Application of indoleacetic acid in preparation of medicine for preventing or treating neuropathic toxicity caused by chemotherapeutic drugs

Abstract

The invention discloses an application of indoleacetic acid in preparation of a medicine for preventing or treating neuropathic toxicity caused by chemotherapeutics. Experimental results show that the indoleacetic acid (IAA) can significantly relieve pain sensitivity of mice caused by neuropathic toxicity (OIPN) caused by oxaliplatin (OXA), improve morphological damage of dorsal root ganglion (DRG) of the mice and reduce the inflammation level in colon of the mice; however, these benefits are significantly blocked when antagonists are used for the aromatic hydrocarbon receptor (AhR). This indicates that the therapeutic effect of IAA may depend on the activation of the aromatic hydrocarbon receptor (AhR) pathway. The invention provides a new method for treating neuropathic toxicity caused by chemotherapeutics, and expands the application of indoleacetic acid at the same time.

Description

Technical Field

[0001] The present invention relates to a medicine for preparing indoleacetic acid for preventing or treating neuropathic toxicity caused by chemotherapy drugs. Background Art

[0002] Currently, chemotherapy drugs used to treat tumors mainly include platinum drugs (such as carboplatin, cisplatin and oxaliplatin), taxane drugs (such as paclitaxel and docetaxel), eboxilone drugs (such as isabendone), vincristine alkaloids (such as vincristine and vinblastine), etc. However, neuropathic toxicity (OIPN) caused by chemotherapy drugs during chemotherapy has become the main obstacle to its clinical application. Long-term chemotherapy can cause patients to have symptoms such as numbness of hands and feet, significant chronic pain, etc., which seriously reduce the quality of life of patients. At present, the research on the case mechanism of OIPN has not been fully clarified. It may involve ion channel dysfunction, abnormal conduction of neural verification signal pathways, mitochondrial damage, oxidative stress, and intestinal microbial flora imbalance. For OIPN, glutathione, calcium magnesium mixture and sodium ion channel blockers are currently mainly used in combination, but the effect is not ideal. [1] Therefore, exploring effective OIPN prevention and treatment strategies is of great significance for improving the treatment effect and survival rate of cancer patients.

[0003] Although relatively little research has been conducted on the role of indoleacetic acid (IAA) in neuroinflammatory and neurodegenerative diseases, existing evidence suggests a link between tryptophan metabolism and neuroimmune responses. Based on this, the inventors believe that IAA has potential therapeutic value in modulating chemotherapy-induced neuropathic pain.

[0004] References:

[0005] [1] Chinese expert consensus on the diagnosis and treatment of chemotherapy-induced peripheral neuropathy (2022 edition): Chinese Journal of Oncology 2022, 44(9): 928-934. Summary of the Invention

[0006] One of the purposes of the present invention is to provide the use of indoleacetic acid in the preparation of a drug for preventing or treating neuropathic toxicity induced by chemotherapy drugs.

[0007] To achieve the above objectives, the present invention provides the following technical solution: the drug contains indoleacetic acid and is used in combination with chemotherapy drugs. The drug is used to relieve pain caused by tumor treatment and significantly reduce the neuropathic toxicity caused by chemotherapy drugs.

[0008] The chemotherapy drug is a cancer chemotherapy drug.

[0009] The cancer chemotherapy drug is selected from one or more of oxaliplatin, cisplatin, carboplatin, paclitaxel, vincristine, and vinblastine.

[0010] The chemotherapy drug is oxaliplatin.

[0011] The symptoms of neuropathic toxicity include one or more of the following: numbness, glove-like sensation, stocking-like sensation, burning pain, tingling in the hands and feet, and weakened or absent deep tendon reflexes.

[0012] The indoleacetic acid acts on the aryl hydrocarbon receptor (AhR).

[0013] This study established an OIPN mouse model using intraperitoneal injection of oxaliplatin. Using both intraperitoneal injection and oral gavage, the effects of IAA on pain sensitivity, dorsal root ganglion (DRG) morphological damage, and colon inflammation caused by OIPN in mice were investigated to explore whether indoleacetic acid could alleviate the pathological conditions caused by OIPN in mice. Furthermore, the effect of an aryl hydrocarbon receptor (AhR) antagonist on the therapeutic effects of IAA was studied using the OIPN mouse model, clarifying the signaling pathways that may underlie IAA's therapeutic effects.

