Use of a rapamycin in the preparation of a medicament for alleviating opiate drug tolerance caused by an anti-angiogenic drug, an alleviating medicament and a drug

Abstract

The application discloses application of rapamycin in preparation of a reliever of opiate drug tolerance caused by an anti-angiogenic drug, the reliever and a medicine, and relates to the technical field of medicine application. It is found that a TSC1-mTOR-autophagy axis plays a role in opiate drug tolerance caused by an anti-angiogenic drug, and the specific embodiment is that the anti-angiogenic drug can cause a decrease in TSC1 content, an mTOR / RPS6 pathway is activated, thereby autophagy flow is blocked, and pain sensation is enhanced; therefore, the opiate tolerance condition can be relieved by adjusting the mTOR / RPS6 pathway through rapamycin.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical application technology, and in particular to the application of rapamycin in the preparation of a relieving agent for opioid tolerance caused by anti-angiogenic drugs, as well as the relieving agent and the drug. Background Technology

[0002] Cancer pain is one of the most common and painful symptoms in patients with advanced cancer, seriously affecting their lives and treatment outcomes.

[0003] Opioid analgesics are currently the gold standard and core drugs for treating moderate to severe cancer pain. However, long-term, high-dose use of opioids can lead to opioid tolerance. Opioid tolerance refers to the gradual weakening of the body's response to opioids (such as morphine, fentanyl, oxycodone, etc.) after long-term or repeated use, resulting in the need for increased doses to achieve the original analgesic or other pharmacological effects. This is a serious clinical problem.

[0004] Anti-angiogenic therapy is a cancer treatment that limits tumor growth and metastasis by inhibiting the formation of new blood vessels in the tumor (i.e., angiogenesis), for example, by inhibiting VEGF (vegetative-growth factor). This treatment is an important part of standard treatment for many patients with advanced cancer. However, anti-angiogenic therapy can exacerbate opioid tolerance. Summary of the Invention

[0005] The main objective of this invention is to propose the use of rapamycin in the preparation of a remedy for opioid tolerance caused by anti-angiogenic drugs, as well as a remedy and a drug, with the aim of improving the problem that anti-angiogenic therapy in the prior art exacerbates opioid tolerance.

[0006] To achieve the above objectives, this invention proposes the use of rapamycin in the preparation of an anti-angiogenic drug tolerance reliever.

[0007] In one embodiment, the rapamycin works by inhibiting the mTOR / RPS6 pathway.

[0008] In one embodiment, the anti-angiogenic drug includes at least one of bevacizumab, ramoximab, and ranibizumab.

[0009] In one embodiment, the opioid drug includes at least one of morphine, fentanyl, pethidine, oxycodone, and methadone.

[0010] The present invention also provides a relief agent for alleviating opioid tolerance caused by anti-angiogenic drugs, said relief agent comprising rapamycin.

[0011] The present invention also provides a medicament comprising the aforementioned reliever, and the medicament further comprising excipients, the excipients comprising at least one of starch, lactose and sucrose.

