CN115364103A - Application of TGF (transforming growth factor) beta pathway inhibitor in preparation of medicine or health-care product for preventing or treating blood brain barrier damage - Google Patents

Abstract

The present invention provides an application of a TGFβ pathway inhibitor in the preparation of medicines or health products for preventing or treating blood-brain barrier damage. In a specific implementation scheme, the TGFβ pathway inhibitor is LY2109761. The present invention finds for the first time that using the TGFβ pathway inhibitor LY2109761 as a drug or health product for preventing or treating blood-brain barrier damage can significantly improve blood-brain barrier damage. In a further experiment, the TGFβ pathway inhibitor LY2109761 was used as a drug or health product to prevent or treat blood-brain barrier damage, and it could also significantly improve the cognitive function of aging mice. The mechanism may be related to the drug's blood-brain barrier Conservation related.

Description

TGFβ pathway inhibitors are used in the preparation of drugs for the prevention or treatment of blood-brain barrier damage or for protection Application in health products

technical field

The invention belongs to the technical field of drugs for treating blood-brain barrier damage, and relates to the application of a TGFβ pathway inhibitor in the preparation of drugs or health products for preventing or treating blood-brain barrier damage.

Background technique

The blood brain barrier (BBB) is a physiological barrier between blood circulation and nerve tissue. It highly selectively transports the beneficial components in the blood into the brain tissue, restricts the passage of harmful substances in the blood, and metabolizes the brain. The generated waste is discharged in time to provide a stable and controllable microenvironment for the maintenance of the normal function of the nervous system. During the aging process, the blood-brain barrier gradually leaks: on the one hand, toxic substances, inflammatory factors or cells in the blood enter the brain, causing damage to nerve cells, microglia, etc.; on the other hand, the metabolism of the brain itself The toxic waste produced cannot be discharged in time and will also cause harm to it. This series of factors will lead to the occurrence and deterioration of geriatric diseases, such as coronary heart disease, ischemic stroke, hypertension, Alzheimer's disease, Parkinson's syndrome and so on. Therefore, the development of an anti-blood-brain barrier drug has important clinical value for the prevention and treatment of senile diseases.

Currently, there is no specific drug for blood-brain barrier protection in clinical practice. The only medically accepted therapeutic agents that can be used to increase the integrity of the blood-brain barrier are glucocorticoids. Glucocorticoids are powerful anti-inflammatory compounds capable of suppressing all stages of the inflammatory response. Common corticosteroids include prednisone, dexamethasone, and hydrocortisone. Although glucocorticoids are widely used to treat various inflammatory conditions, long-term use of glucocorticoids may have side effects such as immunosuppression, humoral shifts, brain changes, and psychological changes.

Finding the repositioning of drugs, that is, the research and development of new uses of existing drugs, "new use of old drugs", has been proved to be a shortcut for the development of new drugs. Compared with traditional new drug discovery, since existing drugs have accumulated a large amount of data in terms of pharmacodynamics, pharmacokinetics, and toxicology, drug repositioning can save a lot of money by developing new uses based on existing drugs. Early research and development investment, while greatly shortening the cycle of drug development. The new drug development method based on drug repositioning has become a new drug development strategy widely adopted in the world.

Contents of the invention

In view of the deficiencies in the prior art, the technical problem to be solved by the present invention is to provide the application of an inhibitor in the preparation of drugs or health products for preventing or treating blood-brain barrier damage, so as to improve blood-brain barrier damage.

To achieve the purpose of the above invention, the present invention provides an application of a TGFβ pathway inhibitor in the preparation of medicines or health products for preventing or treating blood-brain barrier damage.

Specifically, the TGFβ pathway inhibitor is LY2109761, and its structural formula is as follows (I):

Another aspect of the present invention is to provide a pharmaceutical composition or health product for preventing or treating blood-brain barrier damage, wherein, the pharmaceutical composition or health product contains TGFβ pathway inhibitor or its drug as active substance Accept the salt.

