Use of sodium brostallicin for the preparation of a medicament for the prevention and treatment of senile dementia

Abstract

The application belongs to the technical field of pharmaceutical chemistry, and particularly relates to application of brostallicin sodium in preparation of a drug for treating senile dementia. The application relates to application of brostallicin sodium in treatment of senile dementia. The application establishes a dementia model of a dementia rat and a dementia mouse through an in-vivo experiment, and establishes a PC12 cell, a BV2 cell and a HT22 cell damage model through an in-vitro experiment, and finds that brostallicin sodium has a prevention and treatment effect on senile dementia. The brostallicin sodium is expected to be used as a preparation of a drug for treating senile dementia for clinical application.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical chemistry technology and relates to the pharmaceutical application of brozopen sodium, specifically its application in the preparation of drugs for treating Alzheimer's disease. Background Technology

[0002] Brozopine (BZP), also known as sodium 5-bromo-2-(α-hydroxypentyl)benzoate, has the following molecular structural formula:

[0003]

[0004] It is an analogue of butylphthalide (NBP), a drug used to treat cerebral ischemia. Our research team has discovered for the first time that BZP has a good preventive and therapeutic effect on Alzheimer's disease.

[0005] With the increasing severity of aging, the incidence of Alzheimer's disease is rising year by year. Alzheimer's disease can be divided into primary dementia and vascular dementia (VD). The former is also known as Alzheimer's disease (AD). Aβ deposition forming amyloid plaques and neurofibrillary tangles are the main pathological features of AD. Clinical symptoms include memory impairment, cognitive decline, and impaired motor balance. In severe cases, it can lead to mood and personality changes, loss of self-care ability, and even death. Vascular dementia, second only to primary dementia in its prevalence, accounts for 20%-30% of all dementia cases. It is caused by various cerebrovascular diseases such as cerebral infarction, cerebral hemorrhage, and cerebral hypoperfusion, resulting in brain cell damage, neuronal death, and subsequent dementia symptoms, including progressive cognitive decline, mood disorders, and difficulties with language and daily living. However, the available drugs for treating Alzheimer's disease are very limited, and there is an urgent need to develop new drugs for the prevention and treatment of Alzheimer's disease. Summary of the Invention

[0006] In previous work, our research team synthesized sodium 5-bromo-2-(α-hydroxypentyl)benzoate and obtained an invention patent. Through long-term research, we found that it has a good effect in preventing and treating ischemic stroke. Therefore, the purpose of this invention is to provide the application of sodium 5-bromo-2-(α-hydroxypentyl)benzoate in the preparation of drugs for treating Alzheimer's disease.

[0007] To achieve the objective of this invention, the technical solution is as follows:

[0008] This invention establishes dementia models in rats and mice through in vivo experiments and establishes PC12, BV2, and HT22 cell damage models in vitro, demonstrating that brozopen sodium has preventive and therapeutic effects on Alzheimer's disease.

[0009] Advantages of this invention: Through in vitro and in vivo experiments, this invention has shown that BZP can improve the learning, memory, and cognitive functions of VD and AD rats by shortening the average latency, increasing the number of times rats cross the target quadrant, and prolonging the effective time spent in the target quadrant. Furthermore, it was found that BZP can significantly improve the cell survival rate and improve cell morphology of L-Glu-induced excitotoxic cell damage in PC12 cells. The 20 μmol / L BZP group showed a better effect on improving cell survival than the equimolar concentration NBP group (P<0.05). It holds promise for use in the preparation of drugs for the treatment of Alzheimer's disease and for clinical application. Attached Figure Description

[0010] Figure 1 Eight weeks after 2-VO surgery and four weeks after drug administration, rats in each group underwent the final water maze test. A: Latency of vascular dementia (VD) rats in each group from day 58 to 62 after 2-VO surgery; B: Number of times VD rats in each group crossed the target quadrant on day 62 after 2-VO surgery; C: Effective time of rats in each group staying in the target quadrant on day 62 after 2-VO surgery; D: Swimming trajectory diagram of VD rats in each group on day 62 after 2-VO surgery. * P < 0.05 ** P < 0.01 compared to the Sham group; # P < 0.05 ## P < 0.01 compared to the 2-VO group. (mean ± standard deviation, n = 10⁻¹²).

[0011] Figure 2 Four weeks after administration at the same time point, rats in each group underwent the final water maze test. A: Latency period of AD rats in each group from day 58 to day 62 at the same time point; B: Number of times AD rats in each group crossed the target quadrant on day 62; C: Effective time of AD rats in each group in the target quadrant on day 62; D: Swimming trajectory diagram of AD rats in each group on day 62. * P < 0.05 ** P < 0.01 compared to the control group; # P < 0.05 ## P < 0.01 compared with the AD group. (mean ± standard deviation, n = 10⁻¹²).

