Extracranial methods to promote cerebrospinal fluid drainage

Abstract

Facilitating cerebrospinal fluid (CSF) drainage is useful in the prevention and treatment of neurodegenerative diseases, including Alzheimer's disease. This invention provides information on how extracranial approaches and methods can facilitate CSF drainage. This invention encompasses extracranial methods for facilitating CSF drainage through the nasopharyngeal lymphatic plexus (NPLP) and deep cervical lymphatic vessels (dcLVs) to prevent and treat neurodegenerative diseases.

Description

[Technical Field]

[0001] The present invention relates to a method for increasing or restoring the rate or level of outflow of cerebrospinal fluid (CSF) from the central nervous system into the systemic circulation. The present invention also relates to a method for diagnosing and treating neurodegenerative diseases by evaluating the level of CSF outflow and increasing outflow in patients identified as suffering from reduced CSF outflow levels. [Background technology]

[0002] All publications herein are incorporated by reference to the same extent that each individual publication or patent application is specifically and individually indicated as being incorporated by reference. The following description contains information that may be useful in understanding the present invention. Nothing provided herein constitutes prior art, or relates to the invention claimed herein, or constitutes prior art of any publication specifically or implicitly referenced herein.

[0003] The volume range of cerebrospinal fluid (CSF) in adults is 140-200 ml. CSF is produced from the choroid plexus at a rate of 400-600 ml / day and circulates throughout the brain parenchyma and spinal cord (References 1 and 2). Therefore, the fluid undergoes turnover (replacement) 3-5 times per day. The central nervous system (CNS) includes the brain, spinal cord, optic nerve, and retina, which require high energy for their delicate and fine activity. Similarly, the CNS generates waste products and antigenic macromolecules, including synapses and myelin fragments, as well as misfolded proteins such as amyloid and hyperphosphorylated tau. These CNS-derived waste products and macromolecules are dissolved in the interstitial fluid and CSF, which should be adequately removed into the systemic circulation. Excessive accumulation of these waste products and macromolecules, including amyloid and hyperphosphorylated tau, can lead to neurodegenerative diseases such as Alzheimer's disease (Reference 3).

[0004] Traditionally, arachnoid villi and cribriform plates are known to be drainage pathways for CSF (References 1 and 2). However, two research groups have rediscovered meningeal lymphatic vessels (mLVs) in the dura mater of the dorsal skull of mice (References 4 and 5) [the so-called "dorsal mLVs" (Figure 1)], and have proposed them as a novel CSF drainage pathway. They (4, 5) showed that dorsal skull mLVs are phenotypic similar to peripheral lymphoid capillaries. They also showed that dorsal mLVs transport macromolecules and cells along the superior sagittal and transverse sinuses (4, 5). However, Ma and Proulx, and their colleagues (Reference 6), did not find substantial CSF reuptake and drainage through dorsal mLVs using controlled, low-velocity, and volumetric stereotactic CSF tracer infusion. These researchers have suggested that CSF drains along cranial nerves as it leaves the skull (5). In contrast, our group identified basolateral mLVs as one of the major pathways for CSF drainage (Figure 2) and compared morphological and functional differences between dorsal mLVs and basolateral mLVs in adult and aged mice (Figures 3-5) (6). Importantly, basolateral mLVs acquired lymphedema-like features and delayed CSF drainage with aging (Figure 5) (6). These findings may explain the pathological mechanisms of neurodegenerative diseases such as Alzheimer's disease, namely the excessive accumulation of waste products and macromolecules in the brain due to reduced CSF drainage caused by age-related impairment of basolateral mLVs. However, lateral cranial basal LVs are considered to be the drainage pathway for CSF circulating in the posterior part of the brain and spinal cord (Figure 6).

[0005] Therefore, it is not well understood whether millilevins (mLVs) exist to cover cerebrospinal fluid drainage from the central nervous system and the anterior (excluding the cribriform) and middle cranial fossa regions of the skull (Figure 6). If they exist, it is unclear how they connect to extracranial lymphatics. In 2009, Pan et al. (Reference 7) reported lymphatic vessels (LVs) in the human nasal cavity and nasopharynx, but they did not describe the relationship between CSF drainage and these LVs. In 2017, Ma and Proulx and their colleagues (Reference 8) described that in Prox1-GFP mice, a bright signal of the tracer P40D680 injected intracranially into the nasal cavity, and tracers originating from the plexus of lymphatic vessels over the pharynx traced toward the deep cervical lymph node (dcLN), could be observed. Based on these findings, they proposed that CSF can be excreted through pharyngeal LVs. However, the identity of pharyngeal LVs and their CSF excretion was not fully elucidated. In 2022, Jacob and Thomas et al. (Reference 9) published OVA-A 555 After injecting a tracer into the subarachnoid space, we reported an extended anterior mLV network around the cavernous sinus with an exit pathway passing through the foramina of emissary veins, using light-sheet fluorescence microscopy (LSFM) imaging of whole mouse head specimens. Furthermore, we performed real-time vascular wall (VW) magnetic resonance imaging (VW-MRI) after systemic injection of gadobutrol in patients with neurological pathology. However, tracing in fixed tissues and organs was not successful. 555 Because this reflects the distribution of phagocytic macrophages rather than the distribution of mLVs and VW-MRI, this study lacked detailed histological and functional information regarding mLVs thought to transport intracranial CSF to extracranial compartments.

[0006] U.S. Patent Application Publication No. 20190269758, Methods and Compositions for Modulating Lymphatic Vessels in the Central Nervous System (Reference 10), relates to the manipulation of mlVs for the treatment of neurodegenerative diseases. However, this application does not disclose or suggest a method for increasing or restoring the rate or level of CSF outflow from the central nervous system into the systemic circulation by modulating or manipulating the nasopharyngeal lymphatic plexus (NPLP)-deep cervical lymphatic vessel (dcLV) pathway of CSF.

[0007] U.S. Patent Application Publication No. 20210311076, Compositions and Methods for the Diagnosis and Treatment of Neurological Diseases (Reference 11), relates to the manipulation of mLV for the treatment of neurodegenerative diseases. However, this application does not disclose or suggest a method for increasing or restoring the rate or level of cerebrospinal fluid (CSF) outflow from the central nervous system into the systemic circulation by modulating or manipulating the nasopharyngeal lymphatic plexus (NPLP)-deep cervical lymphatic vessel (dcLV) of the CSF. [Overview of the project]

[0008] The following embodiments and aspects thereof are described and illustrated in conjunction with systems, compositions, and methods, which are intended to be illustrative and not to limit the scope.

[0009] Facilitating cerebrospinal fluid (CSF) drainage is useful in the prevention and treatment of neurodegenerative diseases, including Alzheimer's disease. This invention provides information on how extracranial approaches and methods can facilitate CSF drainage.

[0010] The present invention encompasses extracranial methods for promoting CSF drainage through the nasopharyngeal lymphatic plexus (NPLP) and deep cervical lymphatic vessels (dcLV) to prevent and treat neurodegenerative diseases.

[0011] 1. The applicant has newly discovered that the nasopharyngeal lymphatic plexus (NPLP) is a hub for CSF drainage through the skull base, including the cribriform plate.

[0012] 2. The applicant has discovered a novel pathway for CSF drainage from the intracranial cavity to the NPLP.

[0013] 3. The applicant has discovered a novel pathway for CSF drainage from the intracranial cavity to the submucosa of the hard palate.

[0014] 4. The applicant claims that CSF drainage through the nasopharyngeal lymphatic plexus (NPLP) / medial side deep cervical lymphatic vessel (M-dcLV) pathway is performed on the cranial base side mLV ( We newly discovered that the NPLP pathway is larger in volume and faster than the basolateral mLV / lateral (L)-dcLV (L-dcLV: lateral side deep cervical lymphatic vessel) pathway, emphasizing the importance of NPLP.

[0015] 5. The applicant has newly discovered that NPLP is impaired with aging, and that exogenous drugs can restore the increase in lymphatic vessel area with increasing CSF drainage levels and the decrease in lymphatic vessel area with decreasing CSF drainage.

[0016] 6. The applicant has newly discovered that the contraction and relaxation of the circular smooth muscle covering dcLV can be regulated or modulated by stimulation and inhibition of smooth muscle cells, peripheral nerves, neurotransmitters, mechanical stimuli, and gentle massage.

[0017] 7. The inventors of this application have newly discovered that cerebrospinal fluid (CSF) drainage via NPLP can be regulated by exogenous agents via an extracranial approach. Cerebrospinal fluid drainage can be facilitated by extracranial manipulation, drug administration, and associated regulatory factors.

[0018] In one embodiment, the present invention is a method for increasing the outflow of cerebrospinal fluid (CSF) from the central nervous system, comprising repairing or expanding the nasopharyngeal lymph plexus (NPLP), and including: To determine the targets that require increased CSF outflow; The drug is administered to subjects requiring an effective amount of NPLP fluid, such that the amount of the drug repairs or expands the subject's NPLP, thereby achieving an increase in CSF outflow from the central nervous system into the systemic circulation in the subject. Determining a subject who requires increased CSF outflow may include determining that the subject has a neurodegenerative disease or condition, determining that the subject has risk factors for a neurodegenerative disease or condition, or both. The disease or condition may be age-related cognitive decline, Alzheimer's disease, Parkinson's disease, Huntington's disease, or stroke. In particular, the drug may be a VEGFR3 agonist such as VEGF-C or VEGF-D, an analog, variant, or fragment thereof, or any combination thereof. The drug may also be fibroblast growth factor 2 (FGF-2), insulin-like growth factor 1 (IGF-1), hepatocyte growth factor (HGF), endothelin-1 (ET-1), angiopoietin-1, a Tie2 agonist, neuropilin, or prostaglandin E2. The agent may be a gene vector containing a gene encoding a protein or a drug polypeptide. The gene may encode vascular endothelial growth factor-C, angiopoietin-1, or a Tie2 agonist.

[0019] In one form, the agent can be selectively administered to or near the NPLP-dcLV space. The NPLP-dcLV space may be located in the nasopharyngeal mucosa. The agent can be selectively administered to or near the hard palate submucosa - mandibular lymph node space. The hard palate submucosa - mandibular lymph node space may be located in the hard palate mucosa. The agent can be administered intrathecally into the CSF space, or nasally to the nasopharynx, or orally to the hard palate submucosa. In this regard, the subject's central nervous system may contain soluble molecules, and an increase in CSF outflow reduces the amount of soluble molecules in the brain. The subject's central nervous system may contain amyloid-β plaques, and an increase in CSF outflow reduces the amount of amyloid-β plaques in the brain.

[0020] In another form, the present invention relates to a method of increasing the outflow of cerebrospinal fluid (CSF) from the central nervous system, the method comprising increasing the contraction and relaxation of the dcLV, the method comprising determining a subject who requires an increase in CSF outflow, and administering to the subject an effective amount of a dcLV modulator, the amount increasing the contraction and relaxation of the subject's dcLV, thereby increasing the CSF flow from the central nervous system to the systemic circulation in the subject. Determining a subject who requires an increase in CSF outflow may include determining that the subject has a neurodegenerative disease or condition, determining that the subject has a risk factor for a neurodegenerative disease or condition, or both. The disease or condition can be cognitive decline associated with aging, Alzheimer's disease, Parkinson's disease, Huntington's disease, or stroke. The agent may be an agonist of the circular smooth muscle covering the dcLV. The agent may be the G protein-coupled receptor agonist phenylephrine or a nitric oxide donor. In one form, the drug may be administered transcervically, percutaneously, or locally to the cervical muscles, cervical tubercle, or cervical space, or the drug may be mechanically applied to the side of the neck.

[0021] In yet another form, the present invention relates to a method for preventing, treating or improving a neurodegenerative disease or condition in a subject, comprising repairing or expanding the nasopharyngeal lymph plexus (NPLP), the method comprising determining a subject that requires increased CSF outflow from the central nervous system and administering an effective amount of NPLP fluid to the subject that requires such an amount of the drug repairs or expands the NPLP in the subject, thereby increasing CSF flow from the central nervous system into the systemic circulation in the subject. The disease or condition may be age-related cognitive decline, Alzheimer's disease, Parkinson's disease, Huntington's disease, or stroke. In particular, the drug may be a VEGFR3 agonist such as VEGF-C or VEGF-D, an analogue, variant, or fragment thereof, or any combination thereof. The drug may also be fibroblast growth factor 2 (FGF-2), insulin-like growth factor 1 (IGF-1), hepatocyte growth factor (HGF), endothelin-1 (ET-1), angiopoietin-1, a Tie2 agonist, neuropilin, or prostaglandin E2. The agent may be a gene vector containing a gene encoding a protein or a drug polypeptide. The gene may encode vascular growth factor-C, angiopoietin-1, or a Tie2 agonist.

[0022] The drug can be selectively administered to or near the NPLP-dcLV space, the NPLP-dcLV space being located in the nasopharyngeal mucosa. The drug can be administered intrathecally to the CSF space or nasally to the nasopharynx. The drug can be selectively administered to or near the hard palate submucosa-mandibular lymph node space, the hard palate submucosa-mandibular lymph node space being located in the hard palate mucosa. The drug can be administered intrathecally to the CSF space, nasally to the nasopharynx, or orally to the hard palate submucosa. In this regard, the central nervous system in question may contain soluble molecules, and increased CSF outflow reduces the amount of soluble molecules in the brain. The central nervous system in question may also contain amyloid-beta plaques, and increased CSF outflow reduces the amount of amyloid-beta plaques in the brain.

