A pharmaceutical composition for inhibiting microglial inflammatory activation and use thereof

Abstract

The application discloses a medicine composition for inhibiting inflammatory activation of microglial cells and application thereof. 6-gingerol and eucommia alcohol are combined with each other, and the combination has a significant synergistic effect, and can effectively inhibit inflammatory activation of microglial cells.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceuticals, and specifically relates to a pharmaceutical composition for inhibiting inflammatory activation of microglia and its use. Background Technology

[0002] Microglia are mononuclear macrophages residing in the central nervous system (CNS). They are glial cells that support and protect neurons, accounting for 10%–15% of the total number of glial cells in the CNS parenchyma. They are widely distributed in the brain and spinal cord, with significant differences in their distribution across different brain regions. They are most densely packed in the substantia nigra of the midbrain and least densely distributed in the brainstem and cerebellum. Microglia are important immune cells in the brain and play a crucial role in the intrinsic defense system of neural tissue. Under normal physiological conditions, microglia are in a resting state, exhibiting a multibranched morphology, and functioning to monitor the brain's microenvironment. Their physiological surveillance functions include: clearing accumulated or degenerated neurons and other tissue components; dynamically regulating neurons; synaptic pruning; and maintaining overall brain homeostasis. During development, microglia eliminate apoptotic remnants of excess newborn neurons through phagocytosis and promote neurogenesis during brain development. Simultaneously, brain-derived neurotrophic factors derived from microglia are important for the formation of dendritic spines during learning. However, under certain pathological stimuli, microglia are activated and enter a pathological state, morphologically manifesting as amoeboid changes. Activated microglia are generally divided into two types: M1 (pro-inflammatory) or M2 (anti-inflammatory). An imbalance between these two states exacerbates the development of central nervous system diseases. If they remain chronically activated, they exhibit neurotoxicity. As the disease progresses, the number of activated M1 microglia in the substantia nigra increases, leading to more severe damage to dopaminergic neurons. This may be related to oxidative stress, induction of pro-inflammatory cytokine release, immune phagocytic damage, and autophagy dysfunction. Inhibiting excessive activation of M1 microglia has been shown to slow dopaminergic neuronal degeneration. In neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease (PD), the level of microglia activation in disease-associated brain regions is closely related to their pathological progression.

[0003] Previous research by our research group found that Eucommia ulmoides formula has a therapeutic effect on Parkinson's disease. To further explore its effective active ingredient group, our research group conducted preliminary research on the activities of possible active ingredients in Eucommia ulmoides formula, such as 6-shogaol, 6-gingerol, eucommiol, 1-deoxyeucommiol, nobilonine, dendramine, and dendrobiumane A. We also applied network-based methods to the research on the compatibility of traditional Chinese medicine formulas, in order to discover the effective components of Eucommia ulmoides formula that inhibit the synergistic effect of microglial cell inflammatory activation, and to provide a new drug composition for the development of new drugs for Parkinson's disease.

[0004] To develop a drug composition with synergistic active ingredients, it is typically necessary to study drug-drug interactions (DDIs). Drug interactions refer to changes in pharmacodynamics that occur after a patient takes two or more drugs sequentially over a certain period. Clinically, these interactions are generally classified as synergistic or antagonistic. In clinical treatment, combination therapy often has certain advantages over monotherapy.5-6 Synergistic effects can enhance efficacy or reduce adverse drug reactions, while antagonistic effects can lead to decreased efficacy or increased side effects, and may even cause abnormal reactions, interfere with treatment, and worsen the condition. Combination therapy provides better drug efficacy, reduces the dosage of single drugs, and can reduce the risk of adverse reactions. Therefore, combination therapy is widely used in the treatment of many complex diseases, such as hypertension and cancer. Cheng et al. predicted the interaction between two drugs by constructing human protein-protein interaction networks and drug-target networks. Taking a combination of drugs approved by the U.S. Food and Drug Administration (FDA) for the treatment of hypertension as an example, they discovered and verified an effective drug synergistic network relationship, providing new ideas and methods for discovering effective drug combinations and elucidating the effector substances of different efficacy of compound traditional Chinese medicine. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a pharmaceutical composition for inhibiting the inflammatory activation of microglia and its pharmaceutical use. It can effectively inhibit the inflammatory activation of microglia. The combination of 6-shogaol and eucommia ulmoides has a significant synergistic effect and can be used to prevent or treat neurodegenerative diseases related to the inflammatory activation of microglia, especially Parkinson's disease and Alzheimer's disease.

