Glymph - Improvement of lymphatic outflow

Abstract

This disclosure relates to improving the outflow of glymph / lymphophilic solutes from the central nervous system.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 485,278, filed on 16 February 2023. The contents of that application are incorporated herein by reference in their entirety.

[0002] Government interests This invention was made with government support under NS100366 and AG057575, awarded by the National Institutes of Health, and W911NF1910280, awarded by the Army Research Service. The government has certain rights in this invention.

[0003] This disclosure relates to improving the outflow of glymph / lymphophilic solutes from the central nervous system. [Background technology]

[0004] Lymphatic vessels (LVs) play a vital role in returning 4-5 liters of fluid from the interstitial space to the venous circulation daily. Dysfunction of their structure or function can lead to intractable diseases such as lymphedema and contribute to the development of cancer metastasis, autoimmune disorders (e.g., inflammatory bowel disease), glaucoma, obesity, and cardiovascular disease. While LVs are not present in the central nervous system (CNS), recent research suggests that a network of perivascular spaces (glymphatic system) connected by astrocytes facilitates the transport of surrounding meninges to the LVs. 1、2 , emerging along the cranial nerves and spinal nerves 3 It has been shown that the fluid and solute are discharged to the periphery and enter the venous circulation.

[0005] The cervical lymphatic vessels (cLV) in the neck play a role in draining approximately 50% of the cerebrospinal fluid (CSF) from the brain to the cervical lymph nodes (cLN). 4~6 This elimination pathway is involved in Alzheimer's disease. 7 8and have been shown to be involved in the onset of Parkinson's disease and the recovery after stroke and traumatic brain injury. Transport of CNS antigens to the periphery via cLV also affects the onset of multiple sclerosis and modulates the effectiveness of immune checkpoint inhibitors in CNS tumors and amyloid-β immunotherapy. Cervical LV also contributes to fluid homeostasis, and ligation of these increases intracranial pressure 9 and may contribute to cerebral edema 10 suggesting that healthy cLV is essential for efficient fluid and solute clearance from the brain. However, despite their importance, the hydrodynamic properties of these lymphatic vessels have not been directly evaluated in vivo, and their function declines with aging 7、11、12 and coincides with an increased risk of lymphatic-related CNS diseases 5、13、14 therefore this is of particular importance. There is a need to improve glymphatic-lymphatic outflow.

Summary of the Invention

[0006] The present disclosure addresses the aforementioned need in several aspects.

[0007] In one aspect, the present disclosure provides a method for improving glymphatic-lymphatic outflow from the CNS of a subject. The method includes enhancing the flow rate of the cervical lymphatic vessels (cLV) of the subject.

[0008] In some embodiments, enhancing the flow rate includes increasing the contraction of the cLV. In some embodiments, increasing the contraction of the cLV includes administering an agonist of smooth muscle contraction to the subject. In some embodiments, the agonist is a muscarinic agonist, an α1-adrenergic agonist, K ATPThe present invention includes channel inhibitors, NOS inhibitors, soluble guanylate cyclase (sGC) blockers, PKA inhibitors, cyclooxygenase-1 (COX-1) inhibitors, cyclooxygenase-2 (COX-2) inhibitors, prostanoids, prostaglandin E2 receptor 4 (EP4 receptor) antagonists, prostacyclin (PGI2) receptor (IP receptor) antagonists, or nonsteroidal anti-inflammatory drugs (NSAIDs). In some embodiments, the agonist is selected from the group consisting of carbachol, norepinephrine, propranolol, glibenclamide, NG-nitro-L-arginine (L-NNA), ODQ, H-89, KT5720, indomethacin, ketoprofen, AH23848B, GW627368x, CAY10441, pyridostigmine, amifamplidin, neostigmine, edrophonium, and amvenonium. In some embodiments, the agonist includes a prostanoid. In some embodiments, the prostanoid is prostaglandin F 2α (PGF 2α These are lymphoconstrictive prostanoids such as thromboxane A2 (TxA2), PGD2, leukotriene B4, leukotriene C4, leukotriene D4, or TxA2 mimic U46619.

[0009] In some embodiments, improving glymphatic-lymphatic outflow or flow from the CNS of a subject involves administering a phosphodiesterase type 5a inhibitor to the subject.

[0010] In some embodiments, the agonist or inhibitor is administered to the neck of the subject. In some embodiments, the agonist or inhibitor is administered topically.

[0011] In some embodiments, the method further includes increasing the inflow of cerebrospinal fluid (CSF) through the CNS interstitium and sending a message to the neck of the subject. In some embodiments, increasing the inflow of CSF includes administering to the subject an agent selected from the group consisting of a hypertonic solution, a Stat-3 inhibitor, a bone morphogenetic protein (BMP) signaling axis molecule, an AVP (vasopressin) antagonist, an atrial natriuretic peptide (ANP) antagonist, angiotensin II antagonist, an AT2R receptor antagonist, and an AT1 receptor antagonist. In some embodiments, the hypertonic solution includes NaCl or mannitol.

[0012] Another aspect of this disclosure provides a method for facilitating the clearance of material (e.g., waste products) from the CNS interstitium, brain interstitium, and / or spinal interstitium of a subject. The method comprises improving glymph-lymphatic efflux from the CNS of a subject in the manner described above. In some embodiments, the material comprises a fluid or a solute. In some embodiments, the material comprises amyloid-beta (Aβ), tau, or alpha-synuclein. In some embodiments, the material comprises a drug or its metabolites. Examples of drugs include small molecule compounds (e.g., chemotherapeutic compounds), biologics (e.g., proteins, antibodies, nucleic acids, and vectors), and others (e.g., liposomes, nanoparticles).

[0013] Further aspects of this disclosure provide methods for treating cerebral edema, traumatic brain injury, post-traumatic brain injury, neurological disorders, or neurodegenerative diseases in subjects requiring such treatment. The methods include improving glymph-lymph outflow from the CNS of the subject in the manner described above.

[0014] One aspect of the present disclosure provides a method for treating cerebral edema, traumatic brain injury, post-traumatic brain injury, or neurodegenerative disease in a subject in need thereof. The method comprises administering a phosphodiesterase type 5a inhibitor to the subject. In one embodiment, the inhibitor is selected from the group consisting of sildenafil, tadalafil, vardenafil, and avanafil.

[0015] In some embodiments, the neurodegenerative disease is Parkinson's disease (PD), Alzheimer's disease (AD), Lewy body Alzheimer's disease, Lewy body dementia, mixed dementia, vascular dementia, frontotemporal dementia, chronic traumatic encephalopathy (CTE), HIV-related dementia, Lewy body disease, Huntington's disease, or multiple system atrophy. In some embodiments, the method further includes administering a drug to a subject for the treatment of a neurodegenerative disease. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal or human is an aged mammal or human.

[0016] Details of one or more embodiments of this disclosure are described below. Other features, purposes, and advantages of this disclosure will be apparent from this specification and the claims.

[0017] The patent or application file shall contain at least one drawing made in color. A copy of the published patent or patent application containing the color drawing shall be provided by the Patent Office upon request and payment of the necessary fees. [Brief explanation of the drawing]

[0018] [Figure 1-1]This diagram visualizes CSF transport through superficial cervical lymphatic vessels. a. The cLV of anesthetized mice was surgically exposed, and the flow in the exposed vessel was imaged and the movement of the vessel wall tracked using in vivo two-photon microscopy. Heart rate and respiratory rate were recorded simultaneously. Representative time-series images are shown. The cLV was labeled red with dextran injected into the cheek, while microspheres injected into the CM appear green. (Inset) White arrows indicate the direction of each microsphere and show fluid motion. Scale bar: 50 μm b. The red outline represents the vessel wall detected and tracked using a custom MATLAB algorithm to obtain changes in vessel diameter synchronized with flow measurements. c. The iridescent lines represent the superimposed trajectories of the tracked fluorescent microspheres flowing through the vessel. d. The time-averaged velocity field (green arrow) shows the net transport of lymph. e. The boxes show local time-averaged fluid velocity, which is slowest near the vessel wall and fastest near the center of the vessel. [Figure 1-2] This figure visualizes CSF transport through superficial cervical lymphatic vessels. f. Time-averaged velocity profiles measured upstream, downstream, and at the valve show that the highest central velocity is measured at the valve. Average flow velocity profile plotted as a function of distance from the left wall of the vessel. Colored lines in the inset indicate the location of each profile. Scale bar: 50 μm. g. Reynolds number and h. Womasley number of time-averaged flow, mean ± SEM, n=8 mice [Figure 2-1] This shows endogenous pump dysfunction with age. a. Typical images of local time-averaged flow velocity show that lymphatic flow is almost stagnant with age. Scale bar: 50 μm [Figure 2-2]b. This shows intrinsic pump dysfunction with age. The representative temporal variation of median vascular diameter shows an age-dependent decline in vascular function. Maximum and minimum values ​​(diastolic and systolic peaks) are also shown. c. The representative temporal variation of downstream velocity calculated from particle tracking in different age groups shows that lymphatic flow velocity decreases with age. In (b) and (c), dots represent raw data, and solid lines are obtained by smoothing over a 500-millisecond frame. d. Intrinsic velocity (contraction frequency), e. mean downstream velocity, and f. drainage velocity (frequency of the curve is shown in b) show an age-dependent decline. g. Median vascular diameter does not change with age. One-way ANOVA and Tukey's post-hoc test were performed (d-h). Mean ± SEM, n=5-7 / group. h. Linear regression of intrinsic velocity and drainage velocity in 95% confidence intervals for all age groups shows a strong correlation between cervical lymphatic vessel contraction and lymphatic outflow. [Figure 3] This study demonstrates that lymphatic outflow decreases with age. a. Temporal variation of volumetric flow rate Q, calculated from the product of spatially averaged instantaneous downstream velocity and instantaneous cross-sectional lymphangion area. Assuming lymphangions have a circular cross-section, the cross-sectional area was calculated using the median measured pipe diameter. By definition, Q>0 represents forward flow, and Q<0 represents reverse flow. b. The reverse flow rate increased with age. c. Discharge rate, d. Discharge volume, and e. Particle count decreased with age. One-way ANOVA Tukey's post-hoc test was performed (b-e). Mean ± SEM, n=5-7 / group. f. Representative images of valves (outlined by dotted lines) in young and aged mice. Functional valves open and close to regulate flow in young mice, but not in aged mice. Scale bar: 50 μm [Figure 4-1] This shows that phasic contraction is impaired in aging due to the loss of lymphatic smooth muscle actin. (a) Representative images of cervical lymphatic vessels after labeling with nuclei (DAPI), collagen IV (Col IV) 23, and smooth muscle actin 24, 25. The orthogonal section shows the three-dimensional distribution. Scale bar: 100 μm. [Figure 4-2]This shows that phasic contraction is impaired in aging due to the loss of lymphatic smooth muscle actin. (b~c) Collagen IV and SMA fluorescence vary across the tube direction. Tube width was normalized to allow direct comparison between lymphatic vessels. Thick lines represent average intensity and are shown as shaded areas of the SEM, while thin lines represent individual animals. Average fluorescence was calculated when more than two lymphatic vessels were collected from a single individual. Colored dots represent individual animals. Mann-Whitney (inter-animal, Col IV: aged vs. juvenile, p=0.3411, U=6; SMA: aged vs. juvenile, p=0.0328, U=1). Unpaired Student's t-tests were performed, and the bar graphs show the area under the curve calculated for both proteins as mean ± SEM. Juvenile (n=5), aged (n=4). (d) Representative phase-average dilation and contraction of lymphatic vessels in different age groups. Phase meaning was performed over at least 10 cycles. Representative normalized lymphatic wall velocities for different age groups were calculated by distinguishing the curves in (e) and (d). By definition, a positive velocity means expansion and a negative velocity means contraction. [Figure 5-1] This study demonstrates that PGF2α improves lymphatic function by promoting phasic tubular contraction. Representative images of a. time-averaged speed and b. velocity in young mice (top row) and 22-month-old mice (bottom row) before (left column) and after (right column) administration of PGF2α. c. Variation in tubular diameter indicates that PGF2α stimulates the tubules, promoting intrinsic pulsation in the young and aged groups, with greater increases in intrinsic velocity (e) and contraction amplitude Δd(i) observed in aged mice. d. Variation in downstream velocity indicates that PGF2α-induced stimulation of the tubular wall increased outflow at faster mean downstream velocities (panel (f) and outflow velocity (h)). g. Median tubular diameter indicates that PGF2α administration reduces tubular size. Paired t-tests were performed on e-h. Mean ± SEM, n=5. [Figure 5-2]This study demonstrates that PGF2α improves lymphatic function by promoting phasic tubular contraction. Representative images of a. time-averaged speed and b. velocity in young mice (top row) and 22-month-old mice (bottom row) before (left column) and after (right column) administration of PGF2α. c. Variation in tubular diameter indicates that PGF2α stimulates the tubules, promoting intrinsic pulsation in the young and aged groups, with greater increases in intrinsic velocity (e) and contraction amplitude Δd(i) observed in aged mice. d. Variation in downstream velocity indicates that PGF2α-induced stimulation of the tubular wall increased outflow at faster mean downstream velocities (panel (f) and outflow velocity (h)). g. Median tubular diameter indicates that PGF2α administration reduces tubular size. Paired t-tests were performed on e-h. Mean ± SEM, n=5. [Figure 6-1] This study demonstrates that PGF2α restores lymphatic flow and reverses the effects of aging on CSF excretion. (a) Temporal changes in volumetric flow rate (Q) before and after PGF2α administration in young and elderly groups. [Figure 6-2] This study demonstrates that PGF2α restores lymphatic flow and reverses the effects of aging on CSF efflux. b. The reversal rate decreased with PGF2α in both the younger and older age groups. c. Efflux rate, d. efflux volume, and e. particle count were restored with PGF2α at the age-related stage. Paired t-tests were performed for b-e. Mean ± SEM, n=5. [Figure 6-3] This study demonstrates that PGF2α restores lymphatic flow and reverses the effects of aging on CSF efflux. (f) Representative images of OVA-647 signal detected in cervical lymphatic vessels (cLV) before and after PGF2α in young and aged mice. [Figure 6-4] This shows that PGF2α restores lymphatic flow and reverses the effects of aging on CSF efflux. (g) Gradual increase in mean fluorescence intensity measured over 60 minutes, indicating increased efflux after PGF2α. f Quantification of mean pixel intensity (MPI) of the 60-minute in vivo imaging series. Two-way ANOVA with Tukey's multiple comparison test, n=6-7 mice / group. Scale bar: 2mm. [Figure 7]This shows that cervical lymphatic outflow is not regulated by cardiac and respiratory force. (a-d) Representative phase-averaged waveforms of tubal constriction (blue curve) and downstream velocity (orange) compared with changes in measured cardiac signals (red curve) and respiratory signals (green curve). (e-f) Linear regression of intrinsic velocity or efflux velocity with a 95% confidence interval did not reveal any correlation with heart rate or respiratory rate. [Figure 8] This study demonstrates that heart rate and respiratory rate are not affected by PGF2α. The results show a. heart rate and b. respiratory rate before and after PGF2α in young and aged mice. A paired t-test was performed. Mean ± SEM, n=5. [Figure 9] This shows that PGF2α maintains lymphatic drainage to the cervical lymph nodes. (a) Representative images of ex vivo lymph nodes before and after PGF2α administration in young and aged mouse groups. Scale bar = 2 mm. Mean fluorescence pixel intensity (MPI) normalized by the area of ​​lymph nodes SLN (b) and dLN (c). One-way ANOVA with Tukey's post-hoc test, n=4 / group. Bar graphs represent mean ± SEM. [Figure 10-1] This study demonstrates that aging causes loss of smooth muscle actin (α-SMA) coverage in scLV. (a) Representative images of aggregate scLV from 2-month-old and (b) 18-month-old Prox1-GFP animals stained for α-smooth muscle actin (αSMA) and collagen 4a (Col4a). (c) Lymphoid endothelial cells (LECs) within the lymphatic valve along the LV can be visualized with Prox1-GFP. (d) Smooth muscle cells (SMCs) are αSMA-positive, and (e) Col4a is a marker of the collagen basement membrane. (f) The region of interest (ROI) was divided into valve ROI and perivalvular ROI. Quantification of the percentage of area covered by Prox1 in the valve (g) and perivalvular region (h). Area covered by aSMA (i, j) and Col4a (k, l). [Figure 10-2]This study demonstrates that aging leads to loss of smooth muscle actin (a-SMA) coating in scLV. (m)LEC was quantified as DAPI+ and Prox1+. (n)Total DAPI+ cell count and (o)Total DAPI+ / Prox1+ cell count did not decrease in 18-month-old animals compared to young animals. (p)SMC was considered as DAPI+ / aSMA+ and was similarly abundant in young and aged mice (q). (r)Orientation of aSMA and Col4a fibers. (s)Fiber orientation in degrees of aSMA+ and Col4a+ fibers in valve ROI and perivalvular ROI. Orientation was calculated as axial (0°) or longitudinal (+90° or -90°). (t)Coherence of fiber orientation as a percentage of fibers in valve ROI and perivalvular ROI. Scale bar: 100 μm. [Figure 11-1] This study demonstrates that PGF2α reverses the effects of aging on CSF efflux and increases 780 brain clearance. (a) CSF clearance was evaluated by intracisrural injection of ovalbumin conjugated with Alexa 647 (OVA-Alexa 647), and CSF efflux was detected in the cervical lymphatic vessels (cLV) in both young and aged mice, regardless of the presence or absence of PGF2α. [Figure 11-2] (b) This shows that PGF2α reverses the effects of aging on CSF efflux and increases 780 brain clearance. (b) Progressive increase in mean fluorescence intensity measured over 60 minutes, showing increased efflux after PGF2α. Scale bar: 2 mm. (c) Quantification of mean pixel intensity (MPI) over 60 minutes of in vivo imaging series shown in b. Two-way ANOVA with Tukey's multiple comparison test, n=6-7 mice / group. Interstitial fluid (ISF) clearance from brain parenchyma was assessed by injecting Direct Blue 53 (DB53) into the striatum. DB53 is removed from the brain into plasma, where it binds to circulating albumin and is retained for over 2 hours, allowing for stable quantification of brain clearance into plasma. [Figure 11-3](d) Coronal section from anterior (A) to posterior (P), showing brain retention of DB53 after intrastriatal injection. Scale bar: 2 mm. (e) Quantification of brain retention expressed as % area of ​​DB53 fluorescence. Unpaired Student's t-test was performed. Mean ± SEM, n=6-7 / group. (f) Representative imaging of in vivo femoral vein image acquired after injection of 1 μl of 4% DB53 into the striatum. Scale bar: 1 mm. (g) Quantification of plasma clearance measured in the femoral vein. Two-way ANOVA using Sidac's multiple comparison test, n=6-7 mice / group. [Modes for carrying out the invention]

[0019] This disclosure relates to increased glymph / lympholluteolytic outflow from the brain or CNS. Certain aspects of this disclosure relate to the restoration of cervical lymphatic function. Such restoration in aging can relieve cerebrospinal fluid drainage.

