Use of dorsomorphin in the preparation of a drug for treating neuropathic pain caused by paclitaxel

Abstract

The application belongs to the technical field of medicine, and discloses application of Dorsomorphin in preparation of a drug for treating neuropathic pain caused by paclitaxel. The application establishes a paclitaxel-induced mouse neuropathic pain model, and confirms that Dorsomorphin can dose-dependently relieve paclitaxel-induced mechanical hyperalgesia, thermal hyperalgesia and cold hyperalgesia. The mechanism of action includes inhibiting up-regulation of spinal cord NF-kappa B p65 protein expression, inhibiting microglial cell activation, down-regulating Scn1a protein expression, inhibiting up-regulation of lysophosphatidic acid expression and reducing serum pro-inflammatory factor levels. At the same time, Dorsomorphin and paclitaxel are used in combination to synergistically enhance the inhibitory effect on lung adenocarcinoma cell proliferation and migration, and do not weaken the anti-tumor invasion activity of paclitaxel. The application provides a new treatment option for paclitaxel-induced neuropathic pain, and has the dual clinical value of analgesia and synergistic anti-tumor.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of Dorsomorphin in the preparation of drugs for treating paclitaxel-induced neuropathic pain. Background Technology

[0002] Paclitaxel is a widely used anticancer drug in clinical practice, and it has shown significant efficacy in the treatment of various malignant tumors such as lung cancer, ovarian cancer, and breast cancer. However, paclitaxel often causes paclitaxel-induced neuropathic pain, with an incidence rate as high as 30% to 70%. Clinical manifestations include sensory abnormalities, motor disorders, persistent pain, and abnormal temperature sensitivity, which seriously affect the patient's quality of life.

[0003] Paclitaxel inhibits tumor growth by promoting tubulin polymerization, stabilizing microtubule architecture, and interfering with the mitotic cycle of cancer cells. However, it has toxic effects on the peripheral nervous system, and the mechanism by which it induces peripheral neuropathy is not fully understood. Potential mechanisms proposed by existing studies include: inducing changes in mitochondrial membrane potential and calcium ion release, leading to increased levels of reactive oxygen species and oxidative stress damage; regulating inflammatory channel-related signaling pathways or cytokines, promoting the expression of pro-inflammatory factors, and inducing neuroinflammatory responses; regulating the expression of pro-inflammatory cytokines through the NF-κB signaling pathway, promoting glial cell activation; affecting the production of lysophosphatidic acid, activating lysophosphatidic acid receptors and inducing pain; and altering ion channel function, manifested as upregulation of sodium and calcium channels and downregulation of potassium channels, leading to abnormally increased neuronal excitability.

[0004] Currently, there are limited treatment options for paclitaxel-induced neuropathic pain in clinical practice. The American Cancer Society guidelines recommend duloxetine as a first-line treatment, but long-term use may lead to drug dependence and cardiovascular adverse reactions. Other existing analgesics, such as opioids, have limitations in efficacy and adverse reactions, and the clinical need remains unmet.

[0005] Dorsomorphin, also known as Compound C, is a potent, reversible, selective AMPK inhibitor that competes for AMPK's ATP binding site and is also a dual inhibitor of the BMP signaling pathway. Known uses of Dorsomorphin in the prior art include: its role as a research tool for inhibiting AMPK and ALK protein kinases; its antitumor activity in various tumor models; its inhibition of ICAM-1 and VCAM-1 expression in inflammatory responses by suppressing NF-κB activity, PI3K phosphorylation, and p38MAPK; its application in the development of drugs for treating Guillain-Barré syndrome by promoting axonal regeneration in damaged nerve cells; and its application in the development of drugs for suppressing inflammatory pain by inhibiting the p38MAPK / c-fos and NF-κB signaling pathways to achieve analgesia.

[0006] It should be noted that the pathogenesis of paclitaxel-induced neuropathic pain involves neuronal damage caused by paclitaxel's unique microtubule stabilizing effect, which is fundamentally different from the etiology and pathological mechanism of general inflammatory pain. To date, the application of Dorsomorphin in paclitaxel-induced neuropathic pain has not been reported. Therefore, developing a novel drug that can effectively alleviate this condition without affecting the anti-tumor effects of paclitaxel is of significant clinical importance. Summary of the Invention

[0007] To overcome the above-mentioned technical problems, the present invention provides the application of Dorsomorphin in the preparation of drugs for treating paclitaxel-induced neuropathic pain.

