain, and the underlying mechanism remains unclear.
发明内容Contents of the invention
基于此，有必要提供一种基于Wnt/β-catenin信号通路的抑制剂的应用。Based on this, it is necessary to provide an application of an inhibitor based on the Wnt/β-catenin signaling pathway.
此外，还有必要提供一种基于Wnt/β-catenin信号通路的药物组合物。In addition, it is also necessary to provide a pharmaceutical composition based on the Wnt/β-catenin signaling pathway.
一种抑制剂在制备治疗椎间盘源性疼痛的药物的应用，所述抑制剂为β-catenin蛋白与TCF7转录因子结合抑制剂和/或Wnt/β-catenin信号通路抑制剂。An application of an inhibitor in the preparation of a drug for treating intervertebral disc-derived pain. The inhibitor is an inhibitor of the combination of β-catenin protein and TCF7 transcription factor and/or an inhibitor of Wnt/β-catenin signaling pathway.
在一个实施例中，所述抑制剂为具有如下结构式的化合物iCRT14、其立体异构体、其药学上可接受的盐、其溶剂化物或其前药：In one embodiment, the inhibitor is a compound iCRT14 having the following structural formula, its stereoisomer, its pharmaceutically acceptable salt, its solvate or its prodrug:
Figure PCTCN2021136555-appb-000001
Figure PCTCN2021136555-appb-000001
在一个实施例中，所述化合物iCRT14的有效作用浓度为20μM～100μM。In one embodiment, the effective concentration of the compound iCRT14 is 20 μM-100 μM.
在一个实施例中，所述化合物iCRT14的有效作用浓度为50μM。In one embodiment, the effective concentration of the compound iCRT14 is 50 μM.
一种药物组合物，所述药物组合物包括β-catenin蛋白与TCF7转录因子结合抑制剂和/或Wnt/β-catenin信号通路抑制剂。A pharmaceutical composition, which includes an inhibitor of the combination of β-catenin protein and TCF7 transcription factor and/or an inhibitor of Wnt/β-catenin signaling pathway.
在一个实施例中，所述药物组合物包括具有如下结构式的化合物iCRT14、其立体异构体、其药学上可接受的盐、其溶剂化物或其前药：In one embodiment, the pharmaceutical composition includes the compound iCRT14 having the following structural formula, its stereoisomer, its pharmaceutically acceptable salt, its solvate or its prodrug:
Figure PCTCN2021136555-appb-000002
Figure PCTCN2021136555-appb-000002
在一个实施例中，所述化合物iCRT14的有效作用浓度为20μM～100μM。In one embodiment, the effective concentration of the compound iCRT14 is 20 μM-100 μM.
在一个实施例中，所述化合物iCRT14的有效作用浓度为50μM。In one embodiment, the effective concentration of the compound iCRT14 is 50 μM.
在一个实施例中，所述药物组合物为椎间盘源性疼痛的治疗药物组合物。In one embodiment, the pharmaceutical composition is a therapeutic pharmaceutical composition for discogenic pain.
结合实施例部分的数据可以看出，β-catenin信号通路在椎间盘损伤中的激活会造成治疗椎间盘源性疼痛，β-catenin蛋白与TCF7转录因子结合抑制剂可以抑制β-catenin蛋白与TCF7转录因子结合，从而抑制β-catenin信号通路在椎间盘损伤中的激活，同时Wnt/β-catenin信号通路抑制剂可以抑制β-catenin信号通路在椎间盘损伤中的激活。β-catenin信号通路在椎间盘损伤中的激活被抑制后，进而可以抑制下游趋化因子CCL2的表达，从而达到治疗椎间盘源性疼痛的效果。Combined with the data in the Examples, it can be seen that the activation of the β-catenin signaling pathway in intervertebral disc injury will cause the treatment of intervertebral disc-derived pain, and the binding inhibitor of β-catenin protein and TCF7 transcription factor can inhibit the binding of β-catenin protein and TCF7 transcription factor Combined, thereby inhibiting the activation of β-catenin signaling pathway in intervertebral disc injury, and Wnt/β-catenin signaling pathway inhibitor can inhibit the activation of β-catenin signaling pathway in intervertebral disc injury. After the activation of β-catenin signaling pathway in intervertebral disc injury is inhibited, the expression of downstream chemokine CCL2 can be inhibited, so as to achieve the effect of treating discogenic pain.
因此，β-catenin蛋白与TCF7转录因子结合抑制剂和/或Wnt/β-catenin信号通路抑制剂均可以基于Wnt/β-catenin信号通路的抑制达到治疗椎间盘源性 疼痛的效果。Therefore, the combination inhibitor of β-catenin protein and TCF7 transcription factor and/or the inhibitor of Wnt/β-catenin signaling pathway can achieve the effect of treating discogenic pain based on the inhibition of Wnt/β-catenin signaling pathway.
附图说明Description of drawings
图1A为β-catenin过表达转基因小鼠的疼痛阈值检测结果，其中横坐标显示β-catenin过表达转基因小鼠于他莫昔芬诱导β-catenin信号通路异常积累后0、4、8、12、16、20周进行Von Frey机械应力检测后脚掌收缩反应。Figure 1A is the pain threshold detection results of β-catenin overexpression transgenic mice, where the abscissa shows β-catenin overexpression transgenic mice after 0, 4, 8, and 12 days after tamoxifen induced abnormal accumulation of β-catenin signaling pathway , 16, 20 weeks after Von Frey mechanical stress test, paw contraction response.
图1B为β-catenin过表达转基因小鼠的交叉频率检测结果，其中横坐标显示横坐标显示β-catenin过表达转基因小鼠于他莫昔芬诱导β-catenin信号通路异常积累后4、8、12、16、20、24周采用LABROS系统进行行为学检测。Figure 1B is the detection result of crossover frequency of β-catenin overexpression transgenic mice, where the abscissa shows that the abscissa shows that the β-catenin overexpression transgenic mice were abnormally accumulated in the β-catenin signaling pathway induced by tamoxifen 4, 8, At 12, 16, 20, and 24 weeks, LABROS system was used for behavioral testing.
图1C为β-catenin过表达转基因小鼠的站立次数检测结果，其中横坐标显示横坐标显示β-catenin过表达转基因小鼠于他莫昔芬诱导β-catenin信号通路异常积累后4、8、12、16、20、24周采用LABROS系统进行行为学检测。Figure 1C is the test results of standing times of β-catenin overexpression transgenic mice, where the abscissa shows that the abscissa shows that the β-catenin overexpression transgenic mice were abnormally accumulated in the β-catenin signaling pathway induced by tamoxifen 4, 8, At 12, 16, 20, and 24 weeks, LABROS system was used for behavioral testing.
图2A为β-catenin过表达转基因小鼠脊柱椎体μCT扫描代表性图片。Figure 2A is a representative picture of the μCT scan of the vertebral body of the β-catenin overexpression transgenic mouse.
图2B为β-catenin过表达转基因小鼠脊柱椎体的番红O固绿染色代表性图片。Fig. 2B is a representative picture of safranin O fast green staining of vertebral bodies of transgenic mice overexpressing β-catenin.
图3A为β-catenin过表达转基因小鼠背根神经节PGP9.5与Tuj1免疫荧光染色代表图片，其中DAPI用以细胞核染色。Figure 3A is a representative picture of immunofluorescent staining of PGP9.5 and Tuj1 in dorsal root ganglia of transgenic mice overexpressing β-catenin, in which DAPI was used for nuclear staining.
图3B为β-catenin过表达转基因小鼠背根神经节中痛觉相关基因qPCR检测结果其中横坐标Ctr代表Cre阴性对照组，Mut代表β-catenin过表达转基因小鼠组，纵坐标为Mut组qPCR检测基因水平相对于Ctr组qPCR检测基因水平的倍数变化。Figure 3B is the qPCR detection results of pain-related genes in the dorsal root ganglion of β-catenin overexpressed transgenic mice. The abscissa Ctr represents the Cre negative control group, Mut represents the β-catenin overexpressed transgenic mouse group, and the ordinate represents the qPCR of the Mut group The fold change of the detected gene level relative to the gene level detected by qPCR of the Ctr group.
图3C为β-catenin过表达转基因小鼠背根神经节中炎症相关基因qPCR检测结果，其中横坐标Ctr代表Cre阴性对照组，Mut代表β-catenin过表达转基因小鼠组，纵坐标为Mut组qPCR检测基因水平相对于Ctr组qPCR检测基因水平的倍数变化。Figure 3C shows the results of qPCR detection of inflammation-related genes in the dorsal root ganglia of β-catenin overexpression transgenic mice, where the abscissa Ctr represents the Cre negative control group, Mut represents the β-catenin overexpression transgenic mouse group, and the ordinate represents the Mut group The fold change of gene levels detected by qPCR relative to the gene levels detected by qPCR in the Ctr group.
图4A为不同浓度BIO处理的C28/I2细胞(人软骨细胞系)中AXIN2与DKK1表达水平变化，其中横坐标为BIO浓度梯度0，10，20，50μM；纵坐标为各组qPCR检测基因水平相对于BIO浓度0组qPCR检测基因水平的倍数变化。Figure 4A shows the changes in the expression levels of AXIN2 and DKK1 in C28/I2 cells (human chondrocyte line) treated with different concentrations of BIO, where the abscissa is the BIO concentration gradient of 0, 10, 20, and 50 μM; the ordinate is the gene level detected by qPCR in each group Fold change in gene levels detected by qPCR relative to BIO concentration 0 group.