[0020] Cervical lymphatic vessels, aging, and cerebrospinal fluid drainage Cervical lymphatic vessels (cLVs) have been shown to drain solutes and cerebrospinal fluid (CSF) from the brain. However, their hydrodynamic properties have not been previously evaluated in vivo. Certain aspects of this disclosure are based, at least in part, on the unexpected finding that the primary driving factor of cLV flow is intrinsic pumping due to contraction of the lymphatic wall. As disclosed herein, two-photon optical imaging using in vivo particle tracking of a CSF tracer (2P-OPTIC) in mouse cLVs is the first method developed to characterize their flow, and it has been identified that the primary driving factor is intrinsic pumping due to contraction of the lymphatic wall.

[0021] Furthermore, as disclosed herein, contraction frequency and flow rate decreased in aged mice, which was consistent with a decrease in smooth muscle actin. Slow flow in aged mice is associated with prostaglandin F, a prostanoid that increases smooth muscle contractility. 2α (PGF2α Topical application of smooth muscle cell contractile agonists such as ) can be used to provide relief, thereby restoring lymphatic function and increasing net flow back to youthful levels. Since cLVs are important regulators of CSF efflux, restoring their function has been shown to be an effective treatment for improving age-related CSF clearance.

[0022] Collective lymphatic vessels (LVs) in the abdomen and limbs drive flow through two mechanisms: active (endogenous) pumping and passive (exogenous) pumping. Active pumping is generated by the rapid, phasic contraction of smooth muscle cells surrounding the lymphandions, the functional units of the LVs. The lymphandions are separated by unidirectional valves, and their synchronous contractions drain the lymph by restricting backflow and ensuring unidirectional flow. Passive pumping relies on external compression of the LVs by the contraction of surrounding skeletal muscles. Respiratory and cardiac cycles have also been proposed to drive lymph. 15 However, to date, nothing is known about the transport mechanism of cLV. Prior techniques involve quantifying the velocity of labeled tracers, which provides transport flow rates over time but does not provide insight into the spatial characteristics of the flow. Ultrasound has been used to measure velocity and lymphatic contractility, but it cannot resolve different tracers flowing within the lymph. Optical techniques use relative changes in signal intensity of fluorescent dyes (indocyanine green or fluorescent conjugate dextran) to measure lymphandion contraction, but they cannot calculate velocity. In vitro optical imaging of particles flowing within LV in artificially pressurized, isolated lymphatic vessels is proving to be a groundbreaking approach. 16 17 .

[0023] To overcome these limitations and obtain quantitative in vivo velocity data, we developed two-photon optical imaging (2P-OPTIC) using in vivo particle tracking of CSF tracers. This enables high spatiotemporal resolution of flow velocity within a single lymphandion in vivo and allows for simultaneous quantification of LV contractility and CSF-derived solute transport using a dual tracer approach. As disclosed herein, cLV contraction is the primary driving force of fluid flow, and these decline with age, coinciding with the partial loss of lymphoid smooth muscle cells. Furthermore, the age-dependent decline in cLV function is linked to the smooth muscle contractile prostaglandin F 2α (PGF 2α It has been proven that they can be saved by ) 18、19 Therefore, age-induced CLV dysfunction contributes to the development of neurodegenerative and neuroimmunological diseases. 7 8 This may contribute to a blunted response to CNS immunotherapy, which provides opportunities for recovery from acute injury and therapeutic intervention.

[0024] As disclosed herein, we introduced 2P-OPTIC, a novel method for quantifying LV fluid dynamics. These represent the first hydrodynamic measurements of cLVs in vivo in young and aged mice. The cLVs were found to have a diameter of approximately 70 μm and contain a flow velocity of approximately 150 μm / s. The main driving factor of the viscosity-dominant flow within these lymphatic vessels is the intrinsic contraction of lymphandions, and aging impairs cLV function by reducing intrinsic contraction. As with other lymphatic networks, we found a decrease in SMCs and their main contractile protein, smooth muscle actin. In addition, it was found that the dysfunctional valves of aging cLVs are unable to properly regulate unidirectional transport of slower flows, resulting in significant retrograde flow that exacerbates inefficiency in aging cLVs. Furthermore, as disclosed herein, PGF is a compound known to induce contractility of SMCs. 2α Use 19 28This restores cLV pumping by increasing intrinsic contractions without altering heart rate and respiratory rate. This disclosure shows that optimizing cLV function leads to increased CSF efflux in juvenile mice and ultimately restores CSF clearance in aged mice. Rescue of cLV function was validated by both restoration of lymphatic hydrodynamic transport and increased tracer accumulation within cervical lymph nodes.

[0025] 2P-OPTIC provides quantitative, real-time in vivo measurement of CSF emissions via cLV. Previous studies relied on ultrasound-based measurements. 29、30 By optically tracking the lymphocytes in motion 22、31、32 or using non-invasive optical coherence tomography (OCT) 33、34 While lymphatic flow has been measured in vivo, none of these studies have evaluated cLV transport. Several studies have used high-resolution stereomicroscopy to measure cLV dynamics to evaluate the perineural outflow pathway and lymphatic drainage of CSF. 5 35 However, while these methods (as seen in Figure 6f) enable high-volume imaging and robust tracer quantification, they do not provide quantitative information on lymphatic flow dynamics. Two-photon microscopy enables in vivo high spatiotemporal resolution imaging, and optical sectioning allows for accurate particle tracking velocity calculations. The dual-tracer approach disclosed herein has enabled labeling of cLV to track contractile dynamics and CSF to obtain lymphatic flow measurement results with unprecedented detail. The drawbacks of this technique are the need to inject exogenous tracers and surgical exposure of superficial cervical LV, but these can be optimized by labeling of endogenous components of the lymph (i.e., cells or expressed proteins) and transcutaneous two-photon imaging. Although 2P-OPTIC was developed for cLV, it can also be used in other lymphatic networks.

[0026] Unlike the cardiovascular circulation, the lymphatic network lacks a central pump to propel fluid. Therefore, lymph is transported by intrinsic contractions of lymphandions and by extrinsic contractions from surrounding skeletal muscles, respiration, and heartbeats. 15 Regarding the flow of cLV (both scLV and dcLV), the inventors observed a flow approximately 25% lower than the average Re measured in rat mesenteric LV, but a typical average Re (greater than 0.006) in small lymphatic vessels (less than 100 μm). 36 The flow velocity during contraction increases by almost two orders of magnitude (approximately 1500 μm / sec, instantaneous Re approximately 0.6), which is comparable to that of mesenteric lymphatic vessels. 37 Even at higher flow velocities, the flow remains laminar and viscosity-dominated, with little role for inertial effects. The Wo of cLV is approximately 0.03, indicating very little transient inertial effect, an order of magnitude smaller than the Wo of mesenteric lymphatic vessels (0.1), and much smaller than the Wo (Wo=1.4) observed in the human thoracic duct where inertial effects are significant. 38 Interestingly, Re and Wo measured in cLV are comparable to those observed in small arteries and veins of similar size, but also to those observed in the perivascular spaces of the glymphatic system. 39 The cervical LV transported approximately one-third (approximately 70 nL / min) of the flow in mesenteric lymphatic vessels of similar diameter (91 μm, approximately 230 nL / min). 37 The flow is pulsating, and the intrinsic contraction velocity is approximately 0.2 Hz ≈ 12 min -1 It vibrated at a similar frequency (excretion rate) as the mesentery aggregate LV. 40 However, abdominal LV (flank, 6 min -1 It was faster than ).

[0027] The results disclosed herein do not show the influence of extrinsic sources (i.e., cardiac and respiratory cycles) because the frequency of the dominant flow is an order of magnitude smaller than either the heart rate or respiratory rate (2–3 Hz). This finding is intuitive because scLVs are located close to the skin and are relatively unconstrained by cervical muscle tissue, which could otherwise cause extrinsic pumping. Extrinsic pumping may be more significant to the flow in dcLVs because they are surrounded by several muscle groups in the neck. Nevertheless, like venous drainage, cLVs may differ slightly from limb LVs, given their location relative to the heart. This difference is likely to be more pronounced in bipedal animals, but venous drainage in the head and neck of rodents relies on gravity for venous return, compared to the periphery where blood requires negative pressure from the cardiac cycle and extrinsic contraction from adjacent muscle groups to return fluid towards the heart against gravity. This feature and the data disclosed herein suggest that cLVs rely on intrinsic rather than extrinsic contraction to drive lymphatic flow.

[0028] Aging brings about morphological changes (e.g., decreased lymphatic capillary density) and functional changes (e.g., decreased transport capacity of collecting LVs) in the lymphatic system (LV), and it has been shown that all lymphatic networks studied to date, including the thoracic vascular LV, cutaneous LV, meningeal LV, and mesenteric LV, are impaired with age. 41 The underlying mechanisms for this decline are related to decreased production of lymphangiogenic factors and reduced regenerative capacity of lymphoid endothelial cells. Aging also leads to a decrease in smooth muscle actin filament coverage, both of which surround membrane composition, causing increased permeability and decreased contractility. This study shows that cervical lymphoid vessels are suffering from the same aging smooth muscle actin-related changes seen in other collecting lymphatic vessels in the rest of the body, but this is an important observation because these collecting lymphatic chains affect CNS function.

[0029] As disclosed herein, cLV is a downstream promoter of CSF efflux and is a key component of the proposed glymphatic-lymphatic system. The glymphatic system transports intracranial CSF through the perivascular space around the arteries to the extracellular space of the brain, where it mixes with interstitial fluid, collects metabolic waste products, and is then effluxed along the perivenous space. 46 47 The fluid is then collected by the meningeal lymphatic vessels or exits along the cranial and spinal nerves, where it is taken up by the extracranial lumbar collection (LV) and discharged into the cLN via the cLV. 48 49 50 5 The organization of the gliolymphatic pathway means that cLV is important for CNS clearance. 49 This is highlighted by the fact that knocking out Prox-1 (which thereby causes mispatterned leaky LV) and / or surgical ligation of cLV reduces the entry of CSF into the glymphatic pathway and ultimately slows CSF clearance. 7 This indicates that a decrease in downstream (lymphatic) flow leads to a deceleration of upstream (glymphatic) flow due to the conservation of mass, and that ligation increases intracranial pressure. 9 , causes edema 10 This is further supported by the fact that it is possible. A similar effect was reproduced by optically resecting the meningeal lymphatic vessels, which are further upstream components of the pathway. 7 While this disclosure demonstrates that aging dramatically affects cLV, there is also evidence that aging causes meningeal LV regression and plasticity changes, further contributing to delayed CSF efflux. 7 11 In addition to lymphatic changes, aging is also associated with intracranial effects such as decreased CSF production in the choroid plexus 62 and decreased CSF outflow from the cranial foramen. Nevertheless, this disclosure shows that restoring cLV function alone is sufficient to alleviate these age-related effects and provides a novel therapeutic strategy.

[0030] Therapeutic potential for re-establishing CSF clearance has been proposed for Alzheimer's disease, Parkinson's disease, ischemic stroke, and traumatic brain injury. 51In aged mice, meningeal lymphatic drainage (LV) is also impaired, but this effect can be remedied by treatment with the lymphotrophic factor vascular endothelial growth factor-C (VEGF-C), leading to improved lymphatic flow and cognition in older mice. 7 This interaction is mediated by cLV ​​because their surgical ligation negated the beneficial effects. 7 In a mouse model of Alzheimer's disease, meningeal LV excision increases amyloid-beta loading and promotes its deposition. 7 On the other hand, meningeal lymphatic vessel-deficient mice (K14-C-VEGFR-3-Ig gene-transformed mice) also possess larger amounts of tau, such as amyloid-beta (whose presence is correlated with Alzheimer's disease). 52 Aged animals also show a 40% decrease in the clearance of radiolabeled amyloid-beta injected intraparenchymally. Similarly, in a mouse model of Parkinson's disease, ligation of cLV restricted lymphatic outflow and worsened α-synuclein aggregation. 53 This is because mice with meningeal LV dysfunction showed increased infarct volume after transient middle cerebral artery occlusion, potentially leading to acute ischemic stroke. 54 In addition to its effects on disease progression, cLV function also modulates the effectiveness of existing therapies. For example, in mouse models of intracranial glioma and metastatic melanoma, meningeal LV undergo extensive remodeling, suppressing the CD8+ response and leading to reduced dendritic cell transport to cLNs, which diminishes the effectiveness of anti-PD-1 / CTLA-4 checkpoint therapy. 55 56 Restoring LV function using VEGF-C improves the response to immune checkpoint inhibitors targeting anti-amyloid antibodies used to treat CNS tumors and Alzheimer's disease. 55 56 57 .

[0031] This disclosure states that cLV is prostaglandin F 2α PGF is a naturally occurring prostanoid that binds to receptors. 2α This study demonstrates that topical application of (dinoprost) can rescue both ISF and CSF efflux in aged mice. 2αAn analogue of this drug (carboprost) is already used clinically to induce uterine contractions and terminate postpartum hemorrhage. Carboprost is administered by intramuscular injection, but it can be adapted for transdermal administration to the cervical region, which is relatively less invasive compared to techniques targeting the meningeal LV that require VEGF-C gene therapy to the skull or hydrogel-encapsulated VEGF-C application (some of which are already in clinical trials).

[0032] To reiterate, we developed two-photon optical imaging (2P-OPTIC) using in vivo particle tracking of CSF tracers and found that cervical lymphatic pumping is impaired with age. Aging leads to a decrease in the frequency and amplitude of lymphatic contractions due to SMC loss, resulting in slower flow velocities and lower discharge. Furthermore, aged lymphatic valves are unable to open and close properly, which explains the increase in retrograde flow. Since the cLV is the convergence point of multiple CSF outflow pathways, the results disclosed herein provide evidence that improving cLV function can restore CSF efflux in aged animals and provide a therapeutic platform for treating a wide range of neurological disorders.

[0033] Accordingly, one aspect of the present disclosure provides a method for improving glymph-lymph outflow from a target CNS by, in particular, increasing the flow rate of the target cLV.

[0034] In some embodiments, increasing the flow rate includes increasing the contraction of the cLV. In some embodiments, increasing the contraction of the cLV includes administering a smooth muscle contraction agonist to the subject. In some embodiments, the agonist includes a cholinergic muscle stimulant. In some embodiments, the cholinergic muscle stimulant is selected from the group consisting of pyridostigmine, amifamplidin, neostigmine, edrophonium, and amvenonium. In some embodiments, the agonist includes a prostanoid. In some embodiments, the prostanoid is prostaglandin F 2α (PGF 2α ) includes.

[0035] In some embodiments, improving the glymphatic-lymphatic outflow or flow rate of cLV from the target CNS involves administering a phosphodiesterase 5a inhibitor (PDE5 inhibitor) to the subject.

[0036] In some embodiments, the agonist or inhibitor is administered to the neck of the subject. In some embodiments, the agonist or inhibitor is administered topically.