[0008] This invention establishes a paclitaxel-induced mouse model of neuropathic pain, systematically studies the effects of different doses of Dorsomorphin on pain behavior, and elucidates its mechanism of action at the molecular and cellular levels. Simultaneously, in vitro cell experiments verify the effects of combined use of Dorsomorphin and paclitaxel on the biological behavior of tumor cells.

[0009] The specific technical solution is as follows: Animal model construction and dosing regimen A paclitaxel-induced neuropathic pain animal model was established using SPF-grade adult male Kunming mice. Paclitaxel was administered intraperitoneally on days 1, 3, 5, and 7. Different doses of Dorsomorphin were injected intraperitoneally into the model animals on days 2, 4, 6, 8, 10, 12, 14, and 16, and the analgesic effect was evaluated through behavioral experiments.

[0010] Mechanism of action study Differentially expressed genes were screened using transcriptome sequencing and validated using quantitative real-time polymerase chain reaction (qPCR). Western blotting, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA) were employed to detect the expression levels of related proteins and signaling molecules in spinal cord tissue, as well as the levels of inflammatory factors in serum, to clarify the target and regulatory pathway of Dorsomorphin.

[0011] Verification of combined anti-tumor effects Human lung adenocarcinoma A549 cells were used. Cell proliferation was detected by CCK-8 assay, and cell migration and invasion were detected by Transwell assay. The antitumor effects of Dorsomorphin, paclitaxel alone and in combination were evaluated.

[0012] Compared with the prior art, the beneficial effects of the present invention are: Providing new clinical treatment options Dorsomorphin can effectively relieve paclitaxel-induced neuropathic pain, providing a new drug option for the clinical treatment of this condition.

[0013] Multi-target regulation, stable analgesic effect Dorsomorphin exerts its analgesic effect through a multi-target mechanism, including inhibiting NF-κB signaling pathway activation to reduce the release of pro-inflammatory factors, inhibiting spinal microglia activation, downregulating the expression of the sodium channel-associated protein Scn1a, and inhibiting the upregulation of lysophosphatidic acid expression. This multi-target mechanism of action makes its analgesic effect superior to single-target drugs and is expected to avoid the tolerance and dependence problems common to existing analgesics.

[0014] Synergistically enhances the effect of chemotherapy, achieving multiple uses of one drug. Dorsomorphin not only does not weaken the inhibitory effect of paclitaxel on tumor cell invasion, but also synergistically enhances the inhibitory effect of paclitaxel on tumor cell proliferation and migration. This characteristic means that while relieving chemotherapy-induced neuropathic pain, Dorsomorphin can further enhance the anti-tumor efficacy of paclitaxel, demonstrating its clinical value as a single drug with multiple uses.

[0015] It has good drug-like properties and outstanding transformation potential. Dorsomorphin is a small molecule compound with stable physicochemical properties, providing a good foundation for drug development. This invention provides sufficient experimental evidence for the clinical translation of this compound through animal behavioral verification, molecular mechanism elucidation, and cellular functional verification. Attached Figure Description

[0016] Figure 1 DM alleviates PTX-induced mechanical hyperalgesia (MWT changes at different DM doses). The mechanical pain threshold of mice was measured using a Von Frey fiber analgesic at days 2, 4, 6, 8, 10, 12, 14, and 16 after PTX administration, before administration (day 0). Data are expressed as mean ± standard error (n=6–8). Compared with the Vehicle group, the MWT in the PTX group was significantly decreased (#P<0.05, ##P<0.01, ###P<0.001); compared with the PTX group, the MWT in the PTX+DM (4 mg / kg) and PTX+DM (8 mg / kg) groups was significantly increased (*P<0.05, **P<0.01, ***P<0.001). Bonferroni test was used after two-way ANOVA.

[0017] Figure 2DM alleviates PTX-induced thermal hyperalgesia. Mechanical pain thresholds (TWL) in mice were measured using a hot plate test on days 2, 4, 6, 8, 10, 12, 14, and 16 after PTX administration, before administration (day 0). Data are expressed as mean ± standard error (n=6–8). Compared with the Vehicle group, the TWL in the PTX group was significantly decreased (##P<0.01, ###P<0.001); compared with the PTX group, the TWL in the PTX+DM (2 mg / kg), PTX+DM (4 mg / kg), and PTX+DM (8 mg / kg) groups was significantly increased (*P<0.05, **P<0.01, ***P<0.001). Two-way ANOVA was performed with Bonferroni post-hoc test.

