Use of repatinib in the preparation of a medicament for preventing, alleviating or treating chronic pain

Abstract

The present application relates to a new use of repatinib or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing, alleviating or treating chronic pain, and belongs to the technical field of biological medicine. Research has found that repatinib can activate alpha7 nicotinic acetylcholine receptor (alpha7 nAChR), increase the Ca + level and beta-endorphin content in the hypothalamic arcuate nucleus neurons, promote the functional reconstruction of the endogenous opioid analgesic system, and significantly reduce the mechanical pain sensitivity in the trigeminal nerve injury model; and the analgesic effect can be blocked by methyllycaconitine, an alpha7 nAChR specific antagonist, confirming its target dependence. In addition, repatinib shows significant analgesic effect in various chronic pain models such as sciatica and migraine, showing broad-spectrum potential for chronic pain. The present application opens up a new medical use for repatinib and provides a new strategy for the treatment of chronic pain.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the novel use of ripretinib (DCC-2618) or a pharmaceutically acceptable salt thereof in the preparation of medicaments for the prevention, relief or treatment of chronic pain, and the use of ripretinib as an α7 nicotinic acetylcholine receptor activator. Background Technology

[0002] Chronic pain is a common clinical condition that seriously affects patients' physical and mental health. It includes trigeminal neuralgia, sciatica, neuropathic pain, inflammatory pain, migraine, and tumor pain. This type of pain is characterized by recurrent attacks and persistent nature, and clinically manifests in various forms such as mechanical anomalous pain, mechanical hyperalgesia, thermal hyperalgesia, spontaneous pain, persistent pain, or recurrent headaches. Its etiology is complex, often caused by peripheral or central nervous system damage, inflammatory responses, trauma, surgery, metabolic diseases, tumors, drug effects, or other pathological processes.

[0003] Currently, drugs used clinically to treat chronic pain mainly include gabapentin, antidepressants, nonsteroidal anti-inflammatory drugs (NSAIDs), triptans, calcitonin gene-related peptide pathway drugs, local anesthetics, and opioids. However, the mechanisms of pathogenesis and drug sensitivity vary significantly among different types of chronic pain. Existing drugs are usually only effective for some patients or certain types of pain, and long-term use may cause problems such as sedation, gastrointestinal adverse reactions, cardiovascular risks, tolerance, dependence, and addiction. Therefore, the development of novel analgesics that can be widely used for various types of chronic pain is of significant clinical importance and urgently needed.

[0004] The arcuate nucleus (ARC) of the hypothalamus is a core component of the endogenous opioid analgesic system, rich in neurons expressing pro-melanocortin (POMC) and β-endorphin. POMC is sequentially processed by prohormone-converting enzymes to generate β-lipotropin and β-endorphin; this processing is regulated by intracellular calcium... 2+ Regulation of levels. α7 nicotinic acetylcholine receptor (α7 nAChR) is a receptor with high Ca2+ levels. 2+ Permeable homopentamer-gated cation channels, whose activation can increase intracellular Ca²⁺ in neurons. + The level of α7 nAChR in ARC may thus be involved in the regulation of POMC processing and β-endorphin production. Previous studies have reported that the expression level of α7 nAChR in ARC is significantly reduced in neuropathic pain states, but the causal relationship between it and the occurrence and development of chronic pain is still unclear, and no drug intervention strategies targeting this target have been reported.

[0005] Ripretinib (DCC-2618) is a known kinase on / off control inhibitor, currently primarily used to inhibit KIT and PDGFRA-related tumor signaling for the treatment of advanced gastrointestinal stromal tumors. To date, there are no reports of ripretinib being used for the prevention or treatment of chronic pain, nor are there any reports of its analgesic effects in different pain states such as neuropathic pain, inflammatory pain, and migraine. Summary of the Invention

[0006] Addressing the technical challenges of limited efficacy in existing chronic pain treatments, the lack of common and usable pharmacological targets for different pain types, significant adverse reactions with long-term use, and the absence of small molecule drugs capable of restoring the function of the endogenous β-endorphin system, this invention provides a novel use of retrapinib as an α7 nicotinic acetylcholine receptor activator in the preparation of drugs for the prevention, relief, or treatment of chronic pain.

[0007] This invention targets α7 nAChR, using a virtual screening of an FDA-approved drug library, and validated through animal experiments. It was found that targeted injection of retrapinib into the ARC receptor significantly activates this receptor, effectively reversing the analgesic response in model rats. In various chronic pain animal models, including trigeminal nerve injury, sciatica, migraine, and inflammatory pain models, retrapinib demonstrated significant analgesic effects, suggesting its broad-spectrum anti-chronic pain potential. Mechanistic studies further indicate that retrapinib increases intracellular calcium in ARC neurons by activating α7 nAChR. 2+ This increases the level of POMC, thereby promoting the proteolytic processing and β-endorphin production, thus exerting an endogenous analgesic effect. This invention provides new strategies and drug candidates for the development and clinical treatment of drugs for neuropathic pain, inflammatory pain, migraines, and other chronic pain.