[0012] Figure 3 The effect of BZP on hippocampal neurons in VD rats (n=3).

[0013] Figure 4The effect of BZP on neurons in the hippocampus of AD rats (n=3).

[0014] Figure 5 The effect of BZP on the levels of inflammatory factors IL-6 and COX-2 in peripheral blood plasma of VD rats, (A) IL-6; (B) COX-2. ** P<0.01 compared with the Sham group, # P<0.01, ## P<0.01 compared with the 2-VO group (mean ± standard deviation, n=5).

[0015] Figure 6 To investigate the effects of BZP on cell morphology and viability after L-Glu-induced excitotoxic injury in PC12 cells. PC12 cells were incubated for 18 h in complete medium containing BZP (10, 20, 40 μmol / L) and then injured for 24 h with 20 mM L-Glu-low glucose DMEM. ** P<0.05 compared to the control group ## P<0.01 compared with the L-Glu group, ▲ P<0.05 compared with the NBP group (mean ± standard deviation, n=3).

[0016] Figure 7 The effects of BZP on cell morphology and viability of HT22 cells after H2O2-induced oxidative stress injury were investigated. HT22 cells were incubated for 18 h in complete medium containing BZP (10, 20, 40 μmol / L) and then injured for 24 h with 400 μM H2O2-low glucose DMEM.

[0017] Figure 8 To determine the effects of BZP on the expression of inflammatory factors IL-1β, IL-6, COX-2 and TNF-α in L-Glu-induced excitotoxic injury of PC12 cells, (A) IL-1β; (B) IL-6; (C) COX-2; (D) TNF-α. ** P<0.01 compared to the Control group, ## P<0.01 compared with the L-Glu group (mean ± standard deviation, n=3). Figure 9 To determine the effects of BZP on the expression of inflammatory factors IL-1β, IL-6, COX-2 and TNF-α induced by LPS in BV2 cells, (A) IL-1β; (B) IL-6; (C) COX-2; (D) TNF-α. * P<0.05, ** P<0.01 compared to the Control group, # P<0.05, ##P<0.01 compared with the LPS group (mean ± standard deviation, n=3). Detailed Implementation

[0018] The NBP described in this invention is butylphenyl peptide. To better illustrate this invention, the following examples are provided: Example 1

[0019] I. In vivo experiments:

[0020] 1. Water maze behavioral experiment

[0021] 1.1 A modified bilateral common carotid artery (2-VO) method was used to establish a VD rat model. VD rats were screened using a first water maze test, and the final water maze test was used to observe the behavioral improvement effect of BZP on VD rats. The water maze was used to assess rat behavior, including: the average latency of each group of rats in the orientation navigation test and spatial exploration test, and the number of times each group of rats crossed the target quadrant and the effective time spent in the target quadrant in the spatial exploration test. Results are shown in Appendix. Figure 1 .

[0022] Depend on Figure 1 It can be seen that the average escape latency of rats in each group tends to shorten with the extension of training days. Compared with the Sham group, from day 58 to day 62 of the experiment, the average escape latency of rats in the 2-VO group was significantly prolonged (P < 0.01); compared with the 2-VO group, the BZP (12, 24 mg / kg) group significantly shortened the average escape latency of rats (P < 0.05, P < 0.01). Among them, on day 62, the effect of BZP (24 mg / kg) in shortening the average escape latency of rats was significantly better than that of the equimolar dose of NBP group (P < 0.05). Figure 1 As shown in Figure B, compared with the Sham group, the number of times rats in the 2-VO group crossed the target quadrant was significantly reduced (P < 0.01); compared with the 2-VO group, the BZP (12, 24 mg / kg) group dose-dependently increased the number of times rats crossed the target quadrant (P < 0.05). Figure 1 As shown in Figure C, compared with the Sham group, the effective time of rats in the 2-VO group was significantly shortened in the target quadrant (P < 0.05); compared with the 2-VO group, the BZP (12, 24 mg / kg) group dose-dependently prolonged the effective time of rats in the target quadrant (P < 0.05). Figure 1 As shown in the swimming trajectory diagram of the D rats, compared with the Sham group, the effective time spent in the target quadrant and the number of times the target quadrant was crossed were significantly reduced in the 2-VO group, and the swimming trajectory showed obvious marginality. Compared with the 2-VO group, the BZP (12, 24 mg / kg) group showed a dose-dependent increase in the time spent in the target quadrant and the number of times the target quadrant was crossed, and the swimming trajectory showed a certain degree of purposefulness.

