Application of Piezo1 agonist and pharmaceutical preparation for nose

Abstract

The invention discloses an application of a Piezo1 agonist and a pharmaceutical preparation used for a nose, including an application of the Piezo1 agonist in preparation of a drug for promoting removal of cerebrospinal fluid and / or intracerebral metabolic waste through nasal tissues, and an application of the Piezo1 agonist in preparation of a drug for improving a nasal lymphatic vessel drainage function. The invention also provides a nasal drug delivery preparation containing the Yoda1. According to the invention, the Piezo1 agonist is proposed for the first time to eliminate A beta by improving the drainage function of nasal lymphatic vessels so as to improve cognitive and olfactory dysfunctions of the Alzheimer's disease, a nasal administration strategy is provided, and a new drug intervention direction is provided for AD treatment. The Yoda1 activates a signal channel of nasal lymphatic vessel Piezo1, not only can enhance the removal of A beta, but also can act on a meningelymphatic system through a nasal cavity-brain channel to further improve the metabolic balance of A beta in the brain, so that the Yoda1 has a relatively good clinical application prospect.

Description

Technical Field

[0001] The present invention relates to a new application of a drug, in particular to the application of a Piezo1 agonist and a pharmaceutical preparation for the nose. Background Art

[0002] Alzheimer's Disease (AD) is a common neurodegenerative disease, and its characteristic pathological changes include β-amyloid (Aβ) deposition, neuronal loss, synaptic dysfunction, and chronic neuroinflammation. Recent studies have shown that there are many clearance pathways of Aβ in the brain, which not only rely on microglial phagocytosis, but can also be excreted by lymphatic vessels such as meningeal lymphatic vessels through the cerebrospinal fluid interstitial fluid drainage pathway. However, with the progression of AD, the function of lymphatic vessels is impaired and the drainage ability decreases, resulting in an increase in the accumulation of Aβ, thereby accelerating the disease process. At present, the weight of nasal lymphatic vessels in the clearance of Aβ in the brain, the degree of damage to nasal lymphatic vessels in the progression of AD, and the impact of nasal lymphatic vessel injury on the Aβ clearance rate are all unknown. Whether nasal lymphatic vessels can be used as the action target of drugs for the treatment of AD or drugs to enhance the Aβ clearance rate needs to be further studied and confirmed.

[0003] Piezo1 receptors are highly expressed on lymphatic endothelial cells, and the literature reports that the Piezo1 signaling pathway is closely related to the regeneration and repair of lymphatic vessels. Yoda1 is a highly effective Piezo1 agonist, and existing studies have shown that it can regulate cell functions in various disease models. However, the damage sites and degrees of lymphatic vessels in different disease models vary greatly, and there is no evidence that Piezo1 agonists can effectively repair the damage of nasal lymphatic vessels caused by AD. The study on using Piezo1 agonists to enhance the drainage ability of nasal lymphatic vessels and thereby enhance the Aβ clearance rate in the brain has not been reported yet. Summary of the Invention

[0004] Object of the Invention: The object of the present invention is to provide the application of a Piezo1 agonist in the preparation of a drug for promoting the clearance of cerebrospinal fluid and / or intracerebral metabolic wastes through nasal tissues, and to solve the problem of how to promote the clearance of cerebrospinal fluid and / or intracerebral metabolic wastes through nasal tissues. Another object of the present invention is to propose the application of a Piezo1 agonist in the preparation of a drug for enhancing the drainage function of nasal lymphatic vessels, and to solve the problem of how to enhance the drainage function of nasal lymphatic vessels. The third object of the present invention is to propose a pharmaceutical preparation for the nose, and to solve the problem of how to obtain a pharmaceutical preparation for the treatment of Alzheimer's disease for nasal administration.

[0005] Technical Solution: The present invention discloses the application of a Piezo1 agonist in the preparation of a drug for promoting the clearance of cerebrospinal fluid and / or intracerebral metabolic wastes through nasal tissues.