Specifically, the TGFβ pathway inhibitor is LY2109761, and its structural formula is as follows (I):

Specifically, the pharmaceutical composition is in the form of intravenous injection and oral administration.

Specifically, the dosage of the pharmaceutical composition is 1-2 times/day, 30mg/kg-100mg/kg each time.

Specifically, the administration time of the pharmaceutical composition is continuous administration for 6-8 days.

The application of a TGFβ pathway inhibitor provided in the embodiment of the present invention in the preparation of medicines or health products for preventing or treating blood-brain barrier damage, specifically using TGFβ pathway inhibitor LY2109761 as a drug for preventing or treating blood-brain barrier damage Or health care products, by selectively inhibiting the TGFβ/Smad2 signaling pathway, reducing the leakage of the blood-brain barrier, can significantly improve the blood-brain barrier damage, and can also improve the cognitive impairment of the nervous system.

Description of drawings

Figure 1 is an illustration of the results of Western blot detection of brain tissue sections of aging mice in Example 1 of the present invention;

Fig. 2 is a graphical representation of the results of quantitative analysis of immunoblot detection of brain tissue sections of aging mice in Example 1 of the present invention;

Fig. 3 is an illustration of the results of immunofluorescence staining detection of brain tissue sections of aging mice in Example 1 of the present invention;

Fig. 4 is a graphical representation of the quantitative analysis results of immunofluorescence staining detection of brain tissue sections of aging mice in Example 1 of the present invention;

Figures 5a to 5c are structural schematic diagrams of experimental devices for performing cognitive behavioral experiments on aging mice in Example 2 of the present invention;

Fig. 6 is a graph showing test results of cognitive behavioral experiments on aging mice in Example 2 of the present invention.

Detailed ways

In order to make the object, technical solution and advantages of the present invention clearer, the specific implementation manners of the present invention will be described in detail below in conjunction with the accompanying drawings. Examples of these preferred embodiments are illustrated in the accompanying drawings. The embodiments of the invention shown in and described with reference to the drawings are merely exemplary, and the invention is not limited to these embodiments.

Here, it should also be noted that, in order to avoid obscuring the present invention due to unnecessary details, only the structures and/or processing steps closely related to the solution according to the present invention are shown in the drawings, and the related Other details are not relevant to the invention.

As mentioned above, there are currently no targeted drugs for protecting the blood-brain barrier in clinical practice, and the only medically accepted therapeutic drug that can be used to increase the integrity of the blood-brain barrier is glucocorticoids. However, long-term and large-scale use of glucocorticoids has side effects, such as immunosuppression, humoral shifts, brain changes, and psychological changes.

In view of the above problems, the embodiment of the present invention provides an application of a TGFβ pathway inhibitor in the preparation of drugs or health products for preventing or treating blood-brain barrier damage based on the idea of developing new uses of existing drugs. Specifically, the TGFβ pathway inhibitor is LY2109761, and its structural formula is as follows (I):

LY2109761 is a novel selective TGFβ receptor I/II (TβRI/II) dual inhibitor, which has been used in Phase I and Phase II clinical studies of malignant tumors. It has been proven to inhibit the migration and invasion of various tumor cells, The anti-tumor effect of LY2109761 has been confirmed in many aspects.

The present invention discovers a new function of the TGFβ pathway inhibitor LY2109761, which can reduce the leakage of the blood-brain barrier and significantly improve the damage of the blood-brain barrier.

The embodiment of the present invention also provides a pharmaceutical composition or health product for preventing or treating blood-brain barrier damage, wherein, the pharmaceutical composition or health product contains TGFβ pathway inhibitor or pharmaceutically acceptable salt thereof as an active substance . Specifically, the TGFβ pathway inhibitor is LY2109761

In a preferred scheme, the pharmaceutical composition is in the form of intravenous injection or oral administration.

In a preferred scheme, the dosage of the pharmaceutical composition is 1-2 times/day, 30mg/kg-100mg/kg each time.