[0018] Figure 3 DM alleviates PTX-induced cold-induced hypersensitivity. The total score of mice was measured using the acetone assay on days 2, 4, 6, 8, 10, 12, 14, and 16 after PTX administration, before administration (day 0). Compared with the Vehicle group, the total score in the PTX group was significantly higher (#P<0.05, ##P<0.01, ###P<0.001); compared with the PTX group, the total scores in the PTX+DM (2 mg / kg), PTX+DM (4 mg / kg), and PTX+DM (8 mg / kg) groups were significantly lower (**P<0.01, ***P<0.001). Data are expressed as mean ± standard error (n=6–8). Bonferroni test was used after two-way ANOVA.

[0019] Figure 4 DM inhibited the increase in IL-6, IL-1β, and TNF-α induced by paclitaxel. After the behavioral experiment, the serum levels of IL-6, IL-1β, and TNF-α in mice were detected by ELISA. (A) Expression level of IL-1β; (B) Expression level of IL-6; (C) Expression level of TNF-α. Data are expressed as mean ± standard error (n=4). *P<0.05, **P<0.01. Bonferroni test was used after one-way ANOVA.

[0020] Figure 5 DM alleviated the upregulation of Iba1, NF-κB p65, and Scn1a protein expression induced by PTX. (A) Western blot images of Iba1 and β-Actin. (B) Quantitative analysis of Iba1 expression. (C) Western blot images of p65, Scn1a, and β-Actin. (D) Quantitative analysis of p65 and Scn1a expression. Protein levels were calibrated for β-Actin. Data are expressed as mean ± standard error (n=3). *P<0.05, **P<0.01, ***P<0.001. Bonferroni test was used after one-way or two-way ANOVA.

[0021] Figure 6 DM inhibited the upregulation of LPA. Immunohistochemical results of LPA in the vehicle group (A), PTX group (B), and PTX+DM (8 mg / kg) group (C). Data are expressed as mean ± SEM (n=4). ***P<0.001. Bonferroni test was used after one-way ANOVA.

[0022] Figure 7 (AC) Effects of different concentrations of PTX, DM, and the combined use of PTX and DM on cell proliferation; OD values ​​were measured using the CCK-8 assay at 24, 48, and 72 hours. (D) Effects of the combined use of 2.5 nM PTX, 2.5 μM DM, and 2.5 nM PTX and 2.5 μM DM on cell proliferation. (E) Effects of the combined use of 5 nM PTX, 5 μM DM, and 5 nM PTX and 5 μM DM on cell proliferation. (F) Effects of the combined use of 10 nM PTX, 10 μM DM, and 10 nM PTX and 10 μM DM on cell proliferation. Data are presented as mean ± standard error (n=3). Compared with the Vehicle group, *P<0.05, **P<0.01, ***P<0.001 (Figure AF); compared with the 5 nM PTX group, ###P<0.001 (Figure E); compared with the 10 nM PTX group, ###P<0.001 (Figure F). Two-way ANOVA was used, and Bonferroni post-hoc tests were employed.

[0023] Figure 8 Effects of DM, PTX, and PTX+DM on migration and invasion of A549 cells. (A) Representative images (×100x) of crystal violet-stained migration (top) and invasion (bottom) in the Vehicle group, 5nM PTX group, 5μM DM group, and 5nM PTX+5μM DM group. (B) Quantitative analysis of cells in the migration experiment. (C) Quantitative analysis of cells in the invasion experiment. Data are expressed as mean ± standard error (n=3). ns P>0.05, 0.001***P<0.001. Two-way ANOVA was performed with Bonferroni correction. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0025] Example 1: Establishment of a mouse model of neuropathic pain induced by paclitaxel laboratory animals SPF-grade adult male Kunming mice, weighing between 18 and 20 grams, were used and housed in a temperature- and humidity-controlled barrier environment with free access to food and water.

[0026] Model building Mice were randomly divided into a control group and a model group. Mice in the model group received intraperitoneal injections daily on days 1, 3, 5, and 7, with a dose of 8 mg / kg body weight of paclitaxel. Mice in the control group received an equal volume of solvent intraperitoneally during the same period.