[0012] In the technical solution of this invention, it was unexpectedly discovered that the TSC1-mTOR-autophagy axis plays a role in opioid tolerance caused by anti-angiogenic drugs. Specifically, anti-angiogenic drugs lead to a decrease in TSC1 content and activation of the mTOR / RPS6 pathway, thereby blocking autophagic flux and increasing pain. Therefore, regulating the mTOR / RPS6 pathway with rapamycin can alleviate this opioid tolerance. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0014] Figure 1 Figures show the clinical findings and animal validation results of bevacizumab treatment on morphine tolerance; where A is the result of bevacizumab weakening the analgesic effect of morphine and accelerating morphine tolerance in Example 1; B is the experimental flowchart of the animal experiment in Example 1 verifying that inhibiting VEGF signaling weakens morphine analgesia; C is the tail-flick test result in the animal experiment in Example 1 verifying that inhibiting VEGF signaling weakens morphine analgesia; D is the hot plate test result in the animal experiment in Example 1 verifying that inhibiting VEGF signaling weakens morphine analgesia; E is the result of the hot plate test in Example 1. The flowcharts show the maximum probable effect response curves of morphine in the tail-flick test or tail-flick test in Example 1; F is the median effective dose result of morphine in the tail-flick test in Example 1; G is the median effective dose result of morphine in the hot plate test in Example 1; H is the flowchart of the animal experiment in Example 1 verifying the inhibition of VEGF signaling to accelerate morphine tolerance; I is the result of the hot plate test in the animal experiment in Example 1 verifying the inhibition of VEGF signaling to accelerate morphine tolerance; J is the result of the tail-flick test in the animal experiment in Example 1 verifying the inhibition of VEGF signaling to accelerate morphine tolerance. Figure 2Figure 1 shows the proteomic results of the effect of bevacizumab treatment on morphine tolerance. Specifically, A is a volcano plot of differential expression analysis between the IgG1+Saline group and the IgG1+Morphine group in Example 1; B is a volcano plot of differential expression analysis between the IgG1+Morphine group and the Beva+Morphine group in Example 1; C is a volcano plot of differential expression analysis between the IgG1+Saline group and the Beva+Morphine group in Example 1; D is a Venn diagram of differential protein expression analysis between the IgG1+Morphine group and the Beva+Morphine group in Example 1; E is a hierarchical clustering analysis of protein expression in the three treatment groups in Example 1; F is a pathway enrichment analysis result between the IgG1+Saline group and the IgG1+Morphine group in Example 1; G is a pathway enrichment analysis result between the IgG1+Morphine group and the Beva+Morphine group in Example 1; H is a pathway enrichment analysis result between the IgG1+Saline group and the Beva+Morphine group in Example 1; and I is an interaction network diagram of autophagy-related proteins in Example 1. Figure 3 The diagram shows the experimental results of animal experiments confirming the pathway by which bevacizumab induces opioid tolerance. A represents the immunoblotting results of representative proteins from Example 1; B represents the bar chart of relative protein levels of p-mTOR / mTOR in different groups of Example 1; C represents the bar chart of relative protein levels of p-RPS6 / RPS6 in different groups of Example 1; D represents the bar chart of relative protein levels of p62 in different groups of Example 1; E represents the bar chart of relative protein levels of LC3B-I / II in different groups of Example 1; F represents the bar chart of relative protein levels of Beclin-1 in different groups of Example 1; G represents the bar chart of relative protein levels of ATG5 in different groups of Example 1; H represents the bar chart of relative protein levels of ATG5 in different groups of Example 1. Bar chart showing the relative levels of ATG7 protein; I is a bar chart showing the relative levels of ATG16L1 protein in different groups in Example 1; J is an immunofluorescence image of p-mTOR in neurons (NeuN) of the prefrontal cortex (mPFC) in different groups in Example 1; K is an immunofluorescence image of p-RPS6 in neurons (NeuN) of the mPFC in different groups in Example 1; L is an immunofluorescence image of p62 in neurons (NeuN) of the mPFC in different groups in Example 1; M is an ultrastructural diagram showing the changes in autophagic flux after different treatments in Example 1; N is a bar chart showing the count of secondary lysosomes in each cell between the two groups in Example 1; O is a bar chart showing the count of autophagosomes in each cell between the two groups in Example 1. Figure 4Figure 1 shows the experimental results of the effect of rapamycin on the reduction of opioid tolerance to anti-angiogenic drugs. Specifically, A is a schematic diagram of the experimental procedure for the study of the effect of combined intervention of Beva and rapamycin on mice in Example 2; B is a diagram of the tail-flick test after different drug treatments in Example 2; C is a diagram of the hot plate test after different drug treatments in Example 2; D is a diagram of the immunoblotting experiment of representative proteins in Example 2; E is a diagram of the relative protein levels of p-mTOR and mTOR under different treatment conditions in Example 2; F is a diagram of the relative protein levels of p-RPS6 and RPS6 under different treatment conditions in Example 2; G is a diagram of the relative protein levels of p62 under different treatment conditions in Example 2; H is a diagram of the experimental results. Example 2: LC3B-Ⅰ / Ⅱ relative protein levels under different treatment conditions; I: Immunofluorescence of p-mTOR in neurons (NeuN) of mPFC in different treatment groups in Example 2; J: Immunofluorescence of p-RPS6 in neurons (NeuN) of mPFC in different treatment groups in Example 2; K: Immunofluorescence of p62 in neurons (NeuN) of mPFC in different treatment groups in Example 2; L: Ultrastructural diagram of autophagic flux changes after different treatments in Example 2; M: Bar chart of secondary lysosome counts in each cell among different treatment groups in Example 2; N: Bar chart of autophagosome counts in each cell among different treatment groups in Example 2.

[0015] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially. Furthermore, the meaning of "and / or" throughout the text includes three parallel solutions; for example, "A and / or B" includes solution A, or solution B, or a solution where both A and B are satisfied simultaneously. In addition, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] Cancer pain is one of the most common and painful symptoms in patients with advanced cancer, seriously affecting their lives and treatment outcomes.

[0018] Opioid analgesics are currently the gold standard and core drugs for treating moderate to severe cancer pain. However, long-term, high-dose use of opioids can lead to opioid tolerance. Opioid tolerance refers to the gradual weakening of the body's response to opioids (such as morphine, fentanyl, oxycodone, etc.) after long-term or repeated use, resulting in the need for increased doses to achieve the original analgesic or other pharmacological effects. This is a serious clinical problem.

[0019] Anti-angiogenic therapy is a cancer treatment that limits tumor growth and metastasis by inhibiting the formation of new blood vessels in the tumor (i.e., angiogenesis), for example, by inhibiting VEGF (vegetative-growth factor). This treatment is an important part of standard treatment for many patients with advanced cancer. However, anti-angiogenic therapy can exacerbate opioid tolerance.

[0020] In view of this, the present invention provides the use of rapamycin in the preparation of an anti-angiogenic drug tolerance reliever.

[0021] In the technical solution of this invention, it was discovered for the first time that the TSC1-mTOR-autophagy axis plays a role in opioid tolerance induced by anti-angiogenic drugs. Specifically, anti-angiogenic drugs lead to a decrease in TSC1 levels, activation of the mTOR / RPS6 pathway, thereby obstructing autophagic flux and increasing pain. Therefore, regulating the mTOR / RPS6 pathway with rapamycin can alleviate this type of opioid tolerance. This discovery differs from the traditional opioid tolerance pathway, which involves desensitization and downregulation of μ-opioid receptors (MOR). This type of opioid tolerance cannot be alleviated by regulating the mTOR / RPS6 pathway with rapamycin.