[0014] The beneficial effects brought about by the technical solution provided by the present invention include at least:

[0015] 1) There is no effective treatment for OIPN. This study found that indoleacetic acid (IAA) can significantly reduce the neuropathic toxicity (OIPN) caused by chemotherapy drugs and improve patients' quality of life.

[0016] 2) Indoleacetic acid is a metabolite of tryptophan and has no biological toxicity. The present invention expands its new therapeutic uses. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The efficacy of IAA on OIPN symptoms in mice. (A) IAA preventive and therapeutic effects on OIPN in mice (n = 6); (B) Mouse body weight changes; (C) Mechanical pain threshold induced by Von Frey filament stimulation; (D) Escape behavior score induced by 4°C cold stimulation. Data are expressed as mean ± SEM. ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.

[0018] Figure 2 Effects of IAA treatment on DRG morphology in OIPN mice. (A) DRG hematoxylin-eosin staining (630×); (B) DRG nucleolar size statistics.

[0019] Figure 3 Effects of IAA treatment on myelin morphology in OIPN mice.

[0020] Figure 4 The effect of IAA on reducing colonic inflammation in OIPN mice. (A) Colonic hematoxylin-eosin staining (400×); (B) Colonic inflammation pathological score (n=4)

[0021] Figure 5 Effects of AhR antagonism on OIPN symptoms in mice. (A) Effects of AhR antagonism on OIPN symptoms in mice (n = 6); (B) Changes in mouse body weight; (C) Mechanical pain threshold induced by Von Frey filaments; (D) Escape behavior score induced by 4°C cold stimulation. Data are expressed as mean ± SEM. ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.

[0022] Figure 6 To show the effect of AhR antagonism on DRG morphology in OIPN mice. (A) DRG hematoxylin-eosin staining (630×); (B) DRG nucleolar size statistics (n=4).

[0023] Figure 7 To antagonize the effect of AhR receptor on myelin morphology in OIPN mice.

[0024] Figure 8 Antagonism of AhR receptors blocked the effect of IAA in reducing colon inflammation in OIPN mice. (A) Colon hematoxylin-eosin staining (400×); (B) Colon inflammation pathological score (n=4). DETAILED DESCRIPTION

[0025] To make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be described in further detail below.

[0026] Example 1: IAA antagonizes the occurrence of OIPN in mice

[0027] 1.1 Experimental drugs and preparation methods

[0028] (1) Preparation of Oxaliplatin Injection

[0029] Dissolve 5 mg of oxaliplatin powder in 5 mL of 5% glucose solution to a concentration of 1 mg / ml and store in a dark place at -20°C until use.

[0030] (2) Preparation of IAA solution

[0031] Dissolve 100 mg of IAA powder in 1 mol / L NaOH, adjust the pH to 7.4, and dilute to 10 mL with PBS for later use.

[0032] 1.2 Experimental animals

[0033] Experimental animals: C57BL / 6J mice (5 weeks old, weighing 16-18 g, all male, SPF grade, certificate number: A202411250281) were purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd. (license number: SCXK (Su) 2020-0009). The animals were housed in the SPF-grade experimental animal room of Jiangsu Institute of Traditional Chinese Medicine (license number: SYXK (Su) 2021-0025), with an ambient temperature of 22-25°C, a relative humidity of 50-60%, and a light intensity of 8:00-20:00, and were allowed to move freely, eat, and drink water. After one week of adaptive feeding, the mice were randomly divided into 24 groups, with 6 mice in each group. All procedures involving animals were approved by the Animal Care and Use Committee of Jiangsu Institute of Traditional Chinese Medicine.

[0034] 1.3 Experimental methods

[0035] 1.3.1 Animal grouping and modeling:

[0036] Animal model: After all mice were acclimated to a fixed environment for one week and baseline pain sensitivity was measured, they were randomly divided into the following four groups: normal group (Vehicle), model group (OXA), IAA intraperitoneal injection (50 mg / kg) IAAP group, and oral gavage (500 mg / kg) IAAG group.