[0037] In some embodiments, the method further includes increasing the influx of CSF through the CNS interstitium and sending a message to the neck of the subject. In some embodiments, increasing CSF influx includes administering to the subject an agent selected from the group consisting of a hypertonic solution, a Stat-3 inhibitor, a bone morphogenetic protein (BMP) signaling axis molecule, an AVP (vasopressin) antagonist, an atrial natriuretic peptide (ANP) antagonist, angiotensin II antagonist, an AT2R receptor antagonist, and an AT1 receptor antagonist. In some embodiments, the hypertonic solution comprises NaCl or mannitol.

[0038] Another aspect of this disclosure provides, among other things, a method for facilitating the clearance of waste products from the CNS interstitium, brain interstitium, and / or spinal interstitium of a subject by improving gymphatic-lymphatic efflux from the CNS of the subject in the manner described above. In some embodiments, the waste products include amyloid-beta (Aβ), tau, or alpha-synuclein. In some embodiments, the waste products include drugs or their metabolites. Examples of drugs include small molecule compounds (e.g., chemotherapeutic compounds), biologics (e.g., proteins, antibodies, nucleic acids, and vectors), and others (e.g., liposomes, nanoparticles).

[0039] Further aspects of the present disclosure provide, among other things, methods for treating cerebral edema, traumatic brain injury, post-traumatic brain injury, or neurodegenerative disease in a subject by improving glymph-lymph outflow from the subject's CNS in the manner described above. In some embodiments, the neurodegenerative disease is Parkinson's disease (PD), Alzheimer's disease (AD), Lewy body Alzheimer's disease, Lewy body dementia, mixed dementia, vascular dementia, frontotemporal dementia, chronic traumatic encephalopathy (CTE), HIV-related dementia, Lewy body disease, Huntington's disease, or multiple system atrophy.

[0040] In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal or human is an elderly mammal or human.

[0041] Agonist of smooth muscle contraction Certain aspects of this disclosure provide a method for improving glymph-lymph outflow from the CNS of a subject. The method includes enhancing the flow rate of the cLV of the subject by increasing the contraction of the cLV. In some embodiments, increasing the contraction of the cLV includes administering a smooth muscle contraction agonist to the subject.

[0042] As used herein, “smooth muscle contraction agonist” refers to any agent that stimulates the contraction of smooth muscle, such as the smooth muscle of the clLV wall. Examples of such agonists include mamuscarinic agonists (e.g., carbachol), α1-adrenergic agonists (e.g., noradrenaline and propranolol), and K ATPChannel inhibitors (e.g., glibenclamide), NOS inhibitors (e.g., NG-nitro-L-arginine (L-NNA)), sGC blockers (e.g., ODQ), PKA inhibitors (e.g., H-89 and KT5720), COX-1 or COX-2 inhibitors (e.g., indomethacin), anti-inflammatory drugs (e.g., NSAIDs (e.g., ketoprofen)), prostanoids (e.g., thromboxane A2 (TxA2) and PGF2α, PGD2, leukotriene B4) Examples include C4, and D4, thromboxane A2 (TXA2) mimic U46619, EP4 receptor antagonists (e.g., AH23848B and GW627368x), IP receptor antagonists (e.g., CAY10441), and cholinergic muscle stimulants (e.g., pyridostigmine, amifamplidin, neostigmine, edrophonium, and ambenonium), as well as their derivatives and / or pharmaceutically acceptable salts thereof.

[0043] Prostanoids are a family of lipid mediators produced by the action of cyclooxygenase on arachidonic acid, a 20-carbon unsaturated fatty acid. Prostanoids include a family of lipid mediators formed from arachidonic acid (AA) via the prostaglandin (PG)H synthase or cyclooxygenase (COX) pathway. These include PGE2, PGD2, and PGF. 2α This includes prostacyclin (PGI2) and thromboxane A2 (TxA2). Prostanoids such as dinoprost and carboprost are known to modulate biological processes such as smooth muscle tone, vascular permeability, pain, fever, and platelet aggregation. Lymphoconstriction-promoting prostanoids (i.e., those that promote or induce lymphoconstriction, pumping, or transport) are useful in the methods described herein.

[0044] Commercial therapeutic agents of pyridostigmine, amifamplidin, neostigmine, edrophonium, and amvenonium are known in the art. Examples include the following: [Table 1]

[0045] As used herein, the terms “derivative,” “variant,” and “analog” are used interchangeably to refer to compounds having a structure derived from the structure of a parent compound (e.g., a compound disclosed herein, e.g., a prostanoid), whose structure is sufficiently similar to that of the compound disclosed herein, and based on that similarity, a person skilled in the art would expect that it will exhibit the same or similar activity and utility as the compound herein, or, as a precursor, induce the same or similar activity and utility as the compound herein. A derivative or analog may be a prodrug, ester, salt, or metabolite of the compound herein.

[0046] Inhibitors of phosphodiesterase type 5a Certain aspects of the present disclosure provide (i) a method for improving glymph-lymph outflow from the CNS of a subject, or / and (ii) a method for enhancing the flow rate of the cLV of a subject by improving relaxation of the cLV smooth muscle by inhibiting phosphodiesterase type 5a. For example, the method may include administering a phosphodiesterase type 5a inhibitor or a PDE5 inhibitor to the subject.

[0047] The terms "phosphodiesterase inhibitor" or "PDE inhibitor" refer to compounds and their derivatives or analogues (e.g., salts or solvates) that function by inhibiting the activity of the enzyme phosphodiesterase. An exemplary phosphodiesterase is phosphodiesterase type 5 (PDE5). PDE inhibitors can be compounds that reduce PDE activity in vivo and / or in vitro. PDE5 inhibitors increase cGMP levels by inhibiting the degrading action of PDE5 on cGMP.

[0048] Representative PDE5 inhibitors include cyclic guanosine 3',5'-monophosphate type 5 cGMP PDE inhibitors, also known as PDE5 inhibitors. Examples of PDE5 inhibitors include sildenafil, tadalafil, vardenafil, avanafil, rodenafil, udenafil, mirodenafil, zaprinast, P20066 (Ethypharm), SLx-2101 (Kadmon Pharmaceuticals), PF00489791 (Pfizer), INT007 (IntelGenx Technologies), and dasantafil. Additional exemplary PDE5 inhibitors are described in U.S. Patents 11,851,427, 9,387,210, 5,250,534, 5,859,006, 6,362,178, and 7,378,430, and International Patent Publications 2008 / 095835, 2009 / 050554, 2009 / 124119, 2010 / 015589, 2010 / 074783, and 2011 / 015523, each of which is incorporated herein by reference in its entirety.

[0049] Suitable effective dosages may include, but are not limited to, approximately 0.0001 mg / kg to 100 mg / kg, 0.01 to 80 mg / kg, 0.01 to 50 mg / kg, and 0.01 to 5 mg / kg (e.g., 0.001 mg / kg, 0.002 mg / kg, 0.005 mg / kg, 0.01 mg / kg, 0.02 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 5.0 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, and 25 mg / kg) administered daily or as needed. The target population can be administered therapeutic compositions containing the compound / drug in the following dose ranges: sildenafil (25-100 mg / day), tadalafil (2.5-20 mg / day or every other day), vardenafil (5-20 mg / day), or avanafil (50-200 mg / day).

[0050] Composition and Use Various compositions are available for use or therapy as described herein, for example, transdermal formulations such as patches or injections, intramuscular injections, implants, oral tablets, subcutaneous formulations, intranasal formulations, buccal formulations, topical gels and solutions, or topical patches. In some embodiments, the compositions may be in the form of solid medication formulations (e.g., tablets, capsules, granules, powders, sachets, or chewable preparations), solutions, gels, suspensions, emulsions, shampoos, conditioners, creams, foams, gels, lotions, ointments, transdermal patches, films, tinctures, or pastes. Furthermore, methods and uses of the compositions described herein for treating diseases, preventing diseases, treating conditions, and / or preventing conditions are provided herein.

[0051] Formulations of compounds, their derivatives, analogs, or salts may provide a dose sufficient to improve glymph-lymphatic efflux from the central nervous system (CNS). The pharmaceutically effective amount of compounds, their derivatives, analogs, or salts present in the compositions disclosed herein may depend on the patient's condition and mode of administration.

[0052] A pharmaceutical composition containing any of the compounds described herein, or a derivative, analogue, or salt thereof, may further comprise a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition may include, but is not limited to, MESTINON, MESTINON TIMESPAN, REGONOL, RUZURGI, FIRDAPSE, PROSTIGMIN BROMIDE, PROSTIGMI, BLOXIVERZ, REVERSOL, and ENLON, and may be formulated (e.g., using the same proportions and the same excipients, and / or containing the same dose strength) or administered in the same manner as commercially available drug, prodrug, and derivative products. The FDA approval labels for each of these products, including those relating to their formulation, dosage, and administration, are available on the FDA website.

[0053] The above-mentioned compounds and agents and related compositions are useful for (i) improving glymph-lymph outflow from the CNS of a subject, (ii) promoting the clearance of waste products from the CNS interstitium, brain interstitium, and / or spinal interstitium of a subject, and (iii) treating cerebral edema, traumatic brain injury, or neurodegenerative disease in a subject.

[0054] In general, the compounds, drugs, or compositions herein can be administered in therapeutically effective doses by any of the permitted modes of administration. The preferred dosage range depends on a number of factors, including the severity of the disease or condition to be treated, the age and relative health status of the subject, the potency of the compound used, the route and form of administration, the indication for which the administration is intended, and the preferences and experience of the physician involved. A person skilled in the art in treating such diseases or conditions will be able to determine the therapeutically effective dose of the compounds disclosed herein for a given disease or condition, without excessive experimentation, relying on their personal knowledge and the disclosures of this application. Accordingly, the compounds or compositions disclosed herein can be administered as pharmaceutical formulations, including those suitable for topical, oral (including buccal and sublingual), nasal, pulmonary, or parenteral (including intramuscular, intra-arterial, intrathecal, subcutaneous, and intravenous) administration, or in forms suitable for inhalation or inhalation.

[0055] The pharmaceutical compositions described herein can be formulated to suit their intended route of administration. Examples of routes of administration include parenteral (e.g., intravenous, intradermal, or subcutaneous), oral (e.g., inhalation), transdermal (topical), and transmucosal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may contain the following components: sterile diluents such as water for injection, saline solution, fixative oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetic acid, citrate, or phosphoric acid; and agents for adjusting tonicity such as sodium chloride or dextrose. pH can be adjusted using acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be sealed in ampoules, disposable syringes, or glass or plastic multi-dose vials.

[0056] Suitable pharmaceutical compositions for injection include sterile aqueous solutions (if water-soluble) or dispersions, and sterile powders for the immediate preparation of sterile injection solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid enough to allow for easy injection. It must be stable under manufacturing and storage conditions and protected from contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Adequate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it is preferable to include isotonic agents, such as sugars and polyhydric alcohols, such as mannitol, sorbitol, and sodium chloride, in the composition. Sustained absorption of the injectable composition can be achieved by including absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition.

[0057] Sterile injectable solutions can be prepared by incorporating the required amount of active compound or drug, along with one or a combination thereof from the components listed above, into a suitable solvent, and then, if necessary, by sterilizing by filtration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other necessary components of those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, typical preparation methods include vacuum drying and freeze-drying, which yield powders of the active ingredient and any additional desired ingredients from a pre-sterilized filtered solution.

[0058] Oral compositions may generally contain inert diluents or food carriers. For therapeutic oral administration purposes, the active compound or agent may be incorporated with excipients and used in the form of tablets, lozenges, or capsules, such as gelatin capsules. Oral compositions may also be prepared using fluid carriers. Pharmaceutically compatible binders and / or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges, etc., may contain any of the following components: binders such as microcrystalline cellulose, gum tragacanth, or gelatin; excipients such as starch or lactose; disintegrants such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or Sterotes; flow enhancers such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or flavorings such as peppermint, methyl salicylate, or orange flavoring, or compounds having similar properties.

[0059] When administered by inhalation, the active agent or compound can be delivered in the form of an aerosol spray from a pressurized container or dispenser containing a suitable propellant, such as a gas like carbon dioxide or a nebulizer. Such methods include those described in U.S. Patent No. 6,468,798.

[0060] Systemic administration of compounds or drugs may also be carried out by mucosal or dermal means. In the case of mucosal or dermal administration, a penetrating agent suitable for penetration into the barrier is used in the formulation. Such penetrating agents are generally known in the art and, for example, in mucosal administration, include detergents, bile salts, and fusidic acid derivatives. Mucosal administration can be achieved by the use of nasal sprays or suppositories. In the case of dermal administration, the active compound can be formulated into ointments, plasters, gels, or creams, as is generally known in the art.

[0061] Local use In certain embodiments, the mode of administration is topical or transdermal, using a simple daily medication regimen that can be adjusted according to the degree of pain. As used herein, the term “topical” refers to the administration of the composition to the skin and underlying tissues, as well as to the mucous membranes and underlying tissues.

[0062] Specifically, topical and / or transdermal treatments using the compounds, agents, or compositions disclosed herein are preferred for local control of a condition or pathology in a subject, while simultaneously ensuring that undesirable side effects are minimized and suppressed. To achieve this objective, the pharmaceutical compositions of this disclosure may be suitable for topical administration. In this case, the pharmaceutical composition comprises one or more compounds or agents described herein, a pharmaceutically acceptable topical carrier, and optionally a penetration enhancer. In some embodiments, the penetration enhancer may include a base. The base may be present in a concentration sufficient to provide a formulation pH in the range of approximately 7.5 to 13.0. The pharmaceutical composition may be aqueous. The aqueous pharmaceutical composition may be in the form of a cream, gel, lotion, paste, or solution.

[0063] Various skin penetration enhancers are known in the art and can be used in the present invention. Examples of suitable accelerators include ethers such as diethylene glycol monoethyl ether (commercially available as TRANSCUTOL) and diethylene glycol monomethyl ether; surfactants such as sodium laurate, sodium lauryl sulfate, cetyltrimethylammonium bromide, benzalkonium chloride, poloxamer (231, 182, 184), Tween (20, 40, 60, 80), and lecithin (U.S. Patent No. 4,783,450); alcohols such as ethanol, propanol, octanol, and benzyl alcohol; polyethylene glycols and their esters such as polyethylene glycol monolaurate (PEGML; see, for example, U.S. Patent No. 4,568,343); amides and other nitrogenous compounds such as urea, dimethylacetamide (DMA), dimethylformamide (DMF), 2-pyrrolidone, 1-methyl-2-pyrrolidone, ethanolamine, diethanolamine, and triethanolamine; terpenes; alkanones; and organic acids, specifically citric acid and succinic acid. AZONE (registered trademark), DMSO and C 10 Sulfoxides such as MSO can also be used.

[0064] Other suitable accelerators include lipophilic co-accelerators, typically called “plasticizing” accelerators, i.e., accelerators having a molecular weight in the range of about 150 to 1000, a water solubility of about 1% by weight, preferably less than about 0.5% by weight, and most preferably less than about 0.2% by weight. The Hildebrand solubility parameter of plasticizing accelerators is in the range of about 2.5 to about 10, preferably in the range of about 5 to about 10. Such accelerators are described, for example, in U.S. Patent No. 6,586,000 and WO01 / 43775. Preferred lipophilic accelerators are fatty esters, fatty alcohols, and fatty ethers. Examples of particular most preferred fatty acid esters include methyl laurate, ethyl oleate, propylene glycol monolaurate, propyleneglycerol dilaurate, glycerol monolaurate, glycerol monooleate, isopropyl n-decanoate, and octyldodecyl myristate. Examples of fatty alcohols include stearyl alcohol and oleyl alcohol, and examples of fatty ethers include diols or triols, preferably C2-C4 alkanediols or triols substituted with one or two fatty ether substituents. Additional penetration enhancers are known in the field of topical drug delivery. See, for example, Percutaneous Penetration Enhancers, Smith et al., editors (CRC Press, 1995).

[0065] The formulations described herein may be any form suitable for topical application to the skin, for example, the skin on the neck and surrounding tissues. These may include, for example, creams, lotions, solutions, gels, ointments, pastes, plasters, paints, bioadhesives, etc., and / or may be prepared to include liposomes, micelles, and / or microspheres. Such formulations may be aqueous, i.e., may contain water, or may be non-aqueous, and may optionally be used in combination with an occlusive overlayer so that moisture evaporating from the body surface is maintained within the formulation during and after application to the body surface.

[0066] The formulations of the present invention may optionally contain a pharmaceutically acceptable viscosity enhancer and / or film-forming agent. Viscosity enhancers increase the viscosity of the formulation, thereby preventing it from spreading beyond the application site. Balsam Fir (Oregon) is an example of a pharmaceutically acceptable viscosity enhancer. Film-forming agents, upon drying, form a protective film on the application site. This film inhibits the removal of the active ingredient and maintains contact with the treated site. An example of a film-forming agent suitable for use in the present invention is Flexible Collodion USP. As described in p. 1530 of Remington, The Science and Practice of Pharmacy, 19th Ed. (Easton, Pa.: Mack Publishing Co., 1995), collodion is an ethyl ether / ethanol solution containing pyroxylin (nitrocellulose) that evaporates to leave a pyroxylin film. Film-forming agents may also function as a carrier. Solutions that dry to form a film are sometimes called paints.