[0008] Specifically, the following technical solutions are provided: The first aspect of this invention provides the use of retrapinib or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention, relief or treatment of chronic pain, said retrapinib having the following structure: .

[0009] In this invention, the retrapinib is DCC-2618, CAS number 1442472-39-0, and molecular formula C. 24 H 21 BrFN5O2.

[0010] Furthermore, the chronic pain is pain that persists or recurs for longer than the normal tissue healing time, or pain that persists or recurs for at least 3 months.

[0011] Furthermore, the chronic pain includes, but is not limited to, one or more of neuropathic pain, inflammatory pain, migraine, and tumor pain, wherein the neuropathic pain includes one or more of trigeminal neuralgia, sciatica, and spinal nerve injury-related pain.

[0012] Furthermore, the retrinib is used to relieve at least one of the following symptoms: mechanical aberrant pain, mechanical hyperalgesia, thermal hyperalgesia, periorbital hyperalgesia, spontaneous pain, persistent pain, or headache.

[0013] Furthermore, retrinib increases intracellular Ca²⁺ levels by activating α7 nicotinic acetylcholine receptors in arcuate nucleus neurons of the hypothalamus. + The signal promotes the proteolytic processing of pro-okratin and the production of β-endorphin, thereby exerting an analgesic effect.

[0014] Furthermore, the drug comprises 0.1%-100% ripatinib by weight, with the remainder being pharmaceutically acceptable excipients, such as carriers, diluents, excipients, stabilizers, solubilizers, buffers, preservatives, surfactants, liposomes, nanocarriers, or centrally targeted delivery materials.

[0015] Furthermore, the drug can be formulated into a preparation suitable for oral, nasal, intravenous, subcutaneous, intramuscular, intraperitoneal, intrathecal, intraventricular, or intracerebral administration, and the preparation can be a tablet, capsule, granule, oral liquid, injection, nasal preparation, liposome preparation, nanopreparation, implant, sustained-release preparation, or delivery preparation targeting the central nervous system.

[0016] Furthermore, the route of administration of the drug can be selected based on drug brain exposure, pain type, disease condition, and formulation properties, and the specific effective therapeutic dose can be determined based on the route of administration, test subjects, pain model, pharmacokinetics, and safety data.

[0017] A second aspect of the invention provides the use of ripatinib or a pharmaceutically acceptable salt thereof in the preparation of α7 nicotinic acetylcholine receptor activators.

[0018] Furthermore, the retrinib interacts with at least one amino acid residue from Gln139, Ser170, Trp171, Tyr210, or Cys211 in the α7 nicotinic acetylcholine receptor ligand binding pocket.

[0019] Furthermore, the α7 nicotinic acetylcholine receptor activator is used to increase intracellular calcium levels. 2+ Levels and / or β-endorphin levels.

[0020] Furthermore, the α7 nicotinic acetylcholine receptor activator is used to prevent, relieve, or treat chronic pain; the chronic pain includes, but is not limited to, one or more of neuropathic pain, inflammatory pain, migraine, and tumor pain, and the neuropathic pain includes, or more of trigeminal neuralgia, sciatica, spinal nerve injury-related pain, and postherpetic neuralgia.

[0021] Furthermore, the α7 nicotinic acetylcholine receptor activator comprises 0.1%-100% retrapinib by mass, with the remainder being pharmaceutically acceptable excipients, such as carriers, diluents, excipients, stabilizers, solubilizers, buffers, preservatives, surfactants, liposomes, nanocarriers, or centrally targeted delivery materials.

[0022] The present invention has the following beneficial effects: This invention is the first to discover that retrinib can act as an α7 nicotinic acetylcholine receptor (α7 nAChR) activator, by restoring Ca2+ in neurons of the arcuate nucleus of the hypothalamus. 2+ This invention promotes the hydrolysis and processing of eosinogen and the production of β-endorphin, thereby remodeling the function of the endogenous opioid analgesic system. It has shown significant analgesic effects in various chronic pain models, including neuropathic pain (trigeminal neuralgia, sciatica), migraine, and inflammatory pain, demonstrating broad-spectrum anti-chronic pain potential. Its target is clearly defined and receptor-dependent; the α7 nAChR-specific antagonist methyl taurine can completely block its analgesic effect. Molecular docking further confirms that retrapinib can directly interact with key amino acid residues in the α7 nAChR ligand binding pocket. This invention breaks through the traditional understanding of retrapinib as a kinase inhibitor, providing a novel drug strategy for chronic pain treatment with definite efficacy and a clear target, effectively overcoming the clinical shortcomings of existing drugs, such as large differences in efficacy for different types of pain and the tendency to develop tolerance and dependence with long-term use. Attached Figure Description