[0022] In some embodiments, rapamycin exerts its effect by inhibiting the mTOR / RPS6 pathway. Specifically, rapamycin can inhibit the mTOR / RPS6 pathway by suppressing phosphorylated mTOR and phosphorylated RPS6 levels, thereby alleviating opioid tolerance induced by anti-angiogenic drugs.

[0023] In some embodiments, the anti-angiogenic drug includes at least one of bevacizumab, ramucirumab, and ranibizumab. That is, the anti-angiogenic drug can be any one of bevacizumab, ramucirumab, and ranibizumab, or two or three of them, all within the scope of protection of this invention. It should be noted that bevacizumab is a monoclonal antibody that exerts its anti-angiogenic effect by inhibiting vascular endothelial growth factor (VEGF) to block tumor angiogenesis. Ramucirumab is a fully humanized monoclonal antibody whose target is vascular endothelial growth factor receptor 2 (VEGFR2). It specifically binds to the extracellular domain of VEGFR2, blocking the binding of VEGF ligands to the receptor and the activation of downstream signaling pathways. Ranibizumab is also a humanized monoclonal antibody fragment that inhibits angiogenesis by binding to the VEGF-A subtype.

[0024] In some embodiments, the opioid drug includes at least one of morphine, fentanyl, meperidine, oxycodone, and methadone. That is, the opioid drug can be any one of morphine, fentanyl, meperidine, oxycodone, and methadone, or two or three of these drugs, all within the scope of this invention. The above-mentioned opioid drugs have good analgesic effects on cancer pain.

[0025] The present invention also provides a relief agent for alleviating opioid tolerance caused by anti-angiogenic drugs, said relief agent comprising rapamycin. Rapamycin has a good relieving effect on opioid tolerance caused by anti-angiogenic drugs.

[0026] For example, the concentration of rapamycin used in the relief agent is 2 mg / day to 6 mg / day, and the concentration can be any range of two values ​​consisting of 2 mg / day, 3 mg / day, 4 mg / day, 6 mg / day or more.

[0027] The present invention also provides a medicament comprising the aforementioned reliever, and further comprising excipients, the excipients being at least one selected from starch, lactose, and sucrose. It is understood that the excipients may be adjusted according to the actual method of use.

[0028] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0029] Experimental materials Morphine is morphine hydrochloride injection, purchased from Northeast Pharmaceutical Group Shenyang No. 1 Pharmaceutical Co., Ltd., Guangdong Provincial Drug Administration Approval No.: K2025093; Bevacizumab (Beva) was purchased from MedChemExpress, catalog number: HY-P9906; IgG1 protein was purchased from MedChemExpress, catalog number: HY-P70796.

[0030] Example 1: Effect of anti-angiogenic drug (bevacizumab) treatment on opioid (morphine) tolerance 1. Discovery of clinical phenomena This study recruited 20 participants, divided into two groups: patients receiving morphine alone (n=13) and patients receiving bevacizumab combined with morphine (n=7). Both groups were observed for 21 days. The daily analgesic morphine requirement during the observation period was defined as the total morphine dose actually used every 24 hours, including the fixed basal analgesic dose and breakthrough pain rescue dose. If different routes of administration or dosage forms existed, they were uniformly converted to oral morphine equivalents for comparison to determine the morphine requirement. This requirement was then statistically analyzed and summarized. Figure 1 In the middle of A. Figure 1 In the "Morphine" group, "Morphine" refers to the group receiving morphine treatment only, while "Beva+Morphine" refers to the group receiving bevacizumab combined with morphine treatment.

[0031] Depend on Figure 1 As shown in Figure A, during the 21-day observation period, patients receiving morphine alone (n=13) maintained a stable analgesic response, with no significant increase in morphine requirement from baseline. In contrast, patients receiving bevacizumab plus morphine (n=7) exhibited a progressively significant increase in morphine dose requirement starting around day 5 and continuing to rise throughout the study period. By day 21, the mean increase in morphine requirement in the bevacizumab plus morphine group was approximately 5 mg higher than that in the morphine-only group. These results indicate that bevacizumab administration significantly attenuates morphine-induced analgesia, thereby accelerating the development of morphine tolerance.

[0032] 2. Animal experiments verified that inhibiting VEGF signaling weakens morphine analgesia. All experimental mice in this study were provided with free access to food and water and housed in a standard laboratory environment: temperature 25±2℃, humidity 40%-70%, and a 12-hour light-dark cycle. Mice were randomly assigned to groups, with 2-6 mice per cage. The experimental procedures involving the mice were approved by the Ethics Committee of the Third People's Hospital of Shenzhen (Ethics Approval No.: 2025-001-01). The experimental mice were C57BL / 6 mice, purchased from Kingfamtec Co., Ltd.

[0033] like Figure 1 The experimental procedure shown in Figure B is as follows: On day 0, the thermal pain threshold and comprehensive pain response were measured using the tail-flick test and hot plate test, respectively, and recorded as the baseline response (Baleline, abbreviated as BL). On day 1, the mice were first treated with either bevacizumab (5 mg / kg, tail vein injection) or IgG1 (5 mg / kg, tail vein injection) once. The tail-flick test and hot plate test were performed on days 2 and 3, respectively. On day 4, the mice were treated with either morphine (MF, 10 mg / kg) or saline (subcutaneous injection, abbreviated as sc) once, and the tail-flick test and hot plate test were performed at 15 minutes, 30 minutes, 1 hour, 1.5 hours, 2 hours, and 3 hours after the morphine or saline injection.