[0027] Model Validation The mechanical withdrawal reflex threshold of mice was measured using the Von Frey analgesia test before administration and on days 2, 4, 6, 8, 10, 12, 14, and 16. The latency of the thermal withdrawal reflex was measured using the hot plate test, and the cold stimulation pain response was evaluated using the acetone test. Each group consisted of 6 to 8 animals. Experimental data are expressed as mean ± standard error and were analyzed using a two-way ANOVA followed by a Bonferroni post-hoc test. The results showed that compared with the control group, the mechanical and thermal pain thresholds of the model group mice were significantly reduced, while the cold stimulation pain score was significantly increased. These differences were statistically significant, indicating that the paclitaxel-induced neuropathic pain model was successfully established.

[0028] Example 2: The relieving effect of Dorsomorphin on paclitaxel-induced neuropathic pain Experimental grouping and drug administration Mice that successfully modeled the disease were randomly divided into four groups: a paclitaxel model group, a paclitaxel combined with low-dose Dorsomorphin group, a paclitaxel combined with medium-dose Dorsomorphin group, and a paclitaxel combined with high-dose Dorsomorphin group. A normal control group was also included. Each group consisted of 6 to 8 mice. Dorsomorphin was administered at doses of 2, 4, and 8 mg per kilogram of body weight, and was given intraperitoneally once daily on days 2, 4, 6, 8, 10, 12, 14, and 16. The control group and the model group received the same volume of solvent during the same period.

[0029] Behavioral testing Using the same detection method as in Example 1, the mechanical pain threshold, thermal pain threshold, and cold pain response scores of mice in each group were measured at corresponding time points. Experimental data are expressed as mean ± standard error, and Bonferroni post-hoc tests were performed after two-way ANOVA.

[0030] Experimental results Mechanical pain assessment results showed that the mechanical pain thresholds in the paclitaxel combined with medium-dose Dorsomorphin group from day 6 to day 16 and the paclitaxel combined with high-dose Dorsomorphin group from day 4 to day 16 were significantly higher than those in the paclitaxel model group, and exhibited a clear dose-dependent effect.

[0031] The results of thermal pain detection showed that the latency of thermal pain in the paclitaxel combined with medium-dose Dorsomorphin group, the paclitaxel combined with medium-dose Dorsomorphin group from day 8 to day 16, and the paclitaxel combined with high-dose Dorsomorphin group from day 4 to day 16 were significantly longer than those in the paclitaxel model group, and the analgesic effect increased with increasing dose.

[0032] The results of cold pain testing showed that the cold stimulation scores of the paclitaxel combined with low-dose, medium-dose, and high-dose Dorsomorphin groups were significantly lower than those of the paclitaxel model group from day 4 to day 16, which can effectively relieve cold pain hypersensitivity.

[0033] The above results confirm that Dorsomorphin can alleviate various symptoms of hyperalgesia induced by paclitaxel in a dose-dependent manner.

[0034] Example 3: Study on the mechanism of Dorsomorphin in relieving neuropathic pain Sample collection After the behavioral experiment on day 16, serum and spinal cord tissue samples were collected from mice in each group and stored at low temperature for later use.

[0035] Transcriptome sequencing and gene validation Transcriptome sequencing was performed on spinal cord tissues from mice in the solvent control group, paclitaxel model group, and paclitaxel combined with high-dose Dorsomorphin group to screen for differentially expressed genes. RT-qPCR was used to validate the screened differentially expressed genes and clarify the gene expression trends after Dorsomorphin intervention.

[0036] Serum inflammatory factor detection The levels of IL-1β, IL-6, and TNF-α in serum were detected using enzyme-linked immunosorbent assay (ELISA). Four mice were used in each group. Data are expressed as mean ± standard error, and Bonferroni test was performed after one-way ANOVA. Results showed that the levels of the three inflammatory factors in the serum of mice in the paclitaxel model group were significantly increased. After intervention with Dorsomorphin, the levels of the three inflammatory factors were significantly downregulated, indicating that Dorsomorphin can inhibit the systemic inflammatory response induced by paclitaxel.

[0037] Spinal cord tissue protein expression detection The protein expression levels of NF-κB p65, Iba1, and Scn1a in spinal cord tissue were detected by Western blotting. Each group consisted of three mice. Protein levels were calibrated by β-Actin. Data are expressed as mean ± standard error. One-way or two-way ANOVA was performed followed by Bonferroni test. Results showed that the expression of all three proteins was significantly upregulated in the spinal cord of mice in the paclitaxel model group. After Dorsomorphin intervention, the expression levels of all three proteins were significantly reduced, indicating that Dorsomorphin can inhibit NF-κB signaling pathway activation, inhibit microglia activation, and downregulate the expression of sodium channel-related proteins.