[0006] The technical solution of this invention is summarized as follows:

[0007] A pharmaceutical composition for inhibiting inflammatory activation of microglia, said pharmaceutical composition being made from the following raw materials in parts by weight: 0.1-2 parts of 6-shogaol and 1-3 parts of eucommia ulmoides.

[0008] The preferred weight proportions of 6-shogaol are: 0.2 parts, 0.4 parts, 0.5 parts, 0.6 parts, 0.8 parts, 1 part, 1.2 parts, 1.4 parts, 1.5 parts, 1.6 parts, and 1.8 parts.

[0009] The preferred weight proportions of eucommia ulmoides are: 1 part, 1.2 parts, 1.4 parts, 1.5 parts, 1.6 parts, 1.8 parts, 2 parts, 2.2 parts, 2.4 parts, 2.5 parts, 2.6 parts, and 2.8 parts.

[0010] The preferred weight proportions of the two ingredients are: 0.5-2 parts of 6-shogaol and 1-3 parts of eucommia ulmoides alcohol;

[0011] More preferably, the weight parts of the two are: 1-2 parts of 6-shogaol and 1-2 parts of eucommia ulmoides alcohol;

[0012] More preferably, the weight parts of the two are: 1-1.5 parts of 6-shogaol and 1-2 parts of eucommia ulmoides alcohol;

[0013] More preferably, the weight parts of the two are: 1 part of 6-shogaol and 1 part of eucommia ulmoides alcohol;

[0014] More preferably, the weight parts of the two are: 1 part of 6-shogaol and 2 parts of eucommia ulmoides alcohol.

[0015] The concentration of 6-shogaol is 1.0-8 μM, preferably 1.5-7.5 μM, 2.0-7 μM, 2.5-6.5 μM, 3-6 μM, 3.5-5.5 μM, or 4-5 μM.

[0016] The concentration of eucommia ulmoides alcohol is 1.0-10 μM, preferably 1.5-9 μM, 1.5-7.5 μM, 2.0-7 μM, 2.5-6.5 μM, 3-6 μM, 3.5-5.5 μM, 4-5 μM, 4-6 μM, or 4-8 μM.

[0017] The present invention also provides a pharmaceutical preparation comprising the above-described pharmaceutical composition and pharmaceutical excipients.

[0018] The pharmaceutical preparation is selected from oral medications and parenteral medications.

[0019] The pharmaceutical preparation is selected from one of the following: powder, solution, pill, granule, capsule, tablet, and injection.

[0020] The pharmaceutical preparation is selected from oral suspensions, and the pharmaceutical excipients of the oral suspensions include sodium carboxymethyl cellulose, xanthan gum, and water.

[0021] The present invention further provides the use of the above-described pharmaceutical composition and pharmaceutical formulation in the preparation of a medicament for inhibiting inflammatory activation of microglia.

[0022] The present invention further provides the use of the above-described pharmaceutical composition and pharmaceutical formulation in the preparation of a medicament for treating neurodegenerative diseases associated with microglial inflammatory activation.

[0023] The neurodegenerative disease is selected from Parkinson's disease or Alzheimer's disease.