图4B为BIO处理C28/I2细胞(人软骨细胞系)后β-catenin与CCL2蛋白表达，其中BIO处理浓度为1μM，处理时间为12小时。Figure 4B shows the expression of β-catenin and CCL2 proteins after BIO treatment of C28/I2 cells (human chondrocyte line), wherein the BIO treatment concentration was 1 μM and the treatment time was 12 hours.
图4C为BIO与IL-1β对于大鼠原代纤维环(AF)细胞中β-catenin蛋白水平的影响，其中BIO处理浓度为1μM，处理时间为12小时，IL-1β处理浓度为10nM，处理时间为12小时。Figure 4C is the effect of BIO and IL-1β on the level of β-catenin protein in rat primary annulus fibrosus (AF) cells, wherein the BIO treatment concentration was 1 μM, the treatment time was 12 hours, the IL-1β treatment concentration was 10 nM, and the treatment The time is 12 hours.
图4D为BIO与IL-1β对于大鼠髓核(NP)细胞中β-catenin蛋白水平的影响，其中BIO处理浓度为1μM，处理时间为12小时，IL-1β处理浓度为10nM，处理时间为12小时。Figure 4D is the effect of BIO and IL-1β on the level of β-catenin protein in rat nucleus pulposus (NP) cells, wherein the BIO treatment concentration is 1μM, and the treatment time is 12 hours, and the IL-1β treatment concentration is 10nM, and the treatment time is 12 hours.
图4E为不同浓度BIO处理的C28/I2细胞(人软骨细胞系)中CCL2与CCR2表达水平变化，其中横坐标为BIO浓度梯度0，10，20，50μM；纵坐标为各组qPCR检测基因水平相对于BIO浓度0组qPCR检测基因水平的倍数变化。Figure 4E shows the changes in the expression levels of CCL2 and CCR2 in C28/I2 cells (human chondrocyte line) treated with different concentrations of BIO, where the abscissa is the BIO concentration gradient of 0, 10, 20, and 50 μM; the ordinate is the gene level detected by qPCR in each group Fold change in gene levels detected by qPCR relative to BIO concentration 0 group.
图4F为BIO处理C28/I2细胞(人软骨细胞系)后CCL2的免疫荧光代表性图片，其中DAPI用以细胞核染色。Figure 4F is a representative image of immunofluorescence of CCL2 after BIO treatment of C28/I2 cells (human chondrocyte cell line), in which DAPI was used for nuclei staining.
图4G为BIO处理C28/I2细胞(人软骨细胞系)后CCL2免疫荧光的平均光密度统计，其中纵坐标为CCL2免疫荧光的平均光密度，横坐标为C28/I2细胞不同处理方式，DMSO为对照处理，BIO为实验组处理。Figure 4G is the average optical density statistics of CCL2 immunofluorescence after BIO treatment of C28/I2 cells (human chondrocyte line), where the ordinate is the average optical density of CCL2 immunofluorescence, the abscissa is the different treatment methods of C28/I2 cells, and DMSO is Control treatment, BIO treatment for the experimental group.
图5A为TCF7转染的C28/I2细胞(人软骨细胞系)中TCF7表达水平变化。Fig. 5A is the expression level change of TCF7 in C28/I2 cells (human chondrocyte cell line) transfected with TCF7.
图5B为TCF7转染的C28/I2细胞(人软骨细胞系)中AXIN2与DKK1表达水平变化。Figure 5B shows the changes in the expression levels of AXIN2 and DKK1 in C28/I2 cells (human chondrocyte line) transfected with TCF7.
图5C为TCF7转染的C28/I2细胞(人软骨细胞系)后TCF7与CCL2蛋白表达，其中pc-DNA3-TCF7质粒与空载质粒pc-DNA3转染时间均为24小时，浓度为1μg/μL。Figure 5C shows the expression of TCF7 and CCL2 proteins in C28/I2 cells (human chondrocyte line) transfected with TCF7, wherein the transfection time of pc-DNA3-TCF7 plasmid and empty plasmid pc-DNA3 was 24 hours, and the concentration was 1 μg/ μL.
图5D为TCF7转染的C28/I2细胞(人软骨细胞系)中CCL2与CCR2表达水平变化。Figure 5D shows the changes in the expression levels of CCL2 and CCR2 in C28/I2 cells (human chondrocyte line) transfected with TCF7.
图5E为TCF7转染的C28/I2细胞(人软骨细胞系)后CCL2的免疫荧光代表性图片，其中DAPI用以细胞核染色。FIG. 5E is a representative image of immunofluorescence of CCL2 after TCF7 transfection of C28/I2 cells (human chondrocyte cell line), in which DAPI was used for nuclei staining.
图5F为TCF7转染的C28/I2细胞(人软骨细胞系)后CCL2免疫荧光的平均光密度统计，其中纵坐标为CCL2免疫荧光的平均光密度，横坐标为C28/I2 细胞不同处理方式，DMSO为对照处理，BIO为实验组处理。Figure 5F is the average optical density statistics of CCL2 immunofluorescence after TCF7 transfection of C28/I2 cells (human chondrocyte line), where the ordinate is the average optical density of CCL2 immunofluorescence, and the abscissa is the different treatment methods of C28/I2 cells, DMSO was the control treatment, and BIO was the treatment of the experimental group.
图6A为椎间盘退变小鼠在不同治疗后的疼痛阈值检测结果。横坐标为针对椎间盘退变小鼠的不同处理，其中LSI为椎间盘不稳手术造模，塞莱希布(Celebrex)为阳性对照，Celebrex与iCRT14腹腔注射浓度均为50mg/kg，均每3天注射一次，于术后一个月开始注射，共注射6周，于术前1周，术后4，7，10，13周进行Von Frey机械应力检测小鼠后脚掌收缩反应。Figure 6A is the pain threshold detection results of mice with intervertebral disc degeneration after different treatments. The abscissa represents the different treatments for mice with intervertebral disc degeneration, in which LSI is the surgical model for intervertebral disc instability, Celebrex is the positive control, and the intraperitoneal injection concentrations of Celebrex and iCRT14 are both 50 mg/kg, both every 3 days One injection, starting one month after the operation, and a total of 6 weeks of injection, 1 week before the operation, 4, 7, 10, and 13 weeks after the operation, the Von Frey mechanical stress test was performed to detect the contraction response of the hind paw of the mouse.
图6B和图6C为不同浓度iCRT14处理的C28/I2细胞(人软骨细胞系)中AXIN2(6B)与DKK1(6C)表达水平变化，其中横坐标为iCRT14浓度梯度0，25，50μM，处理时间为12小时；纵坐标为各组qPCR检测基因水平相对于iCRT14浓度0组qPCR检测基因水平的倍数变化。其中*号表明与iCRT4浓度0且IL1β浓度为0组的对比，#号表明与iCRT14浓度0且IL1β浓度为20nM组的对比。IL1β预处理C28/I2细胞浓度为20nM，处理时间为4小时。Figure 6B and Figure 6C are the changes in the expression levels of AXIN2 (6B) and DKK1 (6C) in C28/I2 cells (human chondrocyte line) treated with different concentrations of iCRT14, where the abscissa is the iCRT14 concentration gradient 0, 25, 50 μM, treatment time is 12 hours; the vertical axis is the fold change of the gene level detected by qPCR in each group relative to the level of the gene detected by qPCR in group 0 with iCRT14 concentration. Wherein, the * sign indicates the comparison with the iCRT4 concentration of 0 and the IL1β concentration of 0 group, and the # sign indicates the comparison with the iCRT14 concentration of 0 and the IL1β concentration of 20nM. The concentration of IL1β pretreatment of C28/I2 cells was 20nM, and the treatment time was 4 hours.
图6D和图6E为不同浓度iCRT14处理的C28/I2细胞(人软骨细胞系)中CCL2(6D)与CCR2(6E)表达水平变化，其中横坐标为iCRT14浓度梯度0，25，50μM，处理时间为12小时；纵坐标为各组qPCR检测基因水平相对于iCRT14浓度0组qPCR检测基因水平的倍数变化。其中*号表明与iCRT4浓度0且IL1β浓度为0组的对比，#号表明与iCRT14浓度0且IL1β浓度为20nM组的对比。IL1β预处理C28/I2细胞浓度为20nM，处理时间为4小时。Figure 6D and Figure 6E show the changes in the expression levels of CCL2 (6D) and CCR2 (6E) in C28/I2 cells (human chondrocyte line) treated with different concentrations of iCRT14, where the abscissa is the iCRT14 concentration gradient 0, 25, 50 μM, treatment time is 12 hours; the vertical axis is the fold change of the gene level detected by qPCR in each group relative to the level of the gene detected by qPCR in group 0 with iCRT14 concentration. Wherein, the * sign indicates the comparison with the iCRT4 concentration of 0 and the IL1β concentration of 0 group, and the # sign indicates the comparison with the iCRT14 concentration of 0 and the IL1β concentration of 20nM. The concentration of IL1β pretreatment of C28/I2 cells was 20nM, and the treatment time was 4 hours.
图6F为iCRT14处理C28/I2细胞(人软骨细胞系)后CCL2的免疫荧光代表性图片，其中DAPI用以细胞核染色。Figure 6F is a representative image of immunofluorescence of CCL2 after iCRT14 treatment of C28/I2 cells (human chondrocyte cell line), in which DAPI was used for nuclei staining.
图6G为iCRT14处理C28/I2细胞(人软骨细胞系)后CCL2免疫荧光的平均光密度统计，其中纵坐标为CCL2免疫荧光的平均光密度，横坐标为C28/I2细胞不同处理方式，DMSO为对照处理，iCRT14为实验组处理。Figure 6G is the average optical density statistics of CCL2 immunofluorescence after iCRT14 treatment of C28/I2 cells (human chondrocyte line), where the ordinate is the average optical density of CCL2 immunofluorescence, the abscissa is the different treatment methods of C28/I2 cells, and DMSO is Control treatment, iCRT14 treatment for the experimental group.
