Use of wnt5a-ryk signaling pathway inhibitor in assisting opioid analgesia and pharmaceutical composition

Abstract

The application discloses a use of a Wnt5a-Ryk signal path inhibitor in assisting opioid analgesia and a pharmaceutical composition. The application provides a new use of a Wnt5a / Ryk signal path inhibitor in preparing a medicine for enhancing the analgesic effect of an opioid, delaying or preventing analgesic tolerance of the opioid and relieving pain-induced sensitization of the opioid, wherein the Wnt5a-Ryk signal path inhibitor is an active substance capable of blocking the combination of Wnt5a and a Ryk receptor, and the Wnt5a-Ryk signal path inhibitor is used in combination with the opioid. A pharmaceutical composition containing the Wnt5a-Ryk signal path inhibitor, the opioid and a pharmaceutically acceptable carrier is developed. The pharmaceutical composition of the application can safely and effectively enhance the analgesic effect of the opioid, delay or prevent analgesic tolerance and relieve pain-induced sensitization, and has important clinical significance and market value.

Description

Technical Field

[0001] This application relates to the field of adjunctive analgesia technology, and in particular to the use of a Wnt5a-Ryk signaling pathway inhibitor in adjunctive opioid analgesia and a pharmaceutical composition thereof. Background Technology

[0002] Pain is one of the most common clinical symptoms, severely impacting patients' quality of life. μ-opioid receptor (MOR) agonists, represented by morphine, are currently the gold standard for treating moderate to severe pain (especially cancer pain). In the World Health Organization's (WHO) three-step cancer pain management ladder, opioids are the first-line and foundational medications for treating severe cancer pain.

[0003] However, the long-term clinical application of opioids is severely constrained by three core issues: (1) analgesic tolerance, meaning that after long-term and repeated use, the analgesic effect of the same dose of opioids is significantly reduced, and clinical treatment has to continuously increase the dosage; (2) opioid-induced hyperalgesia (OIH), meaning that long-term use of opioids leads to an abnormally increased sensitivity of patients to painful stimuli, manifested as a decreased pain threshold and abnormal pain; and (3) addiction and withdrawal reactions. Among these, analgesic tolerance and hyperalgesia often occur simultaneously and promote each other, forming a vicious cycle, which leads to a decline in the clinical efficacy of opioids and an increase in adverse reactions, severely limiting their long-term use in the treatment of chronic pain.

[0004] Current intervention strategies for opioid tolerance and OIH mainly include adjuvant medications such as NMDA receptor antagonists (e.g., ketamine), COX-2 inhibitors, and calcium channel modulators. However, these strategies have limitations in efficacy and significant side effects, which greatly restrict their long-term clinical use. Therefore, finding novel adjuvant drugs that can safely and effectively enhance the analgesic effect of opioids, delay the onset of tolerance, and alleviate OIH has significant clinical importance and market value.

[0005] Existing research indicates that Wnt5a, as a non-canonical Wnt ligand, is upregulated in neuropathic pain, inflammatory pain, and various chronic pain models. Wnt5a mediates downstream signaling through its receptor Ryk (receptor-like tyrosine kinase). Studies have found that chronic morphine treatment upregulates Wnt5a expression in the dorsal horn of the spinal cord, and the Wnt5a signaling pathway is involved in morphine-induced astrocyte activation and the development of oligopyelitis-induced hypoglycemia (OIH). However, there are currently no reports of combining Wnt5a signaling pathway inhibitors with opioids to enhance analgesic effects, alleviate tolerance, and reduce pain sensitization. Summary of the Invention

[0006] The purpose of this application is to provide a novel use of a Wnt5a-Ryk signaling pathway inhibitor in adjuvant opioid analgesia, and a pharmaceutical composition thereof.

[0007] To achieve the above objectives, this application adopts the following technical solution:

[0008] This application discloses the use of a Wnt5a-Ryk signaling pathway inhibitor in the preparation of a drug that enhances the analgesic effect of opioids. The Wnt5a-Ryk signaling pathway inhibitor is an active substance that can block the binding of Wnt5a to Ryk receptors. The Wnt5a-Ryk signaling pathway inhibitor is used in combination with opioids.

[0009] It should be noted that Wnt5a binds to the extracellular domain of the receptor Ryk, prompting the intracellular domain of Ryk to recruit GRK2 / 3 and β-arrestin1 / 2, thereby promoting phosphorylation and receptor endocytosis of the μ-opioid receptor (MOR) at the Ser375 site, thus inhibiting the Gi / o-mediated G protein analgesic signaling pathway of MOR. The study in this application found that the Wnt5a / Ryk signaling pathway inhibitor can relieve the above-mentioned inhibition of MOR G protein signaling and enhance the analgesic effect of opioids by blocking the binding of Wnt5a to Ryk.

[0010] In this application, the Wnt5a-Ryk signaling pathway inhibitor is at least one of Box-5, Wnt5a neutralizing antibody, Ryk-Fc fusion protein, Wnt5a signaling pathway inhibitory small molecule compound, Ryk antagonistic antibody, Ryk siRNA, Ryk shRNA, and Ryk gene knockout reagent.

