Application of 4-methyleneglutamic acid in pharmacy

Abstract

The invention discloses application of 4-methyleneglutamic acid in pharmacy, particularly relates to application of 4-methyleneglutamic acid in preparation of medicines for treating, improving or preventing cerebral apoplexy, and belongs to the technical field of medicines. Experimental results of the applicant show that when 4-methyleneglutamic acid is applied to preparation of the medicine for treating, improving or preventing cerebral apoplexy (especially hemorrhagic cerebral apoplexy), neuronal cell activity after cerebral hemorrhage can be improved, neuronal cell toxicity can be inhibited, neuronal apoptosis can be inhibited, and cerebral apoplexy can be inhibited. A new medicine choice is provided for preventing and / or treating the hemorrhagic stroke.

Description

Technical Field

[0001] The invention belongs to the technical field of medicines, and in particular relates to application of 4-methyleneglutamic acid in pharmaceutical manufacturing. Background Art

[0002] Stroke, commonly known as stroke, is a disease caused by various reasons that damage cerebral blood vessels and produce focal or overall brain tissue damage. It is a common acute cerebrovascular disease in middle-aged and elderly people. Stroke can be roughly divided into two categories, namely ischemic stroke and hemorrhagic stroke, of which hemorrhagic stroke accounts for 10% to 25%. Hemorrhagic stroke refers to bleeding caused by non-traumatic rupture of blood vessels in the brain parenchyma. Hemorrhagic stroke is characterized by rapid onset, severe condition, high mortality, and survivors often have severe neurological dysfunction. Currently, effective treatment methods are limited to surgical removal of hematoma, dehydration, intracranial pressure reduction, and neuroprotection. From the perspective of efficacy, whether it is surgical treatment or drug intervention, the mortality rate of hemorrhagic stroke is still high, and the recurrence rate, complications, and sequelae are still many. Therefore, it is of great significance to discover new targets for the treatment of hemorrhagic stroke and develop a new drug that can alleviate the condition, can be used for a long time, is inexpensive, and does not produce side effects.

[0003] 4-Methyleneglutamic acid (4-MGA), English name: Glutamic acid, 4-methylene-, alias: 4-methyleneglutamic acid or L-2-Amino-4-methylenepentanedioic acid, etc., CAS number: 7150-74-5, molecular formula: C6H9NO4, the structure is as follows:

[0004]

[0005] There are currently no reports on the use of 4-methyleneglutamate in the treatment, improvement or prevention of stroke. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide the use of 4-methyleneglutamic acid in pharmaceutical preparation.

[0007] Hemorrhagic stroke is a serious central nervous system disease characterized by high morbidity, high disability and high mortality. At present, the treatment methods for hemorrhagic stroke mainly focus on controlling intracranial pressure, hemostasis and surgical intervention, and there is a lack of effective neuroprotective drugs. Metabolomics studies have shown that the metabolic network of the body of hemorrhagic stroke will undergo significant changes, and changes in certain metabolites may be closely related to disease progression and neural damage. The present invention found through non-targeted metabolomics screening that the level of 4-methyleneglutamate was significantly decreased in patients with hemorrhagic stroke, suggesting that it may play an important role in the pathological process of hemorrhagic stroke. It was further confirmed by basic experiments that exogenous supplementation of 4-methyleneglutamate can significantly protect neuronal damage caused by hemorrhagic stroke and inhibit the release of LDH and apoptosis of damaged neurons.

[0008] Based on the above research, the technical solution of the present invention is: use of 4-methyleneglutamic acid in the preparation of drugs for treating, improving or preventing stroke.

[0009] Furthermore, the stroke is selected from hemorrhagic stroke or ischemic stroke, or is hemorrhagic stroke transformed from ischemic stroke.

[0010] The invention also provides a pharmaceutical composition for treating, improving or preventing cerebral stroke, comprising a therapeutically effective dose of 4-methyleneglutamic acid.

[0011] Furthermore, the above-mentioned pharmaceutical composition also contains pharmaceutically acceptable carriers and / or excipients.