[0023] In another embodiment, the present invention relates to a method for preventing, treating or improving a neurodegenerative disease or condition in a subject, comprising repairing or enlarging the nasopharyngeal lymph plexus (NPLP), the method comprising determining a subject requiring increased CSF outflow from the central nervous system and administering to the subject an effective amount of a dcLV fluidizing agent, thereby increasing the contraction of the subject's dcLV, and thereby increasing CSF outflow from the central nervous system into the systemic circulation in the subject. The disease or condition may be age-related cognitive decline, Alzheimer's disease, Parkinson's disease, Huntington's disease, or stroke. The agent may be a stimulant of the circular smooth muscle surrounding the dcLV. The agent may be the G protein-coupled receptor agonist phenylephrine or a nitric oxide donor. The agent stimulating the circular smooth muscle surrounding the dcLV may increase or decrease myosin phosphorylation by activating myosin light chain kinase or myosin light chain phosphatase. The drug may be administered to the target transcervically, percutaneously, locally to the cervical muscles, cervical tubercle, or cervical space, or the drug may be mechanically applied to the side of the neck.

[0024] In another embodiment, the present invention relates to a method for determining a change in CSF outflow rate or quantity in a subject, comprising: obtaining a biological sample; obtaining a sample of nasopharyngeal mucosa or hard palate mucosa at at least two separate times; assaying the presence and amount of CSF or assayable substance in CSF to obtain a value for each assay; comparing the values ​​of the amount of CSF or assayable substance to determine a change in the value of CSF or assayable substance, wherein the change in the value of CSF or assayable substance over time indicates a change in the amount or rate of CSF outflow. The biological sample can be obtained by swab or biopsy. The biological sample may include NPLP or hard palate. The assay can be performed by imaging of NPLP or hard palate, or by protein analysis of the obtained biological sample. [Brief explanation of the drawing]

[0025] This patent or application document includes at least one drawing prepared in color. A copy of this patent or patent application publication containing the color drawing will be provided by the Office upon request and payment of the necessary fees.

[0026] The disclosure of this invention will be best understood from the following detailed description when read in conjunction with the accompanying drawings. It should be emphasized, according to common practice, that various features in the drawings are not proportional to the actual size. Rather, for clarity, the dimensions of various structures have been arbitrarily enlarged or reduced. The drawings include the following figures:

[0027] [Figure 1] Figure 1 shows meningeal lymphatic vessels (dorsal mLVs) on the dorsal side of the mouse skull. Immunofluorescence (IFS) images showing VE-cadherin + LYVE-1 + dorsal mLVs along the superior sagittal sinus and transverse sinus.

[0028] [Figure 2] Figure 2 shows meningeal lymphatic vessels on the basolateral side of the skull of a mouse (basolateral mLVs). The IFS image shows LYVE-1 + basolateral mLVs (arrows) along the pyramidal subsinus and sigmoid sinus with VE-cadherin.

[0029] [Figure 3] Figure 3 shows a schematic diagram illustrating the morphological and functional differences between dorsal and basal cranial molecular volume devices (mLVs) in adult and aged mice.

[0030] [Figure 4] Figure 4 shows a schematic diagram illustrating that lateral cranial base molecular lung cells (mLVs) are a "hot route" for CSF drainage (posterior brain region) and degenerate in aged mice.

[0031] [Figure 5] Figures 5A-5B show that CSF drainage from lateral cranial basal mLs (mLVs) is significantly reduced in aged mice. (A) Lateral cranial basal mLs are visualized by intracranial injection of Alexa 488 (A488)-conjugated LYVE-1 antibody through the cisterna magna (CM). After 12 hours, QD705 is injected into the intracranial cavity, and the injected QD705 is visualized in lateral cranial mLs 15 minutes later using a fluorescence microscope. (B) Comparison of QD705 drainage (arrows) in A488-LYVE-1+ lateral cranial mLs (arrowheads) between 3-month-old adult mice and 27-month-old mice.

[0032] [Figure 6] Figure 6 is a schematic diagram showing known and unknown CSF drainage pathways to the cervical lymph nodes.

[0033] [Figure 7] Figures 7A-7B show the distribution of dextran exogenously injected into the intracranial cavity of adult mice. (A) Schematic diagram showing intracranial injection of TMR-dextran (10kDa) through the cisterna magna of 10-week-old Prox1-GFP mice. The head was sampled 60 minutes later. (B) Images showing the distribution of TMR-dextran in the intracranial and extracranial cavities. Note the strong Prox1+ in the hippocampus, LVs around the nasopharynx and oropharynx, and hard and soft palates.

[0034] [Figure 8]Figures 8A-8C show that in adult Prox1-GFP mice, lymphatic plexuses are present in the nasopharynx but not in the oropharynx, and that CSF-derived TMR-dextran is present. (A) Image showing the distribution of lymphatic vessels (LV) in the head and neck. (B) Image showing lymphatic plexuses in the nasopharynx and oropharynx. (C) Image showing lymphatic plexuses in the nasopharynx (also called the nasopharynx) but not in the oropharynx contains CSF-derived TMR-dextran (reddish-yellow arrowhead). Green arrows indicate lymphatic valves.

[0035] [Figure 9] Figures 9A-9B show vital imaging illustrating the dynamic and active efflux of dextran from the intracranial cavity into the NPLP. (A) Schematic diagram showing intracranial injection of TMR-dextran (10kDa) through the cisterna magna of a 10-week-old Prox1-GFP mouse. Semi-vital imaging was performed 30 minutes later. (B) Video showing TMR-dextran derived from CSF (reddish-yellow arrowhead) dynamically effluxed through the NPLP.

[0036] [Figure 10] Figure 10 shows that NPLP is rich in and selectively contains CSF-derived fluorescent microbeads in adult Prox1-GFP mice. Fluorescent microbeads were injected into the intracranial lumen via the cisterna magna, and NPLP was sampled 6 hours later. The dotted box is enlarged as the right panel.

[0037] [Figure 11]Figure 11 shows that dcLVs have intermittent periodic lymphatic valves (green arrowheads) and lymphangions, and contain TMR-dextran injected into the intracranial cavity via the cisterna magna 30 minutes prior. Note that L-dcLVs are connected to basolateral LVs via the jugular foramen, while M-dcLVs are connected to LVs originating from the nasopharynx. Injected dextran was detected not only in dcLNs but also in M- and L-dcLVs.

[0038] [Figure 12] Figures 12A-12B show the butterfly shape of the NPLP located in the submucosa of the nasopharynx. (A) Images showing the dorsal, ventral, and lateral views of the NPLP. The green lines show the outlines of the major Prox1+ / VEGFR3+ / LYVE1+ lymph nodes (LVs) that make up the NPLP. The flattened and condensed posterior nasal lymphatic plexus is located anterior to the NPLP. (B) Schematic diagram showing the structure and appearance of the nasopharynx.

[0039] [Figure 13] Figure 13 shows a coronal section of NPLP (white arrowhead) in an adult Prox1-GFP mouse.

[0040] [Figure 14] Figure 14 shows the schematic architecture of the NPLP. Dorsal, ventral, lateral, and coronal sections of the NPLP are depicted. The NPLP is divided into the head, body, and tail. Green lines indicate the outlines of the major LVs and valves for CSF outflow.

[0041] [Figure 15] Figure 15 shows a schematic 3D architecture of the NPLP. The green lines and vessels represent the inflow and outflow of the CSF, as well as the main LVs consisting of the NPLP.

[0042] [Figure 16] Figures 16A-16C show the finding that the lymphatic lymphoid (LV) originating from the pituitary gland area runs along the cavernous sinus connected to the NPLP in Prox1-GFP mice. (A) Schematic diagram showing the imaging area (red line). (B) Sequential images showing the LV starting from the pituitary gland area connected to the NPLP. (C) Enlarged view of the red-lined box in (B) showing the lymphatic end of the Prox1+ pituitary gland.

[0043] [Figure 17] Figure 17 is a photograph showing the 3D structure of the LV connecting the pituitary region to the NPLP, as shown in Figure 16.

[0044] [Figure 18] Figures 18A-18B show the finding that the lvium (LV) runs along the pterygopalatine artery (PPA), which connects to the LVs of the NPLP and hard palate. (A) Schematic diagram showing the imaging area (green line). (B) Sequential images showing that the LV runs along the pterygopalatine artery (PPA) and connects to the LVs of the NPLP and hard palate submucosa.

[0045] [Figure 19] Figure 19 shows a photograph illustrating the 3D structure of the pterygopalatine artery (PPA) running along the pterygopalatine artery (PPA), which connects to the NPLP and LVs of the hard palate submucosa, as shown in Figure 18.

[0046] [Figure 20]Figures 20A-20D show that Prox1+ LVs are located below the olfactory epithelium, connecting to both the posterior nasal lymphatic plexus and the nasopharynx (NPLP). (A) Schematic diagram showing the imaging area (green line). (B) LVs in the olfactory mucosa are richly and selectively contained in CSF-derived fluorescent microbeads in Prox1-GFP adult mice. Fluorescent microbeads were injected into the intracranial lumen through the cisterna magna, and the olfactory was sampled 6 hours later. (C) LVs in the olfactory mucosa are richly and selectively contained in CSF-derived QD705 in Prox1-GFP adult mice. QD705 was injected 60 minutes before sampling. (D) Image showing two connected LVs between the olfactory mucosa and the NPLP and posterior nasal lymphatic plexus.

[0047] [Figure 21] Figure 21 shows a summary diagram for detecting three lymphatic routes from the intracranial cavity to the extracranial NPLP. The NPLP is the hub of CSF drainage. (1) The LV, originating from the pituitary gland, runs along the cavernous sinus, which connects to the NPLP; (2) The LV runs along the pterygopalatine artery (PPA), which connects to the NPLP; (3) The LVs are located below the olfactory epithelium, which connects to both the posterior nasal lymphatic plexus and the nasopharynx NPLP.

[0048] [Figure 22]Figures 22A-22B show that lymphatic vessels (LVs) in the submucosa of the hard palate serve as a drainage route for CSF connected to the mandibular lymphatic nucleus (LN). (A) An image showing lymphatic vessels in the submucosa of the hard palate is connected to the mandibular LN. Injected dextran was detected not only in the mandibular LN but also in the lymphatic vessels of the hard palate. (B) An image showing lymphatic vessels under the hard palate mucosa contains CSF-derived -TMR-dextran.

[0049] [Figure 23] Figure 23 shows the lymphatic plexus in the submucosa of the hard palate of the primate cynomolgus monkey. The image panel on the right shows the presence of CSF-derived phosphors in the lymphatic plexus of the hard palate. The red arrows indicate CSF-derived phosphors (0.1 μm).

[0050] [Figure 24] Figures 24A-24B show the four lymphatic branches connecting NPLP and dcLVs in Prox1-GFP mice. (A) Image showing the four lymphatic branches connecting NPLP and dcLVs before and after soft palate resection. (B) Schematic diagram showing the four lymphatic branches connecting NPLP and dcLVs.

[0051] [Figure 25] Figures 25A-25B show the presence of NPLP in the submucosa of the nasopharynx of the primate cynomolgus monkey. (A) Macroscopic view of the sagittal sectioned head and neck of a cynomolgus monkey. (B) Immunofluorescence images of LYVE1+ NPLP and collagen IV+ BVs in the submucosa of the nasopharynx. In the lower panel, the green lining box is magnified. White arrowheads indicate lymphatic vessels.

[0052] [Figure 26] Figures 26A-26B show that CSF is drained in vivo in primates (cynomolgus monkeys) through the NPLP and hard palate. (A) MRI image of a monkey showing contrast-enhanced CSF by Gadspin P injection via the cisterna magna. Red circles indicate regions of interest for measuring signal enhancement by CSF tracers. (B) Quantification of 3-hour signal enhancement from different regions shows the NPLP, and the hard palate is the CSF drainage pathway.

[0053] [Figure 27] Figures 27A-27C show that medial dcLV (M-dcLV) excretes more CSF than lateral dcLV (L-dcLV) in Prox1-GFP adult mice. (A) Schematic diagram showing ligation of L-dcLV or M-dcLV. (B) Optical and fluorescence microscopy images of each dcLV ligation. (C) Comparison of signal intensity in n=9 dcLNs for each group; p-values ​​were calculated by Brown-Forsy and Welch ANOVA tests.

[0054] [Figure 28] Figures 28A-28D show that the medial dcLV excretes CSF faster than the lateral dcLV in adult Prox1-GFP mice. (A) Schematic diagram showing the timing of intracranial injection of TMR-dextran and statistical analysis. (B) Image showing the dextran in each dcLV. (C) Schematic diagram showing the dextran in each dcLV in (B). (D) Comparison of signal intensity in dcLV. P-values ​​were calculated using the Brown-Forsyand Welch ANOVA test.

[0055] [Figure 29]Figures 29A-29D show that intracisternal injection of AAV-mVEGF-C increases the Prox1+ lymphatic vessel area in the nasopharynx of young Prox1-GFP mice. (A) Schematic diagram showing the timing of intracisternal injection and analysis of AAV-mVEGF-C-mCherry. (B), (C) Comparison of representative images and quantitative parameters of NPLP. (D) Representative images and comparison of CSF tracer drainage in dcLN. P-values ​​were calculated by Whitney's test.