[0067] As is well known in the field of pharmaceutical formulations, ointments are typically semi-solid formulations based on petrolatum or other petroleum derivatives. As will be understood by those skilled in the art, the specific ointment base to be used should provide optimal drug delivery and, preferably, other desired properties, such as emollient properties. Like other carriers or vehicles, the ointment base must be inert, stable, non-irritating, and non-sensitizing. As described in pages 1399-1404 of Remington: The Science and Practice of Pharmacy, 19th Ed. (Easton, Pa.: Mack Publishing Co., 1995), ointment bases can be classified into four classes: oily bases, emulsifying bases, emulsion bases, and water-soluble bases. Oily ointment bases include, for example, vegetable oils, fats derived from animals, and semi-solid hydrocarbons derived from petroleum. Emulsifying ointment bases, also known as absorbent ointment bases, contain little to no water and include, for example, hydroxystearin sulfate, anhydrous lanolin, and hydrophilic petrolatum. Emulsifying ointment bases are either water-in-oil (W / O) emulsions or oil-in-water (O / W) emulsions and include, for example, cetyl alcohol, glyceryl monostearate, lanolin, and stearic acid. Preferred water-soluble ointment bases are prepared from polyethylene glycol of various molecular weights; for further information, again, see Remington: The Science and Practice of Pharmacy.

[0068] As is well known in the art, creams are either oil-in-water or water-in-oil viscous liquids or semi-solid emulsions. The cream base is water-washable and contains an oil phase, an emulsifier, and an aqueous phase. The oil phase, also called the "internal" phase, generally consists of petrolatum and fatty alcohols such as cetyl or stearyl alcohol. The aqueous phase usually exceeds the volume of the oil phase, but not necessarily, and generally contains a humectant. The emulsifiers in cream formulations are generally nonionic, anionic, cationic, or amphoteric surfactants.

[0069] As understood by those working in the field of pharmaceutical formulations, a gel is a semi-solid suspension-type system. A single-phase gel contains an organic polymer substantially uniformly distributed throughout a carrier liquid, which is typically aqueous but preferably also contains alcohol and optionally oil. Preferred “organic polymers,” i.e., gelling agents, are cross-linked acrylic polymers such as the “carbomer” family of polymers, e.g., carboxypolyalkylenes, commercially available at CARBOPOL; hydrophilic polymers such as polyethylene oxide, polyoxyethylene-polyoxypropylene copolymer, and polyvinyl alcohol; cellulosic polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and methylcellulose; gums such as tragacanth gum and xanthan gum; sodium alginate; and gelatin are also preferred. To prepare a homogeneous gel, dispersants such as alcohol or glycerin can be added, or the gelling agent can be dispersed by grinding, mechanical mixing, stirring, or a combination thereof.

[0070] A lotion is a formulation that should be applied to the skin surface without friction, and is typically a liquid or semi-liquid formulation in which particles containing the active agent are present in a water or alcohol base. Lotions are usually solid suspensions, and preferably, for this purpose, contain an oil-in-water liquid oily emulsion. Lotions are preferred formulations for treating large body parts because they facilitate the application of more fluid compositions. In general, insoluble substances in lotions need to be micronized. Lotions typically contain a suspending agent to produce a better dispersion, as well as compounds useful for localizing the active agent and retaining it in contact with the skin, such as methylcellulose, sodium carboxymethylcellulose, etc.

[0071] A paste is a semi-solid dosage form in which an active agent is suspended in a suitable base. Depending on the properties of the base, pastes are classified into those made from fat pastes or those made from single-phase aqueous gels. The base in fat pastes is generally petrolatum or hydrophilic petrolatum. Pastes made from single-phase aqueous gels generally incorporate carboxymethylcellulose or similar materials as the base.

[0072] Plaster is composed of a paste-like mixture that is applied to the body either directly or after saturating a substrate such as cloth. A medicinal plaster can be prepared by dissolving or dispersing a drug containing the base of the present invention in the plaster.

[0073] Bioadhesives are preparations that adhere to the surface of body tissues. Polymer bioadhesive formulations are well known in the art; see, for example, Heller et al., “Biodegradable polymers as drug delivery systems,” in Chasin, M. and Langer, R., eds.: Dekker, New York, pp. 121-161 (1990), and U.S. Patent No. 6,201,065. Suitable non-polymer bioadhesives containing certain fatty acid esters are also known in the art (U.S. Patent No. 6,228,383).

[0074] The formulations described in the present invention may also be prepared using liposomes, micelles, and microspheres. Liposomes are microscopic vesicles having a lipid wall containing a lipid bilayer and can also be used as drug delivery systems as described herein. Generally, liposomal formulations are preferred for poorly soluble or insoluble pharmaceutical agents. Liposomal formulations for use in the present invention include cationic (positively charged) formulations, anionic (negatively charged) formulations, and neutral formulations. Cationic liposomes are readily available. For example, N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are available under the trademark name LIPOFECTIN® (GIBCO BRL, Grand Island, NY). Similarly, anionic and neutral liposomes are readily available, for example, from Avanti Polar Lipids (Birmingham, Ala.) or can be readily prepared using readily available materials. Examples of such materials include, in particular, phosphatidylcholine, cholesterol, phosphatidylethanolamine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), and dioleoylphosphatidylethanolamine (DOPE). These materials can also be mixed with DOTMA in appropriate proportions. Methods for producing liposomes using these materials are well known in the art.

[0075] It is known in the art that micelles are composed of surfactant molecules whose polar head groups are arranged to form an outer spherical shell, with hydrophobic hydrocarbon chains oriented toward the center of the sphere, forming a core. Micelles are formed in aqueous solutions containing surfactants at concentrations high enough for micelles to occur spontaneously. Surfactants useful for micelle formation include, but are not limited to, potassium lauryl sulfate, sodium octanesulfonate, sodium decanesulfonate, sodium dodecanesulfonate, sodium lauryl sulfate, sodium doxate, decyltrimethylammonium bromide, dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, tetradecyltrimethylammonium chloride, dodecylammonium chloride, polyoxyl 8 dodecyl ether, polyoxyl 12 dodecyl ether, nonoxynol 10, and nonoxynol 30. Micelle formulations can be used in conjunction with the present invention by being incorporated into either a topical or transdermal delivery system, or into a formulation to be applied to a target site (e.g., the vestibule) and surrounding tissue.

[0076] Similarly, microspheres can be incorporated into the formulation and drug delivery system. Like liposomes and micelles, microspheres essentially encapsulate drugs or drug-containing formulations. Microspheres are generally formed from synthetic or naturally occurring biocompatible polymers, but not necessarily, and may also be composed of charged lipids such as phospholipids. The preparation of microspheres is well known in the art and is described in relevant documents and literature.

[0077] Various additives known in the art may be included in this topical formulation. For example, certain formulation components can be solubilized using a solvent containing a relatively small amount of alcohol. The formulations of the present invention may also include conventional additives such as opacifiers, antioxidants, fragrances, colorants, gelling agents, thickeners, stabilizers, and surfactants. Other agents, such as antimicrobial agents, may be added to inhibit the growth of microorganisms such as bacteria, yeasts, and fungi. Exemplary antimicrobial agents are typically selected from the group consisting of methyl and propyl esters of p-hydroxybenzoic acid (i.e., methyl and propylparaben), sodium benzoate, sorbic acid, imidourea, and combinations thereof.

[0078] The compounds and related compositions described above are useful in methods for treating a variety of disorders or conditions. Diverse types or combinations of these treatments include, but are not limited to, topical / transdermal sprays using a discharge pump dispenser, topical / transdermal ointments / ointments rubbed onto the treatment site, topical / transdermal wound cleansing rinses, topical / transdermal roll-ons for pain relief, impregnated mini-sponges individually sealed with the composition and reconstituted with water, wound powders composed of micronized freeze-dried materials, and sustained-release epidermal / topical patches for the stepwise and continuous delivery of the composition for site-specific application.

[0079] The therapeutic composition may preferably be administered as needed. For example, in severe cases, it can be used approximately 1 to 4 times per day. In addition, the therapeutic composition may be administered once a week, once every two weeks, once every three weeks, once a week, or once a month until the condition is treated or restored as desired. Furthermore, administration may initially be daily, and then transition to once a week, once a month, etc., depending on clinical improvement. The compositions of the present invention may be used not only as therapeutic adjuncts but also to maintain the user in a pain-free state.

[0080] In certain embodiments, an effective dose composition containing one or more compounds / agents described herein can be administered to a patient in a single dose. In certain embodiments, an effective dose composition can be administered to a patient repeatedly. An effective dose composition containing a compound / agent can be administered to a patient in a dose of 0.0001 mg / kg to 100 mg / kg, for example, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 40 mg / kg, or 50 mg / kg. The composition containing the compound / agent can be administered over a period of time such as 5 minutes, 10 minutes, 15 minutes, 20 minutes, or 25 minutes. Administration may be repeated, for example, regularly, once every hour over 3 hours, 6 hours, 12 hours, or longer, or once every two weeks (i.e., every two weeks) over 1 month, 2 months, 3 months, 4 months, or longer. After the initial treatment regimen, treatment can be administered at a less frequent rate. For example, after administration every two weeks for three months, administration can be repeated once a month for six months or more than one year. Administration of a composition containing the compound / drug can reduce the level of a marker or symptom by, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% or more.

[0081] For a particular administration, the composition can be provided in a preferred form containing about 0.001 to about 100 milligrams of the active ingredient for symptomatic adjustment of the dosage to the target to be treated. An effective amount of the drug can be supplied at a dosage level of about 0.0001 mg / kg body weight to about 100 mg / kg body weight per day. The compound, or its derivatives, analogs, or salts, may be provided in gel or cream form at doses of 20 to 200 mg per day. In one embodiment, the compound, its derivatives, analogs, or salts are provided in gel form at doses of 50 to 100 mg / day, specifically 50 mg / day, 75 mg / day, and 100 mg / day. The compound, or its derivatives, analogs, or salts can be delivered using a transdermal patch at doses of 1 to 10 mg per day, specifically 4 to 6 mg / day. The compound, or its derivatives, analogs, or salts, may also be provided using a buccal gel at doses of 10 mg / day to 100 mg / day. In one embodiment, this dose can be in the form of a buccal gel and is 40-80 mg / day. In one class of this embodiment, this dose can be 60 mg / day.

[0082] Increase inflow Methods described herein may further include enhancing the inflow of the glymphatic system through to or into the CNS interstitium, brain interstitium, and / or spinal interstitium. Glymphatic system inflow can be enhanced in several ways. For example, fluid can be pumped through the central nervous system interstitium using methods and agents known in the art, such as those described in WO2020 / 072357 and WO2014 / 130777 (both incorporated by reference). For example, enhancing glymphatic system inflow may include administering to a subject (such as a mammal) an agent that increases glymphatic clearance, such as a Stat-3 inhibitor or a BMP signaling axis molecule. In other embodiments, the drug is an AVP (vasopressin) antagonist such as tolvaptan, conivaptan, or VPA-985; an atrial natriuretic peptide (ANP) antagonist such as ananthin; angiotensin II antagonist such as losartan; an AT2R receptor antagonist such as PD123319; or an AT1 receptor antagonist such as valsartan. In yet another embodiment, the drug is a drug for the treatment of insomnia or for use as a sleep aid, and includes, but is not limited to, those listed below. [Table 2]

[0083] In another embodiment, the agent may be an agent that prevents AQP4 depolarization or loss of AQP4 polarization, such as JNJ-17299425 or JNJ-17306861. In another embodiment, the step of increasing glymph inflow includes the step of pumping fluid through the central nervous system interstitium. Pumping can be achieved by any apparatus or method known in the art, for example, by using a mechanical pump, an injection pump, etc.

[0084] Alternatively, the step of enhancing the influx of the glymphatic system includes administering a hypertonic agent to the subject. Preferably, the hypertonic agent is a hypertonic solution that can be administered to the subject's plasma.

[0085] Each of the above drugs can be used alone or in combination with one or more other drugs.

[0086] As used herein, “hypertonic” and “hypotonic” are relative terms, for example, in relation to physiological osmotic pressure, but may differ as long as the ultimate goal of an osmotic difference or gradient is achieved between two compartments (such as plasma and central nervous system interstitium) to facilitate the inflow of glymphatic fluid into the central nervous system interstitium, cerebral interstitium, and / or spinal interstitium. Accordingly, “hypertonic solution” refers to any physiologically and / or pharmaceutically acceptable solution that is hypertonic with respect to physiological osmotic pressure, including hypertonic saline or glucose solution. As described herein, the preferred hypertonic solutions in the present invention do not cause BBB disruption.

[0087] The methods described herein provide injectable drugs (e.g., pharmaceutical preparations) that are hypertonic with respect to blood. To determine whether a drug is hypertonic with respect to blood, the osmolality of all chemical components of a solution containing a diluent is calculated. Tonicity can be calculated for liquids and dissolved or diluted drugs and is expressed in milliosmolality per liter of fluid (mOsm / L) or per kilogram of solvent (mOsm / kg). These two values ​​are also known as osmolarity and osmolality, respectively. The osmolality of blood ranges from 285 to 310 mOsm / L, and the osmolality of blood ranges from 275 to 299 mOsm / kg.

[0088] The osmolar concentration of a solution is partially based on the concepts of osmoticity and osmotic pressure. Osmoticity is the diffusion of solutes (dissolved particles) or the movement of fluids through semipermeable membranes such as blood vessels or cell membranes. Osmotic pressure, which facilitates the transport of molecules across membranes, is expressed in osmolar concentrations and is referred to as hypotonic, isotonic, or hypertonic when compared to biological fluids such as blood or plasma. The terms "tonicity" and "osmotic pressure" are often considered synonymous.

[0089] Osmotic pressure is the hydrostatic pressure (or water pressure) required to counteract the movement of water through a semipermeable membrane in response to an osmotic gradient (i.e., different particle concentrations on either side of the membrane). Serum osmotic pressure can be measured using an osmometer or calculated as the sum of the concentrations of solutes present in a solution.

[0090] As used herein, tonicity and osmotic pressure should be considered synonymous and should be understood in a broad sense. Tonicity can mean effective osmotic pressure, which is equal to the sum of the concentrations of solutes in a solution that have the ability to exert osmotic force across membranes, including cell membranes. In a strict sense, osmotic pressure is a property of a particular solution and is independent of any membrane. Tonicity is a property of a solution with respect to a particular membrane. However, this disclosure refers to a solution that is isotonic, hypertonic, or hypotonic with respect to a biological fluid such as blood or plasma, and this reference includes the meaning that a particular solution is isotonic, hypertonic, or hypotonic with respect to the cell membranes of blood or plasma or cells in other biological fluids.

[0091] An operational definition of tonicity can be used to explain this term. This can be based on experiments in which a test solution is added to whole blood and the results are observed. If RBCs in whole blood swell and burst, the test solution is said to be hypotonic compared to normal plasma. If the RBCs contract and become crenate, the test solution is said to be hypertonic compared to normal plasma. If the RBCs remain in the same state, the test solution is said to be isotonic with plasma. The RBC cell membrane can be a reference membrane. For example, whole blood placed in physiological saline (i.e., 0.9% sodium chloride) does not swell, so physiological saline is said to be isotonic.

[0092] The methods described herein involve administering to a subject a pharmaceutical solution or preparation that is hypertonic with respect to plasma or blood. Since hypertonic solutions, when injected into blood, can cause fluid migration from cells and various adverse effects, care must be taken to select an appropriate osmotic pressure that is not so hypertonic as to cause serious thrombosis and / or vascular irritation. In one embodiment, the solution / preparation was injected into a subject in the manner described in the following examples, and 30 minutes later, the resulting plasma osmotic pressure was approximately 320 mOsml.kg. -1 It is more than 600 mOsml.kg -1 If it is less than, for example, approximately 340 or 350 mOsml / kg -1 It is greater than and approximately 375, 400, 425, 450, 475, 500, or approximately 575 mOsml.kg -1 If it is less than this, it is considered to have a suitable osmotic pressure. Generally, a hypertonic solution useful in this invention is about 320 mOsml.kg -1 High tension, for example, 340-3,000 (e.g., 500-2,000, 1,000-2,000, 1,500-1,800) mOsml.kg -1 This indicates the tension. Approximately 600 mOsml.kg -1 Solutions with extremely high osmotic pressure should be used with caution when administered by injection.