[0012] Furthermore, the dosage form of the pharmaceutical composition is a pharmaceutically acceptable dosage form, such as tablets, capsules, granules, suspensions, suppositories, injections, powder injections, pills, sustained-release preparations or targeted preparations and other conventional dosage forms.

[0013] The applicant's experimental results show that the application of 4-methyleneglutamate in the preparation of drugs for the treatment, improvement or prevention of stroke (especially hemorrhagic stroke) can improve the vitality of neuronal cells after cerebral hemorrhage, inhibit the release of LDH from neuronal cells, and inhibit neuronal apoptosis, providing a new drug option for the prevention and / or treatment of hemorrhagic stroke. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is the principal component analysis of serum metabolomics of the experimental group (disease group) and the control group (healthy group), where A is the principal component analysis of the types of differential metabolites of serum metabolites, B is the PCA graph of differential metabolites of serum metabolites, and C is the OPLS-DA graph of differential metabolites of serum metabolites. In the figure, Patient represents the disease group and Healthy represents the healthy group.

[0015] Figure 2VIP score analysis of serum metabolites in the experimental group (disease group) and the control group (healthy group).

[0016] Figure 3 It is the ROC curve analysis and expression level of the serum metabolite 4-methyleneglutamate in the experimental group (disease group) and the control group (healthy group), wherein A is the ROC curve of 4-methyleneglutamate, and B is the expression level of the ROC curve of 4-methyleneglutamate.

[0017] Figure 4 4-Methyleneglutamate dose-dependently improves the cell viability of HT22 neuronal cells after hemorrhagic stroke injury model, where 4-MGA represents 4-methyleneglutamate.

[0018] Figure 5 4-Methyleneglutamate dose-dependently reduces cellular LDH release in the HT22 neuronal cell hemorrhagic stroke injury model, where 4-MGA represents 4-methyleneglutamate.

[0019] Figure 6 4-Methyleneglutamate inhibits the expression level of pro-apoptotic protein Cleaved Caspase-3 in HT22 neuronal cell hemorrhagic stroke injury model, wherein 4-MGA represents 4-methyleneglutamate.

[0020] Figure 7 4-Methyleneglutamate inhibits the expression level of pro-apoptotic protein Bax in HT22 neuronal cell hemorrhagic stroke injury model, where 4-MGA represents 4-methyleneglutamate.

[0021] Figure 8 4-Methyleneglutamate increases the expression level of the anti-apoptotic protein Bcl-2 in the HT22 neuronal cell hemorrhagic stroke injury model, where 4-MGA represents 4-methyleneglutamate. DETAILED DESCRIPTION

[0022] In order to better explain the technical solution of the present invention, the present invention is further described in detail below in conjunction with embodiments, but the embodiments of the present invention are not limited thereto.

[0023] Example 1

[0024] This example is a study that non-targeted metabolomics screening revealed that 4-methyleneglutamate can be used as an important marker for hemorrhagic stroke.

[0025] 1. Metabolite sample preparation:

[0026] 1.1 Source of clinical samples: Six patients with hemorrhagic stroke who were hospitalized in the Department of Neurology of the Affiliated Hospital of Guilin Medical College from May 2020 to May 2021 were selected as the experimental group (disease group). Experienced neurologists collected medical history and conducted physical examinations and finally confirmed the diagnosis. Six healthy subjects who underwent physical examinations in our hospital during the same period were selected as the control group (healthy group). Half of them were male and half were female, with an age range of 41 to 81 years. Inclusion criteria included: (1) patients with cerebral hemorrhage met the diagnostic criteria for cerebral hemorrhage and the onset time was within 72 hours; (2) subjects with similar age and gender composition to the disease group were selected as the control group; (3) general information was collected: age, gender, clinical symptoms, and metabolic-related test results. Exclusion criteria were: (1) patients who had undergone surgical treatment after a new cerebral hemorrhage; (2) patients who had used drugs that might affect metabolism, such as statins, gangliosides, edaravone, dextran, and other lipid-lowering, nerve-nourishing, and oxygen free radical-scavenging drugs 2 hours before the blood sample was collected; (3) patients with ischemic stroke; and (4) patients with metabolic diseases such as diabetes, coronary heart disease, and hyperlipidemia.