[0024] Based on long-term research on Eucommia ulmoides formulas, this invention discloses a pharmaceutical composition for treating Parkinson's disease, as disclosed in CN201810895721. This composition is made from Eucommia ulmoides, Dendrobium nobile, Rehmannia glutinosa, and dried ginger. It significantly improves limb motor coordination in PD model mice and increases the content of dopamine and its metabolites dihydroxyphenylacetic acid and homovanillic acid in the striatum, demonstrating a clear therapeutic effect on Parkinson's disease. Further mechanistic and in vivo metabolic studies revealed 6-gingerol, 6-shogaol, dendrobamine, and dendrobone alkaloids as potential active ingredients. Building upon the above research, this invention uses a human protein-protein interaction (PPI) network and quantifies the network relationship between key disease modules and drug targets to identify and predict the effects against the disease. From a large number of potential active ingredients, a combination of 6-shogaol and eucommia ulmoides alcohol with significant synergistic effects was obtained. However, 6-gingerol, which also showed therapeutic activity in previous studies, did not exhibit synergistic effects when combined with other active ingredients such as eucommia ulmoides alcohol.

[0025] Instruction manual illustrations:

[0026] Figure 1 This invention presents a network diagram of drug A and B target modules and disease key modules with synergistic relationships.

[0027] Figure 2 Effects of 6-shogaol and eucommia ulmoides monomers on BV-2 cell viability in this invention

[0028] Figure 3 The effect of 6-shogaol and eucommia ulmoides monomers on NO inhibition rate in this invention

[0029] Figure 4 The present invention describes the inhibitory effect of 6-shogaol combined with eucommia ulmoides on NO release from BV-2 cells.

[0030] Figure 5 Isobologram analysis of the inhibition of NO by the combination of 6-shogaol and eucommia ulmoides in this invention. Detailed Implementation

[0031] The present invention will be further described below with reference to specific embodiments.

[0032] Example 1

[0033] Drug combination screening and synergistic effect prediction based on the relationship between disease network and drug-target network

[0034] Referring to the network-based method proposed by Cheng et al. (see heng, F., Kovács, IA & Barabási, AL. Network-based prediction of drug combinations. Nat Commun 10, 1197 (2019)), this method constructs a scientifically reliable human protein-protein interaction (PPI) network and identifies and predicts effective drug combinations for specific diseases by quantifying the network relationship between key disease modules and drug targets.

[0035] Acquisition of potential disease targets and drug targets

[0036] The single-cell RNA sequencing (scRNA-seq) dataset GSE184950 was downloaded from the GEO database (https: / / www.ncbi.nlm.nih.gov / gene). Differentially expressed genes in microglia from healthy individuals and Parkinson's disease (PD) patients were analyzed and identified as key disease targets. The Traditional Chinese Medicine Systems Pharmacology Analysis Platform (TCMSP) (https: / / old.tcmsp-e.com / tcmsp.php) was used to search for targets of 6-gingerol, 6-shogaol, eucommia ulmoides, and 1-deoxyeucommia ulmoides. The gene names of the collected targets were converted using the Gene database (https: / / www.ncbi.nlm.nih.gov / gene).

[0037] Drug-Disease Network Topology Analysis

[0038] The shortest path length d(x,y) between drug target (X) and disease target (Y) is calculated, and the network topology relationship between drug (X) and disease (Y) is analyzed using the z-value score z=(d-μ) / σ. The sAB value between the two drug-target modules is also calculated to evaluate the distance between the two drug-target modules.

[0039]

[0040]

[0041] According to the target network evaluation method constructed by Cheng et al., six drug-drug-disease target network relationships were found in Eucommia ulmoides formulas: combination A: 6-gingerol and eucommia ulmoides alcohol; combination B: 6-gingerol and 1-deoxyeucommia ulmoides alcohol; combination C: 6-gingerol and 6-shogaol; combination D: eucommia ulmoides alcohol and 1-deoxyeucommia ulmoides alcohol; combination E: eucommia ulmoides alcohol and 6-shogaol; and combination F: 1-deoxyeucommia ulmoides alcohol and 6-shogaol. However, for drug combinations with therapeutic effects, the target modules of the two drugs must overlap with the disease module, and only drug combinations showing complementary exposure to the disease target module have a significant advantage in efficacy. Figure 1 As shown.