[0023] Figure 1 The study presented differences in acetylcholine receptor expression in rats with chronic infraorbital nerve compression injury (CCI-ION). A represents the mechanical pain threshold in CCI-ION rats; B shows high-throughput sequencing results of acetylcholine receptor gene transcripts highly expressed in the ARC tissues of rats in the sham-operated and CCI-ION groups; and C represents the differences in expression levels of three acetylcholine receptor gene transcripts in cerebrospinal fluid samples from healthy subjects and patients with trigeminal neuralgia. Figure 2 The expression of α7 nAChR in ARC tissues of CCI-ION model rats; where A represents the expression of α7 nAChR in ARC tissues of sham-operated and CCI-ION group rats detected by RT-qPCR. Chrna7Expression comparison; B is a schematic diagram of α7 nAChR protein expression in ARC tissues of rats in the sham-operated group and CCI-ION group as detected by Western blotting; C is the quantitative analysis of α7 nAChR protein as detected by Western blotting. Figure 3 Overexpression or activation of α7 nAChR within the ARC can significantly reverse the mechanosensitive pain response in CCI-ION model rats. Specifically, A represents the pain-therapeutic effect of intra-ARC injection of a lentivirus (lenti-hSyn-Chrna7-up, Chrna7-up) carrying a neuron-specific expression element overexpressing α7 nAChR in CCI-ION model rats; B represents the pain-preventive effect of pre-intra-ARC injection of Chrna7-up in CCI-ION model rats; and C represents the pain-therapeutic effect of intra-ARC injection of the α7 nAChR-specific agonist PNU-282987 in CCI-ION model rats. Figure 4 The potential α7 nAChR agonist ripretinib significantly reversed the mechanical analgesia in CCI-ION model rats. A shows a flowchart of the screening process for potential α7 nAChR agonists from the FDA-approved drug library; B shows the chemical structure of the screened drug ripretinib; C shows that ARC-targeted injection of ripretinib significantly alleviated the mechanical analgesia in CCI-ION rats; and D shows that the analgesic effect mediated by ripretinib can be reversed by the α7 nAChR-specific inhibitor methyllycaconitine (MLA). Figure 5 Repatinib can increase the levels of calcium ions and β-endorphin in ARC cells; where A represents the level of calcium ions in ARC cells after CCI-ION modeling; B represents the reversal of the decrease in intracellular calcium ion levels in ARC cells caused by CCI-ION surgery by retapinib; C represents the level of β-endorphin in ARC cells after CCI-ION modeling; D represents the reversal of the decrease in intracellular β-endorphin levels in ARC cells caused by CCI-ION surgery by retapinib; E is a schematic diagram of the docking of retapinib with the α7 nAChR ligand binding domain. Figure 6 Repatinib significantly reversed changes in mechanical pain sensitivity in other animal models; among them, A shows that intra-ARC injection of retapinib significantly reversed changes in mechanical pain sensitivity in SNI model rats; B shows that intraperitoneal injection of retapinib significantly reversed changes in mechanical pain sensitivity in migraine model mice. Detailed Implementation

[0024] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "comprising" or "including" described in the present invention can also be replaced with the closed-form "being" or "consisting of".

[0026] For the experimental methods involved in the following examples, unless otherwise specified, they are all conventional methods, and data analysis adopts conventional statistical analysis methods; where specific techniques or conditions are not indicated in the examples, the procedures are carried out according to the techniques or conditions described in literatures in the art or according to product specifications; the reagents and materials, unless otherwise specified, can all be obtained from commercial channels or prepared by known methods, and the animal experiments comply with the relevant requirements of Soochow University.

[0027] Example 1: Differential expression of acetylcholine receptors in rats with chronic constriction injury of the infraorbital nerve (CCI-ION) model (1) Establishment of chronic constriction injury of the infraorbital nerve (CCI-ION) model Adult male Sprague-Dawley (SD) rats weighing approximately 200 grams were used, provided by the Laboratory Animal Center of Soochow University, with the license number SYXK (Su) 2021-0074. Feeding conditions: 5 rats per cage, free access to food and water, room temperature 22-25°C, relative humidity 50-60%, low-noise barrier environment, and a 12-hour light / 12-hour dark cyclic photoperiod. Experimental animals need to acclimate for at least 3 days in the standard environment before subsequent experiments. Rats were anesthetized by intraperitoneal injection of 4% chloral hydrate. After the rats were completely anesthetized, they were fixed in the supine position. A small incision was made horizontally at the position of the left maxillary first molar with a sterile scalpel, and the surrounding tissues were blunt dissected with a sterile curved forceps to clearly expose the infraorbital nerve. In the trigeminal neuralgia model (CCI-ION) group, two ligations were performed with medical suturing silk at both ends of the nerve, with an interval of approximately 2 millimeters, and excessive force was avoided. Subsequently, the blood was wiped off, and sodium penicillin was applied to the wound to prevent infection. In the sham operation (sham) group, after the infraorbital nerve was exposed, no ligation was performed, and the wound was treated with the same method.

[0028] (2) Determination of mechanical pain threshold in the vibrissal pad of rats This experiment was conducted under double-blind conditions. The minimum stimulus intensity started from 1 gram, and the left barbel pad of the rat was stimulated with a von Frey needle. Each stimulation caused the von Frey needle to bend to 90°, with a 5-second interval between each stimulation, and at least five consecutive stimulations were performed. A positive response (X) was defined as at least three instances of avoidance or scratching behavior in the five stimulations, at which point the stimulus intensity was reduced. Otherwise, a negative response (O) was recorded, and the stimulus intensity was gradually increased. Recording began from the first alternation of negative / positive responses, followed by four more measurements and recordings. The mechanical pain threshold was calculated by substituting the response patterns of the six measurements into the formula. The calculation formula is: 50% pain threshold (gram) = (10 [Xf+Kδ] ) / 10000. Where Xf represents the intensity of the last mechanical stimulus, and K·δ represents the coefficient corresponding to different response modes.