[0027] 1.2 Serum sample processing: After collecting the above blood samples, the blood samples were processed as follows: (1) 200 μL of acetonitrile was added to 50 μL of serum and vortexed; (2) centrifuged (4°C, 12000 rpm, 10 min); (3) the supernatant was transferred to a 1.5 mL centrifuge tube; (4) evaporated in a centrifugal concentrator; (5) reconstituted with 100 μL of 1% acetonitrile and the supernatant was taken for testing.

[0028] 1.3 Non-targeted metabolomics study: The serum supernatants of the above-mentioned hemorrhagic stroke patients and healthy subjects were processed and the peak area, mass-to-charge ratio, and retention time of the primary mass spectrometry raw data were extracted using MarkerView 1.3 (AB Sciex, Concord, ON, Canada) software to generate a two-dimensional data array (filtering out isotope peaks); PeakView 2.2 (AB Sciex, Concord, ON, Canada) was used to extract the secondary mass spectrometry data and compare it with the Metabolites database, HMDB, METLIN, and standards to identify the metabolite ID, and the identified ID was assigned to the corresponding ion in the primary mass spectrometry two-dimensional data array. The identified metabolome data were statistically analyzed and pathway analyzed using a self-written program based on R language. The obtained data were imported into the metabolomics data analysis website (https: / / www.metaboanalyst.ca / ) for further data analysis, including principal component analysis (PCA), VIP score plot analysis, ROC regression curve analysis, and metabolite expression level analysis.

[0029] 2. We first analyzed the types of differential metabolites in serum between the disease group and the control group. The results showed that most of the differential metabolites were amino acids, up to 57.29%. Figure 1 A. In addition, we performed principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) on the differential metabolites of serum metabolites. Figure 1 B shows that the PCA score graph can reflect the intrinsic changes in the metabolite level. From the PCA score graph, it can be seen that the principal component 1 and principal component 2 in the serum metabolites of the experimental group and the healthy group are clearly separated, with little overlap, indicating that there are obvious differences between the principal components. In order to better distinguish different groups, we used OPLS-DA analysis. Like the PCA analysis results, the results of the OPLS-DA score graph also showed that the serum metabolite components of the experimental group and the healthy group were significantly different. The results are shown in Figure 1 As shown in C.

[0030] 3. We further performed biomarker analysis on all differential metabolites and obtained a VIP score map. In the VIP score map, the larger the VIP value, the higher its importance. The data shows that the top 25 metabolites with VIP values ​​greater than 1 include 4-methyleneglutamic acid. The results are as follows Figure 2 shown.

[0031] 4. We performed regression analysis on the screened N-acetylglutamine and detected their expression levels between the experimental group and the healthy group. The results showed that the level of 4-methyleneglutamate decreased significantly after hemorrhagic stroke. The ROC curve results showed that the area under the AUC curve was 1, showing extremely high specificity and sensitivity. Figure 3 shown.

[0032] According to the above experimental results, 4-methyleneglutamate is suitable as a blood metabolic marker for hemorrhagic stroke and for preparing related detection products for the prevention and / or treatment of hemorrhagic stroke.

[0033] Example 2

[0034] This example is a research experiment on the neuroprotective effect of 4-methyleneglutamic acid on HT22 (mouse hippocampal neuronal cells) neuronal hemorrhagic stroke cell model.

[0035] It is worth noting that, unless otherwise specified, the raw materials used in the present invention are all common commercially available products, and their sources are not specifically limited.

[0036] The following sources of raw materials are provided for illustrative purposes:

[0037] 1. Reagents: 4-Methyleneglutamic acid, Shanghai Aladdin Biochemical Technology Co., Ltd., Catalog No.: H353190; Prussian blue dye (Beijing Solebold Technology Co., Ltd.); CCK8 detection kit (Beijing Solebold Technology Co., Ltd.); LDH detection kit (Beijing Solebold Technology Co., Ltd.).