[0011] It should be noted that Box-5, as a competitive antagonist of Wnt5a, specifically blocks the binding of Wnt5a to the extracellular domain of Ryk by competitively binding to the receptor-binding domain of Wnt5a. This reduces Ryk's recruitment of GRK / β-arrestin, decreases MOR phosphorylation and endocytosis levels, and protects the MOR G protein signaling pathway. It is understandable that other Wnt5a signaling pathway inhibitors or Ryk-targeting inhibitors can achieve similar or identical effects to Box-5, namely, enhancing the analgesic effect of opioids.

[0012] In this application, the opioid is at least one of morphine, fentanyl, sufentanil, remifentanil, oxycodone, hydromorphone, and methadone.

[0013] It should be noted that the above-mentioned opioids are all μ-opioid receptor agonists, which produce analgesic effects by activating the MOR. When used in combination with Wnt5a / Ryk signaling pathway inhibitors, they can synergistically enhance the analgesic effect.

[0014] This application also discloses the use of a Wnt5a-Ryk signaling pathway inhibitor in the preparation of a drug for delaying or preventing opioid analgesia tolerance. The Wnt5a-Ryk signaling pathway inhibitor is an active substance capable of blocking the binding of Wnt5a to Ryk receptors, and is used in combination with an opioid. The limitations of the Wnt5a-Ryk signaling pathway inhibitor and the opioid are as described above.

[0015] It should be noted that chronic opioid exposure can upregulate the expression of Wnt5a and Ryk in the dorsal horn of the spinal cord, forming a positive feedback loop, which further exacerbates the desensitization and endocytosis of MOR and drives the development of analgesia tolerance. Inhibitors of the Wnt5a / Ryk signaling pathway can delay or prevent the formation of the above-mentioned analgesia tolerance by blocking this positive feedback loop, thereby delaying or preventing opioid analgesia tolerance.

[0016] This application also discloses the use of a Wnt5a-Ryk signaling pathway inhibitor in the preparation of a drug to alleviate opioid-induced pain sensitization. This Wnt5a-Ryk signaling pathway inhibitor is an active substance capable of blocking the binding of Wnt5a to Ryk receptors, and is used in combination with opioids. The limitations of the Wnt5a-Ryk signaling pathway inhibitor and the opioids are as described above.

[0017] It should be noted that opioid-induced sensitization (OIH) is closely related to the upregulation of the Wnt5a / Ryk signaling pathway. Inhibitors of the Wnt5a / Ryk signaling pathway can alleviate the reduction in the baseline pain threshold caused by OIH by blocking this pathway, thereby alleviating opioid-induced sensitization.

[0018] This application also discloses a pharmaceutical composition comprising a Wnt5a-Ryk signaling pathway inhibitor, an opioid, and a pharmaceutically acceptable carrier, wherein the Wnt5a-Ryk signaling pathway inhibitor is an active substance capable of blocking the binding of Wnt5a to the Ryk receptor. The Wnt5a-Ryk signaling pathway inhibitor and the opioid are as defined above.

[0019] It should be noted that the pharmaceutical composition of this application combines an inhibitor of the Wnt5a / Ryk signaling pathway with an opioid to facilitate simultaneous clinical administration. This not only achieves a synergistic enhancement of analgesia but also delays or prevents opioid analgesia tolerance and alleviates opioid-induced pain sensitization.

[0020] It should also be noted that the pharmaceutical composition of this application can refer to existing opioid administration routes, including but not limited to intrathecal, epidural, subcutaneous, intravenous, oral (with formulation modification), and transdermal routes. Its dosage form can also be designed as a compound injection, compound oral preparation, etc., according to needs. The application scenarios of the pharmaceutical composition of this application include, but are not limited to, various types of pain such as neuropathic pain, inflammatory pain, cancer pain, and postoperative pain. It is understood that the pharmaceutical composition of this application combines a Wnt5a / Ryk signaling pathway inhibitor with an opioid to facilitate simultaneous clinical administration; however, the key to this application lies in the research finding that the Wnt5a / Ryk signaling pathway inhibitor can enhance the analgesic effect of opioids, delay or prevent opioid analgesia tolerance, and alleviate opioid-induced pain sensitization. Generally, the Wnt5a / Ryk signaling pathway inhibitor can be administered before, simultaneously with, or after opioid administration, or it can be administered prophylactically before long-term use of opioids.

[0021] Due to the adoption of the above technical solutions, the beneficial effects of this application are as follows:

[0022] This application provides a novel use of Wnt5a / Ryk signaling pathway inhibitors in the preparation of drugs that enhance the analgesic effect of opioids, delay or prevent opioid analgesia tolerance, and alleviate opioid-induced pain sensitization. These drugs can safely and effectively enhance the analgesic effect of opioids, delay or prevent analgesia tolerance, and alleviate pain sensitization, and have significant clinical significance and market value. Attached Figure Description

[0023] Figure 1 The chemical structural formula of Box-5 in the embodiments of this application is shown below;

[0024] Figure 2 This is a comparative schematic diagram of the percentage of analgesia (MPE%) and area under the curve (AUC) of intrathecal Box-5 and control group under intrathecal or subcutaneous morphine injection conditions using the tail flick and hot plate methods in the embodiments of this application; wherein, Figure A shows the results of intrathecal injection and Figure B shows the results of subcutaneous injection.

[0025] Figure 3 This is a schematic diagram comparing the mechanical retraction threshold and thermal retraction latency of intrathecal Box-5 injection and the control group after intrathecal morphine injection in the bone cancer pain model (TCI 14d) of this application.

