Application of G15 in treatment of white matter injury based on promotion of cerebrovascular generation

Abstract

The invention relates to the technical field of new application of medicines, in particular to application of G15 to treatment of white matter injury based on promotion of cerebrovascular generation, and finds that the G15 has the effect of promoting cerebrovascular generation, can promote myelination insufficiency caused by hypoxia in the early development stage and can improve neurological dysfunction caused by hypoxia, and the G15 has the effect of promoting cerebrovascular generation. Moreover, myelin sheath regeneration of a local demyelination area after the adult can be promoted, so that the effect of directly treating the white matter injury from the pathological aspect is achieved. The invention provides a new application of G15 in treating white matter injury and long-term neurological dysfunction by promoting cerebrovascular generation.

Description

Technical Field

[0001] This invention relates to the field of new uses of pharmaceuticals, and in particular to the application of G15 in the treatment of white matter injury of the brain based on promoting cerebral angiogenesis. Background Technology

[0002] White matter injury (WMI) is a common neurological disorder characterized by damage to the myelin sheath, loss of axons, and abnormalities in glial cells within the white matter region of the brain. It is prevalent in premature infants and demyelinating diseases such as multiple sclerosis. High-risk groups include newborns and the elderly. WMI has become a significant cause of cognitive decline, motor dysfunction, and disability worldwide, significantly impacting both patients and their families.

[0003] Currently, clinical treatments for white matter injury remain significantly limited. Existing treatments primarily focus on symptomatic relief and disease risk control, lacking specific interventions to promote myelin regeneration. Furthermore, there is a lack of effective drugs to improve myelin damage and neurological dysfunction. Recent reports indicate that promoting cerebral angiogenesis can directly or indirectly promote OPC differentiation and myelin formation. For example, our laboratory previously found that angiogenesis in the neonatal brain can indirectly enhance OPC differentiation and myelin formation. Therefore, promoting cerebral angiogenesis in the white matter injury area and reconstructing the local microcirculation network has become a key target for overcoming current treatment limitations and achieving white matter repair.

[0004] As a specific modulator of GPR30, G15 has been shown in previous studies to have certain effects on vasomotor activity in peripheral tissue (such as heart and kidney) ischemic injury models. However, to date, no studies have reported the application of G15 in neurological diseases, especially white matter injury, nor have they mentioned its role and mechanism in targeting cerebral angiogenesis in the white matter region, repairing myelin damage, and improving neurological function. Given the urgent need for treatment of white matter injury and the limitations of existing strategies, exploring the application value of G15 in this field could provide a novel treatment approach for white matter injury that "targets angiogenesis, promotes white matter myelination, and improves neurological function," possessing considerable clinical translational potential. Summary of the Invention

[0005] The purpose of this invention is to provide an application of G15 in the treatment of white matter injury by promoting cerebral angiogenesis, aiming to provide a new use of G15 in the treatment of white matter injury and long-term neurological dysfunction by promoting cerebral angiogenesis.

[0006] To achieve the above objectives, the present invention provides an application of G15 in the treatment of white matter injury based on promoting cerebral angiogenesis; G15 is applied to the treatment of white matter injury. White matter damage mainly includes insufficient myelin formation due to hypoxia in early development and myelin regeneration disorders caused by demyelinating diseases in adulthood.

[0007] The G15 is prepared using G15 powder, DMSO, polyethylene glycol 300 (PEG300), Tween-80, and pure water (ddH2O).

[0008] The configuration steps for the G15 are as follows: Dissolve G15 powder in 100% DMSO solution; The G15 solution was diluted with DMSO, polyethylene glycol 300 (PEG300), Tween-80 and pure water (ddH2O) to prepare a 2 mg / ml clear G15 solution.

[0009] The ratio of DMSO, polyethylene glycol 300 (PEG300), Tween-80, and pure water (ddH2O) is 2%:20%:5%:73%.

[0010] G15 is a specific antagonist of the GPR30 receptor.