[0006] On the other hand, the present invention discloses the application of a Piezo1 agonist in the preparation of a drug for enhancing the function of nasal lymphatic drainage.

[0007] The Piezo1 agonist promotes the permeability of nasal lymphatics by enhancing the mechanosensitivity of lymphatic endothelial cells, and ultimately enhances the drainage function of nasal lymphatics. Enhancing the drainage function of nasal lymphatics can increase the clearance rate of cerebrospinal fluid and brain metabolic wastes, improve lymphatic circulation disorders, and thus slow down the progression of AD.

[0008] Preferably, in the above application, the Piezo1 agonist is Yoda1, and the structural formula of Yoda1 is as follows:

[0009]

[0010] Preferably, the brain metabolic waste is Aβ protein.

[0011] Preferably, the nasal tissue includes nasal lymphatics.

[0012] Yoda1 repairs the function of nasal lymphatics by activating the Piezo1 channel of lymphatic endothelial cells, accelerates the drainage of brain Aβ through nasal lymphatics, thereby reducing brain Aβ deposition and improving AD-related cognitive and olfactory dysfunctions.

[0013] In some embodiments, Yoda1 can reduce the Aβ burden in the olfactory bulb, cortex and hippocampus, thereby improving the cognitive ability and olfactory function of Alzheimer's disease animals.

[0014] The third aspect of the present invention provides a pharmaceutical preparation for the nose, including at least one of a nasal spray, nasal drops, nasal gel, and nasal powder containing a Piezo1 agonist.

[0015] Preferably, the Piezo1 agonist in the above preparation is Yoda1.

[0016] Furthermore, the final concentration of Yoda1 in the above pharmaceutical preparation for the nose is 100 - 1500 μg / mL.

[0017] Preferably, the above pharmaceutical preparation further contains an Alzheimer's disease treatment drug and other pharmaceutically acceptable excipients.

[0018] Preferably, the Alzheimer's disease treatment drug includes at least one of an Aβ clearance antibody, a Tau phosphorylation inhibitor, a cholinesterase inhibitor, and an anti-inflammatory drug. Combining drugs can enhance the synergistic therapeutic effect on Alzheimer's disease.

[0019] Preferably, the Piezo1 agonist is pre-encapsulated by a nano-delivery system. The nano-delivery system includes liposomes, polymeric nanoparticles, microemulsions or nanomicelles, etc., which can improve the absorption efficiency of the drug in the nasal cavity and prolong its action time.

[0020] Advantages: Compared with the prior art, the present invention has the following remarkable advantages:

[0021] The present invention first proposes that the Piezo1 agonist clears Aβ by enhancing the nasal lymphatic drainage function to improve the cognitive and olfactory function disorders of Alzheimer's disease, and provides a nasal drug delivery strategy, providing a new direction for drug intervention in the treatment of AD. Activating the signal pathway of Piezo1 in the nasal lymphatics can not only enhance the clearance of Aβ, but also act on the meningeal lymphatic system through the nasal-brain pathway to further improve the metabolic balance of Aβ in the brain.

[0022] As a Piezo1 agonist, Yoda1 has higher safety, lower effective concentration, stable activity, good specificity compared with other AD treatment drugs, and the preparation process is mature, suitable for clinical use. Nasal drug delivery can bypass the blood-brain barrier, improve drug targeting, and reduce systemic side effects. Description of the Drawings

[0023] Figure 1 Shows the experimental results of Yoda1 improving the cognitive function and olfactory function of 5×FAD transgenic mice;

[0024] Figure 2 Shows the experimental results of Yoda1 significantly reducing the Aβ burden in the brains of 5×FAD transgenic mice;

[0025] Figure 3 Shows the experimental results of Yoda1 repairing the olfactory mucosa structure and reducing Aβ deposition;

[0026] Figure 4 Shows the experimental results of Yoda1 repairing the structure and function of the olfactory mucosa lymphatics;

[0027] Figure 5 Shows the experimental results of the excretion of Aβ in the brains of 5×FAD transgenic mice through the nasal lymphatics;

[0028] Figure 6 Shows the experimental results of the injury of the nasal lymphatics in 5×FAD transgenic mice. Detailed Embodiments

[0029] The technical solutions of the present invention will be further described below with reference to the drawings.