[0026] Figure 4 This is a schematic diagram comparing the mechanical retraction threshold and thermal retraction latency of the intrathecal Box-5 injection and the control group after intrathecal morphine injection in the neuropathic pain model (PSNL 14d) of this application.

[0027] Figure 5 This is a schematic diagram comparing the percentage of analgesia (MPE%) and the change in baseline latency with the number of days of administration in the continuous subcutaneous morphine injection model in this application, using the daily intrathecal Box-5 injection and the control group, as well as the tail-flick and hot plate methods.

[0028] Figure 6 This refers to the detection results of MOR phosphorylation level in the embodiments of this application;

[0029] Figure 7 This is the result of MOR internalization detection in the embodiments of this application;

[0030] Figure 8 This is the in vitro verification result of Box-5 blocking Wnt5a / Ryk binding in the embodiments of this application. Detailed Implementation

[0031] To address the technical challenges of the lack of safe and effective interventions for analgesic tolerance and opioid-induced hyperalgesia (OIH) caused by long-term use of opioids, and the significant side effects and limited efficacy of existing adjuvant drugs (such as NMDA receptor antagonists).

[0032] This application presents an inventive study that reveals that Wnt5a / Ryk signaling pathway inhibitors can enhance the analgesic effect of opioids, delay or prevent opioid analgesia tolerance, and alleviate opioid-induced pain sensitization. Therefore, this application proposes a novel use of Wnt5a / Ryk signaling pathway inhibitors in the preparation of drugs that enhance the analgesic effect of opioids, delay or prevent opioid analgesia tolerance, or alleviate opioid-induced pain sensitization. Based on this, a novel pharmaceutical composition has been developed comprising a Wnt5a-Ryk signaling pathway inhibitor, an opioid, and a pharmaceutically acceptable carrier.

[0033] The novel use and pharmaceutical composition of this application have the following advantages compared with the prior art:

[0034] (1) This application establishes the Wnt5a / Ryk signaling axis as the endogenous negative regulatory target of the MOR G protein signaling pathway for the first time, and proposes an intervention strategy to block the binding of Wnt5a to the Ryk receptor by inhibiting the Wnt5a / Ryk signaling pathway. By blocking this signaling axis, the recruitment of GRK2 / 3 and β-arrestin1 / 2 by the intracellular Ryk domain can be reduced, and the phosphorylation level and receptor internalization of MOR at the Ser375 site can be reduced, thereby protecting the Gi / o-mediated analgesic signaling pathway. In normal physiological state, bone cancer pain model (TCI 14d) and neuropathic pain model (PSNL 14d), the acute analgesic effect of morphine can be significantly enhanced (MPE% and AUC significantly increased in tail flick and hot plate tests, Two-way ANOVA, P<0.01, n=8~10), overcoming the defects of existing adjuvant drugs (NMDA receptor antagonists, etc.) due to target nonspecificity leading to significant side effects.

[0035] (2) This application can simultaneously alleviate analgesia tolerance (significantly slowed down the decrease in MPE%) and pain sensitization (significantly slowed down the decrease in the baseline pain threshold) by blocking the positive feedback loop of Wnt5a / Ryk expression upregulation caused by chronic opioid exposure. This has been experimentally verified in a tolerance model of subcutaneous morphine injection for 7 consecutive days (Two-way ANOVA, P<0.05~0.01, n=8). This achieves the dual benefit of solving the two major clinical problems of analgesia tolerance and OIH with a single intervention, making up for the limitation of existing technologies that can only intervene in one aspect.

[0036] (3) Box-5, as a specific competitive antagonist of Wnt5a, has high target selectivity. When administered alone, it does not produce analgesic effect within the effective dose range of 5-20 ng / animal (intrathecal) and does not affect the basic pain threshold, indicating that it has no obvious non-target pharmacological activity and has good safety. It can play an auxiliary analgesic and synergistic role without interfering with the body's normal pain regulation. Its safety is significantly improved compared with existing NMDA receptor antagonists and other auxiliary drugs.

[0037] In this application, the term "Wnt5a / Ryk signaling pathway inhibitor" refers to an active substance that can block the binding of Wnt5a ligands to the extracellular domain of the receptor Ryk, including but not limited to Wnt5a antagonistic peptides (such as Box-5), Wnt5a neutralizing antibodies, Ryk antagonistic antibodies, and Ryk-Fc fusion proteins.

[0038] The term "μ-opioid receptor agonist" refers to drugs that can activate μ-opioid receptors (MOR) to produce analgesic effects, including morphine, fentanyl, sufentanil, remifentanil, oxycodone, hydromorphone, and methadone.

[0039] In the following examples, Box-5 is a cyclic polypeptide containing the Met-Asp-Gly-Cys-Glu-Leu sequence, protected by an N-terminus tert-butyloxycarbonyl (Boc) group, with a molecular weight of approximately 1.7 kDa, and its chemical structure is as follows: Figure 1 As shown, it is soluble in physiological saline or phosphate-buffered saline (PBS). Morphine sulfate injection is a commercially available product. All experimental animals used were 6-8 week old male C57BL / 6 mice, weighing 20-25 g. All animal experiments were approved by the institution's animal ethics committee.