[0038] 2.1 Construction of HT22 neuronal cell hemorrhagic stroke cell model: For specific experimental methods, please refer to (Molecular neurobiology, 2022, 59 (3), 1381–1397.). The method is briefly described as follows: HT22 neuronal cells were cultured at 37°C and 5% CO2 using Dulbecco's modified Eagle medium (DMEM, Gibco), 10% fetal bovine serum (FBS, Gibco) and 1% penicillin / streptomycin (4 mg / mL; Sigma-Aldrich). After the cells grew to 90%, they were exposed to 100 μM Hemin (hemin chloride, BCBR5047V, Sigma-Aldrich) to induce cell death. 24 hours after treatment, the cells were rinsed with warm phosphate buffered saline (PBS), cell viability was assessed by CCK-8, and cytotoxicity was detected by LDH.

[0039] 2.2 Cell activity detection: Prepare cell suspension, inoculate cell suspension in 96-well plate, about 100μl per well, do not inoculate cells in the peripheral wells, add 100ulPBS, and place the culture plate in the incubator for 1 day (37℃, 5% CO2). Prepare medium containing 4-methylene glutamate, gradually dilute to 1000, 100, 10, 1, 0nmol / L concentration gradient, remove the original medium of 96-well plate, and wash once with sterile PBS. After the cells are modeled with hemin, add different concentrations of 4-methylene glutamate medium, and set 6 replicates at different concentrations. When measuring, add 10ul CCK-8 solution to each well, place the culture plate in the incubator for 1.5h, use an enzyme reader to measure the absorbance value (OD) at 450nm, and then count the cell activity.

[0040] 2.3 LDH detection of cytotoxicity: LDH (lactate dehydrogenase) detection is a commonly used cytotoxicity detection method. Its principle is based on the release of LDH to the outside of the cell when the cell is damaged or dead. The cytotoxicity or mortality rate is evaluated by detecting the LDH activity in the cell culture medium. The specific method is briefly described as follows: After HT22 cells are cultured to the logarithmic growth phase under appropriate culture conditions, the cells are modeled with hemin and then added with different concentrations of 4-methylene glutamate culture medium. Six replicate wells are set at different concentrations. After 24 hours, the cell culture plate is centrifuged at 400g for 5 minutes using a multi-well plate centrifuge. Take 90μL of the supernatant from each well, add 45μL of the freshly prepared LDH detection working solution, mix well, incubate at room temperature in the dark for 30 minutes, and then measure the absorbance at 490nm. Cytotoxicity (%) = (absorbance of treated sample-absorbance of sample control well) / (absorbance of maximum enzyme activity of cells-absorbance of sample control well) × 100%.

[0041] 2.4 Western blot method to detect the levels of apoptosis-related proteins Cleaved Caspase-3, Bax, and Bcl-2: After washing with PBS buffer solution, the cells were scraped and fully lysed after adding 200ul RIPA lysis buffer (containing 0.1% protease inhibitors). The samples were then centrifuged at 4°C, 15000rpm for 15 minutes, and the supernatant was aspirated to determine the protein concentration and then added to 4X protein loading buffer at a ratio of 1:3 for standby use. Prepare gel and prepare electrophoresis buffer, transfer buffer, etc. Use Nanodrop Lite Spectrophotometer (Thermo scientific) to determine the protein concentration, calculate the sample volume, and load the sample; then perform electrophoresis, transfer, and block. Add primary antibodies Cleaved Caspase-3, Bax, and Bcl-2 and place them on a shaker in a 4°C chromatography cabinet overnight; apply secondary antibodies: apply on a shaker at room temperature for 2 hours. Luminescence: Wash the membrane, prepare chemiluminescent solution (solution A: solution B = 1:1), use Azure Biosystems instrument to expose for 3 seconds to obtain the bands, use ImageJ to calculate the protein expression level, and use Prism software for statistical analysis of the results.

[0042] 3. Statistical Analysis

[0043] The experimental data were expressed as Mean ± SEM, and the differences among the three groups were compared using one-way ANOVA test (MW method). Statistical analysis results showed that P < 0.05 was considered to be significantly different, and P < 0.01 was considered to be extremely significantly different.