[0030] Example 1: It was experimentally confirmed that Yoda1 acts on the nasal lymphatics to promote Aβ clearance to improve cognitive function and olfactory function, and the method is as follows:

[0031] 1. Improvement of cognitive function and olfactory function by Yoda1

[0032] In the experiment, 5×FAD mice (five-transgenic familial Alzheimer's disease model mice) were used. A Yoda1 solution with a concentration of 1 mg / mL was instilled into the nose of the mice every day. The solution used a mixture of normal saline, PEG400, and DMSO as the solvent, and the volume ratio of normal saline, PEG400, and DMSO in the solvent was 75:20:5 (the dosage of Yoda1 was 40 mg / kg). The control group was instilled with the above solvent into the nose, for 4 consecutive weeks. The results showed that the performance of the mice in the Yoda1 group was significantly better than that of the control group in the Y-maze and Barnes tests, indicating that Yoda1 could improve cognitive function. The olfactory performance in the food burying experiment and habituation-dishabituation test was better than that of the control group, indicating that Yoda1 could improve olfactory function (see Figure 1 ). Figure 1 In [Figure], Figure A is the flow chart of the animal experiment. 5×FAD mice at 4-5 months of age after birth and wild-type (WT) mice of the same litter were used. After 2 weeks of continuous nasal instillation, behavioral tests were performed. The Y-maze and Barnes maze were used to detect cognitive function, and the food burying experiment and habituation-dishabituation test were used to detect olfactory function. The duration of the behavioral experiment was 2 weeks. During this period, Yoda1 was instilled every day. After the behavioral experiment, the brain tissue and nasal bone tissue of the mice were taken for subsequent experiments; Figure B is the movement trajectory map of each group of mice in the Y-maze test; Figure C is the statistical chart of the number of times each group of mice entered the novel arm in the Y-maze test, N = 10; Figure D is the statistical chart of the time each group of mice entered the novel arm in the Y-maze test, N = 10; Figure E is the movement trajectory map of each group of mice in the Barnes maze test, and the red area is the target hole; Figure F is the change curve of the time latency of each group of mice entering the target hole every day during the training period in the Barnes maze test; Figure G is the statistical chart of the time latency of each group of mice entering the target hole during the test period in the Barnes maze test, N = 10; Figure H is the pattern diagram of food burying; Figure I is the statistical chart of the time latency of each group of mice finding the buried food in the food burying experiment, N = 10; Figure J is the pattern diagram of habituation-dishabituation; Figure K is the change curve and statistical chart of the sniffing time of the mice in the habituation-dishabituation experiment after continuously sniffing the same odor and then changing to a new odor, N = 10.

[0033] 2. Yoda1 reduces Aβ deposition

[0034] Immunohistochemical detection was used, and the results are shown in Figure 2 . Figure 2Figure A shows a schematic diagram of the mouse brain regions. OB is the olfactory bulb, Pir is the piriform cortex, Ctx is the cortex, Hip is the hippocampus, and Ent is the entorhinal cortex. Figure B shows immunohistochemical staining of 6E10 in brain tissues of 5×FAD transgenic mice and WT mice after treatment with Yoda1. The scale bar is 100 μm. Figure C shows a statistical chart of the 6E10 positive area in the olfactory bulb of 5×FAD transgenic mice after treatment with Yoda1, N = 6. Figure D shows a statistical chart of the 6E10 positive area in the piriform cortex of 5×FAD transgenic mice after treatment with Yoda1, N = 6. Figure E shows a statistical chart of the 6E10 positive area in the cortex of 5×FAD transgenic mice after treatment with Yoda1, N = 6. Figure F shows a statistical chart of the 6E10 positive area in the hippocampal bulb of 5×FAD transgenic mice after treatment with Yoda1, N = 6. Figure G shows a statistical chart of the 6E10 positive area in the entorhinal cortex of 5×FAD transgenic mice after treatment with Yoda1, N = 6. Figure 2 It shows that the Aβ plaque areas in the olfactory bulb, cortex, and hippocampal region of the mice in the Yoda1 group were significantly reduced (p < 0.05).