[0056] [Figure 30] Figure 30 shows that aging mice altered their morphology and reduced lymphoid signs during NPLP. Images show NPLP in 10-week-old and 80-88-week-old Prox1-GFP mice.

[0057] [Figure 31] Figures 31A–31C show transcriptomes of lymphatic endothelial cells (LECs) from young and aging NPLP. (A) Five clusters of LECs were conserved in aging NPLP. (BC) The list shows different gene expression in aging NPLP. Different gene expression was involved in apoptosis promotion and inflammatory signaling in aging NPLP.

[0058] [Figure 32] Figures 32A-32B show that phosphorylated tau (ptau) increases in aging NPLP, and the number of apoptotic lymphoid endothelial cells increases in aging NPLP. The p-value was calculated using Whitney's test.

[0059] [Figure 33]Figures 33A-33E show the effect of AAV-mVEGF-C-mCherry infection on NPLP in aging mice. (A) Schematic diagram showing the timing of intracisional injection and analysis of AAV-mVEGF-C-mCherry. (B), (C) Representative images and comparisons showing the expansion of the lymphatic vessel area of ​​NPLP by intracranial administration of AAV-VEGF-C-mCherry. (D) The increase in lymphatic vessels after AAV-mVEGF-C-mCherry infection is the route of CSF tracer administration. (E) Representative images and comparisons of CSF tracer drainage in dcLN. Note that AAV-mVEGF-C-mCherry infection increased CSF drainage after 3 weeks in aging mice. P values ​​were calculated by two-sided Whitney test.

[0060] [Figure 34] Figure 34 shows that dcLVs have well-developed lymphatic valves (green arrowheads) and lymphangions, and are finely covered with circular smooth muscle cells in Prox1-GFP mice.

[0061] [Figure 35] Figure 35 shows that β3-tubulin+ peripheral nerve fibers are abundantly distributed along dcLVs.

[0062] [Figure 36] Figures 36A-36B show that the innervating peripheral nerve fibers are sympathetic nerve fibers. (A) The innervating peripheral nerves are β3-tubulin+ and tyrosine hydroxylase (TH)+. TH is a sympathetic nerve fiber marker. Vesicular acetylcholine transporter (VAChT) is negative in the innervating peripheral nerve fibers along dcLVs. (B) VAChT antibody is validated pulmonary bronchiolar staining.

[0063] [Figure 37]Figures 37A-37B show that the structure of downstream lymphatic vessels of the NPLP did not change with aging. The p-value was calculated using a two-sided Whitney test and ns. p>0.05.

[0064] [Figure 38] Figures 38A-38C show that dcLVs are contracted by potassium chloride solution. (A) Schematic diagram of the experimental procedure. dcLVs in 10-week-old male Prox1-GFP mice were exposed by muscle incision, treated with saline (0.9% NaCl solution), then treated with 0.1M or 1.0M potassium chloride (KCl) after 5 minutes, and washed with saline after 20 minutes. (B) M-dcLVs in Prox1-GFP mice are contracted by KCl (white arrowhead). (C) M-dcLVs in Prox1-GFP mice injected into the intracranial cavity with QD705 2 hours prior to imaging are contracted by KCl (white arrowhead).

[0065] [Figure 39] Figures 39A-39B show that the G protein-coupled receptor agonist phenylephrine or the nitric oxide donor (sodium nitroprusside) causes contraction or expansion of dcLVs. (A) The image panel shows no contraction or expansion with PBS (phosphate-buffered saline) treatment, but phenylephrine or the nitric oxide donor causes contraction or expansion of dcLVs. (B) The graph shows dose-dependent contraction of dcLVs with phenylephrine or expansion with the nitric oxide donor. The amount of CSF tracer decreased with phenylephrine or increased with the nitric oxide donor. P values ​​were calculated by a two-way repeated-measures ANOVA.

[0066] [Figure 40]Figures 40A–40C show that low doses of phenylephrine (10 nM) enhance CSF drainage, as measured by the amount of CSF tracer drainage in deep cervical lymph nodes. At higher doses (5 mM), the amount of CSF tracer decreased, as shown in Figure 36. The nitric oxide donor, sodium nitroprusside, enhanced CSF drainage at 3 μM, but did not enhance or decrease CSF drainage at 30 μM. P-values ​​were calculated by two-way ANOVA followed by Dunnett's T3 multiple comparison post-hoc test.

[0067] [Figure 41] Figure 41 shows the extracranial regulation of cerebrospinal fluid (CSF) drainage.

[0068] [Figure 42] Figure 42 shows that NPLP is a hub for CSF drainage. The direction of CSF outflow is indicated by the green arrow. [Modes for carrying out the invention]

[0069] All references cited herein are incorporated by reference as if they were fully cited. Unless otherwise defined, technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. Definitions of common terms in molecular biology are as follows: Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8). Allen et al., Remington: The Science and Practice of Pharmacy 22 nd ed., Pharmaceutical Press (September 15, 2012); Hornyak et al., Introduction to Nanoscience and Nanotechnology, CRC Press (2008); Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology 3 rd ed., revised ed., J. Wiley & Sons (New York, NY 2006); Smith, March's Advanced Organic Chemistry Reactions, Mechanisms and Structure 7 thed., J. Wiley & Sons (New York, NY 2013); Singleton, Dictionary of DNA and Genome Technology 3 rd ed., Wiley-Blackwell (November 28, 2012); and Green and Sambrook, Molecular Cloning: A Laboratory Manual 4th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012). For instructions on how to prepare antibodies, please refer to: Greenfield, Antibodies A Laboratory Manual 2 nded., Cold Spring Harbor Press (Cold Spring Harbor NY, 2013); Kohler and Milstein, Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion, Eur. J. Immunol. 1976 Jul, 6(7):511-9; Queen and Selick, Humanized immunoglobulins, US Patent No. 5,585,089 (1996 Dec); and Riechmann etal., Reshaping human antibodies for therapy, Nature 1988 Mar 24, 332(6162):323-7; Paul W. Flint et al., Cummings Otolaryngology: Head and Heck Surgery, Elsevier Health Sciences, 2020 (ISBN 978-0323611794); Parviz Janfaza etal., Surgical Anatomy of the Head and Neck, Harvard University Press, 2011 (ISBN 978-0674058033).

[0070] Those skilled in the art will recognize many methods and substances that are similar to or equivalent to those described herein. Other features and advantages of the present invention will become apparent from the following detailed description, together with the accompanying drawings illustrating various features of embodiments of the invention. In fact, the present invention is by no means limited to the methods and materials described. For convenience, specific terms used in this specification, description, examples and appended claims are collected herein.

[0071] Unless otherwise specified or implied by the context, the following terms and phrases include the meanings provided below. Unless otherwise explicitly stated or evident from the context, the following terms and phrases do not exclude the meanings acquired in the art to which the term or phrase pertains. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains. The invention is not limited to, and is therefore subject to change, the specific methodologies, protocols, reagents, etc., described herein. The definitions and terms used herein are provided to aid in the description of specific embodiments and are not intended to limit the claimed invention, as the scope of the invention is limited only by the claims.

[0072] As used herein, “treatment” or “to treat” includes achieving therapeutic and / or preventive benefits. A therapeutic benefit means the eradication or improvement of the underlying disease or condition being treated. For example, in an individual with a common age-related deficit in cognition or Alzheimer’s disease, a therapeutic benefit includes the partial or complete cessation of the progression of the impairment or condition, or the partial or complete reversal of the impairment or condition. A therapeutic benefit is also achieved by eradicating or improving one or more physiological or psychological symptoms associated with the underlying condition, in such a way that improvement is observed in the patient, despite the fact that the patient may still be affected by the condition. Preventive benefits of treatment include the prevention of a condition, the delay of the progression of a condition (e.g., the delay of the progression of Alzheimer’s disease, or possibly the delay of age-related cognitive decline), or a reduction in the likelihood of the condition developing.

[0073] As used herein, the term “effective dose” can mean an amount sufficient to produce a beneficial or desired outcome in the CNS when administered, such as a beneficial or desired clinical outcome, or an enhancement of cognition, memory, mood, or other desired CNS outcome. An effective dose is also an amount that produces a preventive effect, such as delaying, reducing, or eliminating the onset of a pathological or undesirable condition. Such conditions include, but are not limited to, neurodegeneration. An effective dose may be administered in one or more doses.

[0074] As used herein, “subject” or “individual” refers to an animal, such as a mammal. In some embodiments, “subject” or “individual” is a human. In some embodiments, the subject suffers from Alzheimer’s disease or age-related cognitive impairment.

[0075] In some embodiments, the pharmacological composition is “administered peripherally” or “administered peripherally,” but as used herein, these terms do not refer to direct administration to the CNS, i.e., any form of administration of a drug to an individual, such as a therapeutic agent, that brings the drug into contact with the non-brain side of the blood-brain barrier. As used herein, “peripheral administration” includes intravenous, intra-arterial, subcutaneous, intramuscular, intraperitoneal, transdermal, inhalation, transbuccal, intranasal, rectal, oral, parenteral, sublingual, topical, transmucosal, or intranasal.

[0076] In this specification, “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” refers to any carrier that does not induce the production of antibodies harmful to the organism receiving the composition. Such carriers are well known to those skilled in the art. A complete discussion of pharmaceutically acceptable carriers / excipients can be found in Remington's Pharmaceutical Sciences, Gennaro, AR, ed., 20th edition, 2000: Williams and Wilkins PA, USA. Exemplary pharmaceutically acceptable carriers may include salts, such as mineral salts like hydrochloride, hydrobromide, phosphate, and sulfate; and salts of organic acids such as acetate, propionate, malonate, and benzoate. For example, the compositions of the present invention may be provided in liquid form and may be formulated in saline-based aqueous solutions of various pH (5-8) with or without 0.01-1% of surfactants such as polysorbate-80, or carbohydrate additives such as mannitol, sorbitol, or trehalose. Commonly used buffers include histidine, acetate, phosphate, or citrate. "Pharmacologically acceptable carriers" also include any and all solvents, dispersions, coatings, antimicrobial and antifungal agents, isotonic agents, and absorption retarders. The use of such media and active ingredients for pharmaceutically active substances is well known in the industry. Their use in therapeutic compositions is intended unless any conventional media or agent is incompatible with the active ingredient. Supplementary active ingredients may also be incorporated into the composition.

[0077] Exemplary pharmaceutically acceptable carriers for injectable compositions may include salts, such as mineral salts like hydrochloride, hydrobromide, phosphate, and sulfate; and salts of organic acids such as acetate, propionate, malonate, and benzoate. Commonly used buffers include histidine, acetate, phosphate, or citrate. Under normal storage and use conditions, these preparations may contain preservatives to prevent microbial growth. Prevention of microbial action can be achieved by various antimicrobial and antifungal agents, such as parabens, chlorobutanol; phenol, sorbic acid, thimerosal, etc. In many cases, isotonic agents, such as sugars or sodium chloride, are preferred. Sustained absorption of injectable compositions can be achieved by the use of absorption-delaying agents, such as aluminum monostearate and gelatin in the composition.

[0078] For administration to humans, the preparations meet sterility, pyrogenicity, general safety, and purity requirements as required by the standards of the FDA and other regulatory authorities. Active compounds are generally formulated for parenteral administration, for example, for injection via intravenous, intramuscular, subcutaneous, intrafocal, or intraperitoneal routes. The preparation of active ingredients or aqueous compositions containing ingredients is known to those skilled in the art in light of this disclosure. Typically, such compositions can be prepared as either liquid solutions or suspensions for injection; solid forms suitable for use when adding liquids before injection to prepare solutions or suspensions can also be prepared; and preparations can be emulsified.

[0079] Sterile injection solutions are prepared by compounding the required amount of active compound with various other components as exemplified above in a suitable solvent, and subsequently sterilizing by filtration if necessary. Generally, dispersions are prepared by incorporating various sterilized active components into a sterilized medium containing a basic dispersion medium and other components as required from those listed above. In the case of sterilized powders for the preparation of sterilized injection solutions, the preparation method includes vacuum drying and freeze-drying techniques to produce a powder of the active component + any additional desired components from the previously sterilized solution.

[0080] Once formulated, the solution is administered systemically or topically in a manner compatible with the drug formulation, in an amount that is therapeutically effective according to the criteria described herein. The formulation can be readily administered in various dosage forms, such as the injectable solution type described above, but drug-releasing capsules and the like may also be used.

[0081] "Recombinant host cell" or "host cell" means a cell containing an exogenous polynucleotide, regardless of the method used for insertion, e.g., direct incorporation, transduction, f-conjugation, or any other method known in the art for producing a recombinant host cell. The exogenous polynucleotide may be maintained as a non-integrated vector, e.g., a plasmid, or it may be integrated into the host genome.

[0082] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. That is, the description of polypeptides applies equally to the description of peptides and proteins, and vice versa. The term applies to naturally occurring amino acid polymers, as well as amino acid polymers in which one or more amino acid residues are unnaturally occurring amino acids, such as amino acid analogs. As used herein, the term encompasses amino acid chains of any length, including full-length proteins, in which amino acid residues are linked by covalent peptide bonds.

[0083] The term "nucleic acid" refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides, and polymers thereof in either single-stranded or double-stranded forms. Unless otherwise specified, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties to a reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.

[0084] As used herein, “intracranial” refers to matters within the cranium or skull, including the meninges and parenchyma and other structures.

[0085] As used herein, "extracranial" refers to things that are not inside the cranial cavity, such as the nasopharynx and medial deep cervical lymphatic vessel (dcLV).