[0011] This invention relates to the application of G15 in treating white matter injury by promoting cerebral angiogenesis. The invention reveals that G15 has an effect on promoting cerebral angiogenesis, not only improving insufficient myelin formation caused by early developmental hypoxia and alleviating hypoxic-induced neurological dysfunction, but also promoting myelin regeneration in demyelinated areas in adulthood, thereby achieving a direct pathological treatment of white matter injury. This provides a novel use of G15 to treat white matter injury and long-term neurological dysfunction by promoting cerebral angiogenesis. Attached Figure Description

[0012] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0013] Figure 1This study demonstrated that chronic hypoxia during early development, combined with G15 treatment, promotes angiogenesis and improves myelin formation in the brains of developing mice. Figure a shows the modeling timeline, where mice underwent chronic hypoxia with 10% oxygen from day 3 to day 10 after birth, while receiving continuous G15 treatment once daily. Samples were collected on day 11 for observation. Figure b shows that G15 treatment significantly increased the density of blood vessels (CD31+) in multiple brain regions, including the cortex, cingulate gyrus, genu of the corpus callosum, and striatum. Figures c and d show that the density of myelin (MBP+) and oligodendrocytes (CC1+) in the deep cortex and corpus callosum of the G15 group was significantly higher than that of the control group.

[0014] Figure 2 This study demonstrated that G15 significantly promoted angiogenesis and myelin formation in the damaged area of ​​an adult mouse demyelination model. Figure a shows the modeling timeline. Eleven-month-old NG2CreERT;Tau-mGFP mice were induced with tamoxifen for four days (to label newly formed myelin), followed by local stereotactic injection of Lysolecithin into the brain. Then, G15 was administered once daily for 14 consecutive days. After treatment, samples were collected for observation. Figure b shows that the density of blood vessels (CD31+) in the damaged area was significantly increased in the G15 group compared to the control group. Figure c shows that the newly formed myelin (mGFP+) in the damaged area was significantly increased in the G15 group compared to the control group.

[0015] Figure 3 This study demonstrated that G15 treatment significantly improved neurological function in mice with white matter lesions. Figure a shows a timeline, in which mice were given chronic hypoxia (10% oxygen) from day 3 to day 10 after birth, while simultaneously receiving G15 treatment. Behavioral tests were conducted on day 40. Figure b illustrates that G15 treatment significantly reduced the number of times mice slipped on the balance bar test. Figure c shows that in the water maze test, mice in the G15 treatment group traversed the target quadrant more quickly and over a greater distance than the control group, indicating that G15 treatment significantly improved the spatial memory ability of mice. Figure d shows that G15 treatment improved the social abilities of mice in the three-box social behavior test.

[0016] Figure 4 This is a flowchart describing the configuration process of G15. Detailed Implementation

[0017] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0018] Please see Figures 1 to 4This invention provides the application of G15 in the treatment of white matter injury by promoting cerebral angiogenesis; G15 is applied to the treatment of white matter injury. White matter damage mainly includes insufficient myelin formation due to hypoxia in early development and myelin regeneration disorders caused by demyelinating diseases in adulthood.

[0019] Furthermore, the G15 is prepared using G15 powder, DMSO, polyethylene glycol 300 (PEG300), Tween-80, and pure water (ddH2O).

[0020] Furthermore, the configuration steps for the G15 are as follows: Dissolve G15 powder in 100% DMSO solution; The G15 solution was diluted with DMSO, polyethylene glycol 300 (PEG300), Tween-80 and pure water (ddH2O) to prepare a 2 mg / ml clear G15 solution.

[0021] Furthermore, the ratio of DMSO, polyethylene glycol 300 (PEG300), Tween-80, and pure water (ddH2O) is 2%:20%:5%:73%.

[0022] Furthermore, G15 is a specific antagonist of the GPR30 receptor.

[0023] In this embodiment, the present invention provides the application of GPR30 receptor specific antagonist (capable of specifically inhibiting GPR30) – G15 – in the preparation of a drug for treating white matter injury, where white matter injury mainly includes insufficient myelin formation due to hypoxia in early development and myelin regeneration disorders caused by demyelinating diseases in adulthood.

[0024] G15 preparation method: Dissolve G15 powder (purchased from Selleck's official website) in 100% DMSO solution, and then dilute the G15 solution with 2% DMSO, 20% polyethylene glycol 300 (PEG300), 5% Tween-80 and 73% pure water (ddH2O) to prepare a 2 mg / ml clear G15 solution. This solution should be prepared and used immediately.