[0035] 3. Yoda1 repairs the olfactory mucosa structure and reduces Aβ deposition

[0036] Immunofluorescence detection was used, and the results are shown in Figure 3 , Figure 3 Figure A shows a coronal plane schematic diagram of the mouse nasal bone. Figure B shows immunofluorescence staining of 6E10 and olfactory marker protein (OMP) in the coronal plane of the nasal bone of 5×FAD transgenic mice and WT mice after treatment with Yoda1. The scale bar is 100 μm. Figure C shows hematoxylin and eosin (HE) staining of the horizontal plane of the nasal bone of 5×FAD transgenic mice and WT mice after treatment with Yoda1. The upper scale bar is 1 mm, and the lower scale bar is 100 μm. Figure D shows a statistical chart of the 6E10 positive area in the olfactory mucosa of 5×FAD transgenic mice and WT mice in Figure B, N = 6. Figure E shows a statistical chart of the OMP positive area in the olfactory mucosa of 5×FAD transgenic mice and WT mice in Figure B, N = 6. Figure F shows a statistical chart of the epithelial thickness of the olfactory mucosa of 5×FAD transgenic mice and WT mice in Figure C, N = 6, and 6 different positions were selected for statistics in each olfactory epithelium. The Aβ plaque area in the olfactory mucosa region of the mice in the Yoda1 group was significantly reduced (p < 0.01), the expression of OMP in olfactory sensory neurons was significantly increased, and the HE staining results showed an increase in the thickness of the olfactory mucosa of the mice in the Yoda1 group.

[0037] 4. Yoda1 repairs the structure and function of the lymphatic vessels in the olfactory mucosa

[0038] The level of the lymphatic vessel marker lymphatic vessel endothelial hyaluronan receptor 1 (lyve1) was detected by immunofluorescence, and the results are shown in Figure 4 , Figure 4Figure A shows immunofluorescence staining of 6E10 and lymphatic vessel marker Lyve1 in the nasal mucosa of 5×FAD transgenic mice and WT mice after treatment with Yoda1; the scale bar is 100 μm; Figure B is a statistical graph of the 6E10 positive area in the olfactory mucosa of 5×FAD transgenic mice and WT mice in Figure A, N = 6; Figure C is a statistical graph of the Lyve1 positive area in the olfactory mucosa of 5×FAD transgenic mice and WT mice in Figure A, N = 6; Figure D is a statistical graph of the inner diameter of Lyve1 positive lymphatic vessels in the olfactory mucosa of 5×FAD transgenic mice and WT mice in Figure A, N = 6. Figure 4 It shows an increase in the expression of lyve1 in the mice of the Yoda1 group, and the positive area shows the restoration of the structural integrity of the lymphatic vessels.

[0039] 5. Aβ damages the nasal lymphatic vessels during the AD process.