具体实施方式Detailed ways
为使本发明的目的、技术方案和优点更加清楚，下面将结合附图对本发明的实施方式作进一步地描述。In order to make the object, technical solution and advantages of the present invention clearer, the implementation manners of the present invention will be further described below in conjunction with the accompanying drawings.
本发明公开了一实施方式的抑制剂在制备治疗药物或治疗设备领域的应 用。The present invention discloses the application of an inhibitor of one embodiment in the field of preparation of therapeutic drugs or therapeutic equipment.
具体来说，抑制剂为β-catenin蛋白与TCF7转录因子结合抑制剂和/或Wnt/β-catenin信号通路抑制剂。Specifically, the inhibitor is an inhibitor of the combination of β-catenin protein and TCF7 transcription factor and/or an inhibitor of Wnt/β-catenin signaling pathway.
结合实施例部分的数据可以看出，β-catenin信号通路在椎间盘损伤中的激活会造成治疗椎间盘源性疼痛，β-catenin蛋白与TCF7转录因子结合抑制剂可以抑制β-catenin蛋白与TCF7转录因子结合，从而抑制β-catenin信号通路在椎间盘损伤中的激活，同时Wnt/β-catenin信号通路抑制剂可以抑制β-catenin信号通路在椎间盘损伤中的激活。β-catenin信号通路在椎间盘损伤中的激活被抑制后，进而可以抑制下游趋化因子CCL2的表达，从而达到治疗椎间盘源性疼痛的效果。Combined with the data in the Examples, it can be seen that the activation of the β-catenin signaling pathway in intervertebral disc injury will cause the treatment of intervertebral disc-derived pain, and the binding inhibitor of β-catenin protein and TCF7 transcription factor can inhibit the binding of β-catenin protein and TCF7 transcription factor Combined, thereby inhibiting the activation of β-catenin signaling pathway in intervertebral disc injury, and Wnt/β-catenin signaling pathway inhibitor can inhibit the activation of β-catenin signaling pathway in intervertebral disc injury. After the activation of β-catenin signaling pathway in intervertebral disc injury is inhibited, the expression of downstream chemokine CCL2 can be inhibited, so as to achieve the effect of treating discogenic pain.
本发明中，椎间盘源性疼痛是指由椎间盘退变引起的腰背部疼痛。In the present invention, discogenic pain refers to low back pain caused by intervertebral disc degeneration.
结合实施例部分的数据可以看出，β-catenin信号通路在椎间盘损伤中的激活会造成治疗椎间盘源性疼痛，β-catenin蛋白与TCF7转录因子结合抑制剂可以抑制β-catenin蛋白与TCF7转录因子结合，从而抑制β-catenin信号通路在椎间盘损伤中的激活，同时Wnt/β-catenin信号通路抑制剂可以抑制β-catenin信号通路在椎间盘损伤中的激活。β-catenin信号通路在椎间盘损伤中的激活被抑制后，进而可以抑制下游趋化因子CCL2的表达，从而达到治疗椎间盘源性疼痛的效果。Combined with the data in the Examples, it can be seen that the activation of the β-catenin signaling pathway in intervertebral disc injury will cause the treatment of intervertebral disc-derived pain, and the binding inhibitor of β-catenin protein and TCF7 transcription factor can inhibit the binding of β-catenin protein and TCF7 transcription factor Combined, thereby inhibiting the activation of β-catenin signaling pathway in intervertebral disc injury, and Wnt/β-catenin signaling pathway inhibitor can inhibit the activation of β-catenin signaling pathway in intervertebral disc injury. After the activation of β-catenin signaling pathway in intervertebral disc injury is inhibited, the expression of downstream chemokine CCL2 can be inhibited, so as to achieve the effect of treating discogenic pain.
因此，β-catenin蛋白与TCF7转录因子结合抑制剂和/或Wnt/β-catenin信号通路抑制剂均可以基于Wnt/β-catenin信号通路的抑制达到治疗椎间盘源性疼痛的效果。Therefore, the combination inhibitor of β-catenin protein and TCF7 transcription factor and/or the inhibitor of Wnt/β-catenin signaling pathway can achieve the effect of treating discogenic pain based on the inhibition of Wnt/β-catenin signaling pathway.
优选的，抑制剂为具有如下结构式的化合物iCRT14、其立体异构体、其药学上可接受的盐、其溶剂化物或其前药：Preferably, the inhibitor is a compound iCRT14 having the following structural formula, its stereoisomer, its pharmaceutically acceptable salt, its solvate or its prodrug:
Figure PCTCN2021136555-appb-000003
Figure PCTCN2021136555-appb-000003
现有技术显示，iCRT14可以有效降低Dv1表达水平，并抑制Wnt/β-catenin信号通路下游的TCF与DNA结合。并且有研究显示对结肠癌移植小鼠服用iCRT14可以有效抑制肿瘤细胞增殖。然而iCRT14对于椎间盘源性疼痛是否有治疗效果，以及潜在的作用机制却并不清楚。Existing technologies have shown that iCRT14 can effectively reduce the expression level of Dv1 and inhibit the binding of TCF and DNA downstream of the Wnt/β-catenin signaling pathway. And studies have shown that administering iCRT14 to mice transplanted with colon cancer can effectively inhibit tumor cell proliferation. However, whether iCRT14 has a therapeutic effect on discogenic pain and the underlying mechanism is still unclear.
结合具体实施例，本发明通过构建β-catenin过表达的转基因小鼠，明确了Wnt/β-catenin信号通路异常激活促进了椎间盘结构与功能损伤，激活了小鼠背根神经节，导致了小鼠疼痛增加。采用小鼠椎间盘不稳模型，证实了Wnt/β-catenin信号通路抑制剂iCRT14对椎间盘源性疼痛的治疗效果，且明确了iCRT14治疗椎间盘源性疼痛的主要是通过抑制β-catenin/TCF7的相互作用，进而抑制下游的趋化因子CCL2表达，抑制背根神经节中疼痛感觉神经的异常激活，最终缓解椎间盘源性疼痛。In combination with specific examples, the present invention clarified that the abnormal activation of the Wnt/β-catenin signaling pathway promoted the structural and functional damage of the intervertebral disc by constructing a transgenic mouse overexpressing β-catenin, activated the dorsal root ganglion of the mouse, and resulted in small Increased pain in mice. Using a mouse model of intervertebral disc instability, the therapeutic effect of the Wnt/β-catenin signaling pathway inhibitor iCRT14 on disc-derived pain was confirmed, and it was clarified that iCRT14 treats disc-derived pain mainly by inhibiting the interaction of β-catenin/TCF7 function, thereby inhibiting the expression of the downstream chemokine CCL2, inhibiting the abnormal activation of pain sensory nerves in the dorsal root ganglion, and finally relieving discogenic pain.
总结来说，iCRT14通过抑制β-catenin蛋白与TCF7转录因子结合，从而抑制β-catenin信号通路在椎间盘损伤中的激活，进而抑制下游趋化因子CCL2的表达，达到治疗椎间盘源性疼痛的效果。In summary, iCRT14 inhibits the binding of β-catenin protein and TCF7 transcription factor, thereby inhibiting the activation of β-catenin signaling pathway in intervertebral disc injury, and then inhibiting the expression of downstream chemokine CCL2, so as to achieve the effect of treating discogenic pain.
一般来说，化合物iCRT14的有效作用浓度为20μM～100μM。Generally, the effective concentration of the compound iCRT14 is 20 μM-100 μM.
优选的，化合物iCRT14的有效作用浓度为50μM。Preferably, the effective concentration of the compound iCRT14 is 50 μM.
具体来说，本发明中，治疗药物为椎间盘源性疼痛的治疗药物，治疗设备为椎间盘源性疼痛的治疗设备。Specifically, in the present invention, the therapeutic drug is a therapeutic drug for intervertebral disc-derived pain, and the therapeutic device is a therapeutic device for intervertebral disc-derived pain.
本发明还公开了一实施方式的药物组合物。The invention also discloses a pharmaceutical composition according to one embodiment.
具体来说，药物组合物为β-catenin蛋白与TCF7转录因子结合抑制剂和/或Wnt/β-catenin信号通路抑制剂。Specifically, the pharmaceutical composition is an inhibitor of the combination of β-catenin protein and TCF7 transcription factor and/or an inhibitor of Wnt/β-catenin signaling pathway.
结合实施例部分的数据可以看出，β-catenin信号通路在椎间盘损伤中的激活会造成治疗椎间盘源性疼痛，β-catenin蛋白与TCF7转录因子结合抑制剂可以抑制β-catenin蛋白与TCF7转录因子结合，从而抑制β-catenin信号通路在椎间盘损伤中的激活，同时Wnt/β-catenin信号通路抑制剂可以抑制β-catenin信号通路在椎间盘损伤中的激活。β-catenin信号通路在椎间盘损伤中的激活被抑制后，进而可以抑制下游趋化因子CCL2的表达，从而达到治疗椎间盘源性疼 痛的效果。Combined with the data in the Examples, it can be seen that the activation of the β-catenin signaling pathway in intervertebral disc injury will cause the treatment of intervertebral disc-derived pain, and the binding inhibitor of β-catenin protein and TCF7 transcription factor can inhibit the binding of β-catenin protein and TCF7 transcription factor Combined, thereby inhibiting the activation of β-catenin signaling pathway in intervertebral disc injury, and Wnt/β-catenin signaling pathway inhibitor can inhibit the activation of β-catenin signaling pathway in intervertebral disc injury. After the activation of β-catenin signaling pathway in intervertebral disc injury is inhibited, the expression of downstream chemokine CCL2 can be inhibited, so as to achieve the effect of treating discogenic pain.