[0037] Normal group: 5% glucose solution was injected into the abdominal cavity; PBS solution was gavaged every day.

[0038] Model group: 3 mg / kg oxaliplatin 5% glucose solution was intraperitoneally injected at a dose of 5 ml / kg, with 5 days of treatment and 5 days of rest, for a total of two cycles of 20 days; PBS solution was gavaged daily.

[0039] IAAP group: IAA (50 mg / kg) was intraperitoneally injected every day, followed by 3 mg / kg of oxaliplatin in 5% glucose solution at a dose of 5 ml / kg. Oxaliplatin was administered for 5 days and then stopped for 5 days, for a total of two cycles of 20 days.

[0040] IAAG group: IAA (500 mg / kg) was administered orally daily, followed by intraperitoneal injection of 3 mg / kg oxaliplatin in 5% glucose solution at a dose of 5 ml / kg. Oxaliplatin was administered for 5 days and then stopped for 5 days, for a total of two cycles of 20 days.

[0041] The first day of drug treatment was designated as Day 0 (Day 0). Mechanical pain and cold pain scores were performed on Days 0, 5, 10, 15, and 20, and colon, serum, and DRG samples were collected. All behavioral scores were performed by the same person in a double-blind manner. Figure 1 As shown in A.

[0042] 1.3.2 Effect on mouse body weight:

[0043] The body weight of mice was measured at the same time point on days 0, 5, 10, 15, and 20.

[0044] 1.3.3 Effects on mechanical pain perception in mice

[0045] Mechanical allodynia in mice was assessed using the mechanical paw withdrawal threshold (von Frey test). Mice were placed in a 20 x 17 x 13 cm Plexiglas box with a metal mesh bottom for the mouse's toes to contact. After the mice had acclimated to the box for 15 minutes and were at rest, von Frey fibers were used to vertically stimulate the center of their paws. Upon paw withdrawal, the stress value was recorded electronically. Ten measurements were performed on each mouse.

[0046] 1.3.4 Effects on cold pain perception in mice

[0047] Mice were acclimated to the test environment for 30 minutes before measurement. During testing, they were placed on a 5°C cold plate and covered with a transparent plexiglass box. The measurement lasted 60 seconds, during which the escape behavior of the mice was recorded and scored: 0 = no response; 1 = mild cold escape reaction, such as lifting a hind paw or walking backwards; 2 = strong cold escape reaction, such as jumping. The total score within the 60-second measurement period was recorded. Each mouse was measured three times, and the average was calculated.

[0048] 1.3.5 Obtaining Mouse DRG and Sciatic Nerve Tissue: After killing mice, a midline incision was made through the skin. The lumbar segment of the spine was excised and longitudinally dissected along the midline of the vertebral foramen to expose the dorsal root ganglia within the intervertebral foramen. The DRGs were carefully dissected and harvested under a stereoscope, and residual nerve fibers surrounding the DRGs were trimmed. The skin was incised at the femur, and blunt dissection was performed along the linea alba. Using blunt-tipped forceps, the surrounding connective tissue and blood vessels were carefully separated along the course of the nerve to obtain the sciatic nerve. The DRG tissue and sciatic nerve were then immersed in 4% paraformaldehyde and set aside.

[0049] 1.3.6 HE staining

[0050] (1) Dewaxing of paraffin sections: Place the sections in environmentally friendly dewaxing solution for 20 minutes, repeat once, anhydrous ethanol for 5 minutes, repeat once, 75% alcohol for 5 minutes, and wash with tap water.

[0051] (2) Pretreatment: The sections were treated in high-definition constant stain pretreatment solution for 1 min.

[0052] (3) Hematoxylin staining: Stain the sections with hematoxylin solution for 5 min, wash with tap water, differentiate with differentiation solution, wash with tap water, blue with bluing solution, and rinse with running water.

[0053] (4) Eosin staining: Dehydrate the sections in 95% alcohol for 1 min and then stain in eosin solution for 15 s.