[0029] (3) High-throughput mRNA sequencing RNA-seq technology services were provided by Guangzhou Ruibo Biotechnology. Hypothalamic ARC tissues were collected from rats in the sham and CCI-ION groups, and blood was removed by rinsing with PBS. The tissues were then flash-frozen in liquid nitrogen to ensure the stability and integrity of the RNA in the samples. RNA was extracted using the RNeasy Mini Kit, and DNase I was added to the adsorption column to reduce DNA contamination. Sequencing libraries were constructed using the Illumina TruSeq RNA Sample Prep Kit with a total RNA content of 1 μg. Through fragmentation, end repair, and adapter ligation, cDNA fragments with sequencing adapters were generated. High-throughput sequencing was performed on an Illumina HiSeq3000.

[0030] (4) RNA extraction and real-time quantitative PCR RNA was extracted from cerebrospinal fluid or tissue using the Trizol method. The extracted RNA was reverse transcribed into cDNA using a 5 × PrimeScript RT Master Mix with the following parameters: reverse transcription temperature 42°C for 15 minutes; denaturation temperature 85°C for 2 minutes. Subsequently, quantitative real-time PCR was performed with the following cycling parameters: pre-denaturation temperature 95°C for 15 minutes; denaturation temperature 94°C for 15 seconds; annealing temperature 60°C for 30 seconds; extension temperature 72°C for 30 seconds. The number of cycles was 40. Experimental results are presented as follows: -ΔΔCT Calculations were performed. Trizol was purchased from Takara, the reverse transcription kit was purchased from Takara, and the SYBR fluorescent dye was purchased from Bimake. The relevant transcript-specific primers were synthesized by Ribobio, and the primer sequences are as follows:

[0031] like Figure 1As shown in Figure A, 16 SD rats were divided into two groups of 8 each. The first group was the sham-operated group, and the second group was the CCI-ION group. Behavioral results showed that the mechanical pain threshold of the sham-operated group rats did not change significantly at any time point. The mechanical pain threshold of the beard pad in the CCI-ION group rats decreased significantly from day 14 and could be maintained until day 28. p <0.001 vs. (Sham surgery group). For example... Figure 1 As shown in Figure B, six rats from both the sham-operated group and the CCI-ION model group were selected on day 14, with two rats per sample, for a total of three samples per group. ARC tissue samples were collected for high-throughput sequencing. Nineteen acetylcholine receptor subunit transcripts with high expression abundance were identified in the ARC tissue of CCI-ION group rats. Among these, three transcripts showed significantly different expression levels, including… Chrna3 , Chrna7 and Chrna9 .like Figure 1 As shown in C, the results of quantitative real-time PCR showed that, compared with the cerebrospinal fluid of healthy subjects, the cerebrospinal fluid of patients with trigeminal neuralgia contained... Chrna7 Significantly decreased expression ( p <0.01 vs. (Healthy subject group).

[0032] Example 2: Expression of α7 nAChR in ARC tissues of CCI-ION model rats (1) Establishment of the chronic compression injury model of infraorbital nerve (CCI-ION): The specific operation is the same as in Example 1.

[0033] (2) RNA extraction and real-time quantitative PCR: The specific operation is the same as in Example 1, and the primer sequences are as follows:

[0034] (3) Western blot: ARC tissue samples were added to RIPA lysis buffer, mechanically ground, lysed on ice, and sonicated. After centrifugation at 12500 × g for 20 min at 4 ℃, the supernatant was collected and the protein concentration was determined by BCA method. After unifying the protein concentration of each sample, 4× loading buffer was added, heated at 95 ℃ for 3 min for denaturation, and stored at -80 ℃. Equal amounts of protein were taken for SDS-PAGE. Electrophoresis was first performed at a constant voltage of 80 V for about 1 h, and then adjusted to 120 V until the protein was fully separated. After trimming the separating gel, the protein was transferred to a methanol-activated PVDF membrane at a constant current of 300 mA for about 1 h. The transfer process was carried out in an ice-water bath. After the transfer was completed, the membrane was washed with TBST, blocked with 5% skim milk powder at room temperature for 1 h, and then incubated with primary antibody at 4 ℃ for 14-16 h. The primary antibody dilution ratio was generally 1:1500. After washing, the corresponding HRP-conjugated secondary antibody was added, diluted at 1:3000, and incubated at room temperature for about 1 h. After a second cleaning, the sample was developed using ECL chemiluminescent solution, and images were acquired using a chemiluminescence imaging system. The grayscale values ​​of the target bands were analyzed using Clinx ImageAnalysis software. GAPDH was used as an internal control.

