Use of ivabidin for the preparation of a medicament for the prevention and / or treatment of neuroinflammation

Abstract

The present application relates to the use of narrow-leaved ivy ginkgo in the preparation of a medicine for preventing and / or treating neuroinflammation, and belongs to the field of medicine. The narrow-leaved ivy ginkgo is a single component of traditional Chinese medicine in a traditional Chinese medicine plant, is a natural compound, has advantages such as wide source and small side effects, and can prevent, relieve, improve or treat AD / PD diseases or delay the progress of AD / PD diseases by affecting the mechanism of neuroinflammation, thereby providing a new and effective treatment option for AD / PD diseases.

Description

Technical Field

[0001] This invention relates to the use of anisodamine or a pharmaceutically acceptable derivative thereof in the preparation of medicaments for the prevention and / or treatment of neuroinflammation, and belongs to the pharmaceutical field. Background Technology

[0002] Neurodegeneration refers to a pathological condition primarily affecting neurons, characterized by progressive neuronal dysfunction and loss. Clinically, neurodegenerative diseases encompass a wide range of central nervous system (CNS) diseases with prominent clinical and pathological features, including Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and Huntington's disease (HD). Despite the presence of disease-specific protein aggregates (such as amyloid-β and α-synuclein), these aggregates exhibit persistent neuroinflammation characterized by elevated pro-inflammatory cytokines / chemokines, oxidative stress, and impaired neuroprotective mechanisms. This neuroinflammatory condition further exacerbates protein misfolding and directly induces synaptic and vascular damage, creating a vicious cycle that accelerates disease progression. Therefore, neuroinflammation is both a common pathological marker of neurodegenerative diseases and a potential therapeutic target.

[0003] Current clinical treatments have significant limitations: Alzheimer's disease (AD) medications (such as cholinesterase inhibitors) only improve cognitive symptoms; long-term use of disease progression (PD) drugs (such as levodopa) is prone to side effects; and new AD drugs such as Aβ monoclonal antibodies are expensive and carry risks of cerebral hemorrhage and cerebral edema. Existing methods do not target the core aspect of neuroinflammation; therefore, developing new drugs that focus on clearing or inhibiting neuroinflammation could fill a clinical gap, reduce the social burden of healthcare, and has significant medical and social implications.

[0004] Plant-derived bioactive natural compounds remain a major source of new drug discovery. Sesquiterpene lactones are a class of plant secondary metabolites with potent biological activities, including anti-inflammatory, antioxidant, antitumor, and neuroprotective effects. Ivangustin (IVA) is a sesquiterpene lactone isolated from the traditional Chinese medicine herb Flos Inulae. It exhibits moderate inhibitory activity against HCT116 colorectal cancer and QGY7701 hepatocellular carcinoma cells; however, its role in neuroinflammatory and functional targets remains unknown.

[0005] The structural formula of Ivangustin (IVA, Cas: 14164-59-1) is as follows:

[0006] .

[0007] Application No.: CN202511221908.X, Invention Title: Whitening and Spot-Removing Composition, Conditioning Agent, Drug, and Cosmetics, discloses a whitening and spot-removing composition, conditioning agent, drug, and cosmetics. The whitening and spot-removing composition comprises broccoli, Ganoderma lucidum, and Inula japonica; the broccoli includes broccoli raw material and / or broccoli extract, the Ganoderma lucidum includes Ganoderma lucidum raw material and / or Ganoderma lucidum extract, and the Inula japonica includes Inula japonica raw material and / or Inula japonica extract. The Inula japonica extract includes Ivangustin (CAS: 14164-59-1).

[0008] There are currently no literature reports on the use of anisodamine for neuroinflammation. Summary of the Invention

[0009] This invention provides a new use for narrow-leaved iwazidin.

[0010] This invention provides the use of anisodamine or a pharmaceutically acceptable derivative thereof in the preparation of medicaments for the prevention and / or treatment of neuroinflammatory diseases.

[0011] The derivatives are pharmaceutically acceptable salts, prodrugs, hydrates, or solvates of angiotensin.

[0012] The drug mentioned is a medication for treating Alzheimer's disease or Parkinson's disease.

[0013] The aforementioned drugs are those that improve cognitive impairment, neuropsychological symptoms, mental symptoms, behavioral abnormalities, and sleep disorders.