[0040] The mechanism of action of Box-5 is as follows: After Wnt5a binds to the extracellular domain of the receptor Ryk, Ryk recruits GRK2 / 3 and β-arrestin1 / 2 through its intracellular domain, promoting phosphorylation and receptor endocytosis of MOR at the Ser375 site, thereby inhibiting the Gi / o-mediated G protein analgesia signaling pathway.

[0041] Box-5, as a competitive antagonist of Wnt5a, specifically blocks the binding of Wnt5a to the extracellular domain of Ryk by competitively binding to the receptor-binding domain of Wnt5a. This reduces Ryk's recruitment of GRK / β-arrestin, decreases the phosphorylation and endocytosis levels of MOR, thereby protecting the G protein signaling pathway of MOR and enhancing the analgesic effect of opioids.

[0042] Chronic opioid exposure can upregulate the expression of Wnt5a and Ryk in the dorsal horn of the spinal cord, forming a positive feedback loop that further exacerbates MOR desensitization and endocytosis, driving the development of analgesia tolerance and pain sensitization. Box-5 can delay or prevent the formation of analgesia tolerance and alleviate the reduction in the baseline pain threshold caused by pain sensitization by blocking this positive feedback loop.

[0043] The present application will be further described in detail below through specific embodiments. These embodiments are merely illustrative and should not be construed as limiting the present application. In the following embodiments, many details are described to facilitate a better understanding of the present application. However, those skilled in the art will readily recognize that some features may be omitted in different situations, or may be replaced by other devices, materials, or methods. In some cases, certain operations related to the present application are not shown or described in the specification to avoid obscuring the core parts of the present application with excessive description. For those skilled in the art, detailed description of these related operations is not necessary; a complete understanding of the related operations can be obtained from the description in the specification and general technical knowledge in the art.

[0044] Example 1

[0045] This embodiment verifies that intrathecal injection of Box-5 (MCE, Cat. No.: HY-123071, CAS No.: 1206604-29-6) enhances the acute analgesic effect of intrathecal or subcutaneous morphine in normal mice. Experimental results are shown below. Figure 2 .

[0046] Normal male C57BL / 6 mice were randomly divided into two groups: a control group (it Vehicle group) and an experimental group (it 20 ng Box-5 group), with n=8 mice in each group. The experimental group mice were given Box-5 solution (20 ng / mouse, dissolved in 5 μL of physiological saline) via intrathecal injection 30 min before morphine administration; the control group mice were given an equal volume of physiological saline.

[0047] Dosing regimen A: 30 min after Box-5 pretreatment, administer intrathecal morphine (2 nmol / animal, dissolved in 5 μL of physiological saline). Dosing regimen B: 30 min after Box-5 pretreatment, administer subcutaneous morphine (10 mg / kg).

[0048] The analgesic effect was assessed using the tail-flick test: Mice were placed in a restraint device, and the distal end of their tails was vertically immersed in 50°C hot water to start timing. A positive reaction was considered when the mouse flicked its tail or moved its tail out of the water. The tail-flick latency (cutoff time 15 seconds) was recorded. Each mouse was tested 3 times at 3-minute intervals, and the average value was taken.

[0049] The analgesic effect was evaluated using the hot plate test: Mice were placed inside a transparent cover on a 55°C hot plate, and the timer was started. A positive reaction was indicated by the mouse jumping, waving its paw, or licking its paw. The mouse was immediately removed and the timer was stopped. The latency period of the hot plate (cutoff time 40 seconds) was recorded. Each mouse was tested 3 times with an interval of 3 minutes, and the average value was taken.

[0050] The latency period was measured before and after drug injection. The maximum possible percentage of effect (MPE%) was calculated using the following formula: MPE% = 100 × [(latency after administration - baseline latency) / (cutoff time - baseline latency)], and the area under the curve (AUC) for the 0–3 h time period was calculated to assess the cumulative analgesic effect. Statistical analysis was performed using two-way ANOVA, and AUC comparisons were performed using the Unpaired Student's t-test.

[0051] The results showed that, compared with the it Vehicle control group, intrathecal injection of 20 ng Box-5 30 min prior to administration significantly enhanced the MPE% in the tail-flick and hot plate tests within 30 min to 2 h after intrathecal injection of 2 nmol morphine (Rule A) and subcutaneous injection of 10 mg / kg morphine (Rule B), and significantly increased the cumulative analgesic AUC in the 3 h behavioral tests (Two-way ANOVA, *P<0.05, **P<0.01; AUC: **P<0.01, n=8). The baseline latency in the tail-flick and hot plate tests in the Box-5 monotherapy group (without morphine) was not significantly different from that in the Vehicle group, indicating that Box-5 monotherapy does not produce analgesic effects and does not affect the baseline pain threshold.

[0052] Example 2

[0053] This embodiment verifies the analgesic effect of intrathecal morphine in a Box-5 enhanced bone cancer pain model (TCI 14d) mice. Experimental results are available in [link to experimental results]. Figure 3 .

[0054] Preparation of bone cancer pain model: Lewis lung cancer cells were resuspended in PBS and the concentration was adjusted to 1×10⁻⁶. 7 Cells / mL, stored on ice. Mice were induced and maintained anesthetized with 2% isoflurane. After disinfection of the skin at the upper 1 / 3 of the right tibial tuberosity, an incision of about 0.5 cm was made to expose the tibia. 2 μL of cancer cell suspension was slowly injected into the bone marrow cavity using a microsyringe. The wound was sutured after the needle was removed.