[0034] Three groups of mice were obtained: bevacizumab + morphine, IgG1 + morphine, and IgG1 + saline (i.e., control group). The behavioral assessment methods for pain were as follows: (1) Tail flick test: used to assess the thermal pain threshold. Gently immobilize the mouse and immerse approximately 3 cm of the tail tip in a constant temperature water bath at 50 ± 0.5°C. Record the latency period during which the mouse rapidly pulls its tail out of the water (the timer starts the moment the tail touches the hot water and stops the moment the mouse's tail rapidly pulls out of the water; this time is the latency period), setting a cutoff time of 15 seconds to prevent tissue damage. Calculate the maximum possible percentage effect (MPE%) before and after drug administration: Maximum possible effect (%) = 100 × [(post-drug response - baseline response) / (reference response)] Baseline response); The drug-induced response refers to the time it takes for mice to exhibit an avoidance response to a stimulus after drug treatment; that is, the time from when the mouse receives the stimulus to when it begins to avoid it. The baseline response refers to the basic responses of all groups of mice in the behavioral tests (tail-flick test and hot plate test) before drug administration; that is, the time from when the mouse receives the stimulus to when it begins to avoid it. The reference response is defined as a 15-second tail-flick test and a 40-second hot plate test. Test Results Figure 1 As shown in C.

[0035] (2) Hot plate test: used to assess comprehensive pain response. Mice were placed on a hot plate at 53±0.5°C, and the latency period for pain avoidance behaviors such as licking their hind paws or jumping was recorded, with a cutoff time of 40 seconds. The test results are as follows: Figure 1 As shown in D.

[0036] Depend on Figure 1 C and Figure 1 As shown in Figure D, a significant analgesic response was observed in the IgG1+ saline group during both the tail-flick and hot plate tests, characterized by a significantly prolonged latency period. The latency period in the IgG1+ morphine group was significantly longer than that in the IgG1+ saline group. Furthermore, bevacizumab pretreatment (corresponding to Beva+Morphine) significantly attenuated the morphine-induced analgesia, as evidenced by a significantly shortened latency period of 30-180 minutes after morphine injection. These data indicate that bevacizumab inhibits the analgesic effect of morphine in vivo through systemic VEGF blockade.

[0037] Obtain the response curve of morphine's maximum probable effect in the hot plate test or tail-flip test. For example... Figure 1 The experimental procedure shown in Figure E involves treating two groups of mice once on day 1 with either bevacizumab (5 mg / kg, administered via tail vein) or IgG1 (5 mg / kg, administered via tail vein), resulting in IgG1 and bevacizumab groups. On days 3 and 4, mice were subjected to either a hot plate test or a tail-flick test. The test results are shown below. Figure 1 China F and Figure 1 As shown in G. On day 4, mice in the IgG1 group were subcutaneously injected with morphine at doses of 1 mg / kg, 5 mg / kg, and 10 mg / kg, respectively, while mice in the bevacizumab group were subcutaneously injected with morphine at doses of 3 mg / kg, 10 mg / kg, and 30 mg / kg, respectively. Half an hour after morphine injection, the thermal pain threshold and comprehensive pain response were measured using the tail flick test and hot plate test, respectively. Figure 1 China F and Figure 1In G, "sc" refers to "subcutaneous injection". For each mouse, the maximum possible effect (%) was calculated based on the response after administration. Subsequently, a four-parameter logistic nonlinear regression model was used to fit the dose-response curve, with the logarithm of the morphine dose as the x-axis and %MPE as the y-axis. ED 50 Defined as the morphine dose corresponding to the 50% maximum possible effect on the fitted curve, and derived from the logED obtained by fitting. 50 Obtained by anti-coefficient transformation.

[0038] Depend on Figure 1 China F and Figure 1 As shown in the results from G, the morphine dose-response curve was consistent with the above behavioral findings, indicating a significant rightward shift in bevacizumab-treated mice. In the tail-flick test, the median effective dose (ED) of morphine was... 50 The dose was increased from 2.97 mg / kg (IgG1+Morphine) to 9.89 mg / kg (Beva+Morphine); in the hot plate test, the median effective dose (ED) of morphine was... 50 Increasing the dosage from 4.05 mg / kg (IgG1+Morphine) to 8.66 mg / kg (Beva+Morphine) confirmed that bevacizumab could reduce the intensity of the analgesic effect of morphine.

[0039] 3. Animal experiments verified that inhibiting VEGF signaling accelerates morphine tolerance. The experimental procedure is as follows Figure 1 As shown in Figure H, on day 0, the thermal pain threshold and comprehensive pain response were measured using the tail-flick test and hot plate test, respectively, and recorded as the baseline response (Baleline, abbreviated as BL). On day 1, mice were randomly assigned to groups and treated once with either bevacizumab (5 mg / kg, tail vein injection) or IgG1 (5 mg / kg, tail vein injection). On days 2 and 3, the thermal pain threshold and comprehensive pain response were measured using the tail-flick test and hot plate test, respectively, to investigate the effects of bevacizumab and IgG1 on the thermal pain threshold and comprehensive pain response in mice. From day 4 to day 10 (a total of 7 consecutive days), mice were treated daily with either morphine (MF, 10 mg / kg) or saline (subcutaneous, abbreviated as sc).