[0014] The aforementioned medication is used to improve motor impairment or anxiety-related behaviors.

[0015] The drug in question is one that improves learning and memory impairments.

[0016] The drug mentioned is a medication for treating neurodegenerative diseases.

[0017] The drug is prepared into a commonly used pharmaceutical formulation by adding pharmaceutically acceptable excipients or auxiliary ingredients, with the active ingredient being narrow-leaved iwazidin or its pharmaceutically acceptable derivative.

[0018] Practically, the drug may be administered via one or more of the following methods: oral, injection, implantation, spray, and / or inhalation.

[0019] Practically, the dosage form of the drug is selected from one or more of the following: injection, oral liquid, powder, tablet, granule, capsule, syrup, decoction, sustained-release preparation, enteric solvent, aerosol or suspension.

[0020] The preparation mentioned herein is an oral preparation or an injectable preparation.

[0021] The present invention relates to narrow-leaved iwazidin, a single component of traditional Chinese medicine plants. As a natural compound, it has the advantages of wide availability and few side effects. It can prevent, alleviate, improve or treat AD / PD disease by influencing the neuroinflammatory mechanism, or delay the progression of AD / PD disease, thus providing a new and effective treatment option for AD / PD disease. Attached Figure Description

[0022] Figure 1 The preventive and therapeutic effects of different concentrations of angiospermia zeylan on NO release in LPS-stimulated BV2 microglia and its influence on cell viability;

[0023] Figure 2 The preventive and therapeutic effects of different concentrations of anthocyanin on the expression of iNOS and COX-2 proteins in LPS-stimulated BV2 microglia.

[0024] Figure 3 The preventive and therapeutic effects of different concentrations of narrow-leaved iwazidin on the expression of inflammatory factors and chemokine mRNA in LPS-stimulated BV2 microglia.

[0025] Figure 4 The effects of different concentrations of angiotensin on the prevention and treatment of motor dysfunction and anxiety in LPS-induced AD / PD mouse models;

[0026] Figure 5 The preventive and therapeutic effects of different concentrations of angiotensin on cognitive impairment in LPS-induced AD / PD mouse models;

[0027] Figure 6 The preventive and therapeutic effects of different concentrations of narrow-leaved iwazidin on neuronal pathological changes in an LPS-induced AD / PD mouse model. Detailed Implementation

[0028] The narrow-leaved iwaziin of this invention is a commercially available product, purchased from Chengdu Kesten Biotechnology Co., Ltd.

[0029] Example 1: Preventive and therapeutic effects of narrow-leaved iwazidin on nitric oxide (NO) release in lipopolysaccharide-stimulated BV2 microglia.

[0030] Experimental Procedure: BV2 cells were seeded at a density of 5 × 10^3 cells per well in 96-well plates and allowed to adhere overnight. Subsequently, cells were treated with different concentrations of IVA for 24 hours in the presence of LPS (0.5 μg / mL), using BAY as the positive control. The cell culture supernatant was then collected by centrifugation at 1,000 × g for 3 minutes at 4°C, and NO was quantitatively analyzed using Griess reagent (Beyotime, Shanghai, China). To assess cell viability, BV2 cells were incubated with 100 μL of fresh culture medium containing 1% CCK-8 reagent (Beyotime) at 37°C in the dark for 1 hour, followed by measurement of the optical density (OD) of each well at 450 nm using a multi-mode microplate reader (Tecan, Innsbruck, Austria).

[0031] Experimental results: such as Figure 1 As shown, IVA inhibited LPS-induced NO production in BV2 cells in a concentration-dependent manner without significantly affecting cell viability.

[0032] Experimental Results Analysis: To evaluate the anti-neuroinflammatory properties of IVA, we investigated its inhibitory effect on the production of nitric oxide (NO), a key pro-inflammatory biomarker, in LPS-activated BV2 mouse microglia. Compared to the control group (CK), the model group (LPS) showed significantly increased nitric oxide release, indicating successful establishment of the in vitro neuroinflammation model. Treatment with the positive control drug BAY reduced NO release, suggesting the effectiveness of the experimental system. Treatment with different concentrations of IVA significantly reduced LPS-induced NO release, and this inhibitory effect was dose-dependent. Statistical tests showed significant differences. **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.