[0040] As is well known, lymphatic vessels have the functions of drainage and absorption. Different endothelial cell connection methods of lymphatic vessels determine the functions they perform. Among them, the button type is mainly distributed in the lymphatic capillaries. These cell connections are relatively loose, allowing macromolecules such as interstitial fluid and Aβ to enter the lymphatic lumen. It is the main absorption area and highly expresses the lymphatic vessel markers lyve1, VEGFR3, and Prox1. The zipper type is mainly seen in the larger collecting lymphatic vessels. The connection between their endothelial cells is tight and it is not easy for macromolecules to enter. It is mainly responsible for draining the fluid that has entered the lymphatic system and highly expresses CD31, VE-cadherin, VEGFR3, and Prox1 while lowly expressing lyve1. The nasal septum mucosa of 5×FAD mice was dissected for immunofluorescence staining, and the blood vessel marker CD31 and the lymphatic vessel marker lyve1 were fluorescently labeled. The results are as Figure 5As shown, Figure A is the 3D signal maps of each single channel of the lymphatic vessel marker lyve1, the vascular marker CD31, and 6E10, as well as the multi-channel map after merge, in the olfactory mucosa area under the nasal septum and nasal bone of 5×FAD mice. The enlarged area shows the signal results of each dimension after layer scanning the tissue. Figure B is the 3D signal maps of each single channel of lyve1, CD31, and 6E10, as well as the multi-channel map after merge, in the olfactory mucosa near the cribriform plate of the nasal septum of 5×FAD mice. The enlarged area shows the signal results of each dimension after layer scanning the tissue. The purple asterisk in the enlarged area indicates the observed object Aβ, and the purple arrow indicates the positional relationship between this Aβ and the lymphatic vessel. Figure C is the sagittal pattern diagram of the nasal bone of the mouse head, with the names and positions of each structure marked in the figure. OB is the olfactory bulb, Cirbriform plate represents the cribriform plate, the olfactory mucosa is the olfactory mucosa of the nasal septum, and RM is the respiratory mucosa. Figure D is the 3D signal maps of each single channel of the lymphatic vessel marker VEGFR3, 6E10, and DAPI, as well as the multi-channel map after merge, in the olfactory mucosa area near the cribriform plate of the nasal septum of 5×FAD mice. Figure E is the pattern diagram of the olfactory nerve and cerebrospinal fluid drainage pathway at the cribriform foramina of the cribriform plate, with the names and positions of each structure marked in the figure. OB is the olfactory bulb, Cirbriform plate represents the cribriform plate, the olfactory mucosa is the olfactory mucosa of the nasal septum, CSF represents cerebrospinal fluid, the olfactory nerve is the olfactory nerve, OEC is the olfactory ensheathing cell, OSN is the olfactory sensory neuron, NLs is the nasal lymphatic vessel, Dura mater, Arachnoid mater, SLYM, Pia mater are the dura mater, arachnoid mater, subarachnoid lymphoid membrane, and pia mater respectively. Figure F is the 3D signal maps of each single channel of lyve1, the lymphatic vessel marker Prox1, and 6E10, as well as the multi-channel map after merge, at the cribriform plate of the coronal plane of the nasal bone of 5×FAD mice. The enlarged area shows the signal results of each dimension after layer scanning the tissue. Figure G is the 3D signal maps of each single channel of lyve1, Prox1, and 6E10, as well as the multi-channel map after merge, at the cribriform plate of the sagittal plane of the nasal bone of 5×FAD mice. The enlarged area shows the signal results of each dimension after layer scanning the tissue. Figure H is the 3D signal maps of each single channel of lyve1 and 6E10, at the cribriform plate of the sagittal plane of the nasal bone of 5×FAD mice, with the 647 channel provided as the bright field background color and the multi-channel map after merge. The enlarged area shows the signal results of each dimension after layer scanning the tissue. The scale bar is indicated in the figure.

[0041] Figure 5 The fluorescence results in Figure 5As shown in Figure A, 3D imaging was further used to confirm that Aβ was mainly located inside the lymphatic vessels. Further observation of the fluorescence signal in the area near the cribriform plate showed high expression of lyve1 in the lymphatic vessels, and the NLs were mainly button-shaped, suggesting that this was the main absorption site of Aβ, which could absorb Aβ dissolved in the CSF into the lymphatic vessels. In addition, no obvious CD31 signal was detected at the cribriform foramina. Therefore, it was speculated that central Aβ drained into the olfactory mucosa through NLs rather than blood vessels (as shown in Figure 5 Figure B). Considering the limitations of using lyve1 to label lymphatic vessels, the inventor used another lymphatic vessel marker, VEGFR3, to label the lymphatic vessels in this area and obtained the same result, that is, Aβ was mainly drained through lymphatic vessels rather than blood vessels (as shown in Figure 5 Figure D).