[0023] 1.2 Primary Alzheimer's Disease (AD) rats in aged rats were screened using the initial water maze test, and the ameliorative effect of BZP on the behavior of AD rats was observed using the final water maze test. The water maze was used to assess rat behavior, including: the average latency of each group of rats in the orientation navigation test and spatial exploration test, and the number of times each group of rats crossed the target quadrant and the effective time spent in the target quadrant in the spatial exploration test. Results are attached. Figure 2 .

[0024] Depend on Figure 2 It can be seen that the average escape latency of rats in each group tends to shorten with the extension of training days. Compared with the Control group, the average escape latency of rats in the AD group was significantly prolonged from day 58 to day 62 of the experiment (P < 0.01); compared with the AD group, the BZP (12, 24 mg / kg) group significantly shortened the average escape latency of rats (P < 0.05, P < 0.01). Figure 2 As shown in Figure B, compared with the Control group, the number of times rats in the AD group crossed the target quadrant was significantly reduced (P < 0.01); compared with the AD group, the BZP (12, 24 mg / kg) group dose-dependently increased the number of times rats crossed the target quadrant (P < 0.05). Figure 2 As shown in Figure C, compared with the Control group, the effective time of rats in the AD group was significantly shortened in the target quadrant (P < 0.05); compared with the AD group, the BZP (12, 24 mg / kg) group dose-dependently prolonged the effective time of rats in the target quadrant (P < 0.05). Figure 2 As shown in the swimming trajectory diagram of the AD rats, compared with the Control group, the effective time spent in the target quadrant and the number of times the target quadrant was crossed were significantly reduced, and the swimming trajectory showed obvious marginality. Compared with the AD group, the BZP (12, 24 mg / kg) group showed a dose-dependent increase in the time spent in the target quadrant and the number of times the target quadrant was crossed, and the swimming trajectory showed a certain degree of purposefulness.

[0025] The results indicate that BZP can improve the learning, memory, and cognitive functions of VD and AD rats by shortening the average latency, increasing the number of times rats cross the target quadrant, and prolonging the effective time spent in the target quadrant.

[0026] 2. Nissl staining was used to detect changes in the morphology and number of neurons in the CA1, CA3, and DG regions of the hippocampus in VD and AD rats.

[0027] Nissl bodies are a characteristic structure of neuronal cytoplasm, and the density and color of Nissl staining in neuronal cytoplasm can be used to evaluate neuronal damage. The rat hippocampus is mainly composed of CA1, CA3, and DG regions. CA1 and CA3 regions are closely related to learning and memory functions, while the DG region, also known as the dentate gyrus, is an area involved in memory formation, exploration, stress, and depression. It is a key site for communication between the hippocampus and the external environment and is crucial for encoding spatial information.

[0028] The results are as follows Figure 3 As shown in A, B, and C, in the Sham group, cells in the CA1 and CA3 regions were densely packed, with darker cytoplasm, and the DG region was conical and regularly shaped. Compared with the Sham group, the 2-VO group showed more severe neuronal damage in the CA1, CA3, and DG regions, with sparse cells and a significantly reduced number of Nissl bodies. Compared with the 2-VO group, in the BZP (12, 24 mg / kg) group, cells in the CA1 and CA3 regions were restored to a neat arrangement, the number of Nissl bodies was significantly increased, and the DG region was restored to a conical shape and a more regular morphology.

[0029] The results are as follows Figure 4 As shown in A, B, and C, in the Control group, cells in the CA1 and CA3 regions are densely packed, with darker cytoplasm, and the DG region is conical and regularly shaped. Compared with the Control group, in the AD group, neurons in the CA1, CA3, and DG regions are more severely damaged, cells are sparse, and the number of Nissl bodies is significantly reduced. Compared with the AD group, in the BZP (12, 24 mg / kg) group, cells in the CA1 and CA3 regions are restored to a neat arrangement, the number of Nissl bodies is significantly increased, and the DG region is restored to a conical shape and is more regularly shaped.

[0030] 3. Determination of the levels of inflammatory factors IL-6 and COX-2 in peripheral blood plasma of VD rats

[0031] The levels of IL-6 and COX-2 in peripheral blood plasma of VD rats were quantitatively detected using ELISA. Figure 5 As shown in A and B, compared with the Sham group, the 2-VO group had increased levels of IL-6 and COX-2 (P<0.01); while the BZP (12, 24 mg / kg) group had a dose-dependent decrease in plasma IL-6 and COX-2 levels compared with the 2-VO group (P<0.05, P<0.01).