[0086] As used herein, “exogenous agent” refers to a composition or methodology that can be applied to a subject to increase or stabilize the flow of CSF. In particular, exogenous agents affect the flow in NPLP and / or dcLV. In this regard, “flow agent” is synonymous with exogenous agent, particularly when used to increase or stabilize the outflow of CSF through NPLP and / or dcLV.

[0087] As used herein, “NPLP-dcLV” or “NPLP-dcLV pathway” refers to an extracranial area or space of lymph vessel or lymph structural system through which CSF outflow occurs from the NPLP, which arises in the neck along the internal jugular vein with the carotid sheath, to the deep cervical lymph node.

[0088] NPLP and dcLV

[0089] As described herein, the inventors have identified a novel lymphatic system or pathway that functions in the removal of macromolecules and debris from the central nervous system (CNS). In particular, reduced drainage through NPLP-dcLV ducts reduces cerebrospinal fluid (CSF) outflow, exacerbating symptoms of neurodegenerative diseases characterized by increased concentration and / or accumulation of molecules in the central nervous system, such as Alzheimer's disease (AD). Modulating lymphatic vessels to increase flow may alleviate age-related cognitive impairment or symptoms of AD, including the accumulation of amyloid-beta plaques and hyperphosphorylated tau protein.

[0090] The present invention discloses methods for treating, preventing, inhibiting, or improving symptoms of neurodegenerative diseases or conditions associated with increased concentrations and / or accumulation of macromolecules, cells, and debris in the CNS. The methods can increase lymphatic drainage and, consequently, increase CSF flow. The methods of the present invention are advantageous because they include one, some, or all of the benefits of (i) increased CSF outflow and efflux; (ii) reduced accumulation of macromolecules, cells, or synapses and myelin debris in the CNS (e.g., reduced accumulation of amyloid-beta); and (iii) maintenance or improvement of cognitive function (e.g., memory function) in subjects who have age-related cognitive impairment conditions, are suspected of having dementia (e.g., in neurodegenerative diseases), and / or are at risk of dementia.

[0091] Cerebrospinal fluid flow (CSF flow) and drugs to increase or stabilize the flow.

[0092] As used herein, “flow” refers to the rate of perfusion through a region of the central nervous system in question. In some embodiments of the present invention, “flow” can be measured as the rate at which a label or tracer in the CSF perfuses a specific region of the central nervous system. Thus, the flow can be compared between two subjects or two sets of conditions by determining how quickly the injected label or tracer perfuses a specific region or entire volume of the brain and / or other parts of the CNS. Furthermore, “outflow” refers to the drainage of the CSF into the systemic circulation, particularly via the NPLP-cdLV pathway of the present invention.

[0093] As used herein, “exogenous agent,” “agent,” or “flow agent” broadly refers to a class of compositions that can increase the passage of substances into and out of lymphatic vessels, and thus regulate the flow in CSF. In particular, the present invention relates to agents that specifically or generally increase the flow of CSF in NPLP (“NPLP flow agent”) or dcLV (“dcLV flow agent”).

[0094] While not limited by theory, according to some embodiments herein, the removal of macromolecules through NPLP-dcLV lymphatic vessels can keep their concentrations low in the CSF, and a certain gradient is intended to allow for the removal of macromolecules from parenchyma. Furthermore, the higher the fluid flow and drainage velocity in the CNS, the higher the clearance rate and / or the lower the concentration of cells, macromolecules, waste, and debris forming the CNS.

[0095] Exogenous agents may increase the diameter of NPLP-dcLV lymphatic vessels and / or repair them, which increases or stabilizes the velocity or level of drainage and results in increased CSF flow. In some embodiments, exogenous agents enlarge or repair the NPLP and / or enhance the contraction and relaxation of the dcLV, thus increasing net drainage and resulting in increased or stabilized CSF flow.

[0096] NPLP Flow Agent (NPLP Flow Agent)

[0097] Examples of suitable NPLP fluids for increasing CSF flow include, but are not limited to, vascular growth factor C (VEGF-C), vascular growth factor D (VEGF-D), fibroblast growth factor 2 (FGF-2), insulin-like growth factor 1 (IGF-1), hepatocyte growth factor (HGF), endothelin-1 (ET-1), angiopoietin-1, Tie2 agonists, neuropilin, and prostaglandin E2. Further examples include viral vector-mediated gene transfer of VEGF-C, VEGF-D, FGF-2, IGF-1, HGF, ET-1, or angiopoietin-1.

[0098] VEGFR3, also known as FLT4, is a receptor tyrosine kinase whose signaling pathway is involved in embryonic lymphoid development and adult lymphangiogenesis. Upon ligand binding, VEGFR3 dimerizes and is activated by autophosphorylation. Therefore, VEGFR3 agonists are suitable for methods to treat, mitigate, or prevent neurodegenerative diseases associated with the accumulation of molecules in the brain, such as AD, according to some embodiments herein. Accordingly, in some embodiments, such as methods or compositions, where increased drainage and flow are desired, the fluid agent is a VEGFR3 agonist.

[0099] In particular, VEGF-C promotes lymphatic vessel growth (lymphatic vessel formation). It acts primarily on lymphatic endothelial cells (LECs) via its receptor, VEGFR-3, promoting their survival, growth, and migration. Therefore, VEGF-C is suitable as a VEGFR3 agonist.

[0100] The effective dose of a VEGFR3 agonist according to the method of the present invention can be understood in relation to its ability to treat, improve, or prevent various cognitive impairments resulting from aging or neurodegenerative diseases by expanding or repairing NPLP to increase or stabilize CSF flow, or by increasing the clearance of substances from the CNS. Accordingly, the method of the present invention expands NPLP by at least about 2%, for example, at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%, and in the range between any two of the listed values. In some embodiments, an effective dose of the VEGFR3 agonist increases the CSF flow by at least about 2%, for example, at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%, and increases within a range between these two listed values.

[0101] In addition to VEGF-C, a VEGFR3 agonist may include VEGF-D. In one embodiment, VEGF-C and VEGF-D agonize VEGFR3 together and can be provided in a single composition or in separate compositions. In some embodiments, a VEGFR3 agonist includes variants, orthologs, fragments, or analogs, variants, or functional fragments of VEGF-C or VEGF-D, such as cleavage.

[0102] Embodiments of the present invention also utilize exogenous nucleotides encoding VEGFR3 agonists, such as VEGF-C. In one embodiment, a nucleotide encoding VEGF-C or VEGF-D as described herein is expressed in a subject for administration of a VEGFR3 agonist to the subject. For example, an exogenous vector, such as a retrovirus, lentivirus, adenovirus, or adeno-associated virus vector containing nucleic acid encoding a VEGFR agonist as described herein, can be inserted into the host nucleic acid of the subject. In some embodiments, the vector further includes a transcription mechanism for promoting the transcription of the nucleic acid encoding the VEGFR3 agonist.

[0103] In one embodiment, the VEGFR3 agonist may include modifications such as glycosylation or PEGylation. In some embodiments, a composition for use according to the method described herein comprises a VEGFR3 agonist and a pharmaceutically acceptable diluent or carrier.

[0104] The present invention also relates to a method for assaying the flow rate of CSF through an NPLP or NPLP-dcLV pathway by applying a fluidizing agent such as a chemical or biological molecule, wherein the fluidizing agent is considered an effective agent for increasing CSF flow in a region if the flow or outflow is greater than before the application of the fluidizing agent.

[0105] Accordingly, in one embodiment, the present invention relates to a method for determining a CSF agent that increases the outflow of CSF through the NPLP or NPLP-dcLV pathway.

[0106] dcLV flow agent (dcLV fluid agent)

[0107] Some dcLV flow agents cause strong, transient contraction of dcLVs with 0.1 M and 1.0 M potassium chloride (KCl) (Figure 38). Furthermore, the G protein-coupled receptor agonist phenylephrine or the nitric oxide donor (sodium nitroprusside) can contract or expand dcLVs and regulate the amount of CSF tracer (Figure 39). Low doses of phenylephrine (10 nM) increased the amount of CSF excreted, but high doses (5 mM) decreased the amount of CSF excreted as measured by accumulated CSF tracers in dcLN (Figure 40). Low doses of the nitric oxide donor (3 μM) and sodium nitroprusside enhanced CSF drainage, but no change was observed with the 30 μM nitric oxide donor (Figure 40).

[0108] The contraction and relaxation of the circular smooth muscles surrounding dcLVs can be regulated by 1) stimulation and inhibition of smooth muscle cells or peripheral nerves, 2) neurotransmitters, 3) mechanical stimulators, and 4) gentle massage of the neck area near cdLVs. Cerebrospinal fluid (CSF) drainage is promoted by extracranial manipulations, drug administration, and regulatory factors (Figure 41). Given that there are no significant changes in aging dcLVs, extracranial manipulation of dcLVs can enhance the reduction of CSF drainage.

[0109] The administered composition may comprise a single unit dose of a fluidizing agent effective for increasing or restoring fluidity, increasing clearance, or reducing accumulated amyloid-beta plaques. In some embodiments, the effective amount of the fluidizing agent is about 0.00015 mg / kg to about 1.5 mg / kg and includes any other amount or range intended as the therapeutically effective amount of the compounds disclosed herein. The range may be greater or less than the range of about 0.00015 mg / kg to about 1.5 mg / kg.

[0110] Mechanical stimulation may include single or multiple vibrations for increasing or restoring blood flow, or for increasing clearance, or for reducing accumulated amyloid beta plaques or phosphorylated tau in the CSF. In some embodiments, the effective acceleration of the CSF by the dcLV is about 0.001 m / s 2 ~ about 2.5 m / s 2 and includes any other range intended as a therapeutically effective acceleration disclosed herein. The range may be larger or smaller than the range of about 0.001 m / s 2 ~ about 2.5 m / s 2 . Mechanical stimulation is not limited to mechanical vibrations and may include massage or any other mechanical method that allows the dcLV to contract and release the dcLV. The vibration or massage may be performed to increase the flow of CSF through the dcLV. Vibration machines applicable for human use are available in the art.

[0111] The present invention also relates to a method of applying a fluidizing agent such as a biological or chemical molecule or a physical pressure method and then assaying the flow rate of the CSF through the dcLV, where if the flow or outflow in the dcLV region is greater than before the application of the fluidizing agent, the fluidizing agent is considered an effective agent for increasing the flow of CSF in that region. In one form, the present invention is directed to a method of determining a CSF agent that increases the outflow of CSF through the dcLV.

[0112] Route of administration

[0113] The fluidizing agent according to the present invention can be administered to a subject using any of a number of suitable routes of administration, provided that the route of administration conditions the administration of the fluidizing agent to the subject's NPLP-dcLV route.

[0114] When used herein, "administering to the target NPLP-dcLV pathway" does not necessarily require the fluid agent to be administered directly into the NPLP-dcLV pathway space; rather, the term encompasses the direct and / or indirect administration of the fluid agent to the NPLP-dcLV pathway space. In some embodiments herein, administering the fluid agent to create fluid communication with the target NPLP-dcLV pathway space typically involves, optionally, administering the fluid agent into the brain, thereby intending to administer the fluid agent to the NPLP-dcLV pathway space. Thus, in one embodiment, the fluid agent is not administered systemically. In another embodiment, the fluid agent is not administered systemically, but rather to a fluid, tissue, or organ that is in fluid communication with the NPLP-dcLV pathway space. In some embodiments, the fluid agent is not administered systemically, but rather to the CNS. In some embodiments, the fluid agent is administered to the CNS, but not to any organ or tissue outside the CNS. In some embodiments, the fluid agent is not administered into the bloodstream.

[0115] In some embodiments, the fluid is administered nasally to directly affect the NPLP. For example, the fluid can be provided as a nasal spray or in direct contact with the nasal mucosa.

[0116] In some embodiments, the fluid is administered through contact with the target CSF. For example, the fluid can be injected directly into the patient's CSF (e.g., into the subarachnoid space). Suitable devices for injection may include syringes or pumps that are inserted into or implanted in the target and provide fluid communication with the CSF. In another embodiment, the fluid may be a sustained-release gel that is implanted in the target to provide fluid communication with and thus contact the CSF. Local administration, particularly to the neck region, is also intended to affect the muscles surrounding the dcLV. Non-limiting examples of local administration include creams, lotions, gels, sprays, dispersions, suspensions, pastes, and lotions.

[0117] In some forms, the fluid is administered transcranially. For example, a fluid such as a gel formulation can be placed in the outer portion of the subject's skull and can pass through the skull. In some embodiments, the fluid is brought into contact with a thinned portion of the subject's skull to facilitate transcranial delivery.

[0118] In another form, the fluid agent is administered by inducing the expression of a nucleic acid encoding the fluid agent in the subject. A vector containing nucleic acid, such as a retroviral vector, lentiviral vector, or adenovirus vector, or a viral vector such as an adeno-associated virus (AAV), can be administered to the subject described herein, for example, by injection or inhalation. In some embodiments, nucleic acid expression is induced in the subject, for example, via a transcriptional regulator.

[0119] In some embodiments, the fluid is selectively administered to the target NPLP-dcLV pathway space. As used herein, the term “selectively” in “administered” indicates that the fluid is preferentially administered to the indicated target compared to other tissues or organs. Thus, direct injection into the extracranial NPLP-dcLV pathway constitutes “selective” administration, while administration to the CSF via spinal injection generally does not. In some embodiments, the fluid is selectively administered to the NPLP-dcLV pathway space and not to any outside CNS portion, nor to any tissue or organ outside the CNS. In some embodiments, the fluid is selectively administered to the CNS and not to outside tissues or organs such as the peripheral nervous system, muscles, gastrointestinal system, or muscular system.