本发明中，椎间盘源性疼痛是指由椎间盘退变引起的腰背部疼痛。In the present invention, discogenic pain refers to low back pain caused by intervertebral disc degeneration.
因此，β-catenin蛋白与TCF7转录因子结合抑制剂和/或Wnt/β-catenin信号通路抑制剂均可以基于Wnt/β-catenin信号通路的抑制达到治疗椎间盘源性疼痛的效果。Therefore, the combination inhibitor of β-catenin protein and TCF7 transcription factor and/or the inhibitor of Wnt/β-catenin signaling pathway can achieve the effect of treating discogenic pain based on the inhibition of Wnt/β-catenin signaling pathway.
优选的，药物组合物包括具有如下结构式的化合物iCRT14、其立体异构体、其药学上可接受的盐、其溶剂化物或其前药：Preferably, the pharmaceutical composition includes the compound iCRT14 having the following structural formula, its stereoisomer, its pharmaceutically acceptable salt, its solvate or its prodrug:
Figure PCTCN2021136555-appb-000004
Figure PCTCN2021136555-appb-000004
现有技术显示，iCRT14可以有效降低Dv1表达水平，并抑制Wnt/β-catenin信号通路下游的TCF与DNA结合。并且有研究显示对结肠癌移植小鼠服用iCRT14可以有效抑制肿瘤细胞增殖。然而iCRT14对于椎间盘源性疼痛是否有治疗效果，以及潜在的作用机制却并不清楚。Existing technologies have shown that iCRT14 can effectively reduce the expression level of Dv1 and inhibit the binding of TCF and DNA downstream of the Wnt/β-catenin signaling pathway. And studies have shown that administering iCRT14 to mice transplanted with colon cancer can effectively inhibit tumor cell proliferation. However, whether iCRT14 has a therapeutic effect on discogenic pain and the underlying mechanism is still unclear.
结合具体实施例，本发明通过构建β-catenin过表达的转基因小鼠，明确了Wnt/β-catenin信号通路异常激活促进了椎间盘结构与功能损伤，激活了小鼠背根神经节，导致了小鼠疼痛增加。采用小鼠椎间盘不稳模型，证实了Wnt/β-catenin信号通路抑制剂iCRT14对椎间盘源性疼痛的治疗效果，且明确了iCRT14治疗椎间盘源性疼痛的主要是通过抑制β-catenin/TCF7的相互作用，进而抑制下游的趋化因子CCL2表达，抑制背根神经节中疼痛感觉神经的异常激活，最终缓解椎间盘源性疼痛。In combination with specific examples, the present invention clarified that the abnormal activation of the Wnt/β-catenin signaling pathway promoted the structural and functional damage of the intervertebral disc by constructing a transgenic mouse overexpressing β-catenin, activated the dorsal root ganglion of the mouse, and resulted in small Increased pain in mice. Using a mouse model of intervertebral disc instability, the therapeutic effect of the Wnt/β-catenin signaling pathway inhibitor iCRT14 on disc-derived pain was confirmed, and it was clarified that iCRT14 treats disc-derived pain mainly by inhibiting the interaction of β-catenin/TCF7 function, thereby inhibiting the expression of the downstream chemokine CCL2, inhibiting the abnormal activation of pain sensory nerves in the dorsal root ganglion, and finally relieving discogenic pain.
总结来说，iCRT14通过抑制β-catenin蛋白与TCF7转录因子结合，从而抑制β-catenin信号通路在椎间盘损伤中的激活，进而抑制下游趋化因子CCL2的表达，达到治疗椎间盘源性疼痛的效果。In summary, iCRT14 inhibits the binding of β-catenin protein and TCF7 transcription factor, thereby inhibiting the activation of β-catenin signaling pathway in intervertebral disc injury, and then inhibiting the expression of downstream chemokine CCL2, so as to achieve the effect of treating discogenic pain.
一般来说，化合物iCRT14的有效作用浓度可以为20μM～100μM。Generally, the effective concentration of the compound iCRT14 can be 20 μM-100 μM.
优选的，结合具体实施例，化合物iCRT14的有效作用浓度为50μM。Preferably, in conjunction with specific examples, the effective concentration of the compound iCRT14 is 50 μM.
具体来说，本发明中，药物组合物为椎间盘源性疼痛的治疗药物组合物。Specifically, in the present invention, the pharmaceutical composition is a therapeutic pharmaceutical composition for discogenic pain.
以下为具体实施例。The following are specific examples.
实施例中所用方法如无特别说明均为常规方法。The methods used in the examples are conventional methods unless otherwise specified.
实施例中采用的材料如下：C28/I2细胞系购于上海瑾原生物，并由本实验室保存及培养；DMEM培养基，胎牛血清，双抗(青霉素与链霉素)，胰蛋白酶购自Gibico公司；IL-1β，BIO，iCRT14购自Selleckchem公司；β-catenin，CCL2抗体购自CST公司，Actin，α-TubμLin，Tuj1抗体购自Santa Cruz公司；PGP9.5，抗体购自Abcam公司；TCF7质粒购自Addgene公司；qPCR所用引物由生工生物工程(上海)股份有限公司合成。The materials used in the examples are as follows: C28/I2 cell line was purchased from Shanghai Jinyuan Biotechnology, and was preserved and cultivated by our laboratory; DMEM medium, fetal bovine serum, double antibodies (penicillin and streptomycin), and trypsin were purchased from Gibico; IL-1β, BIO, iCRT14 were purchased from Selleckchem; β-catenin, CCL2 antibodies were purchased from CST; Actin, α-TubμLin, Tuj1 antibodies were purchased from Santa Cruz; PGP9.5, antibodies were purchased from Abcam; The TCF7 plasmid was purchased from Addgene; the primers used in qPCR were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.
小鼠行为学采用LABORAS平台(Metris公司)进行检测，痛觉采用von Frey机械应力检测，其余操作均为常规方法。Mouse behavior was tested using the LABORAS platform (Metris Company), pain was detected using von Frey mechanical stress, and the rest of the operations were performed by conventional methods.
实施例1Example 1
转基因小鼠的构建：β-catenin(ex3)flox/flox小鼠分别与Col2-CreER小鼠进行杂交，构建靶向终板与纤维环的软骨细胞β-cateninAct小鼠，并利用Genotyping对小鼠基因型进行鉴定。该小鼠表达Col2的细胞中会存在β-catenin蛋白的异常积累，激活Wnt/β-catenin信号通路。这个小鼠模型模拟了病理状况下椎间盘细胞过表达β-catenin的状况。Construction of transgenic mice: β-catenin(ex3)flox/flox mice were crossed with Col2-CreER mice to construct chondrocyte β-cateninAct mice targeting endplate and annulus fibrosus, and genotyping the mice genotype identification. The cells expressing Col2 in this mouse will have abnormal accumulation of β-catenin protein and activate the Wnt/β-catenin signaling pathway. This mouse model mimics pathological conditions in which disc cells overexpress β-catenin.
小鼠椎间盘退变模型，采用棘上、棘中韧带切除术进行构建。A mouse model of intervertebral disc degeneration was constructed by resection of the supraspinous and medial spinous ligaments.
体外实验，采用C28/I2人软骨细胞系，作为细胞模型，其中转染TCF7质粒，可提高C28/I2软骨细胞中TCF7表达水平；BIO通过抑制GSK-3β，从而抑制β-catenin降解，从而起到激活Wnt/β-catenin信号通路的作用。In vitro experiments, the C28/I2 human chondrocyte cell line was used as a cell model, in which transfection of TCF7 plasmid can increase the expression level of TCF7 in C28/I2 chondrocytes; To activate Wnt/β-catenin signaling pathway.
体外实验中，BIO有效浓度为1μM，处理时间为12小时；iCRT14有效浓度为50μM，处理时间为12小时；TCF7质粒转染浓度为1μg/ml，转染时间为48小时，IL-1β有效浓度为10ng/ml，处理时间为4小时。In vitro experiments, the effective concentration of BIO was 1 μM, and the treatment time was 12 hours; the effective concentration of iCRT14 was 50 μM, and the treatment time was 12 hours; the transfection concentration of TCF7 plasmid was 1 μg/ml, the transfection time was 48 hours, and the effective concentration of IL-1β It is 10ng/ml, and the treatment time is 4 hours.
为了明确Wnt/β-catenin信号通路异常激活对小鼠行为功能上的影响，在他莫昔芬诱导β-cateninAct小鼠β-catenin信号通路异常激活后，我们于不同时间点对小鼠进行痛觉检测，得到图1A。In order to clarify the impact of abnormal activation of Wnt/β-catenin signaling pathway on behavioral function of mice, after tamoxifen induced abnormal activation of β-catenin signaling pathway in β-cateninAct mice, we performed pain perception on mice at different time points detection, to obtain Figure 1A.
由图1A可以看出，β-catenin过表达转基因小鼠的50％后脚掌收缩疼痛阈值(g)显著低于Cre阴性对照组小鼠，这表示更微弱的机械刺激便可以使得β-catenin过表达转基因小鼠后脚掌收缩，从而反应出β-catenin过表达转基因小鼠对于疼痛更加敏感。It can be seen from Figure 1A that the pain threshold (g) of 50% hindpaw contraction of transgenic mice overexpressing β-catenin was significantly lower than that of Cre-negative control mice, which indicated that weaker mechanical stimulation can make β-catenin excessive. The paws of mice expressing transgenic mice contracted, which reflected that transgenic mice expressing β-catenin were more sensitive to pain.