[0025] G15 administration: Administer G15 to C57BL / 6 mice via gavage (oral administration) at a dose of 10 mg / kg / day. For newborn mice (early developmental stage, P3-P10), the dosage is approximately 20 μL / mouse / day. For adult male NG2-CreERT;Tau-mGFP mice, the dosage is based on body weight; for example, a 30g mouse requires 0.15 ml / mouse / day. Treatment should be administered continuously daily without interruption during the modeling period.

[0026] This invention simulates neonatal white matter injury by simultaneously placing mice and their mothers in a 10% O2 hypoxic chamber for one week on the third day after birth to induce chronic hypoxia. Since GPR30 is reported to be a membrane-bound estrogen receptor, to reduce the potential impact of sex-specific hormone level differences on drug effect observation, male adult mice were selected for the study. Stereoscopic injection of lysolecithin into the corpus callosum of adult male mice was used to simulate localized demyelination of adult white matter. This invention uses immunofluorescence staining and Cellsens image analysis software to quantitatively analyze the effects of G15 on cerebral blood vessels, oligodendrocyte lineage cells, and myelin sheath under two white matter injury models. This invention uses water maze, balance bar, and three-box social behavior tests to evaluate changes in spatial memory, motor coordination, and social skills between G15 treatment and control groups. This invention uses Prism 10.1.2 software for statistical analysis of differences between groups.

[0027] Example 1 I. Animals The experimental animals were newborn C57BL / 6 mice (treated with chronic hypoxia from P3 to P10, and perfusion sampling at P11, weighing approximately 6-8g) and adult NG2-CreERT;Tau-mGFP mice (11 months old, weighing approximately 25-30g). The mice were placed in standard acrylic rodent cages with controlled temperature and humidity, under a 12-hour light / 12-hour dark cycle (lights on at 8 am and off at 8 pm), and the mice had free access to food and water.

[0028] II. Drugs G15 was purchased from the Selleck website and dissolved in 100% DMSO solution. It was then prepared in a solution containing 2% DMSO, 20% polyethylene glycol 300 (PEG300), 5% Tween-80, and 73% pure water (ddH2O) to obtain a clear solution of 2 mg / ml G15.

[0029] III. Gavage administration Oral administration was performed directly via gavage using a suitable type of syringe at a dose of 10 mg / kg / day. In a neonatal white matter injury model, administration was continuous for 7 days from P3 to P10 after birth, with tissue samples collected immediately after treatment. In a demyelinating model, lysolecithin was injected. IV. Effects of G15 on White Matter Injury The purpose of this study was to evaluate the effects of G15 on white matter injury in neonatal white matter injury models and adult demyelinating disease models.

[0030] ① Neonatal white matter injury model: On the third day after birth, mice were placed in an oxygen-deficient chamber with a fixed oxygen concentration of 10% O2. The temperature and humidity in the chamber were suitable. Litterctic pups were randomly divided into a G15 group and a vehicle group (n=4 per group) based on sex. From P3 to P10, G15 / vehicle was administered daily at the same time for one week. The solution was diluted to 2 mg / ml with 2% DMSO, 20% polyethylene glycol 300 (PEG300), 5% Tween-80, and 73% pure water (ddH2O). The dosage was 10 mg / kg / day, administered orally or by gavage. Results are as follows: Figure 1 As shown.

[0031] Figure 1 Figure a in the figure describes the entire experimental timeline. Figure b shows that, compared with the vehicle group, the density of blood vessels (CD31+) in the cortex (P=0.0227), cingulate gyrus (P=0.0080), genu of corpus callosum (P=0.0279), and striatum (P=0.0249) of mice in the G15 group was significantly increased. Figures c and d show that the density of myelin sheath (MBP+) (P=0.0195) and oligodendrocytes (CC1+) (P=0.0046) in the deep cortex and corpus callosum of the G15 group was significantly higher than that in the vehicle group.

[0032] ② Adult Demyelination Model: 11-month-old male NG2-CreERT;Tau-mGFP mice were induced by gavage with 30 mg / ml Tamoxifen 0.1 ml / mouse / day for four consecutive days. After induction, the mice were randomly divided into G15 group and vehicle group (n=3 per group). The mice underwent stereotactic injection of lysolecithin into the corpus callosum region (to induce local demyelination in the corpus callosum region), followed by two weeks of continuous G15 / vehicle treatment. The G15 / vehicle solution was diluted to 2 mg / ml using 2% DMSO, 20% polyethylene glycol 300 (PEG300), 5% Tween-80, and 73% pure water (ddH2O). The dosage was 10 mg / kg / day, administered orally via gavage. Results are as follows: Figure 2 As shown.