[0042] The lymphatic vessels of the nasal mucosa are located in the lower layer of the nasal mucosa and are widely distributed in the olfactory epithelial area and around the nasal septum. The nasal lymphatic vessels contain abundant blind-ended lymphatic capillaries, and the loose cell junctions function to absorb the CSF and macromolecules that enter the nasal cavity, such as Aβ, inflammatory factors, drug molecules, etc., and transport them to the deep cervical lymphatic system through lymphatic flow. Therefore, further exploration of the distribution of lymphatic vessels and blood vessels at the cribriform plate and their drainage ability for Aβ upstream along the drainage pathway. By staining the coronal and sagittal sections of the nasal bone tissue of 5×FAD mice, it was found that NLs were enriched at the cribriform foramina and opened at the cribriform foramina (as shown in Figure 5 Figure H). The labeling of lymphatic vessels in this area further clarified the nasal lymphatic drainage pathway for Aβ.

[0043] The inventor further detected the changes in the lymphatic vessels of the nasal mucosa and Aβ during the AD process in 5×FAD mice. The results are as shown in Figure 6 Figure Figure 6 A shows representative immunofluorescence images of the olfactory epithelial tissues of WT and 5×FAD mice at 2.5, 5, and 10 months of age. The staining of 6E10 (white) is for Aβ deposition, the staining of Lyve1 (green) is for lymphatic vessels, and the staining of CD31 (red) is for blood vessels. Figure B shows a statistical graph of the percentage of 6E10-positive areas. Figure C is a co-labeling curve graph of the positive areas of lymphatic vessels and blood vessels near the Aβ-positive area, showing the positional relationship between Aβ and lymphatic vessels and blood vessels. Figure D shows a statistical graph of the percentage of the positive area of lyve1 in the lymphatic vessels in the olfactory epithelium and the statistical graph of the tube diameter (n = 6), and the statistical graph of the percentage of the CD31+ area in the olfactory epithelium and the statistical graph of the blood vessel diameter (n = 6). Figure 6 It shows that with the occurrence of AD disease, the damage of nasal lymphatic vessels gradually worsens, and the deposition of Aβ in the nose becomes more obvious.

Claims

1. Use of a Piezo1 agonist in the preparation of a drug for promoting the clearance of cerebrospinal fluid and / or intracerebral metabolic waste through nasal tissues.

2. Use of a Piezo1 agonist in the preparation of a drug for enhancing the lymphatic drainage function of the nose.

3. The application according to claim 1 or 2, characterized in that, The Piezo1 agonist is Yoda1.

4. The application according to claim 1, wherein The intracerebral metabolic waste is amyloid-β (Aβ) protein.

5. The application according to claim 1, characterized in that The nasal tissues include nasal lymphatics.

6. A pharmaceutical preparation for the nose, characterized in that, It includes at least one of a nasal spray, nasal drops, nasal gel, and nasal powder containing a Piezo1 agonist.

7. The pharmaceutical preparation for the nose according to claim 6, characterized in that, The Piezo1 agonist is Yoda1.

8. The pharmaceutical preparation for the nose according to claim 6, characterized in that, It also contains a drug for the treatment of Alzheimer's disease and other pharmaceutically acceptable excipients.

9. The pharmaceutical preparation for the nose according to claim 8, characterized in that, The drug for the treatment of Alzheimer's disease includes at least one of an Aβ clearance antibody, a Tau phosphorylation inhibitor, a cholinesterase inhibitor, and an anti-inflammatory drug.

10. The pharmaceutical preparation for the nose according to claim 6, characterized in that, The Piezo1 agonist is pre-encapsulated by a nanodelivery system.

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