[0040] Three groups of mice were obtained: bevacizumab + morphine group, IgG1 + morphine group, and IgG1 + saline group (i.e., control group).

[0041] Depend on Figure 1 I and Figure 1As shown in the results, on days 2 and 3, i.e., after bevacizumab or IgG1 treatment and before morphine administration, there were no significant differences in the response latency in the tail-flick and hot plate tests among the three groups of mice, indicating that a single tail vein injection of bevacizumab itself did not significantly alter the baseline thermal pain threshold or overall pain response in mice. From day 4 onwards, mice in the IgG1+Morphine group showed significant analgesic responses in both the tail-flick and hot plate tests, characterized by a significantly prolonged response latency. Specifically, after the first administration of morphine on day 4, the tail-flick and hot plate latency in the IgG1+Morphine group were significantly higher than those in the IgG1+Saline group, suggesting that subcutaneous injection of 10 mg / kg morphine can produce a significant acute analgesic effect. With continuous morphine treatment for 7 days from day 4 to day 10, the response latency in the IgG1+Morphine group gradually decreased. In the tail-flick test, the morphine-induced latency prolongation gradually decreased from its peak on day 4, approaching the level of the IgG1+Saline group in the later stages. In the hot plate test, although the morphine-treated group maintained a higher latency than the saline group from day 5 to day 10, it also showed an overall trend of gradually decreasing from its peak. This indicates that mice gradually developed morphine analgesia tolerance after continuous morphine administration. Compared with the IgG1+Morphine group, the Beva+Morphine group had a lower overall latency in the tail-flick test, and the decrease was more significant. In the hot plate test, the Beva+Morphine group also had a lower latency at multiple time points than the IgG1+Morphine group, suggesting that bevacizumab pretreatment weakens the morphine-induced analgesic response and may accelerate or enhance the formation of morphine analgesia tolerance. Therefore, Figure 1 I and Figure 1 The results from the study showed that bevacizumab alone did not significantly affect the baseline pain threshold, but under continuous morphine administration, bevacizumab could weaken the analgesic effect of morphine and shorten the latency of morphine-induced response, suggesting that systemic VEGF blockade may promote the decline of morphine analgesia or the development of analgesic tolerance.

[0042] 4. Proteomics reveals molecular mechanisms To elucidate the molecular mechanism by which the analgesic effect of morphine is weakened after bevacizumab treatment, this study conducted proteomic analysis on the medial prefrontal cortex (mPFC) tissues of three groups of experimental mice: IgG1+ saline group, IgG1+ morphine group, and bevacizumab+ morphine group.

[0043] The proteomics analysis procedure was as follows: mPFC tissue was collected and homogenized using a lysis buffer containing sodium dodecyl sulfate (SDS) (containing 4% SDS, 100 mM dithiolitol, 100 mM trihydrochloride (pH 8.0), and a protease inhibitor (purchased from MedChemExpress, catalog number: HY-K0010). The homogenate was quantified using the dicaprinic acid protein quantification method (BCA method). The homogenate was digested with trypsin using the filter-assisted sample preparation (FASP) method, and the resulting peptides were labeled with iTRAQ reagent and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS). Differentially expressed proteins associated with pain or target gene function were screened using bioinformatics methods such as volcano plots, Venn diagrams, hierarchical cluster analysis, GO functional enrichment, KEGG pathway analysis, and protein-protein interaction networks. The screening criteria were: fold change > 1.2 and p < 0.05. The test results are as follows: Figure 2 China A Figure 2 B, Figure 2 C, Figure 2 D, Figure 2 E, Figure 2 China F, Figure 2 China G, Figure 2 H, Figure 2 As shown in Figure I.

[0044] Figure 2 China A to Figure 2 The volcano plot in the middle C shows significant proteomic alterations among the groups. Figure 2 As shown in Figure A, compared with the IgG1+ saline group, IgG1+ morphine treatment upregulated 98 proteins and downregulated 80 proteins, indicating that the molecular response induced by morphine was very strong. Figure 2 As shown in Figure B, compared with the IgG1 + morphine group, bevacizumab + morphine treatment resulted in greater changes in protein expression, with 236 proteins upregulated and 54 proteins downregulated. Similarly, from Figure 2 As shown in the middle C, compared with the IgG1+saline group, the bevacizumab+morphine group had 282 proteins upregulated and 86 proteins downregulated, indicating that bevacizumab blocking VEGF significantly altered the morphine-related proteome pattern in mPFC.

[0045] Figure 2 The Venn diagram analysis further confirmed that there was partial overlap but significant differences in the proteomes of the IgG1+morphine group and the bevacizumab+morphine group, indicating that bevacizumab intervention acts on different molecular networks.

[0046] Figure 2 Hierarchical cluster analysis in the middle E model confirmed significant separation among the three treatment groups, reinforcing the significant effect of bevacizumab on protein expression in mPFC.