[0035] like Figure 2 As shown in Figure A, RNA was extracted from the ARC tissues of rats at 7, 14, 21, and 28 days of gestation, with three rats in each group at each time point. Quantitative real-time PCR results showed that, compared to the sham-operated group, RNA was significantly lower in the CCI-ION group after 14 days in the ARC tissues of rats. Chrna7 mRNA expression was significantly reduced and could persist until day 28 post-surgery. p <0.001 vs. (Sham surgery group). For example... Figure 2 As shown in B and C, total protein was extracted from rat ARC tissues at 7, 14, 21, and 28 days post-operation, with 5 rats in each group at each time point. Western blot analysis showed that, compared to the sham-operated group, the expression of α7 nAChR in rat ARC was significantly reduced in the CCI-ION group after 14 days, and this reduction persisted until day 28 post-operation. p <0.001 vs. (Sham surgery group).

[0036] Example 3: Overexpression or activation of α7 nAChR in ARC can significantly reverse the mechanical pain sensitivity in CCI-ION model rats. (1) Establishment of the chronic compression injury model of infraorbital nerve (CCI-ION): The specific operation is the same as in Example 1.

[0037] (2) Measurement of mechanical pain threshold in rat whiskers: The specific operation is the same as in Example 1.

[0038] (3) Stereoscopic injection of ARC in rats: After anesthetizing the rats, fix them on a stereotaxic instrument. Make an incision in the scalp with a scalpel to expose the Bregma and Lambda points of the skull. Using the Bregma point as the origin, the bilateral ARC positioning coordinates are: anteroposterior AP -4.0 mm, medial-lateral ML ±0.5 mm, and dorsoventral DV -9.8 mm. Administer the drug slowly over 5 minutes. After administration, leave the needle in place for 15 minutes before removing it. If the rats exhibit abnormal symptoms such as mania or depression after regaining consciousness, they should be culled. Inject α7 nAChR overexpressing lentivirus (lenti-hSyn-Chrna7-up, Chrna7-up) and its negative control empty vector lentivirus (lenti-hSyn-negative control-up, NC-up), or the α7 nAChR specific agonist PNU-282987 and its control dimethyl sulfoxide (DMSO). The lentivirus titer should be greater than 1×10⁻⁶. 9 TU / ml, PNU-282987 concentration of 0.1 mM, 0.5 μl injected bilaterally into the ARC.

[0039] like Figure 3 As shown in A, 40 normal rats were divided into 4 groups of 10 each. Group 1 was a sham surgery + NC-up injection group; Group 2 was a sham surgery + Chrna7-up injection group; Group 3 was a CCI-ION + NC-up injection group; and Group 4 was a CCI-ION + Chrna7-up injection group. In the CCI-ION group, the mechanical pain threshold significantly increased from day 3 to day 10 after Chrna7-up injection. p <0.001 vs. Sham surgery + NC-up group; ## p <0.01, ### p <0.001 vs. CCI-ION + NC-up group). This indicates that overexpression of α7 nAChR within the ARC can significantly alleviate the pain behavior response induced by the CCI-ION model in rats.

[0040] like Figure 3As shown in Figure B, to elucidate the preventive effect of α7 nAChR overexpression on pain, we pre-injected lentivirally packaged Chrna7-up, and induced CCI-ION three days later. Pain was then measured on days 7, 14, and 21. Twenty-eight rats were divided into four groups of seven each. Group 1 was the NC-up injection + sham-operated group, Group 2 was the Chrna7-up injection + sham-operated group, Group 3 was the NC-up injection + CCI-ION group, and Group 4 was the Chrna7-up injection + CCI-ION group. From 14 to 21 days post-CCI-ION surgery, the mechanical pain threshold in the face of rats pre-injected with Chrna7-up was significantly higher than that of rats pre-injected with NC-up. p <0.001 vs. NC-up + sham surgery group; ### p <0.001 vs. NC-up + CCI-ION group).

[0041] like Figure 3 As shown in C, 32 normal rats were divided into 4 groups of 8 rats each. Group 1 was a sham surgery + DMSO injection group; Group 2 was a sham surgery + PNU-282987 injection group; Group 3 was a CCI-ION + DMSO injection group; and Group 4 was a CCI-ION + PNU-282987 injection group. In the CCI-ION group, rats injected with the α7 nAChR specific agonist PNU-282987 showed a significant increase in mechanical pain threshold 1-3 hours after injection. p <0.001 vs. Sham surgery + DMSO group; # p <0.05, ### p <0.001 vs. CCI-ION + DMSO group).

[0042] The above results indicate that overexpression of α7 nAChR within the ARC can significantly alleviate or prevent pain behavior responses induced by the CCI-ION model in rats. Specific activation of α7 nAChR can also significantly increase the mechanical pain threshold in CCI-ION rats, suggesting that α7 nAChR is an effective target for the treatment and prevention of trigeminal neuralgia.