[0038] Spinal cord lysophosphatidylcholine detection Immunohistochemistry was used to detect the expression level of lysophosphatidylcholine in spinal cord tissue. Four mice were used in each group. Data are expressed as mean ± standard error. One-way ANOVA was performed followed by Bonferroni test. Results showed that lysophosphatidylcholine expression was significantly increased in the spinal cord of mice in the paclitaxel model group. After Dorsomorphin intervention, the expression level of lysophosphatidylcholine significantly decreased, indicating that Dorsomorphin can inhibit paclitaxel-induced activation of the lysophosphatidylcholine pathway.

[0039] Example 4: Verification of the antitumor effect of the combination of Dorsomorphin and paclitaxel Cells and Grouping Human lung adenocarcinoma A549 cells were used to set up a control group, a paclitaxel-only group, a dorsomorphin-only group, and a paclitaxel-dorsomorphin combination group. A gradient of paclitaxel concentrations (2.5, 5, and 10 nM) was established, corresponding to dorsomorphin concentrations of 2.5, 5, and 10 μM. Each group had three replicates.

[0040] Cell proliferation detection Cell proliferation was assessed at 24, 48, and 72 hours using the CCK-8 assay. Data are expressed as mean ± standard error, and Bonferroni post-hoc tests were performed after two-way ANOVA. Results showed that the combined administration of paclitaxel at corresponding concentrations significantly enhanced the inhibitory effect on A549 cell proliferation compared to paclitaxel alone. Specifically, the combination of 5 nM and 10 nM paclitaxel with corresponding concentrations of Dorsomorphin resulted in statistically significant differences in the inhibitory effect, confirming a synergistic inhibitory effect on tumor cell proliferation.

[0041] Cell migration and invasion detection Transwell assays were used to detect cell migration and invasion abilities, with three replicates per group. Data are expressed as mean ± standard error and were analyzed using two-way ANOVA followed by Bonferroni correction. Results showed that the combination of 5 nM paclitaxel and 5 μM dorsomorphin significantly inhibited cell migration compared to paclitaxel alone. Regarding invasion ability, there was no significant difference in inhibitory effect between the combination group and the paclitaxel-alone group, indicating that dorsomorphin does not weaken the inhibitory effect of paclitaxel on tumor cell invasion.

[0042] In summary, the combined use of Dorsomorphin and paclitaxel can synergistically enhance the inhibitory effect on tumor cell proliferation and migration without affecting the anti-invasive activity of paclitaxel, demonstrating clinical potential for combined drug therapy.

[0043] Although embodiments of the present invention have been shown and described, those skilled in the art will understand that various changes, modifications, substitutions and variations can be made to the above embodiments without departing from the principles and spirit of the present invention, the scope of which is defined by the claims and their equivalents.

Claims

1. Application of Dorsomorphin in the preparation of drugs for treating paclitaxel-induced neuropathic pain.

2. The application according to claim 1, characterized in that, The drug is used to relieve at least one of paclitaxel-induced mechanopathic hyperalgesia, thermal hyperalgesia, and cold hyperalgesia.

3. The application according to claim 1, characterized in that, Dorsomorphin exerts its therapeutic effect through at least one of the following pathways: inhibiting the upregulation of NF-κBp65 protein expression in the spinal cord, inhibiting the activation of microglia in the spinal cord, inhibiting the upregulation of Scn1a protein expression, inhibiting the upregulation of lysophosphatidic acid expression in the spinal cord, and reducing the levels of IL-1β, IL-6, and TNF-α in serum.

4. The application according to claim 1, characterized in that, The dosage of Dorsomorphin is 2 to 8 mg per kilogram of body weight, administered once daily.

5. The application according to claim 4, characterized in that, The dosage of Dorsomorphin is 4 to 8 mg per kilogram of body weight.

6. Application of the combination of Dorsomorphin and paclitaxel in the preparation of antitumor drugs.

7. The application according to claim 6, characterized in that, The tumor is lung adenocarcinoma.

8. The application according to claim 6, characterized in that, The combined use of Dorsomorphin and paclitaxel synergistically inhibits the proliferation and migration of tumor cells without weakening the inhibitory effect of paclitaxel on tumor cell invasion.

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