[0042] This invention uses single-cell data related to microglial activation in Parkinson's disease for analysis. Tables 1 and 2 show that the distance between the target sites of 6-shogaol and eucommia ulmoides alcohol conforms to... Figure 1 The network relationship shown, ZDA<0, ZDB<0, sAB>0, suggests that the two may have a synergistic effect in inhibiting the inflammatory activation of microglia in Parkinson's disease.

[0043] Table 1. Z-scores of the brain-entering components of Eucommia ulmoides formula in the drug-target and disease modules.

[0044]

[0045] Table 2. s of the drug-target module and disease module of the two drug combinations AB Value rating

[0046]

[0047] Example 2

[0048] Effects of 6-shogaol, eucommia ulmoides monomers, and combinations thereof on inflammatory activation of microglia.

[0049] Cell culture: Prepare a 10% complete culture medium by adding 10% fetal bovine serum and 1% penicillin antibiotics to DMEM.

[0050] Cell resuscitation: Take one vial of frozen BV-2 microglia from liquid nitrogen and thaw it rapidly in a 37°C water bath. After complete thawing, quickly transfer the cell suspension in the cryovial to a 15mL centrifuge tube containing 1mL of complete culture medium and centrifuge (800rpm, 5min). Discard the supernatant, add 1mL of complete culture medium, gently pipette to disperse the cells, and transfer to a T25 culture flask containing 4mL of complete culture medium. Incubate at 37°C in a 5% CO2 incubator.

[0051] Cell passage: Resuscitate cells and culture them in a 37°C, 5% CO2 incubator. Passage them every other day at a passage ratio of 1:3 to 1:6. Once the cells have stabilized, experiments can be performed. When the cells reach 80% confluence, discard the culture medium, wash twice with 5 mL of PBS, then add 1 mL of 0.25% trypsin to digest until the adherent cells loosen. Add 3 mL of complete culture medium to stop digestion, pipette the adherent cells off the wall, and transfer the cell suspension to a 15 mL centrifuge tube for centrifugation (800 rpm, 5 min). Discard the supernatant, add 4 mL of complete culture medium, mix well, and transfer to a new T75 culture flask for incubation.

[0052] Cell seeding: After resuspending and mixing the cells, take 10 μL for cell counting, and dilute the cell suspension to 1.2 × 10⁻⁶. 5 Inoculate 400 μL / well into 48-well plates and incubate.

[0053] Drug administration: Accurately weigh 1 mg of LPS powder, dissolve it in sterile ultrapure water to 1 mL, and prepare a 1 mg / mL LPS solution. Dilute to 0.1 μg / mL before use. Stimulate cells 24 h after inoculation with the drug, and prepare the following groups: normal group, LPS group, LPS + 6-shogaol group, LPS + eucommiol group, and LPS + 6-shogaol and eucommiol combination group.

[0054] CCK-8 assay for cell viability: 24 h after drug administration, the 48-well plate was removed, the supernatant was aspirated, 100 μL of diluted CCK-8 solution was added, and the plate was incubated at 37 °C for about 20 min. The 48-well plate was then removed, and 100 μL of the solution was transferred to a 96-well plate. The absorbance (OD value) at 450 nm was measured using a microplate reader, and the relative cell viability was calculated.

[0055] Relative cell viability (%) = OD value of experimental group / OD value of blank control group × 100% (Formula 3)

[0056] NO content detection: 24 h after drug administration, remove the 96-well plate and proceed as follows: (1) Remove Griess Reagent I and II and allow to return to room temperature; (2) Dilute the standard with DMEM (1-100 μM) at a concentration gradient of 0, 1, 2, 5, 10, 40, 60, 100 μM; (3) Add the standard and the supernatant of the 48-well plate to the 96-well plate at 50 μL / well; (4) Add room temperature Griess Reagent I to each well at 50 μL / well; (5) Add room temperature Griess Reagent II to each well at 50 μL / well; (6) Measure the absorbance at 540 nm. Calculate the NO inhibition rate according to the following formula, and calculate the sample concentration (IC50 value) corresponding to a NO inhibition rate of 50%.