[0093] Various primary extenders can be used in the preparation of hypertonic solutions / intravenous injection preparations. Examples include ionizing agents such as NaCl, and non-ionizing agents. Examples of non-ionizing extenders include, but are not limited to, mannitol, glycine, sucrose, lactose, other disaccharides, therapeutic proteins or the active ingredients of the preparation itself, or other extenders known to those skilled in the art. The concentration of non-ionizing extenders does not significantly affect whether the solution has sufficient ionic strength. However, their concentrations affect the osmolality, and therefore may affect the tonicity. In certain examples, NaCl or mannitol is used. Osmotic diuretics such as mannitol or hypertonic saline can establish an osmotic gradient between plasma and brain cells, drawing water across the blood-brain barrier into vascular compartments. Exemplary mouse doses are described in the following examples. Human equivalent doses (HEDs) can be obtained using methods known in the art. For example, see Nair AB, Jacob SJ Basic Clin Pharm. 2016 Mar;7(2):27-31.doi:10.4103 / 0976-0105.177703 and the FDA's Guidance for Industry. Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy. For example, in human subjects, NaCl may be administered at doses of 30 mg / kg or higher (e.g., 30-300 mg / kg), and mannitol may be administered at doses of 130 mg / kg or higher (e.g., 130-1300 mg / kg).

[0094] treatment The methods and compositions described herein may be used to increase glymphatic outflow and interstitial waste clearance. Therefore, these methods and compositions can be used to treat a variety of related disorders.

[0095] In some embodiments, methods are provided for treating the development of neurodegenerative diseases in the brain and / or spinal cord (or CNS) of a subject, comprising the step of increasing glymph-lymph outflow and / or clearance. In one embodiment of the method, reactive gliosis is reduced, thereby delaying or preventing the development of neurodegenerative diseases.

[0096] Reactive gliosis reduces or prevents the clearance of interstitial waste products. Reactive gliosis reduces Aqp4-dependent volume flow, decreases the volume of extracellular space, and impedes ISF solute clearance, including waste products from the brain and spinal cord. Reactive gliosis is known in the art to be associated with neurodegenerative diseases such as Alzheimer's disease. Increased gliosis is also observed in the brains of aged mammals. Reactive gliosis is also associated with certain autoimmune inflammatory disorders, particularly multiple sclerosis. This has also been observed in the CNS of individuals with amyotrophic lateral sclerosis (ALS). Therefore, by using increased lymphatic outflow and / or waste product clearance from the CNS, in certain embodiments, reactive gliosis and its neurodegenerative consequences can be delayed, prevented, reduced, or mitigated. In another embodiment of the present method, reactive gliosis is mitigated, delayed, or prevented. In another embodiment, the method includes administering to a subject a therapeutic agent that increases or promotes glymphatic system clearance.

[0097] A method is provided for promoting the clearance of waste products (e.g., brain, spinal cord, or CNS waste products) from the brain interstitial and / or spinal cord interstitial of a subject, comprising the step of administering to the subject an agent that increases or promotes glymphatic outflow and / or clearance, thereby promoting the clearance of waste products from the brain interstitial and / or spinal cord interstitial. The agent may be, for example, a diuretic. In some embodiments of the method, the brain, spinal cord, or CNS waste products are amyloid-beta (Aβ) (e.g., soluble Aβ), tau, or alpha-synuclein. The method is also suitable for promoting the clearance of substantially any brain waste products known in the art. In one embodiment, the method comprises the step of administering to the subject a therapeutic agent that increases or promotes glymphatic outflow and / or clearance.

[0098] The methods and compositions described herein can be used to slow, delay, or prevent the accumulation of cerebral waste products. Therefore, a method is provided for slowing, delaying, or preventing the accumulation of waste products in the central nervous system of a subject, comprising the step of increasing glymphatic outflow, thereby increasing the clearance of waste products from the central nervous system. In one embodiment, the method comprises administering to a subject a therapeutic agent that increases or promotes glymphatic outflow. In one embodiment, the cerebral waste product is amyloid-beta (Aβ) (e.g., soluble Aβ), tau, or alpha-synuclein. The method is also suitable for slowing, delaying, or preventing the accumulation of substantially any cerebral waste product known in the art. In certain embodiments, a method is provided for reducing, decreasing, delaying the onset of, or preventing the accumulation of amyloid-beta (Aβ), tau, and / or alpha-synuclein in the brain interstitium of a subject. The method comprises the step of administering to a subject an agent that increases or promotes glymphatic outflow.

[0099] Methods and compositions described herein can be used to increase the clearance of a therapeutic agent or modifier from the brain interstitial tissue of a target. Thus, a method for increasing the clearance of a therapeutic agent or modifier from the brain interstitial tissue of a target is provided. The therapeutic agent or modifier can be any known in the art, for example, therapeutic or functionalized nanoparticles, chemotherapeutic agents, antitumor agents, immunomodulators, antibody-based therapeutic agents, viral vectors, liposomes, or RNA-based therapeutic constructs. In one embodiment, the method includes a step of increasing glymph efflux.

[0100] In one embodiment, the method further includes administering a drug to a subject for the treatment of a neurodegenerative disease. Examples of drugs for the treatment of Alzheimer's disease include cholinesterase inhibitors, N-methyl-D-aspartate (NMDA) receptor antagonists, and monoclonal antibodies, such as donepezil, galantamine, rivastigmine, memantine, a combination of memantine and donepezil, and aducanumab. An example of a drug for the treatment of Parkinson's disease is suvorexant.

[0101] Patients or subjects may include, but are not limited to, individuals with neurological disorders or neurodegenerative diseases such as Alzheimer's disease (AD), stroke, dementia, muscular dystrophy (MD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), cystic fibrosis, Angelman syndrome, Liddle syndrome, Parkinson's disease, Pick's disease, Paget's disease, cancer, and traumatic brain injury.

[0102] In some embodiments, the neurological disorder is selected from neuropathy, amyloidosis, cancer (including, for example, CNS or brain), eye disease or disorder, viral or microbial infection, inflammation (including, for example, CNS or brain), ischemia, neurodegenerative disease, seizures, behavioral disorders, lysosomal storage disorders, and the like.

[0103] Neuropathic disorders are disorders or abnormalities of the nervous system characterized by inadequate or uncontrolled nerve signaling or absence thereof, and include, but are not limited to, chronic pain (including nociceptive pain), pain caused by injury to body tissues (including cancer-related pain, neuropathic pain (pain caused by abnormalities in the nerves, spinal cord, or brain), and psychogenic pain (related to mental disorders as a whole or largely), headaches, migraines, neuropathy, and symptoms and syndromes that often accompany neuropathic disorders, such as dizziness or nausea.

[0104] Amyloidosis is a group of diseases and disorders associated with extracellular protein deposits in the central nervous system (CNS), including, but not limited to, secondary amyloidosis, age-related amyloidosis, Alzheimer's disease (AD), mild cognitive impairment (MCI), Lewy body dementia, Down syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch type); Guam Parkinson's-dementia complex, cerebral amyloid vascular disease, Huntington's disease, progressive supranuclear palsy, multiple sclerosis; Creutzfeldt-Jakob disease, Parkinson's disease, transmissible spongiform encephalopathy, HIV-associated dementia, amyotrophic lateral sclerosis (ALS), inclusion body myositis (IBM), and ocular diseases associated with β-amyloid deposition (i.e., macular degeneration, drusen-associated optic neuropathy, and cataracts).

[0105] Cancers of the CNS are characterized by the abnormal proliferation of one or more CNS cells (i.e., nerve cells) and include, but are not limited to, gliomas, glioblastoma multiforme, meningiomas, astrocytomas, acoustic neuromas, chondromas, oligodendrogliomas, medulloblastomas, gangliogliomas, schwannomas, neurofibromas, neuroblastomas, and epidural, intramedullary, or intradural tumors. In the case of cancer, neurologic agents, which are chemotherapeutic agents, may be selected.

[0106] Viral or microbial infections of the CNS include, but are not limited to, viruses (i.e., influenza, HIV, poliovirus, rubella), bacteria (i.e., Neisseria, Streptococcus, Pseudomonas, Proteus, E. coli, S. aureus, Pneumococcus, Meningococcus, Haemophilus, and Mycobacterium tuberculosis), and fungi (i.e., yeast, Cryptococcus neoformans), parasites (i.e., toxoplasma gondii), or infections by other microorganisms such as amoebas that result in CNS pathophysiology, including meningitis, encephalitis, myelitis, vasculitis, and abscesses, which may be acute or chronic.

[0107] Inflammation of the CNS includes, but is not limited to, inflammation caused by physical injury to the CNS (i.e., resulting from accidents, surgery, traumatic brain injury, spinal cord injury, or concussion) and injury resulting from or associated with one or more other diseases or disorders of the CNS (i.e., abscesses, cancer, viral or microbial infections).

[0108] As used herein, CNS ischemia refers to, but is not limited to, a group of disorders relating to abnormal blood flow or vascular behavior in the brain or its causes, including, focal cerebral ischemia, global cerebral ischemia, stroke (i.e., subarachnoid hemorrhage and intracerebral hemorrhage), and aneurysms.

[0109] Neurodegenerative diseases are a group of diseases and disorders associated with loss of neuronal function or death of neuronal cells in the central nervous system (CNS), including Parkinson's disease (PD), Alzheimer's disease (AD), Lewy body Alzheimer's disease, Lewy body dementia, and mixed dementia, or those associated with traumatic brain injury or ischemic (e.g., diffuse ischemic) brain injury, vascular dementia, frontotemporal dementia, or chronic traumatic encephalopathy, adrenoleukodystrophy, Alexander disease, etc. This includes, but is not limited to, Alpers' disease, amyotrophic lateral sclerosis, telangiectatic ataxia, Batten's disease, Cockayne syndrome, corticobasal degeneration, degenerative diseases caused by or associated with amyloidosis, Friedreich's ataxia, frontotemporal lobar degeneration, Kennedy disease, multiple system atrophy, multiple sclerosis, primary lateral sclerosis, progressive supranuclear palsy, spinal muscular atrophy, transverse myelitis, Refsum's disease, and spinocerebellar ataxia.

[0110] Kits and manufactured products In another aspect, the Disclosure provides a kit or product comprising materials useful for the above-described method. The product comprises a container and a label or accompanying documentation on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, infusion bags, etc. Containers may be formed from a variety of materials, such as glass or plastic. The container holds the composition, either by itself or in combination with another composition that is effective in (1) improving the delivery of the composition to a target site (e.g., the skin of the neck, the interstitium of the central nervous system, the interstitium of the brain, and / or the interstitium of the spinal cord), or (2) treating, preventing, and / or diagnosing one or more of the above-described conditions. The container may have a sterile access port (e.g., the container may be an intravenous infusion bag or vial with a stopper that can be punctured by a subcutaneous needle). The label or accompanying documentation indicates that the composition is used to treat a selected condition.

[0111] Furthermore, the product may include (a) a first container containing a composition, the composition comprising an agent that enhances the outflow of the glymphatic system; and (b) a second container containing a composition, the composition comprising an agent that enhances the inflow of the glymphatic system. The product may also include a third container containing a composition, the composition comprising a therapeutic agent or a contrast agent.

[0112] The product in this embodiment of the present invention may further include a package insert indicating that the composition may be used to treat a particular condition. Alternatively, or in addition, the product may further include a fourth container containing a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0113] In some embodiments, the kit or product further includes instructional material (e.g., instructions for using the kit to administer a composition) containing instructions (i.e., a protocol) for carrying out the methods described herein. The instructional material typically includes, but is not limited to, written or printed material. Any medium capable of storing such instructions and communicating them to an end user is contemplated by the present invention. Such medium includes, but is not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips), optical media (e.g., CD-ROMs), etc. Such medium may include an address to an internet site providing such instructional material.

[0114] definition "Prodrug" or "pharmaceutically acceptable prodrug" means a compound that, after administration, is metabolized in the host, for example, by hydrolysis or oxidation, to form one of the compounds of this disclosure. This disclosure includes, to the extent of its scope, prodrugs of the compounds described herein. Such examples include, but are not limited to, choline ester derivatives and N-alkylmorpholine esters. Other derivatives of the compounds described herein are active in both their acid and acid derivative forms, but in acid-sensitive forms they often offer advantages in solubility, histocompatibility, or delayed release in mammalian organisms (see Bundgard, H., Design of Prodrugs, pp. 7-9, 21-24, Elsevier, Amsterdam 1985). Examples of prodrugs include acid derivatives well known to practitioners of the art, such as esters prepared by reaction with suitable hydrophilic alcohols, or amides prepared by reaction with substituted or unsubstituted amines of hydrophilic compounds, or acid anhydrides or mixed anhydrides. Simple aliphatic or aromatic esters, amides, and anhydrides derived from pendant acidic groups on the compounds described herein are certain prodrugs. In some cases, it is desirable to prepare double ester prodrugs such as (acyloxy)alkyl esters or ((alkoxycarbonyl)oxy)alkyl esters. C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, aryl, C7-C12 substituted aryl, and C7-C12 arylalkyl esters of the compounds described herein may be preferred. Conventional procedures for selecting and preparing suitable prodrugs are described, for example, in “Design of Prodrugs” Ed. H. Bundgaard, Elsevier, 1985. As used herein, “prodrug” may also refer to a naturally occurring precursor of a drug.

[0115] The term "biologically active metabolite" means a pharmacologically active product produced in the body through the metabolism of certain compounds or salts thereof disclosed herein.

[0116] The term "pharmaceutically acceptable salt" refers to a salt that, within the bounds of sound medical judgment, is suitable for use in contact with human and lower animal tissues without excessive toxicity, irritation, or allergic reactions, and that is commensurate with a reasonable benefit / risk ratio. pharmaceutically acceptable salts are known in the art. For example, Berge et al., in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein by reference, describe pharmaceutically acceptable salts in detail. pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed using inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by other methods known in the art, such as ion exchange. Other pharmaceutically acceptable salts include adipine, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, and 2-hydroxyethanesulfonate. Examples include lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, and valerate. Suitable salts derived from bases include alkali metal salts, alkaline earth metal salts, ammonium salts, and N + (C 1-4 Alkyl)4 -Examples include salts. Typical alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium. Further pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfons, and aryl sulfons.

[0117] As used herein, the term “effective dose” refers to the amount of drug required to alleviate at least one symptom of a disease or disorder, and relates to a sufficient amount of a pharmacological composition to provide the desired effect. Thus, the term “therapeutic effective dose” refers to the amount of drug sufficient to provide a beneficial effect when administered to a typical subject. As used herein, effective doses may also include, in various contexts, an amount sufficient to delay the onset of symptoms of a disease, alter the course of the disease (e.g., slowing the progression of symptoms, but not limited to these), or reverse the symptoms of a disease. Therefore, specifying an exact “effective dose” is generally impractical. However, for any given case, a suitable “effective dose” can be determined by a person skilled in the art using only routine experiments.

[0118] The effective dose, toxicity, and therapeutic efficacy can be determined, for example, by standard pharmaceutical procedures in cell cultures or experimental animals to determine the LD50 (lethal dose for 50% of the population) and ED50 (therapeutic effective dose for 50% of the population). Dosage may vary depending on the dosage form used and the route of administration utilized. The dose ratio between toxic and therapeutic effects is a therapeutic index and can be expressed as the ratio LD50 / ED50. Compositions and methods exhibiting a large therapeutic index are preferred. The therapeutic effective dose can be initially estimated from cell culture assays. Alternatively, the dose can be formulated in animal models to achieve a circulating plasma concentration range including the IC50 determined in cell cultures or appropriate animal models. Plasma levels can be measured, for example, by high-performance liquid chromatography. The effect of any particular dosage can be monitored by suitable bioassays. Dosage is determined by a physician and can be adjusted, if necessary, to suit the observed therapeutic effect.

[0119] The terms “decrease,” “reduce,” “reduce,” and “inhibit” are all used herein to mean a reduction of a statistically significant amount. In some embodiments, “decrease,” “reduce,” “decrease,” or “inhibit” typically mean a reduction of at least 10% compared to a reference level (e.g., the absence of a given treatment), and may include reductions of, for example, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or greater. The reduction may preferably be reduced to a level that is acceptable as within the normal range for an individual without a given disorder.

[0120] The terms “improve,” “increase,” “grow,” “enhance,” or “activate” are all used herein to mean an increase by a statically significant amount. In some embodiments, the terms “improve,” “increase,” “grow,” “enhance,” or “activate” can mean an increase of at least 10% compared to a reference level, for example, an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or an increase up to 100%, or any increase between 10% and 100%, or an increase of at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times, or any increase between 2 times and 10 times or more compared to a reference level. In the context of markers or symptoms, “increase” means a statistically significant increase of such level.

[0121] As used herein, “subject” or “individual” means human or animal. Typically, animals are vertebrates such as primates, rodents, domesticated animals, or game animals. Examples of primates include chimpanzees, crab-eating macaques, spider monkeys, and macaques, such as rhesus macaques. Examples of rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Examples of domesticated and game animals include cattle, horses, pigs, sheep, goats, deer, bison, buffalo, feline species such as domesticated cats, canine species such as dogs, foxes, wolves, avian species such as chickens, emus, ostriches, and fish such as trout, catfish, and salmon. In some embodiments, the subject is a mammal, such as a human or a non-human mammal. Mammals can be, but are not limited to, humans, non-human primates, mice, rats, dogs, cats, horses, or cattle. Non-human mammals can be advantageously used as subjects to represent animal models of the disorder. The terms “individual,” “patient,” and “subject” are used interchangeably herein. A subject may be male or female.