交叉频率(Crossings Frequency)反映了小鼠的运动能力，站立次数(Rearing numbers)同样从侧面反映小鼠运动以及疼痛阈值变化。在他莫昔芬诱导的β-cateninAct小鼠β-catenin信号通路异常激活后，我们于不同时间点对小鼠进行了行为学检测，得到图1B和图1C。Crossings Frequency reflects the exercise ability of mice, and Rearing numbers also reflects the changes of mouse movement and pain threshold from the side. After tamoxifen-induced abnormal activation of the β-catenin signaling pathway in β-cateninAct mice, we performed behavioral tests on the mice at different time points, as shown in Figure 1B and Figure 1C.
由图1B可以看出，小鼠交叉频率(即小鼠在鼠笼中来回穿梭的次数)显著低于Cre阴性对照组小鼠，这提示β-catenin过表达转基因小鼠由于更加疼痛从而减少了运动量。It can be seen from Figure 1B that the crossover frequency of mice (that is, the number of times the mice shuttled back and forth in the cage) was significantly lower than that of the Cre-negative control group mice, which suggested that the β-catenin overexpression transgenic mice were less painful due to more pain. The amount of exercise.
由图1C可以看出，小鼠站立次数(即小鼠在鼠笼中仅靠后肢着地的次数)显著低于Cre阴性对照组小鼠，这提示β-catenin过表达转基因小鼠由于更加疼痛从而降低了单纯靠后肢支撑身体的能力。It can be seen from Figure 1C that the number of times the mice stood (that is, the number of times the mice landed on the hind limbs in the cage) was significantly lower than that of the Cre-negative control group mice, which suggested that the β-catenin overexpression transgenic mice were more painful and thus Reduced ability to support the body solely on the hind legs.
实施例2Example 2
椎间孔狭窄导致的神经压迫是椎间盘退变引起肢体麻木、腰背部疼痛的重要原因。为了评价β-catenin信号通路异常激活对于小鼠椎间孔影响，我们通过μCT重建小鼠脊柱，结果如图2A所示。Nerve compression caused by intervertebral foraminal stenosis is an important cause of numbness and low back pain caused by intervertebral disc degeneration. In order to evaluate the effect of abnormal activation of the β-catenin signaling pathway on the intervertebral foramina of mice, we reconstructed the mouse spine by μCT, and the results are shown in Figure 2A.
由图2A可以看出，β-catenin过表达转基因小鼠椎间孔孔径减小，骨赘形成增多。这可能造成通过椎间孔的脊神经受到压迫，从而导致β-catenin过表达转基因小鼠痛觉异常。It can be seen from Figure 2A that the β-catenin overexpression transgenic mice decreased the diameter of the intervertebral foramen and increased the formation of osteophytes. This may cause compression of the spinal nerve passing through the intervertebral foramen, resulting in allodynia in β-catenin-overexpressing transgenic mice.
为了明确β-catenin信号通路异常激活对于小鼠椎间盘退变病理进程的影响，我们采用番红O固绿染色对β-cateninAct[即β-catenin(ex3)Col2ER鼠]小鼠椎间盘组织进行染色，得到图2B。In order to clarify the influence of abnormal activation of β-catenin signaling pathway on the pathological process of intervertebral disc degeneration in mice, we stained the intervertebral disc tissue of β-cateninAct [ie β-catenin(ex3)Col2ER mouse] mouse by Safranin O Fast Green staining. Figure 2B is obtained.
由图2B以看出，β-catenin过表达转基因小鼠椎间盘出现退变表型，软骨终板软骨层变薄，软骨下生长板软骨细胞凋亡。这提示β-catenin信号通路异常激活促进了小鼠椎间盘退变的病理进程。It can be seen from Figure 2B that the transgenic mice overexpressing β-catenin showed degenerative phenotypes of the intervertebral disc, the cartilage layer of the cartilage endplate became thinner, and the chondrocytes of the subchondral growth plate apoptotic. This suggests that the abnormal activation of β-catenin signaling pathway promotes the pathological process of mouse intervertebral disc degeneration.
实施例3Example 3
目前关于疼痛的机理并没有完全研究清楚，广泛认为是神经末梢受到各种伤害性刺激后，通过传导系统传入中枢，引起伤害性感受。而背根神经节(DRG)在腰背部疼痛信号传导中扮演重要角色，因此我们采用免疫荧光以及qPCR检测评价了β-cateninAct小鼠L4/L5腰椎附近背根神经节激活水平，得到图3A、图3B和图3C。At present, the mechanism of pain has not been fully studied. It is widely believed that after the nerve endings are subjected to various noxious stimuli, they are transmitted to the center through the conduction system, causing nociception. The dorsal root ganglion (DRG) plays an important role in low back pain signal transmission, so we used immunofluorescence and qPCR to evaluate the activation level of DRG near the L4/L5 lumbar spine of β-cateninAct mice, and obtained Figure 3A, Figure 3B and Figure 3C.
由图3A可以看出，β-catenin过表达转基因小鼠背根神经节PGP9.5与Tuj1的荧光信号显著强于Cre阴性对照组小鼠的背根神经节荧光信号。这提示了β-catenin过表达转基因小鼠背根神经节激活程度更高。It can be seen from Figure 3A that the fluorescence signals of PGP9.5 and Tuj1 in the dorsal root ganglia of the β-catenin overexpression transgenic mice were significantly stronger than those of the Cre negative control group mice. This suggests that β-catenin overexpression transgenic mice have a higher degree of activation of dorsal root ganglia.
由图3B可以看出，β-catenin过表达转基因小鼠背根神经节中Trpa1,Ngf,Calca,Scn9a基因mRNA表达水平显著高于Cre阴性对照组。这提示了β-catenin过表达转基因小鼠背根神经节中痛觉相关基因表达上升。It can be seen from Figure 3B that the mRNA expression levels of Trpa1, Ngf, Calca, and Scn9a genes in the dorsal root ganglia of the β-catenin overexpression transgenic mice were significantly higher than those in the Cre-negative control group. This suggested that the expression of pain-related genes in the dorsal root ganglia of β-catenin overexpression transgenic mice increased.
由图3C可以看出，β-catenin过表达转基因小鼠背根神经节中Il1b,Tnfa,Ccl2,Ccr2基因mRNA表达水平显著高于Cre阴性对照组。这提示了β-catenin过表达转基因小鼠背根神经节中炎症相关基因表达上升。It can be seen from Figure 3C that the mRNA expression levels of Il1b, Tnfa, Ccl2, and Ccr2 genes in the dorsal root ganglia of the β-catenin overexpression transgenic mice were significantly higher than those in the Cre-negative control group. This suggests that the expression of inflammation-related genes in the dorsal root ganglia of β-catenin overexpression transgenic mice is increased.
实施例4Example 4
为了进一步明确Wnt/β-catenin信号通路异常激活作用于腰背部疼痛的具体机制，我们利用BIO(一种β-catenin信号通路激动剂)处理C28/I2(人软骨细胞系)或大鼠椎间盘的原代纤维环(AF)细胞或髓核(NP)细胞进行体外实验，得到图4A、图4B、图4C、图4D、图4E、图4F和图4G。In order to further clarify the specific mechanism of abnormal activation of Wnt/β-catenin signaling pathway on low back pain, we used BIO (an agonist of β-catenin signaling pathway) to treat C28/I2 (human chondrocyte cell line) or rat intervertebral disc Primary annulus fibrosus (AF) cells or nucleus pulposus (NP) cells were subjected to in vitro experiments to obtain Figure 4A, Figure 4B, Figure 4C, Figure 4D, Figure 4E, Figure 4F and Figure 4G.
BIO可以抑制β-catenin的降解，从而导致β-catenin信号通路在细胞中异常积累。由图4A可以看出，随着BIO浓度升高，4小时后TCF7基因下游的AXIN2与DKK1表达水平随之升高。这表明BIO可以促进C28/I2人软骨细胞中TCF7激活。BIO can inhibit the degradation of β-catenin, which leads to abnormal accumulation of β-catenin signaling pathway in cells. It can be seen from Figure 4A that as the BIO concentration increased, the expression levels of AXIN2 and DKK1 downstream of the TCF7 gene increased after 4 hours. This suggests that BIO can promote TCF7 activation in C28/I2 human chondrocytes.
由图4B可以看出，BIO处理的C28/I2中β-catenin蛋白水平与CCL2蛋白水平相较于对照组均有所增加。It can be seen from Figure 4B that the protein levels of β-catenin and CCL2 in BIO-treated C28/I2 were increased compared with the control group.
由图4C和图4D可以看出，BIO与IL-1β处理的大鼠原代纤维环细胞以及髓核细胞中β-catenin蛋白水平相较于对照组均有所增加。It can be seen from Figure 4C and Figure 4D that the β-catenin protein levels in rat primary annulus fibrosus cells and nucleus pulposus cells treated with BIO and IL-1β were increased compared with the control group.