[0120] In some embodiments, the fluid is administered to the area surrounding the cervical region of the target NPLP-dcLV pathway. As used herein, the cervical region surrounding NPLP-dcLV refers to the tissues around lymphatic vessels from the NPLP to the deep cervical lymph nodes.

[0121] In some embodiments, the fluid is administered to the surrounding neck area of ​​the mandibular lymph node. As used herein, the surrounding neck area refers to the tissues around the afferent lymphatic vessels of the mandibular lymph nodes. For example, it includes the lymphatic vessels from the hard palate to the mandibular lymph nodes.

[0122] In any of the administration routes listed herein according to the method of the present invention, the liquid agent may be administered in a single dose or in two or more doses, and it is intended that it may be separated for a certain period of time. For example, in some embodiments, the liquid agent described herein may be administered hourly, daily, every other day, every three days, every four days, every five days, every six days, weekly, every other week, monthly, every other month, etc., via the administration routes described herein. In some embodiments, the liquid agent is administered in a single dose and is not administered in any additional doses.

[0123] Some embodiments include methods for preparing compositions or drugs comprising the fluid agents described herein, suitable for administration according to the routes of administration described herein. For example, in some embodiments, the VEGFR3 agonist is prepared for intranasal administration, administration to the CSF, or transcranial administration.

[0124] Particularly preferred methods of administering the drug include, but are not limited to, intraarachnoid approaches to the CSF space, transnasal or transoral approaches to the nasopharynx, and transcervical, percutaneous, or topical approaches to the neck muscles, neck nodes, or neck space.

[0125] Neurodegenerative diseases and conditions

[0126] The present invention is useful for treating, preventing, inhibiting, improving, or alleviating the symptoms of one or more neurodegenerative diseases. These diseases can occur in subjects, such as humans, as well as in non-human animals, such as non-human mammals, and especially non-human primates.

[0127] In some embodiments, neurodegenerative diseases associated with the accumulation of macromolecules, cells, and debris in the CNS can be treated, prevented, inhibited, or reduced by increasing flow, drainage, and / or clearance in NPLP-dcLV lymphatic vessels. Examples of neurodegenerative diseases include age-related cognitive decline, Alzheimer's disease, Parkinson's disease, Huntington's disease, or stroke.

[0128] In some embodiments, neurodegenerative diseases can be preventively prevented, treated, or ameliorated. Therefore, subjects with one or more risk factors for neurodegenerative diseases may be deemed to require treatment methods described herein. For example, a subject may have amyloid-beta plaques accumulating in the CNS and may not yet have an AD diagnosis based on cognitive symptoms, yet may still benefit from increased flow, increased drainage, increased and / or decreased clearance of amyloid-beta plaques. Several risk factors for AD are suitable as risk factors according to the methods, compositions, and uses of some embodiments herein, such as familial AD, genetic markers of AD, or symptoms of AD such as early dementia. The most important risk factor for sporadic AD is aging. However, the increased risk of this form of AD is also attributable to a variety of genetic abnormalities.

[0129] Methods for increasing flow rate

[0130] The method of the present invention may include determining whether the subject requires increased fluid flow in the central nervous system. Furthermore, the method of the present invention may include determining whether the subject requires increased CFS drainage. If the subject requires increased fluid flow or drainage, the method of the present invention may include administering an effective amount of VEGFR3 agonist or mechanical stimulation to the subject's NPLP-dcLV pathway space. Thus, the fluid flow in the subject's central nervous system, as well as the level or velocity of CFS outflow, can be increased. In some embodiments, the VEGFR3 agonist includes VEGF-C or VEGF-D, or analogs, variants, or fragments thereof. Also, in some embodiments herein, FGF2 may be substituted for the indicated VEGFR3 agonist to increase flow, or may be used in addition to the VEGFR3 agonist to increase flow. In some embodiments, mechanical stimulation may be applied to the neck area surrounding the NPLP-dcLV pathway.

[0131] A subject may be determined to require increased fluid flow or outflow by determining whether the subject has age-related cognitive impairment, has a neurodegenerative disease, or is at risk of developing a neurodegenerative disease. The disease may be associated with increased concentration and / or accumulation of molecules, cells, or debris in the CNS, for example, in Alzheimer's disease (AD). In some embodiments, a subject may be determined to be at risk of the disease, for example, by having a familial occurrence of the disease, by having one or more genetic markers associated with the disease, through aging, or by exhibiting symptoms of the disease, such as early dementia in the case of AD.

[0132] As used herein, “advanced age” refers to an age characterized by a decline in memory function, a decrease in CSF production, and a substantial increase in neuronal aging, and in the context of some embodiments, may include at least 65 years in humans, e.g., at least 60, 65, 70, 75, 80, or 85 years, and includes a range between any of these values. In some embodiments, determining whether a subject requires increased fluid flow or outflow includes determining that the subject has a neurodegenerative disease such as AD. In some embodiments, determining whether a subject requires increased fluid flow or outflow includes determining that the subject has a risk factor for neurodegenerative disease associated with increased concentration and / or accumulation of molecules or macromolecules or cells or debris in the CNS, as described herein. In some embodiments, determining whether a subject requires increased fluid flow or outflow also includes determining that the subject has a risk factor and that the subject has the disease itself.

[0133] In some embodiments, the neurodegenerative disease or condition is selected from the group consisting of at least one of the following: cognitive impairment possibly due to aging, Alzheimer's disease (AD), dementia, Parkinson's disease, Huntington's disease, or stroke. In some embodiments, the neurodegenerative disease is Alzheimer's disease. In some embodiments, the risk factors are the risk factors for Alzheimer's disease as described herein. In some embodiments, the VEGFR3 agonist and / or FGF2 is administered to the subject after it has been determined that the subject has risk factors for a neurodegenerative disease (even if the subject does not necessarily have the disease itself), for example, prophylactic treatment or prevention. In some embodiments, the VEGFR3 agonist and / or FGF2 is administered to the subject after it has been determined that the subject has a neurodegenerative disease.

[0134] While not limited by theory, according to some embodiments herein, systemic administration of VEGFR3 agonists and / or FGF2 is intended not to be required to effectively regulate the size and drainage or flow of NPLP-dcLV lymphatic vessels. Accordingly, in some embodiments, VEGFR3 agonists and / or FGF2 are selectively administered to the target NPLP-dcLV pathway space. In some embodiments, VEGFR3 agonists and / or FGF2 are administered to the inside or outside space of the CNS. In other embodiments, VEGFR3 agonists and / or FGF2 are administered to the NPLP-dcLV pathway space but not to the blood. In some embodiments, the VEGFR3 agonist and / or FGF2 is administered to the subject by a route selected from the group consisting of local administration of the subject, intrathecal administration, nasal administration, oral administration, transcranial administration, contact with the subject's cerebrospinal fluid (CSF), pump infusion into the subject's CSF, implantation into the skull or brain, contact between the subject's thinned skull or portion of the skull and the VEGFR3 agonist and / or FGF2, expression of nucleic acids encoding the VEGFR3 agonist and / or FGF2 in the subject, or at least one combination of any of the listed routes. In some embodiments, it is the VEGFR3 agonist that is administered. In some embodiments, the VEGFR3 agonist is selected from the group consisting of at least one of the following VEGF-C, VEGF-D, or their analogues, variants, or functional fragments.

[0135] In some embodiments, administration of a VEGFR3 agonist results in an increase in NPLP-dcLV lymphatic vessel diameter, NPLP-dcLV pathway lymphatic vessel number, NPLP-dcLV pathway lymphatic drainage, or improvement of symptoms of neurodegenerative disease or condition. For example, in some embodiments, administration of a VEGFR3 agonist increases the diameter of NPLP lymphatic vessels by at least about 5%, e.g., at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%, which includes a range between any two of the enumerated values. In some embodiments, the average diameter of the population of NPL-PdcLV lymphatic vessels in the subject increases by the values ​​described herein. In some embodiments, administration of a VEGFR3 agonist includes increasing the fluid perfusion rate throughout the brain region of the subject, and increasing CSF flow in the central nervous system, including CSF outflow in the subject.

[0136] In some embodiments, mechanical stimulation of the surrounding neck area of ​​the NPLP-dcLV pathway results in increased CSF flow or improvement of symptoms of neurodegenerative diseases or conditions. For example, in some embodiments, mechanical stimulation treatment increases CSF outflow in the central nervous system, including CSF outflow in the subject, by at least about 5%, for example, in the range of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%, and between any two of the listed values. In some embodiments, the mean CSF flow in the NPLP-dcLV lymphatic vessels of the subject increases by the values ​​described herein. Here, mechanical stimulation includes, but is not limited to, vibration, ultrasound, light, electrical, magnetic, and thermal stimulation.

[0137] Increased clearance can reduce macromolecules such as amyloid-beta plaques or decrease their accumulation rate. While not limited by theory, it is intended that removing soluble amyloid-beta from the CNS would create a gradient favorable to the solubilization of amyloid-beta plaques, resulting in continued fluid flow in the CNS, continued removal of CNS, and a reduction in the number or rate of increase of amyloid-beta plaques. Therefore, a reduction in amyloid-beta plaques can mean a reduction in the pathogenesis of diseases caused by amyloid-beta plaques.

[0138] The increased fluid flow can reduce the amount of amyloid-beta plaques accumulated in the subject, or reduce the rate of accumulation. In some embodiments, the amount of accumulated amyloid-beta plaques, or the rate of accumulation, is reduced by at least 2%, for example, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, and includes a range between any two of the listed values.

[0139] Detection of changes in CSF flow

[0140] NPLP is a newly discovered hub for CSF drainage through the skull base including a cribriform plate. The hard palate submucosa is a newly discovered administration route for CSF drainage to the mandibular lymph nodes. Therefore, within the scope of this invention, changes in human CSF flow can be assayed at these locations.

[0141] In one form, a swab or biopsy is prepared from the nasopharyngeal mucosa or hard palate submucosa area to obtain a cell specimen that should contain NPLP or hard palate submucosa lymphatic cells. The CSF content of the sample is assayed or measured separately over time and compared over time as the assay is repeated in humans, and the results are compared.

[0142] If a decrease in CSF outflow over time is observed, exogenous drugs can be administered to increase CSF outflow in the subject.

[0143] Assay methods may include imaging NPLP with tracer molecules and observing them through a microscope. Other methods include, but are not limited to, proteomic analysis of CSF-specific proteins. The levels of selected proteins in CSF can be measured from time to time to determine their amounts, and as the amount of selected proteins decreases or increases, it indicates the level and quality of CSF drainage. An example of a selected protein to monitor may be β2-transferrin in the obtained CSF sample. In particular, for example, an increase in the amount of β2-transferrin present in the sample over time indicates improvement in CSF drainage function. A decrease in the amount of β2-transferrin present in the sample over time indicates impairment of CSF drainage function. Other proteins may be included without limitation.

[0144] Promotion of cerebrospinal fluid (CSF) drainage.

[0145] Facilitating cerebrospinal fluid (CSF) drainage is useful in the prevention and treatment of neurodegenerative diseases, including Alzheimer's disease. Herein, the present invention provides means relating to how extracranial approaches and methods can facilitate CSF drainage. The present invention discloses extracranial methods for facilitating CSF drainage through the nasopharyngeal lymphatic plexus (NPLP) and deep cervical lymphatic vessels (dcLV) for the prevention and treatment of neurodegenerative diseases.

[0146] In one embodiment, the present invention relates to a method for preventing or treating central nervous system diseases by improving CSF drainage in humans by repairing or sometimes increasing NPLP. CNS diseases may include Alzheimer's disease, Parkinson's disease, or Huntington's disease. NPLP is restored by administering drugs such as vascular growth factor C (VEGF-C), vascular growth factor D (VEGF-D), fibroblast growth factor 2 (FGF-2), insulin-like growth factor 1 (IGF-1), hepatocyte growth factor (HGF), endothelin-1 (ET-1), angiopoietin-1, Tie2 agonists, neuropilin, prostaglandin E2, or viral vector-mediated gene transfer of VEGF-C or VEGF-D or FGF-2 or IGF-1 or HGF or ET-1 or angiopoietin-1 to a person in need.

[0147] The contraction and relaxation of circular smooth muscles covering dcLVs can be regulated by stimulation and inhibition of smooth muscle cells, and such agents are those that increase or decrease myosin phosphorylation, and are not limited to those that activate myosin light chain kinase, or myosin light chain phosphatase by activating a voltage-gated Ca2+ channel (KCl) or a G protein-coupled receptor agonist (phenylephrine), or by stimulating a cGMP-dependent protein kinase (nitric oxide or acetylcholine).

[0148] The contraction and relaxation of the circular smooth muscle covering dcLVs can be further modulated by stimulation and inhibition of peripheral nerves, and such agents, though not limited to botulinum toxin or tetanus toxin, can interfere with the depolarization or exocytosis of synaptic vesicles.

[0149] The contraction and relaxation of the round smooth muscle covering dcLVs can also be modulated by the stimulation and inhibition of neurotransmitters, and such agents include, but are not limited to, agonists and antagonists of neurotransmitter receptors, or agonists and antagonists of enzymes that break down the round smooth muscle covering dcLVs, such as agonists or antagonists of norepinephrine.