[0044] 4. Test results

[0045] 4.1 The results of the cell viability experiment showed that compared with the control group, hemin treatment could significantly reduce the viability of HT22 cells, and different doses of 4-methylglutamate after modeling could be given, and the cell viability continued to increase. Moreover, 100 and 1000 nM concentrations of 4-methylglutamate could significantly increase the cell viability after modeling, with significant statistical differences, and P values ​​were less than 0.01 and 0.05, respectively. Figure 4 shown.

[0046] 4.2 Cytotoxicity test results showed that compared with the control group, hemin treatment increased the cytotoxicity of HT22 cells, and after modeling, different doses of 4-methylglutamate were given, and the cytotoxicity continued to decrease. In addition, 10 and 100 nM concentrations of 4-methylglutamate significantly reduced the cytotoxicity after modeling, with significant statistical differences, and P values ​​were less than 0.05 and 0.01, respectively. Figure 5 shown.

[0047] 4.3 Based on the above results, we selected 100nM concentration of 4-methyleneglutamate to further detect cell apoptosis after cell damage. Cleaved Caspase-3 is the activated form of Caspase-3, a key cysteine ​​protease that plays a central role in the process of cell apoptosis. Figure 6 As shown: Compared with the control group, hemin treatment significantly increased the expression level of the pro-apoptotic protein Cleaved Caspase-3 in HT22 cells, while after treatment with 100nM concentration of 4-methyleneglutamate, the expression level of the pro-apoptotic protein Cleaved Caspase-3 was significantly decreased.

[0048] 4.4 Based on the above results, we selected 100 nM concentration of 4-methyleneglutamate to further detect cell apoptosis after cell damage. Bax protein, also known as BCL-2-associated X protein, is an important member of the BCL-2 gene family and plays a key role in the process of cell apoptosis. Figure 7 As shown: Compared with the control group, hemin treatment significantly increased the expression level of the pro-apoptotic protein Bax in HT22 cells, while after treatment with 100nM concentration of 4-methyleneglutamate, the expression level of the pro-apoptotic protein Bax was significantly decreased.

[0049] 4.5 Based on the above results, we selected 100nM concentration of 4-methyleneglutamic acid to further detect cell apoptosis after cell damage. As a cell survival promoting factor, BCL-2 has a significant inhibitory effect on cell apoptosis. It regulates cell apoptosis by combining with pro-apoptotic proteins such as Bax to form a dimer complex. When BCL-2 is overexpressed, it mainly forms a heterodimer of BCL-2 and Bax to prevent cell apoptosis. Figure 8 As shown: Compared with the control group, hemin treatment significantly increased the expression level of the anti-apoptotic protein Bcl-2 in HT22 cells, while after treatment with 100nM concentration of 4-methyleneglutamate, the expression level of the anti-apoptotic protein Bcl-2 was significantly decreased.

[0050] The above results indicate that 4-methyleneglutamate can enhance cell viability, reduce cytotoxicity, inhibit cell apoptosis and play a neuroprotective role in HT22 neuron injury after hemorrhagic stroke.

[0051] In summary, the existing 4-methyleneglutamate should be able to improve neuronal damage in hemorrhagic stroke and can be used to prepare drugs for treating, improving or preventing stroke, thereby providing a new source for preparing drugs for preventing, improving or preventing hemorrhagic stroke, and also discovering the new medicinal value of 4-methyleneglutamate.

[0052] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

Use of 1.4-methyleneglutamic acid in the preparation of drugs for treating, improving or preventing cerebral stroke.

2. The use according to claim 1, characterized in that: The stroke is selected from hemorrhagic stroke or ischemic stroke.

3. The use according to claim 1, characterized in that: The cerebral stroke is a hemorrhagic cerebral stroke transformed from an ischemic cerebral stroke.

4. A pharmaceutical composition for treating, improving or preventing stroke, comprising a therapeutically effective dose of 4-methyleneglutamate.

5. The pharmaceutical composition according to claim 4, characterized in that: The pharmaceutical composition also contains a pharmaceutically acceptable carrier and / or auxiliary material.

6. The pharmaceutical composition according to claim 4, characterized in that: The dosage form of the pharmaceutical composition is a pharmaceutically acceptable dosage form.

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