In a preferred scheme, the administration time of the pharmaceutical composition is continuous administration for 6-8 days.

In order to illustrate that the drugs for the prevention or treatment of blood-brain barrier damage provided by the present invention can be used in relevant clinical research, the embodiments of the present invention provide drug efficacy examples of aging mice, including:

[Example 1] The protective effect of LY2109761 on the blood-brain barrier of aging mice

70-week-old C57BL/6 male aged mice weighing about 35 g were randomly divided into two groups: one was the treatment group and the other was the control group.

(1), according to the following methods, the mice in the administration group and the control group were administered respectively:

Administration group: LY2109761 powder was prepared into a suspension with 1% sodium cellulose, the concentration of LY2109761 was 10 mg/mL, and administered to rats by intragastric administration at a dose of 50 mg/kg, twice a day, Continuous administration for 7 days.

Control group: Compared with the administration group, the only difference is that the control group is only given 1% sodium cellulose solution of the same volume, and the administration method, dosage and administration time are all the same as those of the administration group.

(2), after administration is finished, carry out following treatment to the mice of administration group and matched group respectively:

The mice were anesthetized by intraperitoneal injection with 10% chloral hydrate (about 200 μL/mouse). After anesthesia, the abdominal cavity and thorax of the mouse were opened, and the heart was perfused with 60 mL of cold PBS buffer to completely remove the blood residue in the blood vessel. Then the mice were killed by neck dislocation, and the brains of the mice were quickly separated and cross-cut into two 2mm thick brain tissue slices: one was used for western blot analysis, soaked in RIPA lysate; one was analyzed by immunofluorescence staining, Soak in cold 4% paraformaldehyde fixative.

(3) Immunoblotting and immunofluorescence staining were performed on the brain tissue sections obtained in step (2).

Western blot detection indicators: TGFβ1/Smad2 signaling pathway P-Smad2, transcytosis Mfsd2a, blood-brain barrier leakage (including immunoglobulin IgG and albumin Albumin), loading reference protein GAPDH.

Immunofluorescence staining detection indicators: TGFβ1/Smad2 signaling pathway P-Smad2, transcytosis Mfsd2a, blood-brain barrier leakage (including immunoglobulin IgG and albumin).

Fig. 1 is a diagrammatic representation of the results of immunoblotting detection of brain tissue sections of aging mice in this example; Fig. 2 is a diagrammatic representation of quantitative analysis results of immunoblotting detection of brain tissue sections of aging mice in this embodiment; Fig. 3 is a schematic diagram of the results of immunofluorescence staining detection of brain tissue sections of aging mice in this embodiment; Fig. 4 is a quantitative analysis result diagram of immunofluorescence staining detection of brain tissue sections of aging mice in this embodiment Show. Among them, in Figure 2 and Figure 4, "**" corresponds to P<0.01, and "***" corresponds to P<0.001.

Based on Figures 1 to 4, the experimental results show that: in the aging mouse experiment, compared with the control group, LY2109761 was added to the drug administration group, thereby reducing the expression of P-Smad2 and promoting the expression of Mfsd2a , while significantly reducing the protein expression and fluorescence leakage of IgG and Albumin. The experimental results show that: LY2109761 can inhibit the highly activated TGFβ1/Smad2 signaling pathway in the brain tissue of aging mice, up-regulate the expression level of Mfsd2a, reduce the leakage of the blood-brain barrier, and can significantly improve the damage of the aging blood-brain barrier. Barriers are protective.

[Example 2] LY2109761 improves the cognitive behavior (new object recognition) of aging mice.

In 1988, Ennaceur proposed the object cognition experiment method to test the learning and memory ability of animals. Animals distinguish "new and old objects" according to their characteristics, location and background, which has important research and application value for the development mechanism of learning and memory, and cognitive impairment prevention drugs.

In this embodiment, the operation of cognitive behavior experiment on aging mice is as follows:

(1) With reference to the method of step (1) of Example 1, the mice in the administration group and the control group were administered respectively.