[0122] The subjects may be individuals who have been diagnosed or identified as having or having a condition or disability requiring treatment, or one or more complications related to such condition or disability, and who are, at their discretion, already receiving treatment for such condition or disability, or one or more complications related to such condition or disability. Alternatively, the subjects may be individuals who have not been previously diagnosed with a condition or disability, or one or more complications related to such condition or disability. For example, the subjects may be individuals who exhibit one or more risk factors for a condition or disability, or one or more complications related to such condition or disability, or individuals who do not exhibit any risk factors.

[0123] "Persons in need of treatment for a specific condition or disorder" may be those who have that condition or disorder, have been diagnosed with that condition or disorder, or are at risk of developing that condition or disorder.

[0124] As used herein, the term “administer” means to place the drug disclosed herein on a subject by a method or route that results in at least partial delivery of the drug at a desired site. Pharmaceutical compositions containing the drugs disclosed herein can be administered by any suitable route that results in effective treatment of a subject. The terms “administer” and “dosage” mean any method of providing a pharmaceutical preparation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, inhalation administration, nasal administration, topical administration, intraocular administration, intraocular administration, intracerebral administration, sublingual administration, buccal administration, and parenteral administration, including injection, such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent. In various embodiments, preparations can be administered therapeutically, i.e., to treat an existing disease or condition. In further various embodiments, preparations can be administered prophylactically, i.e., to prevent a disease or condition.

[0125] As used herein, the terms “to treat,” “to treat,” “to treat,” or “to achieve remission” refer to therapeutic treatment aimed at reversing, reducing, achieving remission, inhibiting, delaying, or stopping the progression or severity of a condition associated with a disease or disorder. The term “to treat” includes reducing or mitigating at least one adverse effect or symptom of a condition, disease, or disorder associated with a disorder. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of the disease is reduced. That is, “treatment” includes not only improvement of symptoms or markers but also slowing the progression or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical outcomes include, but are not limited to, reduction of one or more symptoms, whether detectable or undetectable; a reduction in the severity of the disease; a state of stabilization (i.e., no worsening) of the disease; a delay or slowing of disease progression; remission or mitigation, remission (whether partial or total) of the disease condition; and / or a reduction in mortality. The term "treatment" of a disease also includes providing relief from the symptoms or side effects of the disease (including palliative care).

[0126] A “therapeutic dose” is an amount sufficient to improve the disease state or symptoms, in particular any condition or symptoms associated with the disease state, or otherwise prevent, interfere with, delay or reverse the progression of any other undesirable symptoms associated with the disease state.

[0127] The "prophylactic effective dose" is the amount of a pharmaceutical composition that, when administered to a subject, has the intended prophylactic effect, for example, preventing or delaying the onset (or recurrence) of a disease state, or reducing the likelihood of the onset (or recurrence) of a disease state or related symptoms. The complete therapeutic or prophylactic effect does not necessarily occur with a single dose, but may only occur after a series of doses. Therefore, the therapeutic or prophylactic effective dose may be administered in one or more doses.

[0128] As used herein, the term “pharmaceutical composition” refers to an activator in combination with a pharmaceutically acceptable carrier, e.g., a carrier commonly used in the pharmaceutical industry. The term “pharmaceutically acceptable” is used herein to mean a compound, material, composition, and / or dosage form that, within the bounds of sound medical judgment, is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response, or other problems, or complications that do not justify a reasonable benefit / risk ratio.

[0129] As used herein, the term “pharmaceutically acceptable carrier or excipient” means a carrier medium or excipient that does not interfere with the efficacy of the biological activity of the active ingredient of the composition and is not excessively toxic to the host at the concentration in which it is administered. In the context of the present invention, pharmaceutically acceptable carriers or excipients are preferably suitable for topical formulations. This term includes, but is not limited to, solvents, stabilizers, solubilizers, isotonic enhancers, structure-forming agents, suspending agents, dispersants, chelating agents, emulsifiers, antifoaming agents, ointment bases, emollients, skin protectants, gel-forming agents, thickeners, pH adjusters, preservatives, penetration enhancers, complexing agents, lubricants, viscous agents, viscosity enhancers, bioadhesive polymers, or combinations thereof. The use of such agents for the formulation of pharmaceutically active substances is well known in the art (see, for example, “Remington's Pharmaceutical Sciences”, EW Martin, 18th Ed., 1990, Mack Publishing Co.: Easton, PA, which is incorporated herein by reference in its entirety).

[0130] "Neurological disorders" refer to diseases or disorders that affect the CNS and / or have a pathogenesis in the CNS. Exemplary CNS diseases or disorders include, but are not limited to, neuropathy, amyloidosis, cancer, eye diseases or disorders, viral or microbial infections, inflammation, ischemia, neurodegenerative diseases, seizures, behavioral disorders, and lysosomal storage disorders. Specific examples of neurological disorders include, but are not limited to, neurodegenerative diseases (including, but are not limited to, Lewy body dementia, post-polio syndrome, Shy-Drager syndrome, olivopontocerebellar atrophy, Parkinson's disease, multiple system atrophy, striatonigral degeneration, tauopathy (including, but not limited to, Alzheimer's disease and supranuclear palsy), prion diseases (including, but not limited to, bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob syndrome, kuru, Gerstmann-Streussler-Scheinker disease, chronic wasting disease, and fatal familial insomnia), bulbar palsy, and motor neuron diseases. This includes diseases and heterodegenerative disorders of the nervous system (including, but not limited to, Canavan disease, Huntington's disease, neuronal ceroid lipofuscinosis, Alexander disease, Tourette syndrome, Menkes's curly hair syndrome, Cockayne syndrome, Haller-Vorden-Spatz syndrome, Lafora disease, Rett syndrome, hepatolenticular degeneration, Resch-Nyhan syndrome, and Unverlicht-Lundborg syndrome), dementia (including, but not limited to, Pick's disease and spinocerebellar ataxia), and cancer (e.g., cancers of the CNS, including brain metastases resulting from cancer elsewhere in the body).

[0131] Where used herein, the terms “a,” “an,” “the,” and similar terms used in the context of the invention (particularly in the context of the claims) should be interpreted to encompass both singular and plural forms, unless otherwise indicated herein or otherwise clearly contradicted by the context. The enumeration of value ranges herein is intended merely as a simplified way of referring individually to each separate value that falls within that range. Unless otherwise indicated herein, each individual value is incorporated herein as if it were individually enumerated herein. All methods described herein can be performed in any preferred order, unless otherwise indicated herein or otherwise clearly contradicted by the context. The use of any and all examples or exemplary language provided herein (e.g., “etc.”) is intended merely to better illustrate the invention and does not limit the scope of the invention as otherwise claimed. No language herein should be interpreted as indicating any unclaimed element essential to the practice of the invention.

[0132] As disclosed herein, several ranges of values ​​are provided. Unless otherwise explicitly indicated by the context, each intermediate value between the upper and lower limits of that range, up to one-tenth of the lower limit, is also understood to be specifically disclosed. Each smaller range between any stated value or intermediate value within the stated range and any other stated value or intermediate value within that stated range is included in the present invention. The upper and lower limits of these smaller ranges may be independently included in or excluded from the range, and each range in which either, neither, or both limits are included within the smaller range is also included in the present invention, subject to any specifically excluded limits within the stated range. Where a stated range includes one or both limits, a range excluding either or both of those included limits is also included in the present invention.

[0133] The term "approximately" means within 10%, preferably 5%, and more preferably 1% of a given value or range. Alternatively, the term "approximately" means within the allowable standard error of the mean, as considered by those skilled in the art.

[0134] As used herein, the terms “comprising” or “comprises” are used with respect to compositions, methods, and their respective components that are essential to the method or composition, but are open to the inclusion of unspecified elements, whether essential or not. The term “consisting of” refers to the compositions, methods, and their respective components described herein, excluding any elements not enumerated in the description of the embodiments. As used herein, the term “essentially consisting of” refers to those elements required for a given embodiment. This term allows for the presence of elements that do not substantially affect the basic, novel, or functional features of the embodiment. [Examples]

[0135] Example 1 This example describes the materials and methods used in Examples 2 to 7 below.

[0136] animal The University Animal Resources Committee at the University of Rochester Medical Center (protocol number 2011-023) approved all experiments. Eight-week-old male C57BL / 6 mice were obtained from Charles River Laboratories (Wilmington, MA, USA). Eighteen-month-old and 22-month-old mice were obtained from the National Institute on Aging (Bethesda, MD, USA).

[0137] drugs Ketamine / xylazine (100 / 10 mg kg-1, intraperitoneal) anesthesia was administered. PGF2α (5 μM in PBS, VWR) was administered locally directly to the cervical lymphatic vessels.

[0138] Surgical procedure Anesthetized mice were placed in a stereotactic frame, and a 30G needle connected to a PE-10 tube filled with artificial (aCSF) was inserted into the cisterna magna as described herein. The animals were then placed supine on a heating pad to maintain body temperature. A surgical midline incision was made from the jaw to the sternum to expose the superficial cervical lymphatic vessels. To expose the deep cervical LV, the superficial cervical fascia was incised, and a deep cervical incision along the lateral boundary of the trachea was achieved by separating it along the medial boundary of the sternocleidomastoid muscle and the posterior belly of the digastric muscle to expose the common carotid artery and internal jugular vein. The deep cervical LV ran superficially over both of these ductal structures and was confirmed to be Prox1-GFP positive. Subsequently, FITC-dextran (3kDa, 1% solid in saline, 20 μl, Invitrogen) was injected into each cheek. Red fluorescent polystyrene microspheres (FluoSpheres®, 1.0 μm, 580 / 605 nm, 0.25% solid in artificial CSF (aCSF), Invitrogen) were briefly sonicated and injected into a cisterna magna catheter at a rate of 2 μL min-1 for 5 minutes using a syringe pump (Harvard Apparatus).

[0139] Measurement of vital signs Heart rate and respiratory rate were measured using a small animal physiological monitoring device 494 (Harvard Apparatus), with measurements taken at 1 kHz and 250 Hz, respectively. These signals were digitized and recorded using a DigiData 1550A digitizer and AxoScope software 496 (Axon Instruments).

[0140] In vivo two-photon laser scanning microscopy A resonant scanner B-scope (Thorlabs) equipped with a Chameleon 498 Ultra II laser (Coherent) was used for two-photon imaging. FITC-dextran and red microspheres were excited at a wavelength of 820 nm. A 20x water immersion objective lens (1.0 NA, Olympus) was used. Images were acquired simultaneously with physiological recordings (3 kHz, ThorSync software) at 60 Hz (ThorImageLS software).

[0141] Image processing 16-bit images were obtained from two-photon microscopy using two channels (red and green) with a spatial dimension of at least 256 × 256. The green channel captured FITC-dextran in lymphatic vessels, and the red channel captured fluorescent microspheres flowing through the lymphatic vessels. Image registration was the first step in the imaging process necessary to account for mouse movement in the background. A strict translation (no rotation or deformation) calculated with 0.2-pixel accuracy was applied using an efficient MATLAB algorithm. At least 10 consecutive images from the record showing the highest stability and least movement were used as a reference for the strict translation. Malcorrelation in the translation (where the algorithm failed) was investigated and manually corrected by linear interpolation. The corrected translation was then sequentially applied to images with edges also filled with zero-value pixels to ensure spatial uniformity across all images without changing the image resolution. Particles in each image were then detected by applying a minimum intensity threshold. These particles typically resolve across 3-4 pixels in an image with an image resolution of 1.29 μm / pixel. The motion artifact index was calculated from time-lapse imaging using an epifluorescence microscope. The difference between consecutive images was calculated as the average of the absolute differences of each pixel.

[0142] Particle tracking velocity measurement The particles detected in each image were tracked using an automated PTV routine implemented in MATLAB 78 and 79. Briefly, this algorithm positions each particle with sub-pixel precision, correlates its motion across the images, and obtains a series of particle positions (particle tracking) over the recording period. The particle velocity is then calculated using Gaussian smoothing and convolution with a differential kernel. Stagnant particles that adhered to the lymphatic vessel walls and no longer followed the lymphatic flow were covered in each image by subtracting a dynamic background image. This image differed from frame to frame and was calculated by averaging the 100 frames before and after a given image.

[0143] Diameter and contraction frequency of lymphatic vessels Lymphatic vessel diameter: Using custom MATLAB code, the median lymphatic vessel diameter was measured over the same time segments used for velocity measurements. First, the centerlines of the imaged lymphatic vessels were identified using an algorithm that applied a spatially varying threshold to each image and then identified edges using MATLAB's "skeletonize" function. The identified centerlines were also manually verified. Next, 20 transverse lines were interpolated, and the change in vessel diameter was measured with sub-pixel precision by identifying the location along the transverse line where the pixel intensity decreased to 20-40% of the maximum value. The medians over space and time were then calculated. Any slight systematic deviations in the medians (due to translation of the two-photon imaging plane or movement in or out of the imaging plane caused by respiratory artifacts) were smoothed by calculating a moving average over a 1-second frame.

[0144] Intrinsic pulsation velocity: The maximum and minimum values ​​of the smoothed tube diameter measurements obtained above were identified using the MATLAB function "findpeaks". Contractions with amplitudes less than 3% of the average diameter or with intervals less than 2 seconds were rejected. The average of the maximum and minimum values ​​was then identified within 4-5 one-minute timeframes, and the average of these two averages was assigned as the contraction frequency per minute.

[0145] Calculation of flow-derived quantities Average clearance velocity: A 5-minute recording segment was manually selected for analysis to ensure an uninterrupted number of continuous particles within each frame. The average clearance velocity was obtained by separating the imaged domain into bins of size 5 or 10 pixels (depending on the number of particles), calculating the time-averaged flow velocity within each bin, and then calculating the spatial average. The inventors ensured a reliable average estimate by using at least 30 distinct spatial measurements, each having at least 7 measurements in each bin.

[0146] Downstream velocity: Spatially averaged mean downstream velocity V 下流 related

number

number

[0147] Volumetric flow rate, discharge rate, backflow rate, and net outflow: Volumetric flow rate Q of each frame, and the calculated downstream velocity V of the pipe. 下流 and approximate cross-sectional area (πD 2 The calculation was performed as the product of ) / 4 (where D is the median tube diameter measured within each frame). Positive values ​​of Q indicate lymphatic fluid discharge from the tube (forward flow), and negative values ​​indicate retrograde flow. The time profile of the volumetric flow rate was calculated for a 1-minute time frame during recording. The area under this curve was calculated in MATLAB to derive quantities related to the volumetric flow rate. The total positive area indicates the amount of lymph discharged from the tube (shaded in green), and the negative area (curve area less than zero) indicates the amount of lymph flowing into the lymphandion upstream of the imaged cross-section. The discharge rate was calculated as the percentage of forward flow (the ratio of the total positive flow rate, shaded in green, to the total amount flowing through the lymphandion), and the retrograde rate was calculated as the percentage of upstream flow (the ratio of the total amount flowing through the lymphandion, shaded in red, to the total amount). The difference between the total positive and negative flow rates within a one-minute timeframe represented the net amount removed from the pipe.

[0148] Immunohistochemical examination Mice were perfused transcardially with 4% paraformaldehyde in 0.1M phosphate buffer (pH 7.4), and the cervical lymphatic vessels were carefully dissected under a dissecting microscope and placed on gelatin-KCr(SO4)2 coated slides. The tubes still attached to each segment were incubated for 2 hours at room temperature in a 24-well microtiter plate (Corning) at room temperature in a blocking solution of 10% serum-PBS containing 0.1% Triton X-100, 0.05 mg / mL Affinipure Fab fragment (Jackson ImmunoResearch, West Grove, PA, US), and 0.2% gelatin, followed by three 5-minute washes with PBS. The tubes were incubated using mesh well inserts, which allowed for incubation and washing of the entire sample (tubes attached to the segments) with the blocking solution.

[0149] Primary antibodies for smooth muscle actin (1:250, A5228, Millipore Sigma), type IV collagen (1:400, 2150-1470, BioRad), and lymphoid endothelial cell marker LYVE1 (1:400, 25-0443-82, Invitrogen) were incubated overnight at 4°C in PBS containing 5% normal donkey serum, 0.1% Triton X-100, and 0.2% gelatin. After washing with PBS, the tissue was incubated for 1 hour at room temperature in PBS containing 5% normal donkey serum, 0.1% Triton X-100, and 0.2% gelatin with a mixture of secondary antibodies—Cy3 conjugate donkey anti-rabbit, Cy-2 conjugate donkey anti-rabbit, and Cy5 conjugate donkey anti-mouse (1:500, Jackson ImmunoResearch, catalog numbers 711165152 and 715175151, respectively)—and DAPI (2.5 μg / mL, D21490, Invitrogen). The samples, washed with PBS, were then mounted on microscope slides in wells prepared with adhesive spacers. Lymphatic vessels were spread from each node and mounted with ProLong® Gold Antifade Mount (P36930, Invitrogen).