[0054] (5) Dehydration and sealing: The sections were placed in anhydrous ethanol for 2 min twice, n-butanol for 2 min once, and xylene for 2 min once, and then sealed with transparent neutral gum.

[0055] (7) Microscopic examination, image acquisition and analysis.

[0056] 1.3.7 Transmission electron microscopy preparation

[0057] (1) Fixation: Transfer the cut tissue pieces to EP tubes filled with electron microscopy fixative and store at 4°C. Rinse with 0.1 M phosphate buffer (PB) (pH 7.4) three times for 15 minutes each time.

[0058] (2) Post-fixation: Fix with 1% osmium hydroxide in 0.1 M phosphate buffer PB (pH 7.4) at room temperature in the dark for 2 h.

[0059] Rinse with 0.1 M phosphate buffer PB (pH 7.4) three times, 15 min each time.

[0060] (3) Dehydration at room temperature: Dehydrate the tissue in ascending steps of 30%-50%-70%-80%-95%-100%-100% alcohol for 20 min each time, and 100% acetone twice for 15 min each time.

[0061] (4) Infiltration embedding: Acetone:812 embedding agent = 1:1 at 37°C for 2-4 hours, Acetone:812 embedding agent = 1:2 at 37°C overnight, Pure 812 embedding agent at 37°C for 5-8 hours. Pour pure 812 embedding agent into the embedding plate, insert the sample into the embedding plate, and incubate in a 37°C oven overnight.

[0062] (5) Polymerization: Place the embedded plate in a 60°C oven for polymerization for 48 hours, and remove the resin block for later use.

[0063] (6) Positioning: The resin block was sliced into 1.5 μm semi-thin sections using a semi-thin microtome, stained with toluidine blue, and positioned under a light microscope.

[0064] (7) Ultrathin sectioning: Resin block is sliced into 60-80nm ultrathin sections using an ultrathin microtome.

[0065] (8) Staining: Stain in 2% uranyl acetate saturated alcohol solution in the dark for 8 minutes; wash three times with 70% alcohol; wash three times with ultrapure water; stain in 2.6% lead citrate solution in the dark for 8 minutes; wash three times with ultrapure water, blot dry with filter paper, and dry overnight.

[0066] (9) Observe under a transmission electron microscope and collect images for analysis.

[0067] 1.3.8 Statistical analysis

[0068] All experimental data are presented as mean ± SEM. Data significance was tested using t-test or ANOVA. In all tests, a P value < 0.05 was considered statistically different, and a P value < 0.01 was considered statistically significant. All experimental data were plotted using GraphPad Prism 9.0 software. Nucleolar area was calculated using SlideViewer software.

[0069] 1.4 Experimental Results

[0070] 1.4.1 Evaluation of IAA in alleviating pain hypersensitivity induced by OIPN in mice

[0071] The body weight, mechanical pain threshold, and cold pain sensitivity scores of the mice were monitored on days 0, 5, 10, 15, and 20. Compared with the OXA group, the abnormal weight loss of mice treated with IAA was significantly restored ( Figure 1 B), the mechanical pain threshold was significantly increased ( Figure 1 C), cold pain sensitivity was significantly improved ( Figure 1 D), with significant differences evident within the first dosing cycle. We also observed that while both administration routes significantly alleviated oxaliplatin-induced pain sensitivity, the intraperitoneal injection route demonstrated significantly superior efficacy in alleviating the lowered mechanical pain threshold compared to the oral route, despite the intraperitoneal injection dose being significantly lower than the oral administration dose. These results suggest that IAA significantly alleviates a range of oxaliplatin-induced allodynia symptoms and has potential for the treatment of OIPN.

[0072] 1.4.2 Evaluation of IAA in alleviating DRG morphological damage in OIPN mice

[0073] HE staining showed that the damage to DRG in OIPN mice treated with IAA was significantly reduced ( Figure 2 A), the neurons were full and plump, the intercellular spaces were not obvious, only a few small vacuoles appeared in the cytoplasm, and the nucleoli were significantly larger than those in the OXA group ( Figure 2 B).