BIO可以抑制β-catenin的降解，从而导致β-catenin信号通路在细胞中异常积累，由图4E可以看出，随着BIO浓度升高，4小时后，C28/I2细胞中CCL2与CCR2表达水平随之升高。这表明BIO诱导的β-catenin信号通路异常激活可以促进C28/I2人软骨细胞中CCL2与CCR2表达升高。BIO can inhibit the degradation of β-catenin, resulting in the abnormal accumulation of β-catenin signaling pathway in cells. It can be seen from Figure 4E that with the increase of BIO concentration, the expression levels of CCL2 and CCR2 in C28/I2 cells after 4 hours It rises accordingly. This indicates that the abnormal activation of β-catenin signaling pathway induced by BIO can promote the expression of CCL2 and CCR2 in C28/I2 human chondrocytes.
由图4F可以看出，BIO处理C28/I2细胞后，CCL2的免疫荧光信号相较于DMSO处理的对照组显著上升。It can be seen from Figure 4F that after BIO treatment of C28/I2 cells, the immunofluorescence signal of CCL2 was significantly increased compared with the control group treated with DMSO.
由图4F和图4G可以看出，免疫荧光同样表明CCL2在BIO处理后显著上调。As can be seen from Figure 4F and Figure 4G, immunofluorescence also showed that CCL2 was significantly up-regulated after BIO treatment.
当前已有报道CCL2/CCR2信号通路参与了骨关节炎疼痛调控，然而CCL2在椎间盘源性疼痛中的作用却并不清楚，以上结果表明了BIO激活β-catenin信号通路后促进了CCL2/CCR2信号通路表达。It has been reported that the CCL2/CCR2 signaling pathway is involved in the regulation of osteoarthritis pain, but the role of CCL2 in discogenic pain is not clear. The above results indicate that BIO activates the β-catenin signaling pathway to promote CCL2/CCR2 signaling pathway expression.
实施例5Example 5
Wnt/β-catenin信号通路激活后通过下游转录因子TCF7进行转录，我们检测了TCF7上调后能否促进介导疼痛的CCL2表达，得到图5A、图5B、图5C、图5D、图5E和图5F。After the Wnt/β-catenin signaling pathway is activated, it is transcribed through the downstream transcription factor TCF7. We detected whether the up-regulation of TCF7 can promote the expression of CCL2 that mediates pain, and obtained Figure 5A, Figure 5B, Figure 5C, Figure 5D, Figure 5E and Figure 5 5F.
由图5A可以看出，pc-DNA3-TCF7质粒转染24小时后TCF7基因相较于空载质粒转染组(pcDNA3组)表达水平显著上调。这表明转染pc-DNA3-TCF7质粒可以有效上调软骨细胞中TCF7基因表达。It can be seen from FIG. 5A that the expression level of the TCF7 gene was significantly up-regulated 24 hours after transfection with the pc-DNA3-TCF7 plasmid compared with the empty plasmid transfection group (pcDNA3 group). This indicated that transfection of pc-DNA3-TCF7 plasmid could effectively up-regulate the expression of TCF7 gene in chondrocytes.
由图5B可以看出，可以看出pc-DNA3-TCF7质粒转染24小时后AXIN2与DKK1基因相较于空载质粒转染组(pcDNA3组)表达水平显著上调。这表明转染pc-DNA3-TCF7质粒可以有效上调软骨细胞中TCF7基因下游的AXIN2与DKK1基因表达。As can be seen from FIG. 5B , it can be seen that the expression levels of AXIN2 and DKK1 genes were significantly up-regulated 24 hours after transfection with the pc-DNA3-TCF7 plasmid compared with the empty plasmid transfection group (pcDNA3 group). This indicated that transfection of pc-DNA3-TCF7 plasmid could effectively up-regulate the expression of AXIN2 and DKK1 genes downstream of TCF7 gene in chondrocytes.
由图5C可以看出，免疫印迹结果显示TCF7转染的C28/I2细胞中TCF7蛋白水平与CCL2蛋白水平相较于对照组均有所增加。It can be seen from Figure 5C that the results of immunoblotting showed that the TCF7 protein level and the CCL2 protein level in the TCF7-transfected C28/I2 cells were increased compared with the control group.
由图5D可以看出，pc-DNA3-TCF7质粒转染24小时后CCL2与CCR2基 因相较于空载质粒转染组(pcDNA3组)表达水平显著上调。这表明转染pc-DNA3-TCF7质粒可以有效上调软骨细胞中TCF7基因下游的CCL2与CCR2基因表达。As can be seen from Figure 5D, the expression levels of CCL2 and CCR2 genes were significantly up-regulated 24 hours after pc-DNA3-TCF7 plasmid transfection compared with the empty plasmid transfection group (pcDNA3 group). This indicated that transfection of pc-DNA3-TCF7 plasmid could effectively up-regulate the expression of CCL2 and CCR2 genes downstream of TCF7 gene in chondrocytes.
由图5E可以看出，pc-DNA3-TCF7质粒转染C28/I2细胞后，CCL2的免疫荧光信号相较于空载质粒pcDNA3组显著上升。It can be seen from Figure 5E that after the pc-DNA3-TCF7 plasmid was transfected into C28/I2 cells, the immunofluorescence signal of CCL2 was significantly increased compared with the empty plasmid pcDNA3 group.
由图5E和图5F可以看出，免疫荧光同样表明CCL2在TCF7质粒转染后显著上调。As can be seen from Figure 5E and Figure 5F, immunofluorescence also showed that CCL2 was significantly up-regulated after TCF7 plasmid transfection.
以上结果表明β-catenin信号通路激活后可通过下游转录因子TCF7促进CCL2表达，从而促进腰背部疼痛进展。The above results indicate that the activation of β-catenin signaling pathway can promote the expression of CCL2 through the downstream transcription factor TCF7, thereby promoting the progression of low back pain.
实施例6Example 6
在明确了Wnt/β-catenin信号通路激活通过TCF7上调CCL2介导疼痛的作用机制后，我们在众多Wnt/β-catenin信号通路抑制剂中选择iCRT14进行进一步实验。iCRT14可以阻断β-catenin与TCF的相互作用从而抑制β-catenin信号通路的异常激活。我们对棘上棘间韧带切除术后小鼠进行治疗，溶剂玉米油作为阴性对照组，经典抗炎药塞莱希布(Celebrex)作为阳性对照组，得到图6A、图6B、图6C、图6D、图6E、图6F和图6G。After clarifying the mechanism of activation of Wnt/β-catenin signaling pathway through TCF7 up-regulation of CCL2 to mediate pain, we selected iCRT14 among many Wnt/β-catenin signaling pathway inhibitors for further experiments. iCRT14 can block the interaction between β-catenin and TCF to inhibit the abnormal activation of β-catenin signaling pathway. We treated the mice after resection of the supraspinous interspinous ligament, solvent corn oil was used as the negative control group, and the classic anti-inflammatory drug Celebrex was used as the positive control group. Figure 6A, Figure 6B, Figure 6C, Figure 6 6D, 6E, 6F and 6G.
由图6A可以看出，LSI术后小鼠疼痛阈值显著降低，而注射Celebrex与iCRT14注射组疼痛程度在术后10周得到缓解，术后13周时iCRT14注射组展现出了相较于Celebrex更好的疼痛缓解作用。It can be seen from Figure 6A that the pain threshold of the mice after LSI was significantly reduced, and the pain degree of the Celebrex and iCRT14 injection group was relieved at 10 weeks after the operation, and the iCRT14 injection group showed more pain than Celebrex at 13 weeks after the operation. Good pain relief.
iCRT14可以抑制β-catenin与下游的TCF7结合，从而抑制β-catenin信号通路传导，由图6B和图6C可以看出，随着iCRT14浓度升高，12小时后，炎症因子IL1β预处理的C28/I2细胞中AXIN2与DKK1表达水平随之降低。这表明iCRT4可以抑制因炎症异常激活的β-catenin信号通路，从而抑制TCF7下游的AXIN2与DKK1表达升高。由图6D和图6E可以看出，CCL2与CCR2基因表达水平也在iCRT14处理后有所下调。iCRT14 can inhibit the combination of β-catenin and downstream TCF7, thereby inhibiting the transduction of β-catenin signaling pathway. It can be seen from Figure 6B and Figure 6C that, with the increase of iCRT14 concentration, after 12 hours, the inflammatory factor IL1β pretreated C28/ The expression levels of AXIN2 and DKK1 in I2 cells decreased accordingly. This indicates that iCRT4 can inhibit the abnormally activated β-catenin signaling pathway due to inflammation, thereby inhibiting the increased expression of AXIN2 and DKK1 downstream of TCF7. It can be seen from Figure 6D and Figure 6E that the expression levels of CCL2 and CCR2 genes were also down-regulated after iCRT14 treatment.
由图6F可以看出，iCRT14处理C28/I2细胞后，CCL2的免疫荧光信号相较于DMSO对照处理组显著上升。It can be seen from Figure 6F that after iCRT14 treatment of C28/I2 cells, the immunofluorescence signal of CCL2 was significantly increased compared with the DMSO control treatment group.
由图6F和图6G可以看出，荧光结果同样显示了iCRT14可以显著抑制CCL2的表达。It can be seen from Figure 6F and Figure 6G that the fluorescence results also show that iCRT14 can significantly inhibit the expression of CCL2.
以上结果显示了iCRT14对于椎间盘源性疼痛有良好疗效。The above results show that iCRT14 has a good effect on discogenic pain.
以上所述实施方式仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。The above-mentioned embodiments only express several embodiments of the present invention, and the descriptions thereof are relatively specific and detailed, but should not be construed as limiting the patent scope of the present invention. It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims.