[0055] Fourteen days post-surgery, mice with bone cancer pain exhibited significant mechanical and thermal hyperalgesia and were randomly divided into two groups (n=10 mice / group). The experimental group received Box-5 (20 ng / mouse) intrathecally 30 minutes before intrathecal morphine injection, while the control group received an equal volume of physiological saline (Vehicle). Subsequently, both groups received intrathecal morphine (2 nmol / mouse).

[0056] The von Frey method was used to assess the mechanical pain threshold: Mice were placed in a transparent acrylic box with a metal mesh bottom, and the soles of their hind limbs were vertically stimulated with von Frey fibers, causing them to flex and hold for 3–5 seconds. Starting from 0.16 g, six stimulations of higher or lower intensities (range 0.02–2.56 g) were administered sequentially using an up-down method. The 50% withdrawal threshold was calculated as the mechanical pain threshold (unit: mN).

[0057] The Hargraves method was used to assess the thermal pain threshold: Mice were placed in a transparent acrylic box on a 30°C constant-temperature heating plate, and the soles of their hind limbs were irradiated with a constant intensity of heat source. Timing was started, and a mouse's rapid withdrawal of its paw or licking of its paw was considered a positive reaction. Irradiation was stopped immediately and the time was recorded (cutoff time 20 seconds). Each mouse was tested 3 times, with an interval of 3 minutes, and the average value was taken.

[0058] The results showed that, compared with the it Vehicle control group, intrathecal injection of 20 ng Box-5 30 min in advance significantly enhanced the analgesic effect of morphine in bone cancer pain model mice, as evidenced by a significant increase in mechanical claw retraction threshold (von Frey method) and thermal claw retraction latency (Hargraves method) within 0-3 h after morphine administration (Two-way ANOVA, *P<0.05, **P<0.01, n=10 mice).

[0059] Example 3

[0060] This embodiment verifies the analgesic effect of intrathecal morphine in a Box-5 enhanced neuropathic pain model (PSNL 14d) mice. Experimental results are available in [link to experimental results]. Figure 4 .

[0061] Preparation of neuropathic pain model: After anesthetizing mice with 2% isoflurane, the skin and muscles of the left thigh were cut to expose the sciatic nerve trunk. The nerve trunk was longitudinally separated with a glass needle and about 1 / 2 of the nerve trunk was ligated with 4-0 sutures. The wound was then sutured, which is the partial sciatic nerve ligation (PSNL) model.

[0062] Fourteen days post-surgery, mice with neuropathic pain developed significant mechanical and thermal hyperalgesia and were randomly divided into two groups (n=10 mice / group). The experimental group received Box-5 (20 ng / mouse) intrathecally 30 min before intrathecal morphine injection, while the control group received an equal volume of physiological saline (Vehicle). Subsequently, both groups received intrathecal morphine (2 nmol / mouse).

[0063] The assessment methods for mechanical pain threshold and thermal pain threshold are the same as those for the von Frey method and Hargraves method in Example 2, with the same operating parameters.

[0064] The results showed that, compared with the it Vehicle control group, intrathecal injection of 20 ng Box-5 30 min in advance significantly enhanced the analgesic effect of morphine in mice with neuropathic pain, as evidenced by a significant increase in mechanical retraction threshold and thermal retraction latency within 0-3 h after morphine administration (Two-way ANOVA, **P<0.01, n=10 mice).

[0065] Example 4

[0066] This embodiment verifies that daily intrathecal injection of Box-5 alleviates analgesia tolerance and pain sensitization induced by continuous subcutaneous morphine injections. The experimental results are shown in [link to experimental results]. Figure 5 .

[0067] Normal male C57BL / 6 mice were randomly divided into two groups (n=8 mice / group): a control group (it Vehicle + subcutaneous morphine) and an experimental group (it 20 ng Box-5 + subcutaneous morphine). For 7 consecutive days, the following administration regimen was used daily: the experimental group received intrathecal Box-5 (20 ng / mouse, dissolved in 5 μL of physiological saline) 30 minutes before subcutaneous morphine injection, while the control group received an equal volume of physiological saline; subsequently, both groups received subcutaneous morphine (10 mg / kg).

[0068] Following daily subcutaneous morphine injections, the tail-flick latency and hot plate latency were measured 0.5 h after administration using the tail-flick test and hot plate test (procedure as in Example 1), respectively, and the MPE% was calculated to assess the analgesic effect on that day. In addition, the baseline tail-flick latency and hot plate latency were measured before daily administration to assess the development of oral sensitization (OIH).

[0069] The results showed that in the control group (it Vehicle), the MPE% gradually decreased with increasing administration days, indicating the formation of analgesia tolerance; simultaneously, the daily basal latency gradually shortened, indicating the gradual development of oral sensitization (OIH). Compared with the control group, the experimental group (it 20 ng Box-5) showed a significantly slower decrease in MPE% and a significantly slower decrease in basal latency (Two-way ANOVA, *P<0.05, **P<0.01, n=8), indicating that daily intrathecal injection of Box-5 can simultaneously alleviate morphine analgesia tolerance and oral sensitization.