(2), adaptation period: the day before the end of the administration, that is, on the sixth day of the administration, after the first administration, all the mice were transferred to the experimental area to allow the mice to adapt to the new environment. The adaptation time was about 3 to 4 hours. The experimental device used in the adaptation period is shown in Figure 5a, and no objects were placed in the box. The behavior track of the mouse was recorded with a video camera, and the recording time was 10 minutes. During the experiment, after the previous mouse finished recording, wipe the inside of the box with 20% ethanol to remove the traces left by the previous mouse's activities, such as urine, hair, flattened particles, etc., so as to avoid damage to the next study. object impact. After all the recordings were completed, the mice were administered the second dose of the day.

(3) Familiarization period and test period: on the last day of administration, the experiment of familiarization period and test period was carried out.

Familiarization period: The experimental device used in the familiarization period is shown in Figure 5b. Two identical items were placed in the box, and then the mouse was put into the box from the same position, and the mouse’s activity track was recorded with a video camera. The recording time was 10 minutes .

Test period: One hour after the end of the familiarization period, the test period experiment was performed on the same mouse. The experimental device used in the test period is shown in Figure 5c. Compared with the familiarization period, the box of the experimental device used in the test period will be One of the items is replaced with a new item. Put the mouse into the box, and record the activity track of the mouse with a video camera, and the recording time is 5 minutes. Finally, the analysis software was used to calculate the mouse's exploration time on the new object, and then divided by the total activity time of the mouse, and this ratio was used to evaluate the new object recognition ability of the mouse. Among them, the exploration activity is defined as: the mouse's nose points to the object at close range or directly sniffs or licks the object. Exploration of an object is not considered when other parts of the body are in contact with the object and the nose is not pointing at or standing on the object.

Fig. 6 is a diagram of the test results of the cognitive behavior (new object recognition) experiment performed on aging mice in this embodiment. Wherein, in Fig. 6, "*" corresponds to P<0.05.

Based on Figure 6, the experimental results show that compared with the control group, the addition of LY2109761 to the drug administration group can significantly improve the cognitive function of aging mice, and the mechanism may be related to the blood-brain barrier protection of the drug.

To sum up, the application of a TGFβ pathway inhibitor provided in the embodiment of the present invention in the preparation of drugs or health products for preventing or treating blood-brain barrier damage, specifically, the TGFβ pathway inhibitor LY2109761 is used as a preventive or therapeutic blood-brain barrier Drugs or health products for brain barrier damage can significantly improve blood-brain barrier damage and neurocognitive impairment by selectively inhibiting the TGFβ/Smad2 signaling pathway and reducing the leakage of the blood-brain barrier.

The above description is only the specific implementation of the present application. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present application, some improvements and modifications can also be made. It should be regarded as the protection scope of this application.

Claims (7)

1. Application of TGFβ pathway inhibitor in the preparation of medicines or health products for preventing or treating blood-brain barrier damage. 2. The application according to claim 1, wherein the TGFβ pathway inhibitor is LY2109761, and its structural formula is as follows (I):

3. A pharmaceutical composition or health care product for preventing or treating blood-brain barrier damage, characterized in that the pharmaceutical composition or health care product contains a TGFβ pathway inhibitor or a pharmaceutically acceptable salt thereof as an active substance. 4. The pharmaceutical composition or health product according to claim 3, wherein the TGFβ pathway inhibitor is LY2109761, and its structural formula is as follows (I):

5. The pharmaceutical composition or health product according to claim 4, characterized in that, the pharmaceutical composition is in the form of intravenous injection or oral administration. 6. The pharmaceutical composition or health product according to claim 5, characterized in that, the dosage of the pharmaceutical composition is 1-2 times/day, 30mg/kg-100mg/kg each time. 7. The pharmaceutical composition or health product according to claim 6, characterized in that, the administration time of the pharmaceutical composition is continuous administration for 6-8 days.

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