Claims (9)

  1. 一种抑制剂在制备治疗椎间盘源性疼痛的药物中的应用，其特征在于，所述抑制剂为β-catenin蛋白与TCF7转录因子结合抑制剂和/或Wnt/β-catenin信号通路抑制剂。An application of an inhibitor in the preparation of a medicament for treating discogenic pain, characterized in that the inhibitor is an inhibitor of the combination of β-catenin protein and TCF7 transcription factor and/or an inhibitor of Wnt/β-catenin signaling pathway.
  2. 根据权利要求1所述的应用，其特征在于，所述抑制剂为具有如下结构式的化合物iCRT14、其立体异构体、其药学上可接受的盐、其溶剂化物或其前药：The application according to claim 1, wherein the inhibitor is a compound iCRT14 having the following structural formula, its stereoisomer, its pharmaceutically acceptable salt, its solvate or its prodrug:
    Figure PCTCN2021136555-appb-100001
    Figure PCTCN2021136555-appb-100001
  3. 根据权利要求2所述的应用，其特征在于，所述化合物iCRT14的有效作用浓度为20μM～100μM。The use according to claim 2, characterized in that the effective concentration of the compound iCRT14 is 20 μM-100 μM.
  4. 根据权利要求3所述的应用，其特征在于，所述化合物iCRT14的有效作用浓度为50μM。The application according to claim 3, characterized in that the effective concentration of the compound iCRT14 is 50 μM.
  5. 一种药物组合物，其特征在于，所述药物组合物包括β-catenin蛋白与TCF7转录因子结合抑制剂和/或Wnt/β-catenin信号通路抑制剂。A pharmaceutical composition, characterized in that the pharmaceutical composition includes an inhibitor of the combination of β-catenin protein and TCF7 transcription factor and/or a Wnt/β-catenin signaling pathway inhibitor.
  6. 根据权利要求5所述的药物组合物，其特征在于，所述药物组合物包括具有如下结构式的化合物iCRT14、其立体异构体、其药学上可接受的盐、其溶剂化物或其前药：The pharmaceutical composition according to claim 5, wherein the pharmaceutical composition comprises the compound iCRT14 having the following structural formula, its stereoisomer, its pharmaceutically acceptable salt, its solvate or its prodrug:
    Figure PCTCN2021136555-appb-100002
    Figure PCTCN2021136555-appb-100002
  7. 根据权利要求6所述的药物组合物，其特征在于，所述化合物iCRT14的有效作用浓度为20μM～100μM。The pharmaceutical composition according to claim 6, wherein the effective concentration of the compound iCRT14 is 20 μM-100 μM.
  8. 根据权利要求7所述的药物组合物，其特征在于，所述化合物iCRT14 的有效作用浓度为50μM。The pharmaceutical composition according to claim 7, wherein the effective concentration of the compound iCRT14 is 50 μM.
  9. 根据权利要求5～8中任意一项所述的药物组合物，其特征在于，所述药物组合物为椎间盘源性疼痛的治疗药物组合物。The pharmaceutical composition according to any one of claims 5-8, characterized in that the pharmaceutical composition is a therapeutic pharmaceutical composition for discogenic pain.
PCT/CN2021/136555 2021-11-30 2021-12-08 Application of inhibitor and pharmaceutical composition WO2023097721A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202111446782.8A CN114259554A (en) 2021-11-30 2021-11-30 Application of inhibitor and pharmaceutical composition
CN202111446782.8 2021-11-30