[0150] The contraction and relaxation of the circular smooth muscle covering dcLVs are not limited to those described above, but can also be regulated by mechanical stimuli such as vibrators, high-frequency or low-frequency electrical stimuli, or optogenetic stimulation with specific wavelengths of light.

[0151] The method of drug delivery may include, but is not limited to, an intraarachnoid approach to the CSF space, a transnasal or oral approach to the nasopharynx, or a transcervical, transcutaneous, or local approach to the cervical muscles, cervical lymph nodes, or cervical space.

[0152] The various methods and techniques described above provide several ways of carrying out the present invention. Of course, it should be understood that not all of the objectives or benefits described can necessarily be achieved according to any particular embodiment described herein. For example, a person skilled in the art will recognize that a method may be carried out in a way that achieves or optimizes one benefit or group of benefits taught herein without necessarily achieving other objectives or benefits taught herein. Various alternatives are referred to herein. It should be understood that some preferred embodiments include one, another, or several features in particular, other embodiments exclude one, another, or several features in particular, but yet other embodiments mitigate certain features by including one, another, or several advantageous features.

[0153] Furthermore, those skilled in the art will recognize the applicability of various features from different embodiments. Similarly, the various elements, features, and steps discussed above, as well as other known equivalents of each such element, feature, or step, can be used in various combinations by those skilled in the art to carry out the method according to the principles described herein. Of the various elements, features, and steps, some are specifically included, while others are specifically excluded in various embodiments.

[0154] Although the present invention is disclosed in the context of specific embodiments and examples, it will be understood by those skilled in the art that embodiments of the present invention extend beyond the specifically disclosed embodiments to other alternative embodiments, and / or their uses, modifications, and equivalents.

[0155] Preferred embodiments of the present invention, including the best modes known to the inventors for carrying out the present invention, are described herein. Variations of these preferred embodiments will be apparent to those skilled in the art by reading the foregoing description. Those skilled in the art can appropriately use such variations, and it is intended that the present invention can be carried out in ways other than those specifically described herein. Accordingly, many embodiments of the present invention include all modifications and equivalents of the subject matter enumerated in the claims attached herein, as permitted by applicable law. Furthermore, unless otherwise indicated herein, or unless it is clearly inconsistent with the context, any combination of the above elements in all possible variations is encompassed by the present invention.

[0156] All patents, patent applications, publications of patent applications, and other materials such as articles, books, specifications, publications, documents, and objects referenced herein are incorporated herein in their entirety for all purposes; except for all related procedural files history, all that contradict or conflict with this document, or all that may have a limited effect on the broadest scope of the claims currently or hereafter related to this document. For example, there may be any contradiction or conflict between the explanation, definition, and / or use of terms relating to any of the incorporated matters and those relating to this document in which the description, definition, and / or use herein prevails.

[0157] It should be understood that the embodiments of the present invention disclosed herein are illustrative of the principles of the embodiments of the present invention. Other modifications that may be used may be within the scope of the present invention. Accordingly, alternative configurations of the embodiments of the present invention may be used in accordance with the teachings herein, not as an example but as an example. Accordingly, the embodiments of the present invention are not limited to those precisely illustrated and described.

[0158] Various embodiments of the present invention are described in the detailed description above. While these descriptions directly illustrate the embodiments described above, it will be understood that those skilled in the art may anticipate modifications and / or variations to the specific embodiments shown and described herein. Any such modifications or variations that fall within the scope of such descriptions are also intended to be included in the present invention. Unless otherwise specified, the terms in the specification and claims are intended to be given to those skilled in the art in their common usage.

[0159] The above descriptions of various embodiments of the present invention, known to the applicant at the time of filing the present application, are presented and are intended for illustrative and explanatory purposes. This description is not intended to be exhaustive or to limit the invention to the exact forms disclosed, and many modifications and variations are possible in light of the above teachings. The described embodiments illustrate the principles of the present invention and its practical applications and help enable those skilled in the art to utilize the invention with various modifications in various embodiments to suit specific intended uses. Accordingly, the present invention is not intended to be limited to the specific embodiments disclosed for carrying out the present invention. [Examples]

[0160] Examples

[0161] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. Where specific substances are mentioned, they are for illustrative purposes only and not intended to limit the invention. Those skilled in the art can replicate equivalent means or reactants without demonstrating the capabilities of the invention and without departing from its scope.

[0162] Example 1: Facilitating cerebrospinal fluid (CSF) drainage is useful in the prevention and treatment of neurodegenerative diseases, including Alzheimer's disease. Herein, the present invention discloses means relating to how extracranial approaches and methods can facilitate CSF drainage.

[0163] The present invention includes an extracranial method for facilitating CSF drainage through the nasopharyngeal lymphatic plexus (NPLP) and deep cervical lymphatic vessels (dcLV) for the prevention and treatment of neurodegenerative diseases.

[0164] Anesthetized mice (10-week-old male Prox1 GFP mice) were placed prone on a stereotactic frame under a microscope equipped with a heating pad. The head was adjusted to a 90° angle to the body axis with the help of a mouthpiece to facilitate access to the cisterna magna. After a skin incision at the midline of the posterior neck, the muscle layer was carefully separated using a microreactor. The atlanto-occipital membrane overlying the cisterna magna was superficially penetrated using a 33-gauge nanofil needle (World Precision Instruments), and then 3 μl of tetramethylrhodamine (TMR)-conjugated 10kDa MW dextran (Invitrogen, D1816) was injected into the subarachnoid space at a rate of 1 μl / min for 3 minutes using a microsyringe (88000, Hamilton) and a microinfusion machine (Fusion 100, Chemyx Inc.) (Figure 7A). After placing the mice in the appropriate location for 5 minutes, the needle was slowly withdrawn to prevent CSF leakage. The muscle layer and neck skin were then sutured with 6-0 black silk (Ailee, SK617). 60 minutes after injection, the heads were dissected without cardiac perfusion or immersion in saline. After decapitation, the heads were cut in half sagittally, and fluorescence images of the sectioned heads were obtained using a fluorescence stereozoom microscope (AxioZoom V16, Carl Zeiss) (Figure 7A).

[0165] As a result, dextran accumulated to a high degree in the olfactory bulb, cribriform plate, dorsolateral side of the skull, upper spinal cord area, and skull base adjacent to the nasopharynx (Figure 7B). Notably, the notable Prox1-GFP signal was detected in the nasopharynx, oropharynx, hard palate, and soft palate (Figure 7B), suggesting the presence of abundant LVs in these regions.

[0166] Example 2: The same procedure as in Example 1 was applied to anesthetized mice (10-week-old male Prox1-GFP mice), and the mice were sacrificed by transecting the abdominal aorta 60 minutes after injection. After dissecting the surrounding muscles with a surgical microscope (SZX16, Olympus), LVs and dextran of the nasopharynx and dcLN were imaged using a fluorescence stereozoic zoom microscope (Figure 8). The inventors found the existence of medial and lateral afferent LVs to dcLN, and named them medial deep cervical LV (M-dcLV) and lateral dcLV (L-dcLV) (Figure 8A). L-dcLVs were connected to basolateral LVs via the jugular foramen, while M-dcLVs were connected to LVs originating from the nasopharynx (Figure 8A). Injected dextran was detected not only in dcLNs but also in M-dcLVs and L-dcLVs (Figure 8A). Detailed examination of the nasopharynx revealed that dextran was highly detected in the upper part of the nasopharynx but not in the lower part of the nasopharynx and oropharynx (Figures 8B and 8C). Importantly, a well-arranged Prox1+ lymphatic plexus surrounding the nasopharynx was found (Figures 8B and 8C), which was named the "nasopharyngeal lymphatic plexus (NPLP)". The NPLP has a valve (Figure 8C, green arrowhead), and injected dextran was highly detected within the NPLP in the upper part of the nasopharynx (Figure 8C, reddish-yellow arrowhead). The NPLP was bound to the oropharyngeal lymphatic plexus (OPLP), but no dextran signal was detected within the OPLP (Figure 8C). These findings indicate that the NPLP acts as a drainage pathway for CSF connected to the dcLN via M-dcLV, but the OPLP does not.

[0167] Example 3: The same procedures as in Examples 1 and 2 were applied to anesthetized mice (10-week-old male Prox1-GFP mice), and then time-course vital imaging in the NPLP was obtained 30 minutes after injection using a fluorescence stereozoic zoom microscope (Figure 9A). The flow of injected dextran was detected in the NPLP in the upper nasopharynx (Figure 9B, reddish-yellow arrowhead).

[0168] Example 4: To verify the above findings, a solution containing 3 μl of fluorescent microbeads (0.5 μm in diameter, F8887, Thermo-Fisher) was injected into the intracranial lumen at a rate of 1 μl / min for 3 minutes using a microsyringe (88000, Hamilton) and an injector (Fusion 100, Chemyx Inc.). After 6 hours, NPLP was collected and it was found that the fluorescent microbeads bound to the NPLP abundantly and selectively (Figure 10), confirming that the NPLP can function as a hub for CSF drainage.

[0169] Example 5: The procedure of Example 1 was applied to anesthetized mice (10-week-old male Prox1-GFP mice), and the mice were then sacrificed by transecting the abdominal aorta 60 minutes after injection. After dissecting the surrounding muscles with a surgical microscope (SZX16, Olympus), LV and dextran in the proximal region of the dcLN were imaged using a fluorescence stereozoic zoom microscope (Figure 11). Medial and lateral afferent LVs were observed for the dcLN, M-dcLV, and L-dcLV (Figure 11). The L-dcLV was connected to basolateral LVs through the jugular foramen, while the M-dcLV was connected to LVs derived from the nasopharynx (Figure 11). Injected dextran was detected not only in dcLNs but also in M-dcLVs and L-dcLVs (Figure 11). Prox1+ dense lymphatic valves (Proxl+ dense lymphatic valves) were periodically distributed within dcLVs (Figure 11, green arrowheads).

[0170] Example 6: Figure 12 shows posterior, ventral, and lateral views of the NPLP (non-nasopharynx lymphatic plexus) of the front part of the nasopharynx and the posterior nasal lymphatic plexus. To obtain these images, immunofluorescence staining (IFS) of mouse NPLP was performed in 10-week-old male Prox1-GFP mice. After anesthesia and right atrial puncture, the left ventricle was perfused with ice-cold phosphate-buffered saline (PBS) to remove blood. After PBS perfusion, the tissue was fixed by injecting a 4% paraformaldehyde solution into the left ventricle. The nasopharyngeal mucosa was isolated by removing the surrounding skull, nerves, and soft tissue using fine forceps and surgical microscises with the help of a surgical microscope (Stemi508, Zeiss). The dorsal and ventral portions of the mucosa were cut in half transversely, and the lateral portion of the mucosa was cut in half sagittally. The collected tissue was fixed in a 2% paraformaldehyde solution at 4°C for 2 hours. After fixation, the nasopharyngeal mucosa was incubated in 5% normal donkey serum (017-000-121, Jackson ImmunoResearch) at room temperature for 1 hour. To label lymphatic markers, the nasopharyngeal mucosa was immersed in 5% normal donkey serum containing VEGFR3 antibody (AF743, R&D) and LYVE1 antibody (11-034, Angiobio) dissolved at 1:400 at 4°C for 12 hours. After washing with PBS, the tissue blocks were incubated in normal donkey serum containing Alexa-594-conjugated anti-goat IgG antibody and Alexa-647-conjugated anti-rabbit IgG antibody (Jackson ImmunoResearch) at 4°C for 12 hours. After washing with PBS, the tissues were covered with mounting medium (H1200, Vector). Images were acquired using a confocal microscope (LSM 800, Zeiss) with a Plan-Apochromat 10x / NA 0.45 lens.

[0171] A cross-sectional view of the NPLP is shown in Figure 13 (white arrow). Except for tissue sampling and sectioning, the method in Figure 12 was applied. The head was collected, perfused, the surrounding skin was peeled off, and after fixation, it was immersed in a 2% PFA solution at 4°C for 12 hours. For decalcification, the head was immersed in a 0.5M EDTA solution at 4°C for 48 hours. After decalcification, the head sample was dehydrated by immersion in a 30% sucrose solution for 48 hours. The head sample was embedded and frozen in frozen sectioning medium (Leica) and cut into 30 μm sections using a Cryocut Microtome (Leica). IFS was performed. The lymphatic vessels from the anterior portion to the nasopharynx constitute the posterior nasal lymphatic plexus. The NPLP has abundant lymphatic vessels in the middle part of the nasopharynx and connects to the lymphatic vessels in the posterior part of the nasopharynx.

[0172] Based on Figures 12 and 13, 2D and 3D schematic diagrams of NPLP are shown in Figures 14 and 15.