[0057] NO inhibition rate (%) = (OD value of LPS group - OD value of drug-treated group) / (OD value of LPS group - OD value of blank control group) × 100% (Formula 4)

[0058] Results of 6-shogaol and eucommia ulmoides monomers on BV-2 cytotoxicity:

[0059] This study used the CCK-8 assay to determine the effects of different concentrations of 6-shogaol and eucommia ulmoides on the cell viability of BV-2 cells after 24 hours of treatment. The results are shown below. Figure 2 The purpose of this experiment was to screen for compounds at concentrations that would not affect normal cell proliferation. 6-Shogaol and Eucommia ulmoides alcohol showed cell viability of over 90% at their respective four concentrations, without exhibiting significant cytotoxicity.

[0060] Inhibitory effects of 6-shogaol and eucommia ulmoides monomers on NO release from BV-2 cells:

[0061] The results showed that the changes in NO content in the cell supernatant of LPS-induced BV-2 cells after 24 h of treatment with 6-shogaol and eucommia ulmoides alcohol within a safe concentration range were determined using the Griess method. The anti-inflammatory activity of the two drugs was investigated using NO content as an indicator. The results are shown in [Figure number missing]. Figure 3 The IC50 values ​​for the two drugs were 2.715 μM and 12.32 μM, respectively.

[0062] In vitro anti-inflammatory effects of 6-shogaol and eucommia ulmoides alcohol

[0063] NO inhibition rate after combining the two:

[0064] When 6-shogaol and eucommia ulmoides are mixed in a 1:1 and 1:2 ratio, as follows: Figure 4 As the concentration of the combined drug increased, the effect of inhibiting NO release from BV-2 cells also increased, and the effect was significantly different from that of 6-shogaol alone. The inhibition rate of NO by 6-shogaol and eucommia ulmoides combined is shown in Table 3, and the actual half-maximal inhibitory concentration (IC50) of the combined group is shown in Table 4.

[0065] Table 3. NO inhibition rate of 6-shogaol combined with eucommia ulmoides alcohol

[0066]

[0067]

[0068] Table 4 IC50 of 6-shogaol in combination with eucommia ulmoides alcohol 50mix

[0069]

[0070] Example 3

[0071] Isobologram analysis was used to study the interaction between 6-shogaol and eucommia ulmoides alcohol.

[0072] The IC50 values ​​for inhibiting NO by 6-shogaol and eucommia ulmoides were 2.715 and 12.32 μM, respectively, with a ratio of 1:4.53. 6-shogaol and eucommia ulmoides were then formulated in ratios of 1:1 and 1:2. The inhibitory effects on NO in each group were determined using the method described above, and the NO inhibition rate and IC50 were calculated. 50mix Plot the IC50 values ​​and 95% confidence limits of 6-shogaol and eucommia alcohol on the X and Y axes, respectively. 50 Connecting the values ​​forms the summation line, and connecting the confidence limits separately yields the 95% confidence limit of the summation line. The intersection of the proportional line and the summation line represents the theoretical half-maximum inhibitory concentration (IC50) of the glycosidic combination. 50add If the two are matched, IC 50mix If the summation line is on the additive line or within the 95% confidence limit, it indicates that the two drugs have an additive effect; if the summation line is to the left of the confidence limit, it indicates that the two drugs have a synergistic effect; if the summation line is to the right of the confidence limit, it indicates that the two drugs have an antagonistic effect.

[0073] Addition lines are fundamental for evaluating the interaction properties of two drugs. Isobologram analysis was performed; the Isobologram of NO inhibition rate is shown below. Figure 5 As shown in the figure, the IC50 values ​​of the two drugs in each group after combination were... 50mix The values ​​are all less than IC. 50add Value, IC50 value of the inhibition rate of NO after the two drugs are combined 50mix The values ​​all fall to the left of the summation line and the 95% confidence limit, indicating that the two drugs have a synergistic effect.