[0070] Comparative Example 1

[0071] This comparative example is the vehicle control group, used to demonstrate that Box-5 (i.e., inhibition of the Wnt5a / Ryk signaling pathway) is a contributing factor to the enhanced analgesic effect in the above embodiments.

[0072] The experimental protocol was the same as in Example 1, except that the experimental group received an equal volume of physiological saline (Vehicle) instead of Box-5 solution for intrathecal injection, followed by intrathecal injection of morphine (2 nmol / animal) or subcutaneous injection of morphine (10 mg / kg) according to the same protocol. The determination methods for MPE% and AUC by tail-flick and hot plate methods were the same as in Example 1.

[0073] The results showed that the MPE% and AUC of the it Vehicle + morphine group were significantly lower than those of the it 20 ng Box-5 + morphine group in both the tail-flick and hot-plate methods (Two-way ANOVA, **P < 0.01, n = 8 animals). The comparison between Example 1 and Comparative Example 1 shows that after intrathecal administration of Box-5, the acute analgesic effect of morphine (peak MPE% and cumulative AUC) was significantly improved compared to the Vehicle control group alone, indicating that the blockade of the Wnt5a / Ryk signaling pathway is a key factor in enhancing the analgesic effect, and this enhancement was unexpected.

[0074] Comparative Example 2

[0075] This comparative example is the Vehicle control group in a continuous morphine tolerance model, used to demonstrate the alleviating effect of Box-5 on analgesia tolerance and pain sensitization.

[0076] The experimental protocol was the same as in Example 4, except that the experimental group received an equal volume of physiological saline (Vehicle) instead of Box-5 solution for daily intrathecal injection, followed by daily subcutaneous injection of morphine (10 mg / kg) for 7 consecutive days. The methods for determining MPE% and daily basal latency using the tail-flick method and hot plate method were the same as in Example 4.

[0077] The results showed that in the it Vehicle group, the MPE% continuously decreased with increasing administration days, and by day 7, the MPE% was significantly lower than that on day 1. Simultaneously, the daily basal latency period continued to shorten, indicating significant analgesia tolerance and pain sensitization. A comparison between Example 4 and Comparative Example 2 showed that daily intrathecal administration of Box-5 significantly slowed the rate of decrease in MPE% and the rate of shortening of the basal latency period (Two-way ANOVA, *P<0.05, **P<0.01, n=8), demonstrating that sustained blockade of the Wnt5a / Ryk signaling pathway can simultaneously delay analgesia tolerance and alleviate pain sensitization; this dual effect was unexpected.

[0078] Example 1

[0079] The main experimental results of Examples 1-4 and Comparative Examples 1-2 are summarized in Table 1.

[0080] Table 1 Summary of the main experimental results of each embodiment and comparative example

[0081] Dosing regimen Key performance indicators result Example 1 (Intrathecal Morphine) it 20 ng Box-5+it 2 nmol morphine MPE% and AUC of the drift method / hot plate method Significantly higher than the Vehicle group, **P<0.01, n=8 Example 1 (Subcutaneous Morphine) it 20 ng Box-5+sc 10 mg / kg morphine MPE% and AUC of the drift method / hot plate method Significantly higher than the Vehicle group, **P<0.01, n=8 Example 2 (Bone Cancer Pain) it 20 ng Box-5+it 2 nmol morphine Mechanical shrink claw threshold / heat shrink claw latency Significantly higher than the Vehicle group, **P<0.01, n=10 Example 3 (Neuropathic Pain) it 20 ng Box-5+it 2 nmol morphine Mechanical shrink claw threshold / heat shrink claw latency Significantly higher than the Vehicle group, **P<0.01, n=10 Example 4 (Tolerance / OIH Model) Daily 10 ng Box-5+ Daily 10 mg / kg Morphine MPE% decrease rate / rate of reduction in basal latency Significantly slower than the Vehicle group, *P<0.05, **P<0.01, n=8 Comparative Example 1 it Vehicle + Morphine MPE% and AUC of the drift method / hot plate method Significantly lower than Box-5 group Comparative Example 2 Daily IT Vehicle + Daily SC Morphine MPE% / Basal Latency Period Tolerance and OIH significantly formed

[0082] The above results indicate that the combined use of Box-5, an inhibitor of the Wnt5a / Ryk signaling pathway, and opioids can significantly enhance the acute analgesic effect of morphine under various conditions, including normal physiological state, bone cancer pain, and neuropathic pain. Furthermore, in a continuous dosing model, it can simultaneously alleviate analgesic tolerance and pain sensitization, demonstrating an unexpected dual analgesic synergistic effect.

[0083] Test Example 1

[0084] MOR phosphorylation level detection (Western blot):

[0085] Normal male C57BL / 6 mice from Example 1 were used. After pretreatment with Box-5 (20 ng) intrathecally for 30 min, DAMGO (0.1 nmol) was injected intrathecally. The control and experimental groups were obtained in the same way as in Example 1. The control group Vehicle was given an equal volume of physiological saline. The Vehicle+DAMGO group was given DAMGO (0.1 nmol) in addition to the control group. The Box-5+DAMGO group was pretreated with Box-5 intrathecally for 30 min and then injected intrathecally with DAMGO (0.1 nmol).