[0173] Example 7: As shown in Figure 16, new lymphatic vessels originating from the pituitary gland area connected to the NPLP were discovered. Ten-week-old male Prox1-GFP mice were used for tissue removal. The heads were sampled, fixed, and decalcified according to the method in Figure 12. After decalcification, the samples were incubated in CUBIC-L (T3740, Tokyo Chemical Industry Co. Ltd.) solution at 37°C for one week to remove the tissue. After the tissue removal step, the samples were washed with PBS for one day. To label the lymphatic vessels, the samples were immersed in 5% normal donkey serum dissolved with LYVE1 antibody (11-034, Angiobio) in a 1:200 ratio at room temperature for seven days with gentle shaking. After primary antibody incubation and PBS washing, the samples were incubated in normal donkey serum containing Alexa-594-conjugated anti-rabbit IgG antibody (Jackson ImmunoResearch) and lysed at a 1:100 ratio at room temperature for 4 days with gentle shaking. After secondary antibody incubation and washing with PBS, the samples were immersed in D-PROTOSS solution (Ku et al., Nature methods, 2020) for 48 hours to match the refractive index. The cleared samples were imaged using an optical sheet microscope (LightSheet7, Zeiss) with an EC Plan Neofluar 5x / 0.16 lens. Blunt ends of LVs were observed around the pituitary gland (Figure 16). These LVs ran along the cavernous sinus and connected to the NPLP (Figure 16B). A 3D video of these connected LVs is shown in Figure 17 (green arrow).

[0174] Example 8: We newly discovered connections between lymphocytes (LVs) around the pterygopalatine artery (PPA) and the posterior nasal lymphatic plexus connected to the NPLP (Figure 18). The procedure of Example 7 was applied to 10-week-old male Prox1-GFP mice. Here, Prox1+ / LYVE1+ LVs were located along the pterygopalatine artery (PPA), had smooth ends in the basal meninges, and connected to the posterior nasal lymphatic plexus. Lymphatic valves (white arrowheads, high Prox1+ signal) were located at the connection points (Figure 18). Furthermore, some of these LVs passed through the greater palatine foramen and connected to the lymphatic plexus in the submucosa of the hard palate (Figures 18 and 19).

[0175] Example 9: Prox1+ LVs located beneath the olfactory epithelium connect to the posterior nasal lymphatic plexus, which is connected to the NPLP (Figure 20, reddish-yellow and white arrowheads). These findings indicate that CSF can be drained through the LVs of the olfactory mucosa and the posterior nasal lymphatic plexus. A schematic diagram of three novel lymphatic delivery routes from the intracranial cavity to the extracranial NPLP is shown in Figure 21. These are (1) LVs originating from the pituitary region connected to the NPLP, (2) LVs along the pterygopalatine artery (PPA) connected to the NPLP, and (3) LVs located at the anterior skull base along the foramen of the cribriform plate connecting the posterior nasal lymphatic plexus and lymphatic vessels in the olfactory mucosa connected to the NPLP. Thus, the NPLP functions as a hub for CSF drainage.

[0176] Example 10: The same procedure as in Example 1 was applied to anesthetized 10-week-old male Prox1 GFP mice, which were then sacrificed by transecting the abdominal aorta 60 minutes after injection. LVs and dextran in the hard palate submucosa and mandibular LN were imaged using a fluorescence stereozooming microscope (Figure 22). LVs in the hard palate submucosa were bound to the mandibular LN. Injected dextran was detected not only in the mandibular LN but also in the lymphatic vessels of the submucosa of hard palate (Figures 22A and 22B).

[0177] Fluorescent microspheres (ThermoFisher, F8801) were injected into primates (cynomolgus monkeys) under isoflurane anesthesia via the cisterna magna (2 mL, 25 μL / min). Six hours after injection, blood was perfused with PBS and 2% PFA, and then the hard palate was collected. Fluorescent microspheres derived from CSF were found to be located within the lymphatic vessels of the hard palate (Figure 23). These findings indicate that LVs in the hard palate act as a drainage pathway for CSF connected to the mandibular LN. In addition, these LVs can be modified to facilitate CSF drainage.

[0178] Example 10: IFS analysis revealed four lymphatic branches connecting NPLP and dcLVs in Prox1-GFP mice (Figure 24). Removal of the soft palate clearly revealed the four lymphatic branches connecting NPLP and dcLVs (Figure 24), suggesting that CSF flows to dcLVs via NPLP.

[0179] Example 11: In collaboration with the National Primates Center of the Korea Biotechnology Research Institute, nine cynomolgus monkeys were obtained in 2022. The samples were perfused with ice-cold saline. Using 4% paraformaldehyde (PFA), decapitated head samples emerged after 2 hours at 4°C. Subsequently, the samples were emerged (emerged) in 2% PFA at 4°C for 12 hours. Then, retropharyngeal lymph nodes (LNs) equivalent to those in mice were collected. After lymph node removal, the heads emerged (emerged) after 3 weeks at 4°C using 0.5M EDTA, pH 8.0 (Welgene). The EDTA was replaced with fresh EDTA solution every 4 days. After decalcification, the heads were trimmed. The anterior boundary was the choana, and the posterior boundary was the occipital bone. The dorsal boundary was the optic nerve, and the ventral boundary was the uvula. Next, the trimmed head was cut in half sagittally. The brain was removed from the skull. The sample was immersed in a 30% sucrose solution for 72 hours.

[0180] The nasopharynx was located directly below the clivus, one of the elements of the skull base (Figure 25). The ventral part of the nasopharyngeal mucosa is clearly distinguishable from the soft palate (Figure 25). The retropharyngeal lymph node was thought to be located in the white dotted circle in Figure 25.

[0181] To obtain IFS images, fixed and dehydrated nasopharynx tissue was coronally cut using a blade, embedded and frozen in frozen section medium (Leica), and then cut into 30 μm sections using a Cryocut Microtome (Leica). To obtain images of the entire attached tissue, the nasopharyngeal mucosa was carefully separated from the skull base and soft palate. After washing with PBS, the samples were incubated in 5% normal donkey serum (017-000-121, Jackson ImmunoResearch) at room temperature for 1 hour. To label lymphatic markers, the nasopharyngeal mucosa was immersed in 5% normal donkey serum in which LYVE-1 antibody (DP3500, OriGene) and type IV collagen antibody (AB769, Sigma) were dissolved at 1:400 at 4°C for 12 hours. After washing with PBS, tissue blocks were incubated with normal donkey serum containing Alexa-594-conjugated anti-goat IgG antibody and Alexa-647-conjugated anti-rabbit IgG antibody (Jackson ImmunoResearch) at 4°C for 12 hours. After washing with PBS, tissue blocks were incubated with mounting medium (H1200, Vector). Images were acquired using a confocal microscope (LSM 800, Zeiss) with a Plan-Apochromat 10x / NA 0.45 lens.

[0182] Similar to mice, NPLP is present in the submucosa of the nasopharynx in cynomolgus monkeys (Figure 25).

[0183] Furthermore, contrast-enhanced MRI images were acquired in cynomolgus monkeys after injection of the MR contrast agent, Gadspin P, through the cisterna magna. The injection needle was positioned using computed tomography (CT) for Gadspin P injection. After confirming the needle position, Gadspin P (10% dissolved in saline, 25 μL / min) was injected. Images were acquired using FLAIR T2 MRI (3T, Phillips Medical System) for 3 hours and 30 minutes before and after Gadspin P injection. The primates were under isoflurane anesthesia, and physiological monitoring was performed by a veterinarian. Increased signal intensity within the nasopharynx was observed in the primates (Figure 26).

[0184] Example 12: Functional research: To estimate which side of the dcLV excretes more CSF, ligation of one side of the dcLV was performed in bisexual 10-week-old Prox1-GFP mice. After incising the sternocleidomastoid muscle, both sides of the dcLVs were carefully ligated, with one side of the dcLV ligated with 10-0 polypropylene suture (W2794, Ethicon) and the other side sham-ligated. One day after ligation, TMR-dextran was injected into the cisterna magna according to the method of Example 1. Here, compared to the dextran signaling in sham surgery, M-dcLV ligation significantly reduced the dextran signaling in the dcLN, while L-dcLV ligation only slightly reduced the dextran signaling in the dcLN (Figure 27). These results suggest that CSF drainage via the NPLP / M-dcLV pathway is greater than that via the basolateral mLV / L-dcLV pathway, highlighting the importance of NPLP. P-values ​​were calculated using a one-way ANOVA test followed by Turkey's post-hoc test.

[0185] The same intracranial cavity injection was applied to 10-week-old Prox1-GFP mice of both sexes, and the rate of CSF efflux to dcLV was measured (Figure 27). Dextran signaling was measured in dcLV. Compared to dextran signaling in L-dcLV, dextran signaling was 7.9 times, 5.2 times, and 2.1 times higher at 30, 60, and 120 minutes post-injection (n=10-12 for each group) (Figure 28). These results suggest that CSF drainage via the NPLP / M-dcLV pathway is faster than via the basolateral mLV / L-dcLV pathway, highlighting the importance of NPLP. P-values ​​were calculated by one-way ANOVA test and subsequent Turkey post-hoc test.

[0186] Example 13: To test whether NPLP can be modified by exogenous drugs, 1.1x10 13 GC / mL of AAV-mVEGF-C-mCherry was injected into the intracranial cavity via the cisterna magna of 10-week-old Prox1-GFP female mice at a rate of 1.0 μl / min over 3 minutes. The same volume of empty AAV vector was injected as a control. Three weeks after injection, the NPLP of both groups was compared. Compared to the empty AAV group, the AAV-mVEGF-C group showed an increase in Prox1+ area (Figure 29). These findings suggest that NPLP can be modified by exogenous agents. The amount of CSF drained into the dcLN was enhanced in the AAV-VEGF-C group (Figure 29). This finding suggests that altering the CSF lymphatic drainage route, including NPLP, can enhance CSF drainage.

[0187] Example 14: To investigate whether aging alters NPLP, the inventors performed IFS on NPLP in adult and aged mice. Compared to adult (10-week-old male) mice, aged (80-88 week-old) mice showed 1) a decrease in lymphatic valves in NPLP, 2) regression of Prox1+ lymphatic vessels, 3) an increase in LYVE1, and 4) fragmentation of the dorsal NPLP at the skull base portion (Figure 30).

[0188] Furthermore, the transcriptome of lymphoid endothelial cells (LECs) in young and aging NPLP was analyzed. After anesthesia, mice were perfused with ice-cold PBS, and nasopharyngeal submucosa was isolated and pooled in DMEM / F12 medium (Gibco). The nasopharyngeal submucosa was cut into small pieces and incubated at 37°C for 30 minutes, gently inverting every 10 minutes, in a dissociation buffer containing 1 mg / ml collagenase IV (Roche), 1 mg / ml dispase (Gibco), and 0.1 mg / ml DNase I (Gibco). The digested samples were filtered through a 70 μm strainer, and digestion was stopped by adding 2% FBS. The cells were centrifuged at 500 x g for 8 minutes and resuspended in PBS for washing. To eliminate dead cells, 1:1000 Ghost dye (TONBO bioscience) was added to the resuspended cells at 4°C for 15 minutes. Next, PBS was added for washing, and then the cells were stained with phycoerythrin / Cy7 anti-mouse CD326 (Ep-CAM, 118216, Biolegend) antibody, APC anti-mouse podoplanin antibody (127410, Biolegend), and phycoerythrin-labeled anti-mouse CD31 antibody (102508, Biolegend). CD31+PDPN+ cells were considered LECs and sorted by FACS Aria Fusion (Beckton Dickinson). The sorted LECs were placed directly into each well of a 96-well plate containing lysis buffer. The plate was rapidly frozen in liquid nitrogen and stored at -80°C. Smart-Seq3 protocol plate-based single-cell libraries were prepared. Immediately, mRNA from the lysed cells was reverse transcribed, cDNA was amplified, and purified with Ampure XP beads (Beckman Coulter). Purified cDNA was diluted (100 pg / μl) and tagged using Tn5 tagmentation mix (Illumina). The tagged products were amplified using custom index primers and then pooled in single tubes. After final cleanup with Ampure XP beads, the libraries were analyzed by tape station for quality control.Libraries that passed quality control were sequenced on the Illumina High-X platform. The sequenced libraries were demultiplexed and aligned to the mouse reference genome (mm10) using STAR (version 2.7.9.a). Next, the aligned files were merged using the featureCount (version v2.0.1) function of the Subread package to create an unprocessed read count matrix. For cell quality control, cells detected with fewer than 2,000 genes and cells with more than 10% of total reads mapped to mitochondrial genes were considered low-quality / dead cells and discarded. At the gene level, genes expressed in fewer than 3 cells were removed from the expression matrix. The R package Seurat (version 4.1.0) was used for single-cell clustering and visualization. Briefly, Iog2 normalization was 1 × 10⁻¹⁶. 4 The process involved dividing each count for a gene in a given cell by the total number of counts in that cell, with the addition of a pseudocount of 1. The resulting expression matrix was then transformed to have values ​​similar to the logarithmically transformed counts per million. Next, the top 2,000 genes with the highest variability in each dataset were selected using the FindVariableFeatures function: selection method="vst". These highly variable genes were scaled and centered while devolving confounding variables such as the total count and the percentage of reads mapped to mitochondrial genes. Furthermore, module scores for dissociation-inducing and ribosomal genes were calculated using the AddModuleScore function and regressed.

[0189] For visualization in 2D space, principal component analysis was performed, and the top 15 principal components were used as input for Uniform Manifold Approximation and Projection (UMAP). For neighborhood identification and cluster assignment, a shared nearest neighbor graph was constructed using the top 15 principal components, and the Louvain algorithm was applied. To identify genes differentially expressed between cells, the FindMarkers function in Seurat was used with the options: test.use="MAST", logfc. Thresholds=0.3, min.pct=0.3. During differential expression testing, the inventors excluded dissociation-inducible genes, mitochondrial genes, and ribosomal genes. When merging adult and aging mouse datasets, no significant batch effect was observed with respect to clustering, so batch correction methods were not used. The inventors found that although five LEC clusters were conserved, genes involved in apoptosis and inflammation were expressed differently, and that senescent NPLP can be interpreted as being more pro-apoptotic and pro-inflammatory (Figure 31).

