Methods and agents for glaucoma

Abstract

The present invention relates to methods and compositions for detecting, diagnosing, and treating glaucoma diseases. In addition, the present invention discloses compositions and methods for affecting intraocular pressure and increasing aqueous humor outflow in glaucoma. Compositions are provided for treating glaucoma diseases in a subject, reducing intraocular pressure in a subject, and / or reducing extracellular vesicle complexes in the eye of a subject.

Description

Technical Field

[0001] The present invention relates to methods and compositions for the detection, identification, and treatment of glaucoma. More specifically, the present invention discloses compositions and methods that affect intraocular pressure and increase ocular outflows in glaucoma. Background of the Invention

[0003] Glaucoma is a leading cause of vision loss worldwide, affecting an estimated 70 million people. Glaucoma is a permanent blinding disease that is asymptomatic until the patient experiences advanced vision loss. The diagnosis of glaucoma is often delayed.

[0004] The forms of glaucoma are described as open-angle glaucoma or closed-angle glaucoma. Primary open-angle glaucoma (POAG) is the most prevalent, accounting for approximately 75% of cases. Narrow-angle glaucoma and other less common forms account for the other 25%. In POAG, the anterior chamber angle appears healthy and open, and intraocular pressure (IOP) is elevated without an underlying disease.

[0005] Risk factors for POAG include elevated IOP, increasing age, family history, African descent, myopia, and association with diabetes or hypertension. The pathophysiology causing glaucoma is associated with increased resistance to aqueous humor outflow, but the direct mediators of this process remain unknown. Little is known about the causes of glaucoma, and factors leading to its progression have not been identified.

[0006] Signs and symptoms of glaucoma include damaged optic nerve, retinal ganglion cell degeneration, changes in the optic nerve head, and corresponding visual field loss. Elevated IOP is associated with retinal ganglion cell (RGC) death and ultimately visual field (VF) loss. IOP-related optic nerve damage is important in the pathogenesis of POAG. Patients with POAG and ocular hypertension (OHT) have elevated IOP. Elevated IOP is an important risk factor for progression from OHT to POAG. In many secondary glaucomas, elevated IOP is the only common clinical finding. Reducing IOP has been shown to reduce the risk of progression of NTG. In animal models, elevated IOP precedes glaucomatous nerve damage. Generally, the cause of elevated IOP may be due to reduced aqueous humor outflow.

[0007] Medical treatment of glaucoma aims to reduce IOP, which may slow the progression of the disease in some patients. Disadvantages of current treatments include lack of efficacy and drug side effects.

[0008] There is a need for effective methods and compositions for glaucoma, as well as methods for reducing IOP and improving ocular outflows.

[0009] There is an urgent need for methods, kits, and compositions for the detection, identification, and treatment of glaucoma. SUMMARY OF THE INVENTION

[0010] The present invention provides methods, compositions, devices, kits, and reagents for detecting, diagnosing, and treating glaucoma diseases.

[0011] In some aspects, the present invention provides methods and compositions for reducing intraocular pressure and increasing aqueous humor outflow in glaucoma subjects. Multiple aspects of the present invention can reduce the formation and presence of aggregation features and structures in aqueous humor.

[0012] In other aspects, the present disclosure provides therapeutic compositions for glaucoma.

[0013] Multiple embodiments of the present invention provide devices for measuring and characterizing glaucoma aggregation features, as well as intraocular pressure and aqueous humor outflow.

[0014] Other aspects of the present disclosure include methods for diagnosing and screening glaucoma. Other embodiments include kits and reagents for performing the foregoing.

[0015] Multiple embodiments of the present invention include the following:

[0016] An aqueous pharmaceutical composition for ophthalmic use, comprising an active agent selected from cetylpyridinium chloride, polymyxin B sulfate, mycin, and heparin sodium. Mycin can be selected from neomycin, salinomycin, azithromycin, rapamycin, gentamycin, erythromycin, adriamycin, bleomycin, dactinomycin, mitomycin, plicamycin, dihydrostreptomycin, kanamycin, natamycin, rifamycin, and tobramycin. The active agent can account for 0.01 - 2% w / v of the composition. The active agent can account for 0.01 - 0.2% w / v of the composition. The composition can have a pH of about 7.3.

[0017] The above composition can reduce intraocular pressure when administered to the eye.

[0018] When administered to the eye, the above composition can reduce the ocular extracellular vesicle complex.

[0019] When administered to the eye, the above composition can effectively treat glaucoma.