[0043] Example 4: Repaitinib, a potential α7 nAChR agonist, significantly reversed the mechanical pain hypersensitivity response in CCI-ION model rats. (1) To screen small molecule activators of α7 nicotinic acetylcholine receptor (α7 nAChR) with clinical translational potential, this invention employs a virtual screening strategy based on receptor structure to screen an FDA-approved drug library. The three-dimensional structure of α7 nAChR is obtained from publicly available structure databases or through homology modeling using AlphaFold, and preprocessed using the Protein Preparation Wizard module in Schrödinger software, including removing water molecules, adding missing hydrogen atoms, repairing abnormal side chains, and distributing protonated states and partial charges based on the OPLS2005 force field. The ligand binding pocket is determined based on the reported cryo-electron microscopy structures of human α7 nAChR bound to selective agonists. Subsequently, FDA-approved compounds are processed using the LigPrep module, and possible ionization states, tautomers, and stereoisomers are generated using Epik at pH 7.0 ± 2.0. Molecular docking was performed using Glide's ultra-high precision (XP) mode, and compounds were ranked based on docking score, predicted binding conformation, and their ability to form stable interactions with key amino acid residues in the α7 nAChR binding pocket. Ultimately, compounds with favorable docking conformations, established clinical safety profiles, and the potential to regulate α7 nAChR-mediated Ca2+ binding were prioritized. 2+ The signaling candidate drugs will be used for subsequent experimental validation.

[0044] (2) Establishment of the chronic compression injury model of infraorbital nerve (CCI-ION): The specific operation is the same as in Example 1.

[0045] (3) Measurement of mechanical pain threshold of rat whiskers: The specific operation is the same as in Example 1.

[0046] (4) Stereotactic injection of ARC in rats: The specific operation was the same as in Example 3. CCI-ION rats were stereotactically injected with ripretinib (DCC-2618) and the control drug (placebo) at a concentration of 0.5 mM. 0.5 μL was injected into both ARCs over 5 minutes and the needles were left in place for 15 minutes. The α7 nAChR specific antagonist methyllycaconitine (3 mg / kg) and the control drug (physiological saline) were injected intraperitoneally.

[0047] like Figure 4 As shown in A, this invention screened a library of 2588 small molecule compounds approved by the U.S. Food and Drug Administration (FDA). Based on their ability to activate α7 nAChR, a promising candidate drug—ripretinib (DCC-2618)—was initially identified according to the screening process. Its chemical structure is shown below. Figure 4 As shown in B in the diagram.

[0048] like Figure 4 As shown in Figure C, 21 rats were divided into three groups of seven each: the CCI-ION group, the CCI-ION + control drug group, and the CCI-ION + retipinib group. The drug was administered on day 14 post-CCI-ION surgery. Results showed that intra-ARC injection of retipinib significantly alleviated mechanical pain in the rat face. p <0.001 vs. CCI-ION + control drug group).

[0049] like Figure 4 As shown in D, 24 CCI-ION rats were divided into 4 groups of 6 rats each: control drug + saline, control drug + methyl taurine, retiprinib + saline, and retiprinib + methyl taurine. The drugs were injected on day 14 post-CCI-ION surgery. Results showed that intra-ARC injection of retiprinib significantly relieved facial mechanical pain in rats, and this effect was blocked by methyl taurine. p <0.01, p <0.001 vs. Control group (drug + saline); ## p <0.01, ### p <0.001 vs. (Repatinib + saline group). These results indicate that retapinib achieves its analgesic effect by specifically targeting and activating α7 nAChR.

[0050] Example 5: Repatinib can increase the levels of calcium ions and β-endorphin in ARC cells. (1) Establishment of the chronic compression injury model of infraorbital nerve (CCI-ION): The specific operation is the same as in Example 1.

[0051] (2) Stereoscopic injection of rat ARC: The specific operation is the same as in Example 4.

[0052] (3) Intracellular Ca 2+Determination of calcium levels: 30 mg of ARC tissue was washed twice with pre-cooled PBS, resuspended in 500 μL of calcium detection buffer, and homogenized thoroughly by sonication. The homogenate was then centrifuged at 4 °C, 15,000 × g for 5 min, and the supernatant was collected. The calcium concentration in the supernatant was determined according to the instructions of the Abcam calcium ion detection kit. 2+ Content, to assess intracellular Ca in ARC tissue cells 2+ level.

[0053] (4) ELISA assays were performed according to the kit instructions. The rat β-endorphin (β-EP) ELISA kit was purchased from BBILife Sciences. 50 μL of ARC tissue homogenate sample or β-EP standards of different concentrations were added to the corresponding wells and incubated for 45 min. After washing the plate, biotin-labeled β-EP antibody working solution, HRP-labeled streptavidin working solution, and TMB chromogenic substrate were added sequentially, and each step was incubated for 30 min. Finally, 2 M HCl was added to stop the reaction, and the absorbance of each well was measured at 450 nm using a TD-20 / 20 detector (Turner BioSystems, USA).

[0054] (5) The three-dimensional structure of α7 nAChR was predicted using AlphaFold, and preprocessed using AutoDockTools 1.5.7, including the removal of water molecules, the addition of polar hydrogen atoms, and charge distribution. After energy optimization, a docking grid was established with the known ligand binding region of α7 nAChR as the center, and molecular docking was performed using AutoDock Vina. The optimal docking mode was screened based on the binding energy and binding conformation, and the interaction between retrapinib and key amino acid residues of α7 nAChR was predicted. Finally, PyMOL was used for visualization, with α7 nAChR displayed as a cartoon model and retrapinib and its binding site displayed as a rod-like structure.