[0074] Example 4

[0075] Statistical analysis of the interaction between 6-shogaol and eucommia ulmoides alcohol

[0076] After the two drugs were combined, the half-maximal inhibitory concentration (IC50) was measured to be 100%. 50mix The theoretical half-maximal inhibitory concentration is IC50. 50add If IC 50mix <IC 50add This indicates a synergistic effect between the two drugs; if IC 50mix IC 50add This indicates an antagonistic effect between the two drugs. Statistical analysis of the interaction between 6-shogaol and eucommia ulmoides alcohol showed that the IC50 value was... 50add The calculation formula is as follows:

[0077] IC 50add =IC 50A / (P A +RP B )

[0078] In the formula IC 50add R represents the theoretical half-maximal inhibitory concentration (IC50) after the two drugs are combined, and R is the potency ratio of drugs A and B. R = IC50 50A / IC 50B IC 50A IC 50B ICs for A and B respectively 50 Value; P A P B These represent the proportions of A and B in the matching.

[0079] The interaction index (γ) can evaluate the interaction between 6-shogaol and eucommia ulmoides alcohol. If γ < 1, it indicates a synergistic effect, with lower γ values ​​indicating better synergy; if γ > 1, it indicates an antagonistic effect; if γ = 1, it indicates an additive effect. The formula for calculating the interaction index (γ) is as follows:

[0080] γ=IC50 Amix / IC 50A +IC 50Bmix / IC 50B

[0081] The results show that the IC of the matched group 50add The values ​​of γ are shown in the table below. As can be seen from Table 5, the values ​​of γ are all less than 1. Therefore, the 1:1 and 1:2 ratios of 6-shogaol with eucommia ulmoides alcohol have a synergistic effect in inhibiting NO release from BV-2 cells, and the synergistic effect of the 1:2 ratio of 6-shogaol with eucommia ulmoides alcohol is stronger.

[0082] Table 5. Statistical analysis results of the compatibility of 6-shogaol and eucommia ulmoides alcohol.

[0083]

[0084] Example 5

[0085] Oral suspension of 6-shogaol and eucommia ulmoides

[0086]

[0087] Dissolve 6-shogaol and eucommia ulmoides in ethanol, disperse sodium carboxymethyl cellulose and xanthan gum in water, then mix the two solutions thoroughly and sterilize.

[0088] Example 6

[0089] Oral suspension of 6-shogaol and eucommia ulmoides

[0090]

[0091]

[0092] Dissolve 6-shogaol and eucommia ulmoides in ethanol, disperse sodium carboxymethyl cellulose and xanthan gum in water, then mix the two solutions thoroughly and sterilize.

Claims

1. A pharmaceutical composition for treating Parkinson's disease or Alzheimer's disease, characterized in that, The pharmaceutical composition is made from the following raw materials in parts by weight: 1 part 6-shogaol and 1 part eucommia ulmoides alcohol.

2. A pharmaceutical composition for treating Parkinson's disease or Alzheimer's disease, characterized in that, The pharmaceutical composition is made from the following raw materials in parts by weight: 1 part 6-shogaol and 2 parts eucommia ulmoides alcohol.

3. A pharmaceutical preparation, characterized in that: It consists of the pharmaceutical composition according to any one of claims 1-2, and pharmaceutical excipients.

4. The pharmaceutical preparation according to claim 3, characterized in that: The pharmaceutical preparation is selected from oral medications and parenteral medications.

5. The pharmaceutical preparation according to claim 4, characterized in that: The pharmaceutical preparation is selected from one of the following: powder, solution, pill, granule, capsule, tablet, and injection.

6. Use of the pharmaceutical composition according to any one of claims 1-2 or the pharmaceutical preparation according to any one of claims 3-5 in the preparation of a medicament for treating Parkinson's disease or Alzheimer's disease.

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