[0033] Experimental Example 2: Preventive and therapeutic effects of narrow-leaved iwazidin on the expression of iNOS and COX-2 proteins in lipopolysaccharide-stimulated BV2 microglia.

[0034] Experimental procedure: BV2 cells were seeded at a density of 20 × 10^5 cells per well in 24-well plates and allowed to adhere overnight. Subsequently, the cells were treated with different concentrations of IVA for 24 hours in the presence of LPS (0.5 μg / mL), with BAY as the positive control.

[0035] After treatment with DMSO or IVA, BV2 cells were lysed on ice for 15 minutes in RIPA lysis buffer (APExBio) containing 1% protease / phosphatase inhibitor. The supernatant was collected after centrifugation (15,000g, 5 minutes) at 4°C, mixed with 5× loading buffer, and boiled at 100°C for 10 minutes. Protein samples were separated by SDS-PAGE and transferred to ethanol-activated polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The membranes were blocked at 25°C for 1 hour with TBST buffer containing 5% skim milk powder, followed by incubation overnight at 4°C with primary antibody (diluted 1:1000 with 5% bovine serum albumin). After TBST washing, the cells were incubated at 25°C for 1 hour with horseradish peroxidase-labeled secondary antibody (1:5000, SAB, Nanjing, China). Signals were detected on a MiniChemi™ 610 imaging system (Sage, Beijing) using a chemiluminescent reagent (Oriscience Biotechnology), and band intensity was quantitatively analyzed using ImageJ software (NIH, USA).

[0036] Experimental results: such as Figure 2 As shown, Western blot analysis confirmed that IVA treatment significantly inhibited the abnormal increase in LPS-induced iNOS and COX-2 proteins in a concentration-dependent manner, with statistically significant differences.

[0037] Analysis of experimental results: iNOS is a key inducible enzyme in NO synthesis, and COX-2 is another inducible enzyme involved in inflammation induction. Compared with the control group (CK), the expression of iNOS and COX-2 proteins in the model group (LPS) was significantly increased, indicating the successful establishment of the in vitro model of neuroinflammation. After treatment with the positive control drug BAY, the expression of iNOS and COX-2 proteins was significantly reduced, indicating the effectiveness of the experimental system. Treatment with different concentrations of IVA significantly reduced the abnormally high expression of iNOS and COX-2 proteins induced by LPS, and this inhibitory effect was dose-dependent. Statistical tests showed that the differences were significant. **: p<0.01, ***: p<0.001, ****: p<0.0001.

[0038] Experimental Example 3: Preventive and therapeutic effects of narrow-leaved iwazidin on the mRNA expression of inflammatory factors and chemokines in lipopolysaccharide-stimulated BV2 microglia.

[0039] Experimental Procedure: After LPS stimulation for 12 hours, total RNA was extracted from BV2 cells using the Steady Pure RNA Extraction Kit (Accurate, Changsha, China). cDNA synthesis was performed using the Evo M-MLV Reverse Transcription Kit (Accurate) to convert the extracted RNA into cDNA. Subsequently, qRT-PCR was performed on a LineGene 9600 Plus system (Bioer Technology, Hangzhou, China) and a SYBR Green Pro Taq HS qPCR mixture (accurate). Amplification was performed using custom-designed primers (Qingke, Beijing, China). Target gene expression levels were normalized to GAPDH as an internal reference, and 2- ΔΔ Quantitative analysis was performed using the Ct method.

[0040] Experimental results: such as Figure 3 As shown, IVA treatment effectively inhibited the mRNA expression of LPS-induced inflammatory factors (IL-1β, IL-6, and TNF-α) and chemokines (MCP-1, CXCL1, and CXCL9) in a dose-dependent manner, and the differences were statistically significant.