[0074] 1.4.3 Evaluation of IAA in improving sciatic nerve myelin morphological damage in OPIN mice

[0075] Transmission electron microscopy results showed that the myelin sheath structure in the cross section of the sciatic nerve of normal mice was uniform and dense. The myelin sheath in the model group was significantly damaged, with obvious lamellar separation. Compared with the model group, the myelin sheath lamellar separation phenomenon was significantly improved and the damage was reduced in OIPN mice treated with IAA gavage and intraperitoneal injection ( Figure 3 1.4.4 Evaluation of IAA in reducing DRG colon inflammation in OIPN mice

[0076] The colon of mice in the normal group, OXA group and IAA group was stained with HE and the colon inflammation score was calculated ( Figure 4 A), and found that the intestinal inflammation pathology score of mice in the IAA treatment group was significantly reduced ( Figure 4 B) IAA reduces colonic tissue inflammation and may have the ability to protect the intestinal barrier.

[0077] The above results show that IAA significantly alleviated the pathological state of mice caused by OIPN. After oral or intraperitoneal administration of IAA, OIPN mice showed significant improvement in pain sensitivity tests. There was obvious pathological morphological damage to the DRG and sciatic nerve myelin sheath of OIPN mice, and these pathological signs were significantly improved after IAA treatment. In addition, OIPN in mice is not only manifested as nervous system damage, but also accompanied by local inflammation of the colon. Experimental data showed that the pathological level of colon inflammation in mice in the IAA-treated group was significantly lower than that in the OXA group, suggesting that IAA can significantly inhibit local inflammatory responses. At the same time, the dose in the experiment was proven to be non-toxic in actual studies and did not produce obvious side effects. Its safety provides a good basis for further research.

[0078] Example 2: IAA-dependent activation of the AhR pathway alleviates OIPN in mice

[0079] 2.1 Experimental drugs and preparation methods

[0080] (1) Preparation of Oxaliplatin Injection

[0081] Same as Example 1

[0082] (2) Preparation of IAA solution

[0083] Same as Example 1

[0084] (3) Preparation of AhR antagonist solution

[0085] 4 mg of AhR antagonist was dissolved in 1 mL of PBS, sonicated, and stored at 4°C until use.

[0086] 2.2 Experimental animals

[0087] Same as Example 1

[0088] 2.3 Experimental methods

[0089] 2.3.1 Animal grouping and modeling

[0090] After all mice were acclimated to a fixed environment for one week and the baseline of pain sensitivity was measured, they were randomly divided into the following four groups: normal group (Vehicle), model group (OXA), IAA group, and AhR antagonist group (AhR).

[0091] The normal group and the model group were the same as in Example 1.

[0092] IAA group: IAA (50 mg / kg) was intraperitoneally injected every day, followed by intraperitoneal injection of 3 mg / kg of oxaliplatin in 5% glucose solution at a dose of 5 ml / kg. Oxaliplatin was administered for 5 days and rested for 5 days, for a total of two cycles of 20 days.

[0093] AhR group: AhR antagonist (20 mg / kg) and IAA (50 mg / kg) were intraperitoneally injected every day, and then 3 mg / kg of oxaliplatin 5% glucose solution was intraperitoneally injected at a dose of 5 ml / kg. Oxaliplatin was administered for 5 days and then stopped for 5 days, for a total of two cycles of 20 days.

[0094] The first day of drug treatment was designated as Day 0 (Day 0). Mechanical pain and cold pain scores were performed on Days 0, 5, 10, 15, and 20, and colon, serum, and DRG samples were collected. All behavioral scores were performed by the same person in a double-blind manner. Figure 5 As shown in A.