[0055] like Figure 5 As shown in A, after CCI-ION surgery, Ca in ARC 2+ The level decreased significantly ( p <0.05 vs. (Sham surgery group). For example... Figure 5 As shown in B, in CCI-ION rats, after targeted injection of retrapinib into the ARC, the intra-ARC calcium level... 2+ The level has rebounded significantly. p <0.05 vs. CCI-ION + control drug group).

[0056] like Figure 5As shown in C, after CCI-ION surgery, the β-EP content in the ARC decreased significantly ( p <0.01 vs. (Sham surgery group). For example... Figure 5 As shown in D, in CCI-ION rats, the β-EP content in the ARC significantly increased after targeted injection of retrapinib into the ARC. p <0.05 vs. CCI-ION + control drug group).

[0057] like Figure 5 As shown in E, molecular docking revealed the predicted binding of retrapinib to α7 nAChR, with retrapinib located near key residues such as Gln139, Ser170, Trp171, Cys211, and Tyr210.

[0058] The above results indicate that ripatinib can restore intracellular calcium ion levels and β-endorphin synthesis by specifically targeting and activating α7 nAChR.

[0059] Example 6: Repatinib can significantly reverse changes in mechanoreparinib in other animal models. (1) Establishment of a rat model of sciatica: A rat model of sciatica was established using the classic selective sciatic nerve injury (SNI). The sciatic nerve was exposed by cutting through the biceps femoris muscle. The tibial nerve and common peroneal nerve were then ligated and cut separately with 5-0 silk sutures, while the sural nerve was left intact. At the end of the operation, the muscle and skin were sutured in two layers with 5-0 silk sutures, and lidocaine gel was then applied to the wound. For sham surgery, the sciatic nerve was exposed in the same manner as above, without ligation or cutting of the nerve.

[0060] (2) Plantar mechanical pain detection: The sole of the foot is stimulated with von Frey fibers for 1-2 seconds each time. The minimum intensity at which a positive response is obtained is recorded as the mechanical withdrawal threshold (PWT).

[0061] (3) Stereoscopic injection of rat ARC: The specific operation is the same as in Example 4.

[0062] (4) Establishment of a mouse NTG chronic migraine model: Mice were placed in a behavioral testing environment for 72 hours before the experiment. Before each administration, a 10% nitroglycerin (NTG) stock solution dissolved in propylene glycol was freshly diluted with physiological saline to prepare a working solution containing 1% propylene glycol. Mice in the model group were intraperitoneally injected with NTG at a dose of 10 mg / kg, with an injection volume of 10 mL / kg, once every 2 days for a total of 5 injections; the control group was intraperitoneally injected with physiological saline containing 1% propylene glycol at the same time and volume. On each day of behavioral testing, the pain threshold was measured first, followed by the injection of NTG or the solvent.

[0063] (5) Periorbital mechanical pain detection: The periorbital region of mice was stimulated with von Frey fibers for 1–2 seconds each time. The minimum intensity at which a positive response was obtained was taken as the periorbital mechanical pain threshold.

[0064] (6) Intraperitoneal injection in mice: Repatinib was dissolved in the appropriate solvent at the predetermined dose, and the volume of administration was calculated based on the weight of the mice. After gently fixing the mice, an intraperitoneal injection was performed in the lower quadrant of the abdomen; the control group was injected with an equal volume of solvent in the same manner.

[0065] like Figure 6 As shown in A, 24 normal rats were divided into 4 groups of 6 rats each. Group 1 was a sham surgery + control drug injection group; Group 2 was a sham surgery + retrapinib injection group; Group 3 was a SNI + control drug injection group; and Group 4 was a SNI + retrapinib injection group. In the SNI model group, rats injected with retrapinib showed a significant recovery of the mechanical pain threshold, which could persist for up to 2 hours post-injection. p <0.001 vs. sham surgery + control drug group; ## p <0.001, ### p <0.001 vs. SNI + control drug group).

[0066] like Figure 6 As shown in B, 24 normal mice were divided into 4 groups of 6 mice each. Group 1 was a sham surgery + control drug injection group; Group 2 was a sham surgery + retrapinib injection group; Group 3 was an NTG + control drug injection group; and Group 4 was an NTG + retrapinib injection group. In the NTG model group, the mechanical pain threshold significantly increased after retrapinib injection and could persist for up to 3 hours post-injection. p <0.001 vs. sham surgery + injection of control drug group, ### p<0.001 vs. NTG + control drug injection group).

[0067] The above results indicate that retrinib can exhibit significant analgesic effects in other pain model animals, such as the SNI model and the NTG migraine model.