[0041] Experimental Results Analysis: We used qRT-PCR to assess the transcriptional levels of inflammatory and pro-inflammatory factors. Compared to the control group (CK), LPS stimulation significantly upregulated the gene expression of pro-inflammatory cytokines, including IL-1β, IL-6, and TNF-α. Figure 3 A- Figure 3 These cytokines were inhibited in a dose-dependent manner by IVA. Furthermore, our results indicate that LPS exposure enhanced the mRNA expression of chemokines, including monocyte chemokine-1 (MCP-1), CXC motif chemokine (CXCL1), and CXCL9, while IVA treatment effectively inhibited LPS-induced expression of these chemokines in BV-2 cells. Figure 3 D- Figure 3 F). Meanwhile, BAY, a known NF-κB pathway inhibitor, also significantly inhibited LPS-induced expression of cytokines and chemokines. These findings collectively demonstrate that ivangustin exhibits potent anti-neuroinflammatory activity by inhibiting the expression of inflammatory factors and chemokines. **: p<0.01, ***: p<0.001, ****: p<0.0001.

[0042] Experimental Example 4: Preventive and therapeutic effects of narrow-leaved iwazidin on LPS-induced motor impairment and anxiety behavior in AD / PD mice.

[0043] Experimental steps:

[0044] 1. Animal and Ethical Review: Ten-week-old male C57BL / 6J mice (SPF grade, 26±2 g) were purchased from Huafukang Biotechnology Co., Ltd. (Beijing, China, SCXK-JING-2024-0003). Mice were housed in plastic cages at 25±1°C for a one-week acclimatization period under a 12-hour light / dark cycle, with free access to food and water. The experimental protocol was approved by the Ethics Committee of Chengdu University of Traditional Chinese Medicine (Approval No. 2025110). An AD / PD mouse model was established by intraperitoneal injection of LPS.

[0045] 2. Induction of Neuroinflammation and Drug Treatment in Mice: Mice were randomly assigned to six groups based on body weight: control group (carrier), LPS group (carrier), LPS + dimethyl fumarate group (DMF, 100 mg / kg, Topscience, Shanghai, China), and three dosage groups of LPS + IVA (5, 10, and 20 mg / kg). All groups received the corresponding treatment via intraperitoneal injection daily for four consecutive days (carrier: 5% DMSO + 30% PEG400 + 65% saline). On day 4, all groups except the carrier control group received intraperitoneal injection of LPS (2.5 mg / kg). Body weight was monitored throughout the experiment.

[0046] 3. Open Field Test: Mice were acclimatized to the environment for 24 hours prior to behavioral assessment. The test was conducted under quiet conditions with adequate lighting and the experimenter out of sight. Behavioral assessment was performed 16 hours after LPS injection. For the open field test, mice were placed in the center of a 50 × 50 cm open square area, and their movement trajectory, total distance traveled, average speed, and entry / distance to the center area were recorded over 5 minutes to assess locomotion and exploratory behavior.

[0047] Experimental results: (e.g.) Figure 4 As shown, IVA not only significantly improved the reduction in total distance and mean velocity caused by LPS, but also effectively mitigated the reduction in the number of crossings and the moving distance in the center caused by LPS. Statistical tests showed that the differences were significant and statistically significant.

[0048] Analysis of Experimental Results: The above behavioral results indicate that IVA can effectively reverse LPS-induced behavioral abnormalities in mice. The significant improvement in total distance traveled and average speed suggests that IVA alleviated the overall motor impairment and lethargy induced by LPS. More importantly, IVA significantly increased the frequency of mice entering the central area and the distance they traveled within it. Since rodents are naturally inclined to prefer darkness and peripheral areas, their activity level in the central area of ​​an open field is a key indicator for assessing anxiety-like behavior and exploratory desire. LPS treatment led to a decrease in this indicator, reflecting anxiety-like behavior induced by neuroinflammation. Therefore, IVA can effectively alleviate LPS-induced anxiety-like behavior and enhance their courage to explore unfamiliar environments, strongly suggesting that IVA has a clear ameliorative effect on LPS-induced neuroinflammation and its resulting behavioral deficits. *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001.

[0049] Experimental Example 5: Preventive and therapeutic effects of narrow-leaved iwazidin on LPS-induced cognitive impairment in AD / PD mice.

[0050] Experimental procedures: Animal and ethical review, induction of neuroinflammation in mice, and drug treatment were the same as before.