[0095] 2.3.2 Animal behavior experiment: same as Example 1

[0096] 2.3.3 Obtaining mouse DRG and sciatic nerve tissue: Same as Example 1

[0097] 2.3.4 HE staining: same as Example 1

[0098] 2.3.5 Statistical analysis: same as Example 1

[0099] 2.4 Experimental Results

[0100] 2.4.1 Evaluation of the effect of AhR receptor antagonism and IAA on alleviating pain sensitivity induced by OIPN in mice

[0101] The body weight, mechanical pain threshold and cold pain sensitivity scores of the mice were monitored on days 0, 5, 10, 15 and 20. Compared with the IAA group, AhR antagonism caused IAA to lose the effect of restoring the body weight of OIPN mice ( Figure 5 B), no improvement, or even worsening of mechanical pain threshold ( Figure 5 C), the improvement effect of cold pain sensitivity is almost lost ( Figure 5 D) The experimental results indicate that AhR is crucial for IAA to alleviate pain sensitivity caused by OIPN, and antagonizing AhR blocks the therapeutic effect of IAA.

[0102] 2.4.2 Evaluation of AhR receptor antagonism and IAA to alleviate DRG morphological damage in OIPN mice

[0103] HE staining showed that the protective effect of IAA on neurons was lost after AhR antagonism, and the neurons shrank and the nucleolus area did not improve ( Figure 6 A), the nucleolus area was significantly smaller than that of the IAA group ( Figure 6 B).

[0104] 2.4.3 Evaluation of the effect of AhR antagonism on IAA in alleviating sciatic nerve myelin morphological damage in OIPN mice

[0105] Electron microscopy results showed that compared with the IAA-administered group, the sciatic nerve in the AhR antagonist-followed IAA-administered group showed edema and local dissolution, and the myelin sheath was significantly damaged, indicating that IAA lost its protective effect on the sciatic nerve myelin sheath and aggravated myelin damage compared with the model group ( Figure 7 ).

[0106] 2.4.4 Evaluation of AhR receptor antagonism and IAA blockade to reduce colon inflammation in OIPN mice

[0107] Comparison of colon HE staining between AhR antagonist group and IAA group mice ( Figure 8 A) and colon inflammation score results showed that AhR antagonism not only made IAA lose the effect of alleviating colon inflammation, but also aggravated intestinal inflammation, and some scores even exceeded those of the OXA group ( Figure 8 B)

[0108] From the above results, it can be seen that IAA can upregulate the expression of IL-22 after activation through AhR. This cytokine plays a key role in maintaining intestinal barrier function and promoting tissue repair, which is consistent with the experimental observation that IAA can reduce colon inflammation.

[0109] The DRG cells in the IAA-treated mice showed relatively intact morphology, with neuronal cell body size, nuclear and nucleolar structure close to normal. However, in the AhR antagonist-treated group, these protective changes were significantly reversed, with DRG cells exhibiting significant atrophy and structural abnormalities, further demonstrating the role of the AhR signaling pathway in neuroprotection. Similarly, in the AhR antagonist-treated group, the myelin sheath of the sciatic nerve of mice also showed increased damage.

[0110] In terms of inflammatory regulation mechanisms, AhR activation can induce specific downstream signaling cascades, such as the regulation of NF-κB and STAT3 signaling pathways. These signaling pathways play a core role in inflammatory responses, and maintaining their balance is important for alleviating local and systemic inflammation in mouse OIPN.

[0111] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Use of indoleacetic acid in the preparation of drugs for preventing or treating neuropathic toxicity induced by chemotherapy drugs.

2. The use according to claim 1, characterized in that: The drug contains indoleacetic acid and is used in combination with chemotherapy drugs. The drug is used to relieve pain caused by tumor treatment and significantly reduce neuropathic toxicity caused by chemotherapy drugs.

3. The use according to claim 1, wherein the chemotherapy drug is a cancer chemotherapy drug.

4. The use according to claim 3, wherein the cancer chemotherapy drug is selected from one or more of oxaliplatin, cisplatin, carboplatin, paclitaxel, vincristine, and vinblastine.

5. The use according to claim 1, wherein the chemotherapy drug is oxaliplatin.

6. The use according to claim 1, wherein the symptoms of neuropathic toxicity include one or more of the following: numbness of hands and feet, glove-like sensation, stocking-like sensation, burning pain, tingling in hands and feet, and weakened or absent deep tendon reflexes.

7. The use according to claim 1, wherein the indoleacetic acid acts on the aryl hydrocarbon receptor (AhR).

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