[0047] Figure 2 Enrichment analysis of the KEGG pathway in the middle F group showed that differentially expressed proteins between the IgG1+Saline group and the IgG1+Morphine group were not enriched in the autophagy pathway, but Figure 2 China G and Figure 2 Enrichment analysis of the KEGG pathway in H1N1 revealed that differentially expressed proteins between the Beva+Morphine group and the IgG1+Morphine group, as well as between the IgG1+Saline group and the Beva+Morphine group, were primarily enriched in the autophagy pathway. Specifically, bevacizumab plus morphine treatment was associated with upregulation of proteins related to autophagy, endocytosis, immune activation, complement cascade, and amino acid metabolism, while proteins related to neuronal signaling and synaptic plasticity were downregulated. Importantly, Figure 2 The protein-protein interaction (PPI) network mapping in the middle I model highlights TSC1 (tuberous sclerosis complex 1) as a key pivot protein in the bevacizumab regulatory network. TSC1 was significantly downregulated in the bevacizumab + morphine group, and this protein interacts strongly with proteins involved in the mTOR signaling pathway and autophagy-related proteins. Given that TSC1 is a key inhibitor of mTOR activity, the reduced TSC1 expression level in the bevacizumab + morphine group compared to the IgG1 + morphine group suggests that bevacizumab may reduce the analgesic effect of morphine by inhibiting mTOR-dependent autophagy in the medial prefrontal cortex (mPFC).

[0048] These findings suggest that bevacizumab induces extensive proteomic remodeling of the mPFC during morphine exposure, and the inhibited autophagy pathway may be the mechanism underlying the observed reduced analgesic effect and accelerated tolerance in vivo.

[0049] 4. Animal experiments have confirmed the pathway by which bevacizumab induces opioid tolerance. This study investigated whether bevacizumab could mediate changes in mTOR signaling pathway regulation and autophagy levels by inhibiting TSC1 expression in the medial prefrontal cortex (mPFC). Multiscale analysis was performed on morphine-treated mice using Western blotting, immunofluorescence staining, and transmission electron microscopy. Specific tests are as follows: (1) Western blot: Mice were sacrificed, and mouse mPFC tissue was collected. Proteins were extracted from the mPFC tissue using RIPA lysis buffer, and the concentration of the proteins was determined by the BCA method. Subsequently, the proteins were separated by SDS-PAGE electrophoresis and transferred to a membrane. Since mTOR is a known negative regulator of autophagy, classical autophagy markers were then detected. Specifically, the proteins were incubated with specific primary antibodies against target proteins (such as LC3B-I, LC3B-II, p62 autophagy, BECN1, ATG5, ATG7, and ATG16L1), and then developed with horseradish peroxidase (HRP)-labeled secondary antibodies. Gray-scale quantitative analysis was performed using ImageJ software, with Tubulin as the baseline. The protein immunoblotting images are shown below. Figure 3 As shown in Figure A, the three columns on the left represent the results of three parallel samples in the IgG1 + Morphine group, and the three columns on the right represent the results of three parallel samples in the Bevacizumab + Morphine group; subsequently... Figure 3 Quantitative analysis of protein A was performed, and the results are as follows: Figure 3 B, Figure 3 C, Figure 3 D, Figure 3 E, Figure 3 China F, Figure 3 China G, Figure 3 H, Figure 3 As shown in Figure I. In the figure, p-mTOR refers to phosphorylated mTOR; p-RPS6 refers to phosphorylated ribosomal protein S6, and RPS6 refers to ribosomal protein S6; LC3B microtubule-associated protein 1 light chain 3β; LC3B-I is the cytoplasmic soluble form of LC3B, present in the cytoplasm of normal cells; LC3B-II is the membrane-bound form of LC3B after phosphatidylethanolamine modification, which anchors to the autophagosome membrane, and its expression level is positively correlated with the number of autophagosomes. In experiments, it is often measured by detecting the ratio of LC3B-I / LC3B-II. The LC3B-I / II ratio is used to assess the level of autophagy activity. p62 refers to SQSTM1 protein (Sequestosome-1), BECN1 refers to autophagy-related gene 6 homolog, ATG5 refers to autophagy protein 5, ATG7 refers to ubiquitin-like modification activator enzyme, ATG16L1 refers to autophagy-related protein 16-1, and Tubulin refers to tubulin. p-MTOR: MTOR represents the ratio of p-MTOR to MTOR levels and is an important indicator of mTOR signaling pathway activation. p-RPS6: RPS6 represents the ratio of p-RPS6 to RPS6 levels and is an important indicator of RPS6 signaling pathway activation.

[0050] Depend on Figure 3 China A Figure 3 China B and Figure 3As shown in Figure C, bevacizumab significantly increased the phosphorylation levels of p-mTOR (phosphorylated mTOR) and its downstream effector p-RPS6. These results indicate that upregulation of TSC1 leads to an increase in p-mTOR and p-RPS6, meaning that TSC1 upregulation specifically inhibits mTOR pathway activity.

[0051] also, Figure 3 China A Figure 3 China D and Figure 3 The study also showed that bevacizumab treatment led to a decrease in LC3B-I / II levels and an increase in p62 levels, indicating that the impaired autophagic flux was due to impaired autophagosome degradation rather than formation inhibition. Figure 3 China A Figure 3 China F to Figure 3 As can be seen from the data in section I, in contrast to the changes in LC3B-I / II and p62, the protein levels of key autophagy initiation and extension components, such as autophagy protein 5 (ATG5), ubiquitin-like modification activator ATG7 (ATG7), autophagy-associated protein 16-1 (ATG16L1), and BECN1, remained unchanged after bevacizumab treatment.