[0150] Immunolabeled lymphatic vessels were imaged using confocal microscopy (FV3000RS, Olympus, MA,US). Multichannel Z-stacks (20x magnification, step size 1 μm) across the full width of the lymph node were acquired for all samples using the same acquisition parameters. To evaluate protein distribution, the central plane of the acquired Z-stacks was analyzed using the plot profile function in Image J (version 1.53c, NIH, Bethesda,MD,USA) to plot a 200 μm wide line perpendicular to the lumen and obtain a linear fluorescence distribution. Due to the variability in lymphatic vessel width, the X-axis of the obtained graph was normalized by its own diameter. The resulting distribution had the raw fluorescence distribution on the Y-axis and the width ratio on the X-axis, allowing for direct comparison between them. To evaluate differences in protein expression, the area under the curve was calculated for all samples and both proteins. Coverage area was calculated by using an automated thresholding step and calculating the percentage of pixels within a manually drawn lymphatic region of interest (ROI). Using Prox1-GFP images, a ROI was generated around the valve, and a perivalvular ROI was generated by combining the anterior and posterior valve regions of the LV. DAPI+ nuclei were manually counted using CellCounter in Fiji. To quantify lymphoid endothelial cells (LECs), DAPI+ cells that were also Prox1+ were considered LECs. LECs were expressed as a percentage of all DAPI+ nuclei. Smooth muscle cells (SMCs) were quantified from DAPI+ channel images after subtracting LEC nuclei. Remaining DAPI+-positive nuclei that were also positively stained for SMA were considered SMCs and expressed as a percentage of DAPI+ nuclei. Fiber orientation was calculated using OrientationJ in Fiji. Although the orientation was in the range of -90 to 90°, functionally it has longitudinal orientation, so we calculated the absolute value of all negative orientations and expressed the orientation in the range of 0 to 90°. The OrientationJ plugin also calculated fiber orientation coherence and evaluated it for both valve ROI and perivalve ROI. Representative videos were generated from confocal high-magnification Z-stacks (60x, step size 0.4 μm, FV3000RS, Olympus, MA,US) acquired using the same parameters.

[0151] Reflected fluorescence microscopy Ovalbumin conjugated with Alexa Fluor 647 (OVA-647, 0.5 w / v%, 45 kDa, Invitrogen, 10 μL, 2 μL / min) was injected into the cisterna magna via a 30 G needle connected to a PE-10 tube using a syringe pump (Harvard Apparatus). The cervical lymphatic vessels were surgically exposed as described above and imaged using Metamorph software with a fluorescence macroscope (MVX10, Olympus) equipped with a PRIOR Lumen 1600-LED light source and an ORCA Flash 4.0 digital camera (Hamamatsu). Images at 20x magnification from the far-red emission channel (647 nm) were collected at 1-minute intervals for 60 minutes after the start of injection. Exposure time was kept constant across all experimental groups. One microliter of PBS, with or without 5 μM PGF2a, was directly applied to the cervical lymphatic vessels simultaneously with the injection of OVA-647 into the cisterna magna.

[0152] Lymph node imaging One microliter of PBS with or without 5 μM PGF2a was administered, and OVA-647 (0.5 w / v%, 45 kDa, Invitrogen, 10 μL, 2 μL / min) was injected directly into the cervical lymphatic vessels over a 90-minute period, simultaneously with the injection into the cisterna magna. The superficial and deep cervical lymph nodes were then dissected and placed on microscope slices (Fisherbrand). These lymph nodes were placed under a microscope (MVX10 Research Macro Zoom Microscope, 633 Olympus). Images were acquired from the far-red emission channel (647 nm).

[0153] Clearance assay A clearance assay was performed using our published method (Pla, V. et al. A real-time in vivo clearance assay for quantification of glymphatic efflux. Cell Rep 40, 111320 (2022)). Briefly, anesthetized mice were mounted on a stereotactic device and small burr holes were created (AP + 0.6 mm, ML - 2.0 mm). A guide cannula (26G, C315G SPC, pedestal < 4.5 mm) and a dummy cannula (33G, C315DC / SP, projection 0.1 mm) were placed at a DV of 3.3 mm. After 48 hours, the dummy cannula was replaced with an internal cannula (33G, C315I / SP, projection 0.1 mm) connected by a PE10 tube to a 10 μL Hamilton syringe containing 4% (w / v) DB53 in sterile aCSF. The left femoral vein was exposed by skin excision and placed under a fluorescence microscope (microscope: MVX10, Olympus; light: PRIOR Lumen 1600-LED; camera: Flash 4.0 digital, Hamamatsu). Pump infusion (1 μL, 0.2 μL / min) was induced once every 15 minutes for 2 hours for intraparenchymal delivery, simultaneously with imaging across the vein. PBS (1 μl) with or without 5 μM PGF2a was applied directly to the bilateral superficial cervical lymphatic vessels for the duration of imaging. At the end of this experiment, the animals were decapitated and their brains were collected for postmortem analysis. Quantitative imaging analysis of the femoral vein and ex vivo within the brain was performed as described in Pla, V. et al. A real-time in vivo clearance assay for quantification of glymphatic efflux. Cell Rep 40, 111320 (2022).

[0154] Statistics information All statistical analyses were performed using GraphPad Prism 8 and MATLAB. All graph data are plotted as mean ± standard error (SEM). The chosen tests were selected based on the dataset and are reported in the figure legend. Analysis methods include Sidac's two-way ANOVA (multiple comparison test), Tukey's one-way ANOVA (multiple comparison test), and paired two-tailed t-tests. P<0.05 was considered statistically significant. All bar graph data are presented as mean ± standard error (SEM).

[0155] Example 2: Quantifying cervical lymphatic flow. The cervical lymph nodes (LVs) are divided into superficial cervical (scLV) and deep cervical (dcLV) chains, which together drain fluid from the central nervous system (CNS). However, they also play a role in draining lymph from all other extracranial structures of the head and neck. To distinguish the transport characteristics of CNS-specific solutes, we used a tracer mapping approach called 2P-OPTIC (Figure 1a). Both scLVs and dcLVs were surgically exposed to image the cLVs and reduce photon scattering. The superficial cervical LVs are located just beneath the skin and can be seen through the superior cervical fascia, draining into the superior cervical lymph nodes. Because the dcLVs are located medial to the common carotid and internal jugular veins, accessing them for visualization requires access to the trachea and medial depressions of the medial muscle tissue (sternohyoid and omohyoid muscles) as well as the lateral depressions of the lateral muscle tissue (sternocleidomastoid and posterior belly of the digastric muscle).

[0156] LVs were labeled by intramuscular buccal injection of FITC-dextran (3 kDa). Two-photon resonance scanning microscopy was used to achieve single-focus plane 60 Hz frame rate imaging of cLVs. Dextran labeled the draining lymphatic vessels, allowing for measurement of lymphatic vessel diameter (Figure 1a). However, to analyze transport from extracranial drainage to the CNS, 1 μm fluorescent spheres were injected into the CSF via the cisterna magna. 20 Next, particle tracking velocity measurements were used to quantify the flow velocity within the cLV. The mouse cardiac and respiratory cycles were also recorded, which allowed for the simultaneous measurement of flow velocity, contractility (Figure 1b), and cardiac and respiratory cycles (Figure 7).

[0157] In each experiment, more than 1000 particles were tracked during a 10-minute recording (Figure 1c). The pre-lymph node cLV in 2-month-old young adult mice was 72.4 ± 5.5 mm in diameter. Particle tracking was nearly uniform, with increased concentration near the center of the tube as particles passed through the valve. While the fluid rapidly flowed into the downstream lymphatic valve, the lower lymphatic valve remained closed, the tube remained filled, and in some cases, further expanded the tube. Subsequently, with the downstream tube closed, retrograde fluid motion occurred within the lymphatic valve, causing particles to flow backward and closing the upstream valve. The lymphatic valve then contracted, thereby draining the fluid along the LV into the next lymphatic vessel downstream.

[0158] The spatial and temporal average distribution of CSF flow velocity within the lymphatic vessel (Figure 1e) showed an average velocity of 151.7 ± 28.4 mm / sec, which was highest near the center of the channel and lowest near the wall, as expected for pressure-driven flow within the channel (Figure 1f).

[0159] In experiments visualizing the flow through the valve, the highest central velocity was measured at the valve, consistent with the fluid accelerating through a smaller cross-sectional area to maintain a constant flow rate (Figure 1f). The Reynolds (Re) number is a dimensionless ratio between inertial and viscous forces, where Re>1 indicates that inertial forces are dominant and Re<1 indicates that viscous forces are dominant. The Re number for cLV was 0.0064±0.0008 (Figure 1g), which supports the idea that viscous forces are dominant and the fluid is propelled by the pressure difference created by the contraction of the pipe wall.

[0160] Another way to characterize the flow is to calculate the Wo number, which explains the ratio of pulsating flow frequency to viscous effects. The Wo number of cLV, 0.039 ± 0.003, is well below the Wo = 0.1 limit (Figure 1h), suggesting that transient inertial effects are minimal and the fluid is driven primarily by lymphandion contractility. These provide the first explanation for the hydrodynamic properties of CSF discharge via cervical lymphatic vessels in living rodents.

[0161] Deep cervical LVs exhibited similar flow characteristics compared to scLVs, but their visualization was affected by significant motion artifacts due to their proximity to the carotid artery and trachea. The frequency of the vibration artifacts was approximately 3.18 Hz, consistent with the mouse cardiac cycle frequency, suggesting that the heartbeat was the primary source of motion. Nevertheless, the mean downstream flow velocity in dcLVs was comparable to that observed in scLVs (151.7 ± 28.4 μm / sec vs. 166.2 ± 31.6 μm / sec, P = 0.7423), and the flow in both lymphatic chains was viscosity-dominant laminar (Re << 1). The total number of microspheres derived from the CNS was also similar across these two LV chains (dcLV: 125.2 ± 22.0 particles / min vs. scLV: 129.6 ± 28.0 particles / 136 min, P = 0.9069), suggesting that both superficial and deep cervical cLVs play similar roles in CSF efflux.

[0162] Example 3: Lymphatic drainage is impaired with age. CSF outflow along the cervical lymphatic vessels is reduced in aged mice. 5 However, it has not been explained whether this is due to a delay in clearance from the intracranial compartment or to the endogenous effects of aging on LV function. The inventors evaluated the structure and function of cLV using 2P-OPTIC in mice aged 2 months, 18 months, and 22 months (Figure 2).

[0163] The Re and Wo numbers (0.037±0.003 and 0.033±0.003, P=0.344, respectively) at 18 months (0.0052±0.0011) and 22 months (0.0041±0.0008, P=0.2194 (compared to 2 months)) were comparable to those observed in younger animals, indicating a viscosity-dominant flow and minimal inertial force. Tube diameter was measured over 10 minutes at 20 different spatial locations along the tube length, and lymphatic flow dynamics were simultaneously measured (Figure 2b). There was no difference in the median cLV diameter across age, suggesting that cLV reaches adult size at 2 months (Figure 2g). Despite similar sizes, the mean downstream velocity (V) was similar. 下流 Figures 2c and 2e show a decrease of approximately 40% at 18 months of age (90.9 ± 29.1 mm / sec, P=0.21 (compared to 2 months of age)) and a significant decrease of approximately 63% at 22 months of age (56.6 ± 5.7 mm / sec, P=0.02 (compared to 2 months of age)). The cervical lymphatic vessels rapidly expanded and contracted in young mice (13.2 ± 0.8 contractions / min, Figure 2d), with each contraction cycle lasting approximately 4.6 seconds (Figure 2d). This is consistent with previous studies measuring the contractile dynamics of collecting lymphatic vessels in the lower limbs and flanks of young mice under ketamine-xylazine anesthesia. 21 The endogenous velocity decreased sharply in 18-month-old mice (50.0%, P=0.0003) and further decreased in 22-month-old mice (65%, P<0.0001) (Figure 2d), suggesting an age-dependent delay in cLV contractility.

[0164] To evaluate lymphatic function, an assay was performed to quantify the efflux rate or frequency of fluid efflux from lymphandione (Figure 2f). While this efflux rate does not necessarily coincide with the intrinsic contraction rate, it effectively represents the frequency of valve opening and closing and can be used to evaluate the efficiency of valve function. The inventors observed a significant decrease in efflux rate between 2–18 months (-27.8%, P<0.0001) and 18–22 months (-29.3%, P=0.0001) (Figure 2f). These decreases in efflux rate were associated with the intrinsic contraction rate [coefficient of determination (R)]. 2)=0.8045, strongly correlated with the impairment shown in Figure 2h, meaning that lymphandion contraction drives the majority of the flow. However, in contrast to previous in vitro studies using isolated LVs, we did not observe a consistent one-to-one relationship between tube contraction and flow oscillation. 17 22 These findings suggest that upstream and downstream lymphandions influence flow within the lymphandions in vivo, implying that synchronization between smooth muscle cells is crucial for driving regulated flow along the vessels. Interestingly, lymphatic flow in the cLV was independent of heart rate or respiratory rate (Figure 7). These experiments demonstrate that with aging, cervical lymphatic vessels have significantly lower contraction frequencies, resulting in reduced lymphatic transport, suggesting that solute transport from the CNS to the cLN and to the periphery slows down in aged animals.

[0165] Example 4 Lymphatic outflow decreases with age To evaluate the instantaneous volumetric flow rate (Q), the inventors calculated the product of the instantaneous downstream velocity and the instantaneous cross-sectional area (calculated using the median instantaneous lymphatic vessel diameter, assuming a circular cross-section, Figure 3a). The maximum flow rate reached 724.2 nL / min in young mice, while the flow rate decreased dramatically in aged mice (18 months: 423.2 nL / min and 22 months: 180.9 nL / min).

[0166] Next, the inventors calculated the discharge rate, which is the percentage of forward flow through the lymphandion, and the reverse flow rate, which corresponds to the percentage of reverse flow (Figures 3b-d). These two rates are also known as the forward flow rate, which is defined as the fraction of time the fluid flows away from the skull, and the reverse flow rate, which is defined as the fraction of time the fluid flows backward.

[0167] Unsurprisingly, 2-month-old mice exhibited the highest discharge rates, expelling 85.9±1.3% of the fluid downstream, with only 13.9±1.3% of the flow moving retrogradely, indicating that lymphatic flow in juvenile mice is predominantly unidirectional, consistent with regulation by a functional unidirectional valve between lymphandions. Conversely, aged mice showed a reduction in discharge rates of approximately 20% compared to juvenile mice, with 18-month-old and 22-month-old mice pumping only 68.8±1.4% (P<0.0001 (compared to 2-month-old)) and 68.6±2.0% (P<0.0001 (compared to 2-month-old)) of the fluid forward, respectively (Figure 3c). The decrease in anterograde flow was consistent with a striking 2.2-fold increase in retrograde flow in 18-month-old mice (31.2±1.4%, P<0.0001 (compared to 2-month-old mice)) and 22-month-old mice (31.4±2.0%, P<0.0001 (compared to 2-month-old mice)) (Figure 3b). Further investigation of flow in aged mice revealed that the valve was not completely closed (Figure 3f), causing particles to vibrate back and forth without being taken up by lymphandione. This suggests that the aged valve is unable to restrict regurgitation, resulting in the increased retrograde rate and decreased efflux rate measured in aged mice.

[0168] The inventors functionally characterized the degree of impairment by calculating the discharge rate, which is the amount of fluid effectively transported downstream of the tube per minute. In aged mice, lower frequency contractions, combined with a higher retrograde rate and lower discharge rate, resulted in significantly less discharge in 18-month-old mice (20.8 ± 2.3 nL / min, P < 0.0001) and 22-month-old mice (16.9 ± 4.9 nL / min, P < 0.0001) compared to 2-month-old mice (70.6 ± 5.6 nL / min) (Figure 3d). For comparison, this is equivalent to aged cLV transporting only about a quarter (about 24.3 μl / day) of the lymph fluid discharged by juvenile cLV (about 102 μl / day). We hypothesized that this decrease in flow rate would also reduce the arrival of antigens and leukocytes to the cLN. To test this idea, the inventors calculated the number of 1 μm particles transported per minute as a surrogate for solute transport in the micron range (Figure 3e). This analysis revealed that the particle count was also significantly reduced in 18-month-old mice (44.5 ± 7.5) and 22-month-old mice (62.4.5 ± 16.1) compared to 2-month-old mice (104.8 ± 19.1) (63.9% and 49.4%, respectively), indicating a significant impairment of lymphatic outflow in the senescent ducts.