[0033] Figure 2 Figure a in the diagram describes the entire experimental timeline. Figure b shows that the whole-brain vascular density (CD31+) was significantly increased in the G15 group, especially in the non-injury area, where the vascular density was significantly higher than that in the vehicle group. Figure c shows that the newly formed myelin sheath (mGFP+) in the injury area of ​​the G15 group was significantly more than that in the vehicle group.

[0034] V. Effects of G15 treatment on functional impairment caused by white matter injury The purpose of this study was to evaluate the effects of G15 treatment during neonatal hypoxia on long-term neurological function.

[0035] The neonatal white matter injury model described above was divided into two groups: a G15 group and a vehicle group, with 10 mice in each group. During hypoxia, the mice were administered G15 / vehicle, respectively. At day 10, the mice were removed from the hypoxia chamber and placed in a normal, suitable environment for C57BL / 6 mice. At day 40, the mice in both groups, which received different treatments, underwent balance bar, water maze, and three-box social behavior tests to assess their motor coordination, spatial memory, and social abilities. The results are as follows: Figure 3 As shown.

[0036] Figure 3 Figure a shows the experimental timeline. Figure b shows that the number of times the G15 group mice slipped on the balance bar was significantly lower than that of the vehicle group (P < 0.0001). Figure c shows that the time (P = 0.0233) and distance (P = 0.0067) for mice treated with G15 to enter the target quadrant in the water maze experiment were significantly higher than those of the control group, indicating a significant improvement in learning ability. The performance in the three-box social test is shown in Figure d. In the first round of testing (vehicle: P = 0.6691; G15: P = 0.7270), the mice showed almost the same tendency when faced with two identical empty cages, indicating that there was no positional preference between the two groups of mice, and the experimental results are reliable. However, the G15 group mice showed significantly better social performance than the vehicle group in the second round (vehicle: P = 0.2714; G15: P = 0.0071) and the third round (vehicle: P = 0.5035; G15: P = 0.0013).

[0037] All measurements in this experiment used independent samples. Data are expressed as mean ± SEM (mean standard error). Statistical analysis was performed using GraphPad Prism version 10.1.2 (GraphPad Software, San Diego, CA, USA). The t-test was used to compare the means of the independent sample groups. The significance level for all tests was set at 0.05.

[0038] The above-disclosed embodiments are merely preferred embodiments of the present invention G15 based on promoting cerebral angiogenesis to treat white matter injury. Of course, they should not be construed as limiting the scope of the present invention. Those skilled in the art can understand that all or part of the processes of the above embodiments can be implemented, and equivalent changes made in accordance with the claims of the present invention are still within the scope of the invention.

Claims

1. The application of G15 in the treatment of white matter injury by promoting cerebral angiogenesis, characterized in that, G15 is used in the treatment of white matter injury in the brain; White matter damage mainly includes insufficient myelin formation due to hypoxia in early development and myelin regeneration disorders caused by demyelinating diseases in adulthood.

2. The application of G15 in treating white matter injury based on promoting cerebral angiogenesis as described in claim 1, characterized in that, The G15 is prepared using G15 powder, DMSO, polyethylene glycol 300 (PEG300), Tween-80, and pure water (ddH2O).

3. The application of G15 in treating white matter injury based on promoting cerebral angiogenesis as described in claim 2, characterized in that, The configuration steps for the G15 are as follows: Dissolve G15 powder in 100% DMSO solution; The G15 solution was diluted with DMSO, polyethylene glycol 300 (PEG300), Tween-80 and pure water (ddH2O) to prepare a 2 mg / ml clear G15 solution.

4. The application of G15 in treating white matter injury based on promoting cerebral angiogenesis as described in claim 3, characterized in that, The ratio of DMSO, polyethylene glycol 300 (PEG300), Tween-80, and pure water (ddH2O) is 2%:20%:5%:73%.

5. The application of G15 in treating white matter injury based on promoting cerebral angiogenesis as described in claim 1, characterized in that, G15 is a specific antagonist of the GPR30 receptor.

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