[0051] Y-maze test: Mice were acclimatized to the environment for 24 hours before behavioral assessment. The test was conducted under quiet conditions with adequate lighting and the experimenter out of sight. Behavioral assessment was performed 16 hours after LPS injection. In the Y-maze test, mice freely explored the three arms (120° angle, 30×5×15 cm) for 5 minutes, and the entry sequence and frequency were recorded. Spontaneous alternation rate (%) = [number of correct alternations / (total number of arm entries - 2)] × 100%, reflecting the level of spatial working memory.

[0052] Experimental results: (e.g.) Figure 5 As shown, IVA significantly improved the correct spontaneous alternation rate in the Y maze test and improved LPS-induced short-term memory impairment in mice. Statistical tests showed that the differences were significant and statistically important.

[0053] Experimental Results Analysis: The Y-maze test results clearly showed that IVA significantly improved LPS-induced short-term memory impairment in mice. Spontaneous alternation rate is a key indicator reflecting working memory and spatial exploration ability; its decrease means the animal cannot remember the arm sequence it just explored, a typical manifestation of cognitive impairment. LPS exposure led to a significant reduction in spontaneous alternation rate, confirming that neuroinflammation successfully induced a cognitive impairment model in mice. The significant recovery of this indicator after IVA treatment indicates that IVA effectively reversed LPS-induced cognitive impairment and enhanced the spatial working memory ability of mice. This result strongly demonstrates from a behavioral perspective that IVA has positive therapeutic potential for LPS-induced neuroinflammation and related cognitive impairment.

[0054] Experimental Example 6: Preventive and therapeutic effects of narrow-leaved iwazidin on LPS-induced neuronal pathological changes in LPS-induced AD / PD mice.

[0055] Experimental procedures: Animal and ethical review, induction of neuroinflammation in mice, and drug treatment were the same as before.

[0056] Histological examination: Mice were anesthetized 24 hours after LPS injection by intraperitoneal injection of sodium pentobarbital (50 mg / kg), euthanized by cervical dislocation, and brain tissue was removed and fixed in 4% paraformaldehyde for 24 hours. The brain tissue was then embedded in paraffin and sectioned. Hematoxylin-eosin (HE) staining was performed on the brain sections to assess the effect of IVA on LPS-induced neuropathology.

[0057] Experimental results: such as Figure 6 As shown, IVA significantly improved the neuronal shrinkage, degeneration and necrosis in the CA1 and CA3 regions of the hippocampus induced by LPS, and increased the number of healthy surviving neurons. Statistical tests showed that the difference was significant and statistically significant.

[0058] Analysis of Experimental Results: HE staining results showed that IVA had a significant protective effect against LPS-induced hippocampal neuronal damage. The CA1 and CA3 regions of the hippocampus are key brain regions for learning and memory function, and are particularly sensitive to ischemic, hypoxic, and inflammatory stimuli. LPS-induced neuronal shrinkage, degeneration, and necrosis are direct morphological evidence of neuroinflammation leading to cell damage. IVA treatment significantly alleviated these pathological changes and increased the number of healthy neurons, directly confirming at the tissue morphology level that IVA can effectively inhibit the neurotoxicity of neuroinflammation and protect neurons from damage. This result corroborates the behavioral improvements (i.e., improvement in motor impairment, reduction in anxiety-like behavior, and improvement in cognitive function) observed in the open field and Y-maze experiments, forming a complete chain of evidence from cell protection to functional improvement, strongly demonstrating that IVA, through its neuroprotective effect, ultimately improves LPS-induced learning and memory impairment.

[0059] The above-mentioned trials have demonstrated that narrow-leaved iwazidin can significantly inhibit microglial cell activation and neuroinflammatory responses, thereby preventing, alleviating, improving or treating Alzheimer's disease and Parkinson's disease, or delaying the progression of Alzheimer's disease and Parkinson's disease. It is a promising candidate drug for the treatment of Alzheimer's disease and Parkinson's disease.

Claims

1. Use of anisodamine in the preparation of a medicament for the prevention and / or treatment of neuroinflammation, wherein the neuroinflammation is Alzheimer's disease or Parkinson's disease.

2. The use as described in claim 1, characterized in that: The drug is prepared into a commonly used pharmaceutical formulation by adding pharmaceutically acceptable excipients or auxiliary ingredients, with the active ingredient being narrow-leaved iwazidin or its pharmaceutically acceptable derivative.

3. The use according to claim 2, characterized in that: The preparation is an oral or injectable preparation.

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