[0086] After euthanizing mice, DRG tissue was extracted and prepared on ice using RIPA lysis buffer containing protease and phosphatase inhibitors. The homogenate was centrifuged, and the supernatant was collected. 2×SDS loading buffer was added, followed by separation by 10% SDS-PAGE and transfer to a PVDF membrane. The membrane was blocked with TBST containing 5% BSA and then incubated overnight at 4°C with primary antibodies containing MOR (1:1000, abcam, Cat# ab134054), p-MOR (Ser375) (1:1000, Cell Signaling Technology, Cat# 3451S), and β-actin (1:1000, Cell Signaling Technology, Cat# 4970) (4°C overnight). After primary antibody incubation, the membrane was washed three times with TBST and then incubated with HRP-labeled secondary antibody (1:10000, room temperature, 1 h). After washing away the secondary antibody, the membrane was developed using ECL chemiluminescence imaging and photographed using a chemiluminescence imaging system. The total MOR protein level and Ser375 phosphorylation level of MOR in the DRG of mice in the Vehicle, Vehicle+DAMGO, and Box-5+DAMGO groups were detected, and the results are as follows: Figure 6 As shown. Figure 6 Figure A shows the results of Western blotting analysis of total MOR protein levels and Ser375 phosphorylation levels of MOR in the DRG of mice in the Vehicle, Vehicle+DAMGO, and Box-5+DAMGO groups. Figure B shows the results of Western blotting analysis of the band grayscale in Figure A. Statistical analysis was performed using two-way ANOVA, with n = 3 mice per group. *P < 0.05, **P < 0.01, compared with the Vehicle group; #P < 0.05, ##P < 0.01, compared with the Vehicle+DAMGO group.

[0087] Figure 6 The results showed that intrathecal injection of Box-5 (20 ng) 30 min in advance blocked the activation of MOR phosphorylation by intrathecal injection of DAMGO (0.1 nmol).

[0088] MOR endocytosis extent detection (immunofluorescence or flow cytometry):

[0089] HEK-293 cells were cultured in DMEM containing 10% FBS and 1% penicillin antibiotics (37℃, 5% CO2). Lentiviral vectors carrying a C-terminal 3×FLAG tag for human MOR (MOR-FLAG) and a C-terminal 3×HA tag for human RYK (RYK-HA) were constructed and ligated with the puromycin resistance gene (MOR) and the hygromycin resistance gene (RYK), respectively. Cells were first transfected with MOR-FLAG lentivirus and selected for 7 days with puromycin (2 μg / mL) to obtain cell lines stably expressing MOR. Then, RYK-HA lentivirus was transfected and double-selected for 7 days with puromycin (2 μg / mL) and hygromycin (200 μg / mL) to establish MOR / RYK bistable cell lines. Subsequent culture was maintained at half the concentration of each antibiotic (1 μg / mL puromycin, 100 μg / mL hygromycin). Immunofluorescence was used to detect the cellular localization of MOR and RYK after treatment with Vehicle or Morphine (5 μM). Cells were cultured in 35 mm confocal glass dishes and fixed with 4% PFA at room temperature for 30 min after reaching 40% confluence. After washing with PBS, cells were blocked by permeabilization with blocking buffer containing 0.3% Triton X-100, 5% BSA, and 5% donkey serum at room temperature for 2 h. Primary antibody (1:500, overnight at 4°C) was then added, followed by washing with PBS and adding fluorescent secondary antibody (Alexa Fluor 488 / 555, 1:500, room temperature in the dark for 2 h). After washing with PBS, cells were counterstained with DAPI (1 μg / mL) and observed using a laser confocal microscope. Results are as follows: Figure 7 As shown.

[0090] Figure 7 The results showed that overexpression of human RYK protein in HEK-293 cells enhanced basal levels and the endocytotic membrane of human MOR after morphine activation.

[0091] In vitro validation of Box-5 blocking Wnt5a / Ryk binding:

[0092] Vehicle or Box-5 mice from Example 1 were obtained. After anesthesia and euthanasia, DRG tissue was extracted and prepared using RIPA lysis buffer containing protease and phosphatase inhibitors on ice. The homogenate was then centrifuged to collect the supernatant. The supernatant was mixed with 2 μg of Ryk monoclonal antibody (ThermoFisher, Cat# MA1-615) and 20 μL of Protein A / G agarose beads and incubated overnight at 4°C. The supernatant was discarded after centrifugation. The beads were washed 5 times with pre-chilled lysis buffer, followed by centrifugation to discard the washings. 2×SDS loading buffer was added to the beads, and the mixture was boiled for 10 min. The supernatant was then centrifuged to collect the supernatant for Western blot detection of Wnt5a content. The Western blot procedure was the same as above, and the Wnt5a primary antibody was Novus, Cat# NBP2-24752. Results are as follows. Figure 8 As shown. Figure 8 Figure A shows the results of Co-IP and Western Blot analysis of Wnt5a content in DRG samples from mice treated with Vehicle or Box-5 after IP-Ryk; Figure B shows the results of Western Blot band grayscale homogenization analysis in Figure A. Statistical analysis was performed using unpaired t-tests, n = 3 mice / group. **P < 0.01, compared with the Vehicle group.

[0093] Figure 8 The results showed that intrathecal injection of Box-5 (20 ng) 30 min reduced the binding of Wnt5a and Ryk in mouse DRG.