[0190] The inventors also performed IFS in aging NPLP using the same procedure as described in Example 6 to measure phosphorylated tau (ptau) and apoptotic lymphoid endothelial cells. Ptau was detected using anti-phospho-tau (mouse monoclonal (AT8) antibody, MN1020, Thermo) and increased in aging NPLP (Figure 32). The number of apoptotic lymphoid endothelial cells was detected using the TUNEL kit (12156792910, Merck) and increased with aging (Figure 32). These results suggest that increased apoptosis and associated changes (upregulation of type I interferon signaling) may be caused by an increase in CSF ptau.

[0191] Example 15: To test whether senescent NPLP can be modified by exogenous drugs, 1.1x10⁻¹⁰ AAV-VEGF-C-mCherry was administered via the cisterna magna of 75-78 week old Prox1-GFP female mice. 13 GC / mL was injected into the intracranial cavity (1.0 μl / min for 3 minutes). As a control, the same amount of empty AAV vector was injected. Three weeks after injection, the NPLP of both groups was compared. Compared to the AAV-empty group, the AAV-VEGF-C-mCherry group showed 1) Prox1 + The study showed an increase in the affected area and enhanced CSF efflux in dcLNs (Figure 33). Furthermore, CSF tracers were observed in lymphatic vessels after AAV-mVEGF-C infection. These findings suggest that age-related NPLP impairment may be modified by exogenous agents.

[0192] Example 16: IFS analysis reveals that dcLVs possess well-developed semi-luminal shaped lymphatic valves and lymphatic vessels, and are finely covered with circular smooth muscle cells (Figure 34). Peripheral nerve fibers are distributed along the dcLVs (Figure 35). The peripheral nerve fibers innervating them are sympathetic nerve fibers, not parasympathetic nerve fibers (Figure 36). In aging Prox1-GFP mice, there are no significant changes in the structure of surrounding smooth muscle cells or the number of lymphatic valves (Figure 37).

[0193] dcLVs contract strongly and transiently with 0.1 M and 1.0 M potassium chloride (KCl) (Figure 38). Furthermore, the G protein-coupled receptor agonist phenylephrine or the nitric oxide donor (sodium nitroprusside) can contract or expand dcLVs and regulate the amount of CSF tracer (Figure 39). In addition, low doses of phenylephrine (10 nM) increased the amount of drained CSF, but high doses (5 mM) decreased the amount of drained CSF as measured by the accumulated CSF tracer in dcLN (Figure 40). Low doses of the nitric oxide donor (3 μM) and sodium nitroprusside enhanced CSF drainage, but no change was observed with a 30 μM nitric oxide donor (Figure 40).

[0194] These findings suggest that the contraction and relaxation of the circular smooth muscle surrounding dcLVs can be regulated by 1) stimulation and inhibition of smooth muscle cells or peripheral nerves, 2) neurotransmitters, 3) mechanical stimulants, and 4) gentle massage. Essentially, cerebrospinal fluid drainage is facilitated by extracranial manipulation, drug administration, and regulatory factors (Figure 41). Given the lack of significant changes in aging dcLVs, extracranial manipulation of dcLVs can enhance the reduction of CSF drainage.

[0195] In summary, these examples provide information on where CSF drainage occurs and how CSF drainage can be facilitated by extraneurological approaches and methods. The present invention discloses that 1) NPLP is a newly discovered hub for CSF drainage through the skull base and cribriform dish; 2) the hard palatine submucosa is a newly discovered pathway for CSF drainage to mandibular LNs; 3) age-related impairment of CSF drainage can be enhanced by rescuing the CSF drainage pathway with exogenous agents such as VEGF-C; and 4) enhanced contraction and relaxation of the round smooth muscle covering dcLVs can be modulated by stimulation and inhibition of smooth muscle cells, peripheral nerves, neurotransmitters, and mechanostimulants, as CSF drainage can be facilitated by extraneurological procedures, drug administration, and relevant modulologics for the prevention and treatment of neurodegenerative diseases, including Alzheimer's disease (Figure 42).

[0196] While specific embodiments of the present invention have been shown and described, it will be apparent to those skilled in the art that modifications and alterations can be made based on the teachings herein without departing from the present invention and its broader forms, and that the appended claims are intended to encompass within their scope all such modifications and alterations that fall within the true spirit and scope of the present invention.

[0197] Cited references 1. Ransohoff, RM, & Engelhardt, B. (2012). The anatomical and cellular basis of immune surveillance in the central nervous system. Nat Rev Immunol, 12(9), 623-635. https: / / doi.org / 10.1038 / nri3265 2. Proulx, S. T. (2021). Cerebrospinal fluid outflow: a review of the historical and contemporary evidence for arachnoid villi, perineural routes, and dural lymphatics. Cell Mol Life Sci, 78(6), 2429-2457. https: / / doi.org / 10.1007 / s00018-020-03706-5 3. Bettcher, B. M., Tansey, M. G., Dorothee, G., & Heneka, M. T. (2021). Peripheral and central immune system crosstalk in Alzheimer disease - a research prospectus. Nat Rev Neurol, 17(11), 689-701. https: / / doi.org / 10.1038 / s41582-021-00549-x 4. Aspelund, A., Antila, S., Proulx, S. T., Karlsen, T. V., Karaman, S., Detmar, M., Wiig, H., & Alitalo, K. (2015). A dural lymphatic vascular system that drains brain interstitial fluid and macromolecules. J Exp Med, 212(7), 991-999. https: / / doi.org / 10.1084 / iem.20142290 5. Louveau, A., Smirnov, I., Keyes, T. J., Eccles, J. D., Rouhani, S. J., Peske, J. D., Derecki, N. C., Castle, D., Mandell, J. W., Lee, K. S., Harris, T. H., & Kipnis, J. (2015). Structural and functional features of central nervous system lymphatic vessels. Nature, 523(7560), 337-341. https: / / doi.org / 10.1038 / naturel4432 6. Ahn, J. H., Cho, H., Kim, J. H., Kim, S. H., Ham, J. S., Park, I., Suh, S. H., Hong, S. P., Song, J. H., Hong, Y. K., Jeong, Y., Park, S. H., & Koh, G. Y. (2019). Meningeal lymphatic vessels at the skull base drain cerebrospinal fluid. Nature, 572(7767), 62-66. https: / / doi.org / 10.1038 / s41586-019-1419-5 7. Pan, W. R., Suami, H., Corlett, R. J., & Ashton, M. W. (2009). Lymphatic drainage of the nasal fossae and nasopharynx: preliminary anatomical and radiological study with clinical implications. Head Neck, 31(1), 52-57. https: / / doi.org / 10.1002 / hed.20926 8. Ma, Q., Ineichen, B. V., Detmar, M., & Proulx, S. T. (2017). Outflow of cerebrospinal fluid is predominantly through lymphatic vessels and is reduced in aged mice. Nat Commun, 8(1), 1434. https: / / doi.org / 10.1038 / s41467-017-01484-6 9. Jacob, L., de Brito Neto, J., Lenck, S., Corey, C., Benbelkacem, F., Geraldo, L. H., Xu, Y., Thomas, J. M., El Kamouh, M. R., Spajer, M., Potier, M. C., Haik, S., Kalamarides, M., Stankoff, B., Lehericy, S., Eichmann, A., & Thomas, J. L. (2022). Conserved meningeal lymphatic drainage circuits in mice and humans. J Exp Med, 219(8). https: / / doi.org / 10.1084 / jem.20220035 10. Kipnis, J., Louveau, A., Da Mesquita, S. (2019). US 2019-0269758 Al, University of Virginia Patent Foundation (UN162191): U.S. Patent and Trademark Office 11. Kipnis, J. Da Mesquita, S. (2021), US 2021-0311076 Al, University of Virginia Patent Foundation (U N 162191): U.S. Patent and Trademark Office

Claims

1. A fluid pharmaceutical for the nasopharyngeallymphatic plexus (NPLP) used to increase the outflow of cerebrospinal fluid (CSF) from the central nervous system, A drug comprising administering an effective amount of NPLP to a subject requiring increased CSF outflow, wherein the amount of the drug repairs or expands the subject's NPLP, thereby increasing CSF outflow from the central nervous system into the systemic circulation in the subject.

2. A fluid-releasing drug for deep cervical lymphatic vessels (dcLV) used to increase cerebrospinal fluid (CSF) outflow from the central nervous system, The treatment involves administering an effective dose of a drug containing dcLV to a subject requiring increased CSF outflow, wherein the dose increases the contraction and relaxation of the subject's dcLV, thereby increasing CSF outflow from the central nervous system into the systemic circulation in the subject.

3. A liquid NPLP used to prevent, treat, or improve a neurodegenerative disease or condition in a subject, The treatment involves administering an effective dose of a NPLP drug to a subject, thereby increasing the amount of the drug to repair or expand the subject's NPLP, and thereby increasing the outflow of CSF from the central nervous system into the systemic circulation in the subject.

4. A liquid pharmaceutical for dcLV used to prevent, treat, or improve a neurodegenerative disease or condition in a subject, The treatment involves administering an effective dose of a drug containing dcLV to a subject, thereby increasing the contraction and relaxation of the subject's dcLV, and thereby increasing the outflow of CSF from the central nervous system into the systemic circulation in the subject.

5. The pharmaceutical product according to claim 1 or 2, wherein the subject requiring increased CSF outflow has a neurodegenerative disease or condition, a risk factor for a neurodegenerative disease or condition, or both.

6. The pharmaceutical product according to claim 3 or 4, wherein the neurodegenerative disease or condition is age-related cognitive decline, Alzheimer's disease, Parkinson's disease, Huntington's disease, or stroke.

7. The aforementioned pharmaceutical is as follows: (i) A VEGFR3 agonist, optionally wherein the drug is VEGF-C or VEGF-D, an analogue, variant, or fragment thereof, or any combination thereof. (ii) Fibroblast growth factor 2 (FGF-2), insulin-like growth factor 1 (IGF-1), hepatocyte growth factor (HGF), endothelin-1 (ET-1), angiopoietin-1, Tie2 agonist, neuropilin, or prostaglandin E2, (iii) A gene vector having a gene encoding a protein or pharmaceutical polypeptide, optionally wherein the gene encodes an angiopoietin-C, angiopoietin-1, or a Tie2 agonist. The pharmaceutical product according to claim 1 or 3.

8. The aforementioned pharmaceutical is selectively administered to the following sites or their vicinity: (i) NPLP-dcLV space, optionally, the NPLP-dcLV space is located in the nasopharyngeal mucosa, (ii) Submandibular lymph node space beneath the hard palate mucosa, optionally, the said submandibular lymph node space beneath the hard palate mucosa is located within the hard palate mucosa. The pharmaceutical product according to claim 1 or 3.

9. The pharmaceutical product according to claim 1 or 3, wherein the pharmaceutical product is administered to the subject intrathecally into the CSF space, or transnasally into the nasopharynx, or transorally to the hard palate submucosa.

10. The central nervous system of the subject includes the following: (i) Soluble molecules, increasing the CSF outflow reduces the amount of soluble molecules in the brain, or (ii) Amyloid-β plaques, increasing CSF outflow reduces the amount of amyloid-β plaques in the brain. The pharmaceutical product according to claim 1 or 3.

11. The aforementioned pharmaceutical is as follows: (i) G protein-coupled receptor agonist phenylephrine or nitric oxide donor, (ii) A stimulant for the circular smooth muscle covering the dcLVs, optionally a pharmacopoeia that stimulates the circular smooth muscle covering the dcLVs, increases or decreases myosin phosphorylation by activating myosin light chain kinase or myosin light chain phosphatase, and / or The pharmaceutical agent is administered to the subject transcervically, percutaneously, locally to the cervical muscles, cervical tubercle, or cervical space, or the pharmaceutical agent is mechanically applied to the side of the neck. The pharmaceutical product according to claim 2 or 4.

12. An inspection method for determining a change in the amount or rate of CSF outflow in a target, comprising: The process involves assaying the presence and amount of CSF or analyte-table substances in CSF at least twice separately on nasopharyngeal mucosa or hard palate mucosa specimens previously collected from biological samples, and obtaining values ​​each time. A step of comparing the values ​​of CSF or the amount of analyzable substance, and A process for measuring the change over time in the value of CSF or an analyzable substance. Here, the change in the value of CSF or analyzable substance over time indicates the changed CSF flow rate or velocity.

13. The method according to claim 12, (i) The biological sample is obtained by swab or biopsy, (ii) The biological sample includes NPLP or hard palate.

14. The method according to claim 12, wherein the assay is performed by imaging of NPLP or the hard palate, or by protein analysis of the obtained biological sample.

15. A method for increasing CSF outflow from the central nervous system, comprising: (a) Repairing or expanding NPLP, including: Identify targets that require increased CSF outflow, and To the subject in need, an effective amount of NPLP in liquid form is administered, thereby the effective amount of the drug repairs or expands the subject's NPLP, thereby increasing the outflow of CSF from the central nervous system into the systemic circulation in the subject, or (b) Increasing the contraction and relaxation of dcLV, including: Identify targets that require increased CSF outflow, and An effective amount of dcLV in liquid form is administered to the subject, thereby increasing the contraction and relaxation of the subject's dcLV, and thereby increasing the outflow of CSF from the central nervous system into the systemic circulation in the subject.

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