[0052] (2) Immunofluorescence staining: Mice underwent cardiac perfusion with phosphate-buffered saline (PBS) containing 4% (w / v) paraformaldehyde (PFA). After perfusion, brain tissue was separated and frozen sections were prepared. The brain sections were permeabilized, blocked, and incubated overnight with specific primary antibodies (such as anti-TSC1 and anti-NeuN). The secondary fluorescent antibodies included Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) and Goat Anti-Mouse IgG H&L (Alexa Fluor® 647). The nuclei were counterstained with DAPI. The localization and expression levels of target proteins (p-mTOR, p-RPS6, NeuN) in specific neurons of the mPFC were observed using laser confocal microscopy. The results are as follows: Figure 3 J, Figure 3 K Figure 3 As shown in L, DAPI (4',6-diamidino-2-phenylindole) represents nuclear staining, which appears as a blue signal under a fluorescence microscope. Its purpose is to mark all cell nuclei in the image.

[0053] Depend on Figure 3 China J and Figure 3 The study revealed that in the bevacizumab + morphine group, the expression of p-mTOR and p-RPS6 in neurons (NeuN) of the medial prefrontal cortex (mPFC) was significantly increased in NeuN-positive cells. These data support neuron-specific inhibition of the mTOR / RPS6 pathway.

[0054] In addition, by Figure 3 As shown in Figure L, immunofluorescence imaging further revealed a significant increase in neuronal p62 accumulation, reinforcing the view that autophagic flux is blocked during the degradation phase.

[0055] (3) Transmission electron microscopy observation: mPFC tissue from mice treated with bevacizumab was obtained, fixed with glutaraldehyde and osmium tetroxide, dehydrated, embedded in epoxy resin, and then ultrathin sections were prepared and stained with uranyl acetate and lead citrate. The ultrastructural changes of organelles such as autophagosomes and mitochondria in neurons were observed under a transmission electron microscope. The results are as follows: Figure 3 As shown in Figure M; subsequently, quantitative analysis was performed on the number of secondary lysosomes (SL) and autophagosomes, and the results are shown in Figure M. Figure 3 N and Figure 3 As shown in the middle O.

[0056] Depend on Figure 3 As can be seen from the M, transmission electron microscopy provided ultrastructural evidence of autophagic flux disruption. Compared with the IgG1+Morphine group, neurons in mice treated with bevacizumab+Morphine showed a significant reduction in the number of secondary lysosomes and an increase in the number of autophagosomes in the medial prefrontal cortex (mPFC).

[0057] Depend on Figure 3 N and Figure 3 As can be seen from the results, quantitative analysis confirmed that the number of secondary lysosomes increased significantly after treatment with bevacizumab + Morphine, while the number of autophagosomes decreased. Figure 3 Cellular diagram of M and Figure 3 The statistical results of O in the study showed that autophagosome maturation and lysosomal degradation were enhanced.

[0058] Based on the above findings, bevacizumab treatment leads to downregulation of TSC1 expression and activation of the mTOR / RPS6 pathway, while blocking the late stage of autophagy. This may be due to lysosomal dysfunction or impaired proteolytic activity.

[0059] Example 2: The effect of rapamycin on anti-angiogenic drug therapy on opioid tolerance To pharmacologically verify the role of the mTOR signaling pathway in bevacizumab-induced morphine analgesia and neuronal autophagy, healthy C57BL / 6 mice were divided into three groups: "Beva+Saline+Saline", "Beva+Saline+Morphine", and "Beva+Rapa+Morphine", with eight mice in each group. "Rapa" refers to rapamycin, purchased from MedChemExpress, catalog number HY-10219.

[0060] In the "Beva+Saline+Saline" group, mice were treated with bevacizumab (5 mg / kg, 0.1 ml / 10 g body weight, via tail vein injection) on day 1, followed by saline (0.1 ml / 10 g body weight, via intraperitoneal injection) 0.5 h later. On days 2 and 3, they were treated with saline (0.1 ml / 10 g body weight, via intraperitoneal injection). On day 4, after intraperitoneal injection of saline, the mice were treated with saline (0.1 ml / 10 g body weight, via subcutaneous injection) 0.5 h later and 0.5 h later. Mice in the “Beva+Saline+Morphine” group were treated with bevacizumab (5 mg / kg, 0.1 ml / 10 g body weight, via tail vein injection) on day 1, followed by saline (0.1 ml / 10 g body weight, via intraperitoneal injection) 0.5 h later. On days 2 and 3, they were treated with saline (0.1 ml / 10 g body weight, via intraperitoneal injection). On day 4, after intraperitoneal injection of saline (0.1 ml / 10 g body weight) and 0.5 h later, they were treated with morphine (10 mg / kg, 0.1 ml / 20 g body weight, via subcutaneous injection). Mice in the “Beva+Rapa+Morphine” group were treated with bevacizumab (5 mg / kg, 0.1 mL / 10 g body weight, via tail vein injection) on day 1, followed by rapamycin (2 mg / kg, 0.1 mL / 10 g body weight, via intraperitoneal injection) 0.5 h later. Rapaycin was administered on days 2 and 3 (2 mg / kg, 0.1 mL / 10 g body weight, via intraperitoneal injection) on day 4. After intraperitoneal injection of rapamycin (2 mg / kg, 0.1 mL / 10 g body weight), morphine (10 mg / kg, 0.1 mL / 20 g body weight, via subcutaneous injection) was administered 0.5 h later. according to Figure 4 The experimental design in the study A evaluated the behavioral and molecular results of pain: On day 0, the thermal pain threshold and comprehensive pain response were measured using the tail-flick test and hot plate test, respectively, and recorded as the baseline response (Baleline, abbreviated as BL). On day 1, the mice were divided into groups and treated with the corresponding drugs. The tail-flick test and hot plate test were performed on days 2 and 3, respectively. On day 4, the mice were treated with the corresponding drugs, and the tail-flick test and hot plate test were performed at 15 minutes, 30 minutes, 1 hour, 1.5 hours, 2 hours, and 3 hours after the injection of morphine or saline.