[0094] It is understood that the Wnt5a / Ryk signaling pathway inhibitors of this application are not limited to Box-5, but can also be Wnt5a neutralizing antibodies, Ryk antagonistic antibodies, Ryk-Fc fusion proteins, or other active substances capable of blocking the binding of Wnt5a to Ryk receptors. All of these inhibitors can protect the MOR G protein signaling pathway through the same molecular mechanism, thereby exerting an adjuvant analgesic effect to opioids. Similarly, μ-opioid receptor agonists are not limited to morphine, but can also be other MOR agonists such as fentanyl, sufentanil, remifentanil, oxycodone, hydromorphone, or methadone. All of these drugs produce analgesic effects by activating MOR, and when used in combination with Wnt5a / Ryk signaling pathway inhibitors, they can synergistically enhance the analgesic effect. The route of administration in this application is not limited to intrathecal injection, but may also include epidural injection, subcutaneous injection, intravenous injection, or oral administration after formulation modification; the time of administration is not limited to 30 minutes before opioid administration, but may also be given simultaneously with opioids; the frequency of administration may be a single dose or multiple doses at the same frequency as opioids.

[0095] Obviously, the applicable pain types of the Wnt5a / Ryk signaling pathway inhibitors in this application are not limited to bone cancer pain and neuropathic pain, but can also be applied to other types of pain that require opioid analgesia, such as inflammatory pain and postoperative pain. Under the above-mentioned pathological pain conditions, the expression of the Wnt5a / Ryk signaling pathway is upregulated, and the intervention effect of the Wnt5a / Ryk signaling pathway inhibitors can also be achieved.

[0096] The above description, in conjunction with specific embodiments, provides a further detailed explanation of this application and should not be construed as limiting the specific implementation of this application to these descriptions. Those skilled in the art to which this application pertains can make several simple deductions or substitutions without departing from the concept of this application.

Claims

1. The use of Wnt5a-Ryk signaling pathway inhibitors in the preparation of drugs that enhance the analgesic effect of opioids, characterized in that: The Wnt5a-Ryk signaling pathway inhibitor is an active substance that can block the binding of Wnt5a to the Ryk receptor. The Wnt5a-Ryk signaling pathway inhibitor is used in combination with opioid drugs.

2. The use according to claim 1, characterized in that: The Wnt5a-Ryk signaling pathway inhibitor is at least one of Box-5, Wnt5a neutralizing antibody, Ryk-Fc fusion protein, Wnt5a signaling pathway inhibitory small molecule compound, Ryk antagonistic antibody, RyksiRNA, Ryk shRNA, and Ryk gene knockout reagent.

3. The use according to claim 1 or 2, characterized in that: The opioid is at least one of morphine, fentanyl, sufentanil, remifentanil, oxycodone, hydromorphone, and methadone.

4. The use of Wnt5a-Ryk signaling pathway inhibitors in the preparation of drugs that delay or prevent tolerance to opioid analgesia, characterized in that: The Wnt5a-Ryk signaling pathway inhibitor is an active substance that can block the binding of Wnt5a to the Ryk receptor. The Wnt5a-Ryk signaling pathway inhibitor is used in combination with opioid drugs.

5. The use according to claim 4, characterized in that: The Wnt5a-Ryk signaling pathway inhibitor is at least one of Box-5, Wnt5a neutralizing antibody, Ryk-Fc fusion protein, Wnt5a signaling pathway inhibitory small molecule compound, Ryk antagonistic antibody, RyksiRNA, Ryk shRNA, and Ryk gene knockout reagent.

6. The use according to claim 4 or 5, characterized in that: The opioid is at least one of morphine, fentanyl, sufentanil, remifentanil, oxycodone, hydromorphone, and methadone.

7. The use of Wnt5a-Ryk signaling pathway inhibitors in the preparation of drugs to alleviate opioid-induced pain sensitization, characterized in that: The Wnt5a-Ryk signaling pathway inhibitor is an active substance that can block the binding of Wnt5a to the Ryk receptor. The Wnt5a-Ryk signaling pathway inhibitor is used in combination with opioid drugs.

8. The use according to claim 7, characterized in that: The Wnt5a-Ryk signaling pathway inhibitor is at least one of Box-5, Wnt5a neutralizing antibody, Ryk-Fc fusion protein, Wnt5a signaling pathway inhibitory small molecule compound, Ryk antagonistic antibody, RyksiRNA, Ryk shRNA, and Ryk gene knockout reagent. Optionally, the opioid is at least one of morphine, fentanyl, sufentanil, remifentanil, oxycodone, hydromorphone, and methadone.

9. A pharmaceutical composition, characterized in that: The invention comprises a Wnt5a-Ryk signaling pathway inhibitor, an opioid, and a pharmaceutically acceptable carrier, wherein the Wnt5a-Ryk signaling pathway inhibitor is an active substance capable of blocking the binding of Wnt5a to the Ryk receptor.

10. The pharmaceutical composition according to claim 9, characterized in that: The Wnt5a-Ryk signaling pathway inhibitor is at least one of Box-5, Wnt5a neutralizing antibody, Ryk-Fc fusion protein, Wnt5a signaling pathway inhibitory small molecule compound, Ryk antagonistic antibody, Ryk siRNA, Ryk shRNA, and Ryk gene knockout reagent; Optionally, the opioid is at least one of morphine, fentanyl, sufentanil, remifentanil, oxycodone, hydromorphone, and methadone.

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