[0020] The above composition may further comprise one or more of a solubilizer, a surfactant, a tonicifier, and a preservative. The solubilizer may be selected from phosphates, citric acid monohydrate, trisodium citrate, and combinations thereof. The surfactant may be selected from phospholipids, polyglycerol esters, propylene glycol esters, polyethylene glycol esters, copolymer esters, polyoxyethylene sorbitan esters, cyclodextrins, polyvinyl alcohol, polyvinylpyrrolidone, hydroxypropyl methylcellulose, poloxamers, carboxymethylcellulose, hydroxyethylcellulose, polyacrylates, and combinations thereof. The tonicifier may be selected from sodium chloride, trehalose, mannitol, sorbitol, dextrose, potassium chloride, and combinations thereof. The preservative may be selected from benzalkonium chloride, polyquaternium-1, benzododecinium bromide, sorbic acid, methylparaben, propylparaben, chlorobutanol, benzyl alcohol, phenethyl alcohol, oxychloro complex, thimerosal, sodium perborate, disodium edetate, and combinations thereof.

[0021] The above composition can be used for medical treatment, treating the human or animal body, reducing intraocular pressure in the human or animal body, reducing the ocular extracellular vesicle complex in the human or animal body, or preparing or manufacturing a medicament for preventing, ameliorating, or treating glaucoma-related diseases or conditions in a subject in need thereof.

[0022] Multiple embodiments of the present invention further contemplate a method for treating glaucoma, reducing intraocular pressure, or reducing the ocular extracellular vesicle complex in a subject in need thereof, the method comprising administering the above composition to the eye of the subject. Administration can be by injection. The ocular extracellular vesicle complex can be an aggregate of extracellular vesicles having a diameter greater than about 300 nanometers.

[0023] Brief Description of the Drawings

[0024] Figure 1 A diagram showing the anatomical structure of a normal eye (left) compared to a glaucomatous eye (right).

[0025] Figure 2 An electron micrograph of a trabecular meshwork sample (left) and a diagram of aqueous humor flow in the trabecular meshwork of the eye (right) are shown.

[0026] Figure 3 A diagram showing an embodiment of an apparatus for detecting the flow rate and pressure of a fluid composition in a channel. The channel may comprise a meshwork having properties like those of the trabecular meshwork of the eye. The channel meshwork may have a uveal meshwork portion, a corneoscleral meshwork portion, and a justacanalicular meshwork portion. The channel may be a microfluidic channel.

[0027] Figure 4 A diagram showing an embodiment of a series of test channels for detecting the effects of various compounds, compositions, and substances on the flow rate and pressure of a fluid composition in a channel.

[0028] Figure 5 A diagram showing an embodiment of an apparatus and system for detecting the flow rate and pressure of a fluid composition in a channel. The channel may comprise a meshwork having properties like those of the trabecular meshwork of the eye. Alternatively, the apparatus may utilize a reservoir of a test sample of the fluid composition. The channel may be a microfluidic channel.

[0029] Figure 6Shows inefficient EV imaging using conventional fixation techniques due to the inability of EVs to attach. Imaging was surprisingly improved using irreversible crosslinkers. (a) Representative micrographs of isolated bovine vitreous EVs loaded with 4 million, fixed on copper grids with glutaraldehyde and subsequently with UA and lead citrate solutions show a few negatively stained EVs (arrows) at low (left) magnification, and in other pictures, no EVs were observed (middle and right). Negatively stained EVs are shown with a signal (black) around the EV perimeter and a lower signal (white or gray) in the center. (b) Schematic shows that EVs in solution applied to the surface of an electron microscopy grid fail to attach. EVs are present in the discarded solution (black box), and we quantified the size and number of EVs using nanoparticle tracking analysis (NTA). (c) NTA shows the size and concentration of EVs applied to the surface of a TEM grid. (d) NTA shows the size and concentration of EVs present in the discarded solution. The numbers represent the size and number of EVs that could not attach to the surface of the TEM grid. (g) Graph comparing the amount of EVs applied to the surface of an electron microscopy grid (black bars) with the size and concentration of EVs present in the discarded solution (gray bars, n = 3). (f) Representative TEM micrographs of isolated bovine vitreous EVs after EDC-glutaraldehyde fixation, negative staining, and TEM imaging reflect significantly more EVs visualized at low (left), medium (middle), and high (right) magnifications. (g) Graph of the mean and ± standard deviation (log2) shows that significantly more EVs (350-fold) were counted per image from EDC-fixed specimens compared to Glut-fixed grids (n = 3, averaging 7 images counted for each biological replicate, *p < 0.05). (h) Representative TEM micrographs of isolated aqueous humor after EDC-glutaraldehyde fixation show robust negative staining around the EV boundary. (i) TEM images of human aqueous humor show multiple EVs present in situ without EV isolation. Scale bars are (a) 1000 nm (left), 600 nm (middle), 125 nm (right); (f) 600 nm (left), 125 nm (right); (h) 500 nm; (i) 1 μm.