[0169] Example 5: Age-induced smooth muscle cell loss impairs lymphatic contractility. Aged aggregated LV exhibits reduced contractility due to loss of the smooth muscle cell (SMC) layer covering the lymphandion, decreased expression of contractile proteins in the SMCs, and changes in the composition of the basement membrane. Aging is also associated with hyperplasia and dysmorphism in LV. 11 Therefore, the inventors aimed to determine whether age-dependent changes in basement membrane properties may drive biomechanical differences that explain changes in flow. The inventors performed immunohistochemical staining of cLV-labeled collagen IV (Col IV), a major structural protein of the lymphoid basement membrane. Quantification of basement membrane thickness and collagen IV expression showed no significant difference between young and aged animals (P=0.3411, Figure 4a-b), suggesting that the structural scaffold of cLV is maintained during aging.

[0170] Next, we asked whether the loss of the smooth muscle cell (SMC) layer, which plays a role in the spontaneous synchronous contraction of LV, could contribute to the decrease in contractility. SMCs were stained for α-smooth muscle actin, the primary contractile protein present in these cells. 24 24 This was identified by [method / analysis]. SMA fluorescence was 37.5% lower in 22-month-old mice compared to their younger counterparts (P<0.0328), suggesting a significant loss of SMC investiture and contractility. In summary, these results suggest that the loss of SMC due to decreased SMA expression can explain the observed decrease in fluid transport seen in aged tubes. This decrease in SMA expression is consistent with what was seen in mesenteric lymphatic vessels during aging. 13 This may support the view that systemic loss of lymphatic SMCs exists.

[0171] Additional immunohistochemical staining assays of cLV in 2-month-old and 18-month-old Prox1-GFP animals (Figures 10a-b) identified lymphoid endothelial cells (LECs, Figure 10c), α-smooth muscle actin in SMCs (αSMA, Figure 10d), and collagen 4a (Col4a, Figure 10e), the main structural protein of the lymphoid basement membrane. Significant loss of Prox1 and αSMA expression regions was observed in both the lymphatic valve and perivalvular regions of scLV in aged mice. LECs (Prox1-positive regions) covered approximately 12% less of the lymphatic valve in 18-month-old animals compared to young controls (97.2±1.3% compared to 85.8±2.9%, P=0.0075) and 74% less of the perivalvular region of LV (65.5±3.3% compared to 17.1±3.1%, P<0.0001). The SMC (αSMA-positive region) covered 54% less of the valve (72.6±7.1 compared to 33.6±5.1, P=0.0021) and 35% less of the perivalvular region (86.2±3.5 compared to 55.9±5.2, P=0.0013) (Figures 10f-j). A similar relationship regarding SMC coverage was observed in 22-month-old animals. αSMA fluorescence was 37.5% lower in 22-month-old mice (97.5±5.6 arbitrary units, AU) compared to their younger counterparts (156.1±19.0 AU, P=0.0328) (Figures 10a and 10c), suggesting a significant loss of αSMA and contractility. Interestingly, aging led to a 1.5-fold higher loss of SMC coverage in the valve compared to the perivalvular region. Valve αSMA is thought to actively regulate the movement of valve lobes in other rodent lymphatic chains, and its loss may increase regurgitation and decrease lymphatic outflow, as we observed in aged mice.

[0172] Quantification of basement thickness and Col4a expression showed no significant differences between 2-month-old and 18-month-old animals, nor between 22-month-old animals (Figures 10k-l and 4a-b), suggesting that the structural footing of cLV is maintained during aging.

[0173] Next, we investigated whether the decrease in Prox1 and αSMA expression was a result of overall cell loss or rather a result of cell-specific processes. LECs play a role in channel flow, and SMCs drive spontaneous phasic contraction of LVs. A decrease in either of these could reduce contractility. LECs were identified as DAPI+ / Prox1+ cells, and SMCs were identified as DAPI+ / αSMA+39. The results showed that the total cell number, LEC number, and SMC number were comparable between 2-month-old and 18-month-old animals (total cell number: P=0.3854, LEC: P=0.9485, SMA: P=0.7626) (Figure 10m~q).

[0174] Assays were performed to investigate whether abnormal fiber orientation of both αSMA and Col4a fibers may contribute to the abnormal contractility observed in aged animals (Figure 10r). However, fiber orientation and orientation coherence of both markers were found to be preserved in the valve and perivalvular regions during aging (orientation: P=0.1539, coherence: P=0.1466) (Figures 10s and 10t).

[0175] In summary, these results suggest that a decrease in αSMA and Prox1 coverage, rather than a reduction in LEC or SMC, can explain the observed decrease in fluid transport seen in aging ducts. This decrease in SMA expression is consistent with what was observed in aging mesenteric lymphatic vessels and may support the view that this is a systemic process rather than a region-specific event.

[0176] To further characterize the wall dynamics of cLVs, the inventors calculated the phase-averaged tube diameter waveform and the average contraction amplitude over the contraction period (Figure 4d). The inventors observed a 20% decrease in contraction amplitude and a 30% longer systolic period in 22-month-old tubes compared to 2-month-old tubes (P<0.0001, Figure 4d). 2-month-old cLVs achieved twice the wall velocity of their aged counterparts, demonstrating faster and more efficient pumping (P<0.0001, Figure 4e).

[0177] In summary, these results suggest that it is not changes in the basement membrane, but rather a decrease in SMA fiber coverage or SMC loss, that coincides with slower and smaller endogenous pulsations, leading to a decrease in contraction frequency (Figure 2d) and a lower average volume flow rate in old age (Figure 3d).

[0178] Example 6 PGF 2α restores cLV pumping by improving lymphatic contractility Since endogenous pulsation is the main driver of cLV transport and both the number of LECs and SMCs are maintained in old cLV, the question was asked whether salvaging the function of the remaining SMCs in old cLV could restore lymphatic drainage. To test this, prostaglandin F 2α (PGF 2α ) was applied locally (Figure 5a - d). PGF 2α is a prostanoid shown to induce LV contraction by increasing intracellular Ca 2+ concentration in SMCs 19、26、27 .

[0179] PGF 2α was found to dramatically increase the endogenous pulsation rate (Figure 5e) not only in young mice (50.7%, P = 0.0216) but also in old mice (152.0%, P = 0.0136), restoring the endogenous pulsation rate back to levels seen in young animals before treatment (P = 0.487). The increase in pulsation rate also led to a faster downstream velocity in young mice (103.1%, P = 0.0497), but in old mice, PGF 2α exposure induced a 3.5 - fold increase in flow velocity (P = 0.0020), rescued the flow, and even exceeded the baseline velocity in young animals (P = 0.016, Figure 5f). Notably, PGF 2αdecreased the median tube diameter in 2-month-old mice (P = 0.0329) and 22-month-old mice (P = 0.0327) (Figure 5g), but also increased the contraction amplitude (Δd, P < 0.0001 compared to the previous, Figure 5i), suggesting that in addition to the increased phasic activity, the tonic activity was also enhanced. However, there was no change in the ejection rate in 2-month-old mice (P = 0.1220) or 22-month-old mice (P = 0.8362) (Figure 5h), PGF 2α mainly stimulates SMC contraction but does not affect valve dynamics. Reassuringly, local application of PGF 2α induced no changes in heart rate or respiratory rate (Figure 8). These experiments confirmed that PGF 2α rescued the intrinsic pumping ability of aged tubes by amplifying the contraction amplitude (leading to an increase in volume change) and increasing the contraction frequency (promoting faster fluid ejection). These observations further support the idea that age-induced cLV dysfunction can be reversed by optimizing SMC function.

[0180] PGF 2α To quantify whether enhancing tube function using PGF 2α directly leads to an improvement in cLV transport, the inventors calculated the volume flow rate and characterized the ejection and retrograde rates after treatment (Figure 6). After PGF 2α it was observed that the lymph flow was mainly unidirectional and there was little retrograde flow (Figure 6a). After PGF 2α the ejection rate increased by 7.0% in 2-month-old mice (90.8 ± 0.8 compared to 97.1 ± 0.6, P = 0.0045) and by 31.7% in 22-month-old mice (P = 0.0005, 72.5 ± 2.1 compared to 95.5 ± 0.5) (Figure 6c), while the retrograde rate decreased by 64.2% in 2-month-old mice (8.1 ± 1.0 compared to 2.9 ± 0.6, P = 0.0061) and by 83.6% in 22-month-old mice (27.5 ± 2.1 compared to 4.5 ± 0.4, P = 0.0005) (Figure 6b). The decrease in retrograde flow was in both 2-month-old mice (148.6%, P = 0.0021) and 22-month-old mice (181.4%, P = 0.0215) PGF 2αThis later resulted in significantly larger emissions (Figure 6d). More importantly, PGF 2α Treatment using PGF overcame age-induced lymphatic stasis, increased drainage rates, and reduced retrograde rates compared to untreated young controls (P=0.0012 and P=0.0133, respectively). 2α This also helps to alleviate the lower discharge levels in aged animals (P=0.0017), suggesting that with appropriate therapy, the function of aged lymphatic vessels can be restored to youthful levels.

[0181] Example 7: Restoring cervical LV function in aged animals relieves CSF efflux impairment. cLV plays a role in removing CSF from the CNS, therefore PGF 2α The study investigated whether restoring cLV function through local administration enhances CSF clearance.

[0182] After delivery of fluorescent microspheres into a large vessel, the number of particles detected by cLV ​​was quantified as a surrogate for solute transport. PGF 2α Following administration, CSF efflux in 2-month-old mice increased by 35.4% (P=0.0355, Figure 6e). Surprisingly, treated aged mice had nearly four times more solute reaching the cLN compared to untreated aged controls (P=0.0017). Cervical LV stimulation alone was sufficient to enhance CSF efflux in aged mice to levels exceeding those of young, untreated mice (P=0.0208). Furthermore, since the cLV transports fluids by volumetric motion, the increase in CSF clearance should apply to all macromolecular solutes (e.g., proteins and cells) immersed in fluid. 2α To test whether PGF can increase protein efflux in CSF, ovalbumin conjugated with Alexa Fluor 647 (OVA647) was injected into the cisterna magna. Since 2P-OPTIC can only quantify transport along a single cLV, net transport of protein-sized tracers (45 kDa) from CSF was quantified using an epifluorescence microscope to include all superficial neck cLVs. 2αThe treatment time was extended to 60 minutes to determine if the effect could be sustained for a longer period. CSF clearance was estimated from the change in fluorescence intensity of the OVA647 tracer after large-cell injection (Figure 6f).

[0183] PGF 2α However, it was found to promote robust CSF efflux in both young mice (P<0.0001) and aged mice (P<0.0001) (Figure 6f), and to rescue the stunted CSF efflux observed in 22-month-old animals by restoring it to the level of 2-month-old animals. Ex vivo analysis of cLN showed that PGF in 2-month-old and 22-month-old mice 2α Later, a significantly larger accumulation of OVA647 was observed (Figure 9).

[0184] Because the efflux of protein tracers delivered into the cisterna magna can bypass the brain parenchyma, to further evaluate the role of enhancing cLV transport in brain clearance, we labeled intrastriatal interstitial fluid (ISF) by injecting Direct Blue 53 (DB53, also known as T-1824 or Evans Blue) (Figure 11a). DB53 was chosen because it is a small (960.8 Da) fluorescent tracer that reliably tracks ISF. DB53 can be removed from the blood, which has a high affinity for plasma albumin, and can remain in circulation at stable concentrations for up to 135 minutes. The amount of DB53 ultimately retained in the brain was significantly reduced after PGF2a (6.3 ± 0.9 compared to 10.1 ± 1.3, P = 0.0314) (Figures 11d-e). Therefore, cerebral clearance could be dynamically quantified by evaluating the DB53 signal in the femoral vein, which dramatically increased after PGF2a compared to controls (P<0.0001) (Figures 11f-g). DB53 signal was significantly higher in PGF2a compared to controls at 75 minutes (8.3±1.3 compared to 4.6±0.8, P=0.0351), 90 minutes (10.3±1.5 compared to 5.5±0.6, P=0.0014), 105 minutes (10.8±1.4 compared to 6.6±0.7, P=0.0070), and 120 minutes (11.45±8.3 compared to 6.8±0.7, P=0.0023) (Figure 11g).

[0185] In summary, restoring cLV function in both young and aged mice increased CSF efflux and the arrival of CSF-derived proteins to the cLN. Furthermore, it is suggested that cervical lymphatic vessels are a crucial driving factor for CSF clearance and act as a significant barrier to CSF ​​outflow in aging.

[0186] The descriptions of the above-mentioned examples and preferred embodiments should be construed as illustrative, not limiting, the disclosure as defined by the claims. For ease of understanding, numerous variations and combinations of the features described above can be utilized without departing from the disclosure as defined in the claims. Such variations are not considered to deviate from the scope of the disclosure, and all such variations are intended to be included within the scope of the following claims. All references cited herein are incorporated by reference in their entirety.

Claims

1. A method for improving glymph-lymph outflow from the central nervous system (CNS) of a subject, comprising increasing the flow rate of the cervical lymphatic vessels (cLV) of the subject.

2. The method according to claim 1, wherein increasing the flow rate includes increasing the contraction of the cLV.

3. The method according to claim 2, wherein increasing the contraction of the cLV comprises administering a smooth muscle contraction agonist to the subject.

4. The agonist is a muscarinic agonist, an α1-adrenergic agonist, or a K ＡＴＰ The method according to claim 3, comprising a channel inhibitor, an NOS inhibitor, a soluble guanylate cyclase (sGC) blocker, a PKA inhibitor, a cyclooxygenase-1 (COX-1) inhibitor, a cyclooxygenase-2 (COX-2) inhibitor, a prostanoid, a prostaglandin E2 receptor 4 (EP4 receptor) antagonist, a prostacyclin (PGI2) receptor (IP receptor) antagonist, or a nonsteroidal anti-inflammatory drug (NSAID).

5. The method according to claim 4, wherein the agonist is selected from the group consisting of carbachol, norepinephrine, propranolol, glibenclamide, NG-nitro-L-arginine (L-NNA), ODQ, H-89, KT5720, indomethacin, ketoprofen, AH23848B, GW627368x, CAY10441, pyridostigmine, amifanplidine, neostigmine, edrophonium, and amvenonium.

6. The method according to claim 4, wherein the agonist comprises a lymphangioconstriction-promoting prostanoid.

7. The aforementioned prostanoid is prostaglandin F ２α (PGF ２α ), thromboxane A ２ (TxA ２ ), PGD ２ leukotriene B4, leukotriene C4, leukotriene D4, or TxA ２ The method according to claim 6, including imitation U46619.

8. A method for improving glymph-lymph outflow from a target CNS, comprising administering a phosphodiesterase type 5a inhibitor to the target.

9. The method according to any one of claims 3 to 8, wherein the agonist or the inhibitor is administered to the neck of the subject.

10. The method according to claim 9, wherein the agonist or the inhibitor is administered locally.

11. below, To increase the inflow of cerebrospinal fluid (CSF) through the CNS interstitium, and The method according to any one of claims 1 to 10, further comprising one or more of the following: sending a message to the neck of the subject.

12. The method according to claim 11, wherein increasing the influx of CSF involves administering to the subject a drug selected from the group consisting of a hypertonic solution, a Stat-3 inhibitor, a bone morphogenetic protein (BMP) signaling axis molecule, an AVP (vasopressin) antagonist, an atrial natriuretic peptide (ANP) antagonist, angiotensin II antagonist, an AT2R receptor antagonist, and an AT1 receptor antagonist.

13. The method according to claim 12, wherein the hypertonic solution comprises NaCl or mannitol.

14. A method for promoting the clearance of material from the interstitium of a target CNS, the interstitium of the brain, and / or the interstitium of the spinal cord, comprising improving glymph-lymph outflow from the target CNS according to any one of claims 1 to 13.

15. The method according to claim 14, wherein the substance comprises a fluid or a solute.

16. The method according to claim 14, wherein the substance comprises amyloid-beta (Aβ), tau, or alpha-synuclein.

17. The method according to claim 14, wherein the substance comprises a drug or a metabolite thereof.

18. A method for treating cerebral edema, traumatic brain injury, post-traumatic brain injury, or neurodegenerative disease in a subject, comprising improving glymph-lymph outflow from the CNS of the subject according to any one of claims 1 to 12.

19. A method for treating cerebral edema, traumatic brain injury, post-traumatic brain injury, or neurodegenerative disease in a subject in need thereof, comprising administering a phosphodiesterase type 5a inhibitor to the subject.

20. The method according to any one of claims 8 to 19, wherein the inhibitor is selected from the group consisting of sildenafil, tadalafil, vardenafil, and avanafil.

21. The method according to any one of claims 18 to 20, wherein the neurodegenerative disease is Parkinson's disease (PD), Alzheimer's disease (AD), Lewy body Alzheimer's disease, Lewy body dementia, mixed dementia, vascular dementia, frontotemporal dementia, chronic traumatic encephalopathy (CTE), HIV-related dementia, Lewy body disease, Huntington's disease, or multiple system atrophy.

22. The method according to any one of claims 17 to 21, further comprising administering a drug for treating the neurodegenerative disease to the subject.

23. The method according to any one of claims 1 to 22, wherein the subject is a mammal.

24. The method according to claim 23, wherein the mammal is a human.

25. The method according to claim 23 or 24, wherein the mammal or human is an elderly mammal or human.

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