Publications (1)

Publication Number Publication Date
WO2023097721A1 true WO2023097721A1 (en) 2023-06-08

Family

ID=80826125

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/136555 WO2023097721A1 (en) 2021-11-30 2021-12-08 Application of inhibitor and pharmaceutical composition

Country Status (2)

Country Link
CN (1) CN114259554A (en)
WO (1) WO2023097721A1 (en)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009097113A2 (en) * 2008-01-28 2009-08-06 New York University Oxazole and thiazole compounds as b-catenin modulators and uses thereof
US20120264744A1 (en) * 2008-01-28 2012-10-18 Ramanuj Dasgupta Oxazole and thiazole compounds as beta-catenin modulators and uses thereof
WO2017044302A1 (en) * 2015-09-09 2017-03-16 Rush University Medical Center Compositions and methods for treating bone diseases
WO2019157085A2 (en) * 2018-02-06 2019-08-15 Vanderbilt University INHIBITION OF WNT/β-CATENIN SIGNALING IN THE TREATMENT OF OSTEOARTHRITIS
CN111073979A (en) * 2019-12-31 2020-04-28 上海交通大学 Gastric cancer treatment method for blocking CCL28 chemotactic pathway

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009097113A2 (en) * 2008-01-28 2009-08-06 New York University Oxazole and thiazole compounds as b-catenin modulators and uses thereof
US20120264744A1 (en) * 2008-01-28 2012-10-18 Ramanuj Dasgupta Oxazole and thiazole compounds as beta-catenin modulators and uses thereof
WO2017044302A1 (en) * 2015-09-09 2017-03-16 Rush University Medical Center Compositions and methods for treating bone diseases
WO2019157085A2 (en) * 2018-02-06 2019-08-15 Vanderbilt University INHIBITION OF WNT/β-CATENIN SIGNALING IN THE TREATMENT OF OSTEOARTHRITIS
CN111073979A (en) * 2019-12-31 2020-04-28 上海交通大学 Gastric cancer treatment method for blocking CCL28 chemotactic pathway

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
"Doctoral Dissertation", 1 March 2018, LIAONING UNIVERSITY OF TRADITIONAL CHINESE MEDICINE, CN, article XIE, WANQING: "Analysis of Molecular Mechanism of "Dumeridian-Kideny-Bone" in Lumbar Degenerative Osteoarthropathy from Wnt/β-catenin Signaling Pathway", pages: 1 - 108, XP009546072 *
XIE HUANXIN, JING YONGBIN, XIA JINGJUN, WANG XINTAO, YOU CHANGCHENG, YAN JINGLONG: "Aquaporin 3 protects against lumbar intervertebral disc degeneration via the Wnt/β-catenin pathway", INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE, SPANDIDOS PUBLICATIONS, GR, vol. 37, no. 3, 27 March 2016 (2016-03-27), GR , pages 859 - 864, XP093069842, ISSN: 1107-3756, DOI: 10.3892/ijmm.2016.2470 *
XINGCHAO SHEN, XIA BINGJIANG, LING YILONG, WEI JINZHONG, XU YANG: "A Clinical Study of Wufu Yin Jiajian for Treatment of Discogenic Back Pain", THE JOURNAL OF TRADITIONAL CHINESE ORTHOPEDICS AND TRAUMATOLOGY, vol. 32, no. 2, 29 February 2020 (2020-02-29), pages 23 - 29, XP093069836 *
ZHOU YA-QUN, TIAN XUE-BI, TIAN YU-KE, MEI WEI, LIU DAI-QIANG, YE DA-WEI: "Wnt signaling: A prospective therapeutic target for chronic pain", PHARMACOLOGY & THERAPEUTICS, ELSEVIER., GB, vol. 231, 1 March 2022 (2022-03-01), GB , pages 107984, XP093069832, ISSN: 0163-7258, DOI: 10.1016/j.pharmthera.2021.107984 *

Also Published As

Publication number Publication date
CN114259554A (en) 2022-04-01

Similar Documents

Publication Publication Date Title
Bajpayee et al. Sustained intra-cartilage delivery of low dose dexamethasone using a cationic carrier for treatment of post traumatic osteoarthritis
Gaub et al. The histone acetyltransferase p300 promotes intrinsic axonal regeneration
JP4913072B2 (en) Methods and compositions for treating, inhibiting and ameliorating intervertebral disc disease
Hu et al. Optimization of puncture injury to rat caudal disc for mimicking early degeneration of intervertebral disc
Nevo et al. Fibrosis inhibition and muscle histopathology improvement in laminin‐α2‐deficient mice
Park et al. Chronic low back pain: a mini-review on pharmacological management and pathophysiological insights from clinical and pre-clinical data
Sanchez et al. Targeting the β2-integrin LFA-1, reduces adverse neuroimmune actions in neuropathic susceptibility caused by prenatal alcohol exposure
Stover et al. Multiplex epigenome editing of dorsal root ganglion neuron receptors abolishes redundant interleukin 6, tumor necrosis factor alpha, and interleukin 1β signaling by the degenerative intervertebral disc
Zheng et al. LncRNA MALAT1 exhibits positive effects on nucleus pulposus cell biology in vivo and in vitro by sponging miR-503
WO2023097721A1 (en) Application of inhibitor and pharmaceutical composition
US11077110B2 (en) Compositions and methods for treating and preventing metabolic disorders
Jhang et al. Pathogenesis evidence from human and animal models of detrusor underactivity
Sun et al. Therapeutic evaluation of acupoint stimulation with needle-scapelon on rat model of degenerative cervical intervertebral discs
Kidd et al. How does neuropathophysiology affect the signs and symptoms of spinal disease?
Fan et al. Senescent-like macrophages mediate angiogenesis for endplate sclerosis via IL-10 secretion in male mice
Shapiro et al. A Randomized Trial of Instrumented Posterior Lumbar Interbody Fusion Using Machined Cortical Wedges/Local Bone with or without rhBMP2 in the Treatment of Degenerative Lumbar Spondylolisthesis with Stenosis: 809
da Costa et al. A Single-Center, Prospective Analysis of the Natural History of Hemorrhage from Brain Arteriovenous Malformations with or without Associated Aneurysms: 805
Okun et al. Management of referred deep brain stimulation failures: A retrospective analysis from two movement disorder centers: 818
Diringer et al. Effects of Recombinant Activated Factor VII on Perilesional Edema in Patients with Acute Intracerebral Hemorrhage: 801
Ghogawala et al. Spinal Experts’ Review Significantly Increases Patient Consent to Randomization in the SLIP Study: An Ongoing Prospective, Multicenter Surgical Trial: 810
Spears et al. Current Trends in Management of Ruptured Intracranial Aneurysms in Toronto after the International Subarachnoid Trial (ISAT): 804
McCall et al. Neurosurgery Work-hour Restrictions: Impact on Resident Training: 832
Williams et al. Neuronal Correlates of Associative Learning in the Primate Striatum and Its Selective Enhancement by Electrical Stimulation: 819
Tseng et al. Statins Ameliorate Cerebral Vasospasm, Maintain Autoregulation, and Reduce Ischemic Deficits and Mortality after Aneurysmal Subarachnoid Hemorrhage: A Randomized Controlled Trial: 800
Tomlin et al. Revitalizing the Resident Clinic: Developing Clinical Competencies through Continuity of Care: 837

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21966147

Country of ref document: EP

Kind code of ref document: A1