[0030] Figure 7 Shows aqueous humor from healthy individuals (control, patient #1), showing a diffuse distribution of unaggregated EVs. (a-c) Representative micrographs of diluted human healthy control aqueous humor EVs (EVs not separated by ultracentrifugation, only diluted with buffer saline) fixed on copper grids with EDC, glutaraldehyde, and then stained with UA solution. Photographs were captured and recorded using transmission electron microscopy. The images show many negatively stained EVs of various sizes, and most EVs exist independently in the fluid without aggregation. One EV aggregate was observed and imaged in a few samples. Scale bars are marked on the photographs.

[0031] Figure 8 Shows aqueous humor from a healthy person (control, patient #2), showing a diffuse distribution of unaggregated EVs. (a-d) Representative micrographs of diluted human healthy control aqueous humor EVs (EVs not separated by ultracentrifugation, only diluted with buffer saline) fixed on copper grids with EDC, glutaraldehyde and then stained with UA solution. The photographs were captured and recorded using transmission electron microscopy. The images show many negatively stained EVs of various sizes. No EV aggregates larger than 2 μm were observed, nor were large EV masses. Scale bars are marked on the photographs.

[0032] Figure 9 Shows aqueous humor from POAG, and shows large glaucoma-related EV aggregates in patient #1. (a-c) Representative transmission electron micrographs showing POAG aqueous humor samples (EVs not separated by ultracentrifugation, only diluted with buffer saline) fixed on copper grids with EDC, glutaraldehyde and then stained with UA solution. The photographs were captured and recorded using transmission electron microscopy. The diluted POAG specimens show evidence of large-sized glaucoma-related EV aggregates in the fluid. The images show few free EVs observed (a-c). Scale bars are marked on the photographs.

[0033] Figure 10 Shows aqueous humor from POAG, and shows large glaucoma-related EV aggregates in patient #2. (a-c) Representative transmission electron micrographs from a second POAG sample (EVs not separated by ultracentrifugation, only diluted with buffer saline) fixed on copper grids with EDC, glutaraldehyde and then stained with UA solution. The photographs were captured and recorded using transmission electron microscopy. The second POAG aqueous humor specimen shows evidence of fairly large glaucoma-related EV aggregates and larger free EVs. Scale bars are marked on the photographs.

[0034] Figure 11 Shows that the known glaucoma treatment bimatoprost reduces the size of POAG-related EV complexes in the aqueous humor of glaucoma patients when compared to untreated glaucoma samples. (a-b) Representative transmission electron micrographs show that human aqueous humor collected from POAG patients who have never received treatment (placebo, control, buffer saline) shows evidence of extracellular matrix and glaucoma-related EV complexes in the aqueous humor. The images show large collagen-like matrices in all insets, and the size of these electron-dense structures is several micrometers. (c-d) Representative photographs show POAG aqueous humor samples treated with the glaucoma drug bimatoprost and do not show evidence of large extracellular matrix and glaucoma-related EV aggregates. At high magnification imaging, there seem to be larger unaggregated spheres. Scale bars are marked in the figures.

[0035] Figure 12 showed that treatment of aqueous humor from glaucoma patients with buffered saline had no effect on POAG-related EV complexes in a second subject diagnosed with glaucoma.( Figure 12 ) Representative transmission electron microscopy images showed that human aqueous humor collected from POAG patients who had never been treated (placebo, control, buffered saline) showed evidence of glaucoma-related EV complexes. Images showed large electron-dense glaucoma-related EV complexes in all panels. Placebo treatment had no effect on glaucoma-related EV complexes. Scale bars are marked in the figure.

[0036] Figure 13 showed that treatment with bimatoprost decreased the size of POAG-related EV complexes in the aqueous humor of a second subject diagnosed with glaucoma when compared to control-treated glaucoma samples.( Figure 13 ) Representative images showed that POAG aqueous humor from Subject #2 samples treated with bimatoprost showed disruption of large electron-dense glaucoma-related EV aggregates. The size of glaucoma-related EV aggregates was smaller when compared to control (shown in Figure 12 ). At high magnification imaging, we did not observe many aggregated EVs. Scale bars are marked in the figure.