[0068] This invention first used high-throughput sequencing, real-time quantitative PCR, and Western blotting to investigate the expression changes of the acetylcholine receptor family in the arcuate nucleus (ARC) of the hypothalamus in CCI-ION model rats, and then validated this in the cerebrospinal fluid of patients with trigeminal neuralgia (Examples 1 and 2). The results showed that after CCI-ION modeling, the expression of acetylcholine receptors in the ARC tissue... Chrna7 Both mRNA and α7 nicotinic acetylcholine receptor (α7 nAChR) protein expression were significantly reduced, and the cerebrospinal fluid of trigeminal neuralgia patients showed... Chrna7 The expression level also decreased significantly, suggesting that α7 nAChR may be an important target for chronic pain intervention. Based on this, this invention demonstrated, through ARC-targeted injection of α7 nAChR-overexpressing lentivirus or its specific agonist PNU-282987 (Example 3), that overexpression or pharmacological activation of α7 nAChR can significantly increase the mechanical pain threshold in CCI-ION model rats and produce therapeutic or preventative effects on pain behavior caused by trigeminal nerve injury, further confirming the analgesic function of this target.

[0069] Furthermore, this invention used α7 nAChR as a target to conduct virtual screening of an FDA-approved drug library, and verified this through animal experiments. For the first time, it was discovered that retrapinib can significantly alleviate facial mechanical pain hypersensitivity in CCI-ION model rats; this analgesic effect can be completely blocked by methyl taurine, a selective α7 nAChR antagonist, indicating that retrapinib mainly exerts its analgesic effect by activating α7 nAChR (Example 4). Further mechanistic studies showed that retrapinib can restore calcium levels in ARC tissue induced by CCI-ION modeling. 2+ The levels and β-endorphin content were reduced; and molecular docking results showed that retrapinib could interact with key amino acid residues such as Gln139, Ser170, Trp171, Tyr210 and Cys211 in the α7 nAChR ligand binding region, supporting its direct regulation of α7 nAChR at the molecular level (Example 5).

[0070] Furthermore, this invention further investigated the effects of retrapinib on rats with SNI-induced sciatic nerve injury-induced pain and mice with nitroglycerin-induced chronic migraine (Example 6). The results showed that ARC injection of retrapinib significantly increased the plantar mechanical pain threshold in SNI-model rats, and intraperitoneal injection of retrapinib significantly improved periorbital mechanical pain sensitivity in migraine-model mice, indicating that retrapinib has significant analgesic effects on different types of chronic pain and possesses broad-spectrum anti-chronic pain potential.

[0071] In summary, this invention is the first to discover that retrinib can increase intracellular calcium levels by activating α7 nAChR. 2+ This drug promotes the processing of pro-retropine and the production of β-endorphins, thereby significantly alleviating mechanical hyperalgesia associated with chronic pain. Repatinib or a pharmaceutically acceptable salt thereof can be used to prepare medicines for the prevention, relief, or treatment of chronic pain, including but not limited to trigeminal neuralgia, sciatica, spinal nerve injury-related pain, neuropathic pain, inflammatory pain, migraine, and tumor pain.

[0072] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. Use of retrapinib or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention, relief or treatment of chronic pain, wherein the structure of retrapinib is shown below: 。 2. The use according to claim 1, characterized in that, The chronic pain is defined as pain that is persistent or recurrent for at least 3 months.

3. The use according to claim 1 or 2, characterized in that, The chronic pain is selected from one or more of neuropathic pain, inflammatory pain, migraine, and tumor pain; The neuropathic pain includes one or more of the following: trigeminal neuralgia, sciatica, spinal nerve injury-related pain, and postherpetic neuralgia.

4. The use according to claim 1 or 2, characterized in that, The aforementioned retrapinib is used to relieve at least one of the following symptoms: mechanical aberrant pain, mechanical hyperalgesia, thermal hyperalgesia, periorbital hyperalgesia, spontaneous pain, persistent pain, or headache. And / or, the retrapinib increases intracellular Ca2+ levels in hypothalamic arcuate nucleus neurons by activating α7 nicotinic acetylcholine receptors. 2+ It promotes the proteolytic processing of pro-okratin and the production of β-endorphin, thereby exerting an analgesic effect.

5. The use according to claim 1, characterized in that, The drug is formulated for oral, nasal, intravenous, subcutaneous, intramuscular, intraperitoneal, intrathecal, intraventricular, or intracerebral administration.

6. The use according to claim 5, characterized in that, The formulation is a tablet, capsule, granule, oral liquid, injection, nasal formulation, liposome formulation, nanoformulation, implant, sustained-release formulation, or delivery formulation targeting the central nervous system.

7. Use of ripatinib or a pharmaceutically acceptable salt thereof in the preparation of α7 nicotinic acetylcholine receptor activators.

8. The use according to claim 7, characterized in that, The retrapinib interacts with at least one amino acid residue from Gln139, Ser170, Trp171, Tyr210, or Cys211 in the α7 nicotinic acetylcholine receptor ligand binding pocket.

9. The use according to claim 7, characterized in that, The α7 nicotinic acetylcholine receptor activator is used to increase intracellular Ca2+. 2+ Levels and / or β-endorphin levels.

10. The use according to claim 7 or 9, characterized in that, The α7 nicotinic acetylcholine receptor activator is used to prevent, relieve or treat chronic pain; the chronic pain is selected from one or more of neuropathic pain, inflammatory pain, migraine and tumor pain, and the neuropathic pain includes one or more of trigeminal neuralgia, sciatica and spinal nerve injury-related pain.

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