[0061] The pain behavior testing methods were the same as those in Example 1, specifically (1) the tail-flick test and (2) the hot plate test, and the results were as follows: Figure 4 China B and Figure 4 As shown in C. The molecular testing methods are the same as those in Example 1, 4: (1) Western blotting, (2) immunofluorescence staining, and (3) transmission electron microscopy. The results are as follows. Figure 4 D, Figure 4 E, Figure 4 China F, Figure 4 China G, Figure 4 H, Figure 4 Middle I, Figure 4 J, Figure 4 K Figure 4 L, Figure 4 M and Figure 4 As shown in N.

[0062] Depend on Figure 4 China B and Figure 4 The behavioral results of pain analysis in the C-cell study showed that bevacizumab significantly attenuated the analgesic effect in the morphine-induced hot plate and tail-flick tests, as evidenced by a shortened withdrawal latency. Notably, compared to the "Beva+Saline+Morphine" group, the "Beva+Rapa+Morphine" group exhibited significantly prolonged latency in both tests, indicating that rapamycin can improve bevacizumab-induced morphine analgesia impairment.

[0063] Figure 4 In the Western blot analysis results of the protein samples from the "Beva+Saline+Saline" group, the left four columns represent the results of four parallel samples from the "Beva+Saline+Morphine" group, the middle four columns represent the results of four parallel samples from the "Beva+Saline+Morphine" group, and the right four columns represent the results of four parallel samples from the "Beva+Rapa+Morphine" group. The results indicate that: Figure 4 D, Figure 4 E, Figure 4 China F and Figure 4 As shown in the data from G, rapamycin treatment reversed the bevacizumab-induced elevation of p-mTOR and p-RPS6 levels without affecting total protein levels. Furthermore, from... Figure 4 H and Figure 4 As shown in the first part, rapamycin reduced the accumulation of p62 and enhanced the expression of LC3B-I / II, indicating that autophagic flux was restored and pain was relieved.

[0064] Depend on Figure 4 J, Figure 4Immunofluorescence staining of mPFC in mice showed that, compared with the "Beva+Saline+Morphine" group, the expression of p-mTOR, p-RPS6 and p62 in the neurons of mice in the "Beva+Rapa+Morphine" group was reduced, which supports the inhibition of mTOR pathway activity and the normalization of autophagy at the cellular level.

[0065] Depend on Figure 4 Transmission electron microscopy revealed significant changes in the autophagic structure of neurons in all groups. In the "Beva+Rapa+Morphine" group, lysosomal abundance was restored and the number of lysosomal bodies (LCs) decreased, indicating that rapamycin treatment effectively activated the autophagic degradation process. Figure 4 M and Figure 4 Quantitative analysis of N in the brain confirmed this finding: compared with the "Beva+Saline+Morphine" group, the number of neuronal autophagosomes in the "Beva+Rapa+Morphine" group was significantly increased, supporting the recovery of lysosomal function. These results indicate that the autophagic degradation function impaired by the combination therapy of bevacizumab and morphine was effectively restored.

[0066] In summary, rapamycin treatment effectively restored the analgesic effect of morphine by inhibiting the mTOR / RPS6 signaling pathway in the medial prefrontal cortex (mPFC) and reactivated the autophagic degradation process that had been impaired by the combined treatment of bevacizumab and morphine.

[0067] It should be noted that all quantitative data are expressed as mean ± standard error. Statistical software such as GraphPad Prism was used, and a two-tailed Student was selected according to the experimental design. t The analysis of differences between groups should be performed using either a two-way or one-way ANOVA (for comparisons between multiple groups), combined with post-hoc tests (such as Bonferroni correction). "*" indicates a significant difference (P < 0.05); "**" indicates a highly significant difference (P < 0.01); "***" indicates an extremely significant difference (P < 0.001); "****" indicates an extremely significant difference (P < 0.0001). Similarly, "#" indicates a significant difference (P < 0.05); "##" indicates a highly significant difference (P < 0.01); "###" indicates an extremely significant difference (P < 0.001); and "####" indicates an extremely significant difference (P < 0.0001).

[0068] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the patent protection scope of the present invention.

Claims

1. The use of rapamycin in the preparation of an anti-angiogenic drug-induced opioid tolerance reliever.

2. The use of rapamycin as described in claim 1 in the preparation of an anti-angiogenic drug tolerance reliever, characterized in that, Rapamycin works by inhibiting the mTOR / RPS6 pathway.

3. The use of rapamycin as described in claim 1 in the preparation of an anti-angiogenic drug tolerance reliever, characterized in that, The anti-angiogenic drugs include at least one of bevacizumab, ramoximab, and ranibizumab.

4. The use of rapamycin as described in claim 1 in the preparation of an anti-angiogenic drug tolerance reliever, characterized in that, The opioid drugs include at least one of morphine, fentanyl, pethidine, oxycodone, and methadone.

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