[0032] II. In vitro experiments

[0033] 1. Effects of BZP on cell morphology and cell viability after L-Glu-induced excitotoxic cell damage in PC12 cells

[0034] After grouping and drug treatment, the morphology of PC12 cells in each group was observed and the survival rate of PC12 cells in each group was calculated: The morphology of cells in each group under an inverted microscope is shown in the figure below. Figure 6 As shown in Figure A, PC12 cells in the Control group were morphologically intact and neatly and tightly arranged. L-Glu-induced PC12 cell numbers decreased significantly, cell debris was produced in greater quantities, and the cells exhibited atrophic changes, synaptic retraction, and irregular arrangement. BZP10, 20, and 40 μmol / L groups and the NBP pre-incubation group all improved PC12 cell morphology to varying degrees, with plump cell bodies, clear outlines, and significantly increased synapses and cell numbers compared to the L-Glu group. Cell viability was also detected using the CCK8 assay, and the results are shown below. Figure 6 As shown in Figure B, compared with the Control group, the survival rate of PC12 cells in the L-Glu group was significantly reduced (P<0.01); compared with the L-Glu group, 10, 20, and 40 μmol / L BZP increased cell survival rate in a concentration-dependent manner (P<0.01). These results indicate that BZP can significantly improve cell survival rate and cell morphology, with the 20 μmol / L BZP group showing a greater effect on improving cell survival rate than the equimolar concentration NBP group (P<0.05).

[0035] 2. Effects of BZP on cell morphology of H2O2-induced oxidative stress-induced cell damage in HT22 cells

[0036] After treatment with different drugs in different groups, the morphology of HT22 cells in each group was observed: The morphology of cells in each group under an inverted microscope is shown below. Figure 7 As shown, the HT22 cells in the Control group were morphologically intact and arranged neatly and tightly. The number of HT22 cells induced by H2O2 decreased significantly, more cell debris was produced, and the cells showed atrophy-like changes, synapse retraction, and irregular arrangement. The BZP10, 20 and 40 μmol / L groups and the NBP pre-incubation group all improved the morphology of HT22 cells to varying degrees. The cells were plump, with clear outlines, and the number of synapses and cells was significantly increased compared with the H2O2 group.

[0037] 3. BZP inhibits the expression of inflammatory factors related to L-Glu-induced excitotoxic injury in PC12 cells.

[0038] L-Glu-induced PC12 cells undergo a secondary inflammatory response, characterized by increased secretion of inflammatory cytokines such as interleukin (IL)-1β, IL-6, cyclooxygenase 2 (COX-2), and tumor necrosis factor (TNF-α).

[0039] like Figure 8As shown in A, B, C, and D, compared with the Control group, the L-Glu group upregulated the expression of IL-1β, IL-6, COX-2, and TNF-α (P<0.01); while the 10, 20, and 40 μmol / L BZP groups downregulated the expression of IL-1β, IL-6, COX-2, and TNF-α in a concentration-dependent manner compared with the L-Glu group (P<0.05, P<0.01), among which the 20 μmol / L BZP group showed a better downregulation effect on IL-1β, IL-6, COX-2, and TNF-α than the NBP group (P<0.01).

[0040] 4. BZP inhibits the expression of LPS-induced inflammatory factors in BV2 cells.

[0041] LPS can chemotactically convert BV2 cells into an M1-type inflammation model in vitro. The main M1-type inflammatory cytokines include interleukin (IL)-1β, IL-6, cyclooxygenase 2 (COX-2), and tumor necrosis factor (TNF-α).

[0042] like Figure 9 As shown in A, B, C, and D, compared with the Control group, the LPS group showed upregulated expression of IL-1β, IL-6, COX-2, and TNF-α (P<0.05, P<0.01); while the 10, 20, and 40 μmol / L BZP groups showed concentration-dependent downregulated expression of IL-1β, IL-6, COX-2, and TNF-α in cells compared with the LPS group (P<0.05, P<0.01).

[0043] BZP is expected to be used in the preparation of drugs for the treatment of Alzheimer's disease and to be applied clinically.

Claims

1. The pharmaceutical application of sodium 5-bromo-2-(α-hydroxypentyl)benzoate having the following molecular structural formula, characterized in that, It is used as an active ingredient in the preparation of drugs for the treatment or prevention of Alzheimer's disease.

2. The pharmaceutical application of sodium 5-bromo-2-(α-hydroxypentyl)benzoate as described in claim 1, characterized in that, It is prepared as an active ingredient and pharmaceutical excipients into oral or injectable dosage forms.

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