[0037] Figure 14 showed that aqueous humor from glaucoma patients contained larger electron-dense structures that were not present in the aqueous humor of healthy controls. The figure depicts the size and number of unidentified material (which we refer to as "glaucoma-related EV aggregates") in healthy controls or POAG specimens. We obtained healthy control or POAG human aqueous humor, fixed the samples with EDC, and imaged the specimens using transmission electron microscopy. The images were analyzed, and the number of glaucoma-related EV aggregates was determined and plotted relative to each variable. In aqueous humor obtained from a single healthy control subject, the data showed that most EVs did not have glaucoma-related EV aggregates. The data showed that three subjects diagnosed with POAG had a large number of glaucoma-related EV aggregates that were several microns in size. These aggregates were not observed in healthy controls. The figure shows a significant difference in the size of glaucoma-related EV aggregates in POAG compared to healthy controls.

[0038] Figure 15It shows that the aqueous humor of glaucoma patients contains EVs that aggregate and contact each other, compared with healthy controls with fewer EVs contacting each other. The graph description shows the number of EVs contacting each other (X-axis) and the counting frequency (percentage of the total). We obtained healthy control or POAG human aqueous humor, fixed the samples with EDC, and imaged the specimens using transmission electron microscopy. The photos were analyzed, and the number of EVs contacting each other was determined for each variable. In the aqueous humor obtained from a single healthy control subject, the data showed that most EVs were free and not present in aggregates. The data showed that the aqueous humor of two subjects diagnosed with POAG contained EVs that contacted a large number of other EVs. The graph shows a significant difference in the number of EVs contacting each other in POAG compared with healthy controls.

[0039] Figure 16 It shows Figure 15 a graph of the data in. The aqueous humor of glaucoma patients contains EVs that aggregate and contact each other, compared with healthy controls with fewer EVs contacting each other.

[0040] Figure 17 It shows that extracellular vesicles are present in human aqueous humor obtained from healthy control patients, and the major population of EVs is between 100 and 200 nm. The graph description shows the EV population in human aqueous humor obtained from a single healthy control patient. The EV counting frequency is shown as a function of size. We fixed healthy control human aqueous humor and then measured the EV diameter after fixing the human aqueous humor from healthy controls (fixed with EDC and imaged using transmission electron microscopy). The data showed that healthy control human aqueous humor contained EVs. Most of the EVs in this patient had diameters between 100 - 200 nm. All visualized EVs did not contact other EVs (non-aggregated).

[0041] Figure 18 It shows that the extracellular vesicles in the aqueous humor of Subject #1 diagnosed with POAG are located within "glaucoma-related EV aggregates", showing all EVs located within glaucoma-related EV aggregates. (a) Graphical description of the EV population in human aqueous humor obtained from Subject #1 diagnosed with POAG. We fixed the aqueous humor with EDC and imaged it using transmission electron microscopy. The photos were analyzed, and the EV size and number were quantified and plotted. We observed that EVs were present within aggregates (defined as EVs contacting each other or glaucoma-related EV aggregates). The data show the number and size of individual EVs present in glaucoma-related EV aggregates (gray bars) as a function of the total number of counted EVs. A large number of EVs had sizes between 36 nm and 300 nm, and there was a relatively large number of EVs with sizes between 100 - 200 nm. In the aqueous humor of Subject #1, we did not observe free EVs (EVs not contacting each other).

[0042] Figure 19 Shows extracellular vesicles in aqueous humor obtained from human subjects diagnosed with primary open-angle glaucoma (POAG) contacting each other to form aggregates, or EVs existing as free EVs (unaggregated). (a-b) Illustration of the EV population in human aqueous humor obtained from a single subject diagnosed with POAG. We fixed the aqueous humor with EDC fixation and imaged it using transmission electron microscopy. The photographs were analyzed, and the size and number of EVs were quantified. We observed EVs present within aggregates (defined as EVs contacting each other) or free EVs (EVs not contacting each other). The data show a population of EVs not contacting each other (unaggregated EVs). A large number of EVs are between 100 - 500 nm, and some are larger in size.

[0043] Figure 20 Shows a graph representing the size and frequency of EVs within "glaucoma-related EV aggregates" of patients who are Figure 19 shown contacting other EVs and located within. A large number of EVs are between 36 nm and 300 nm, and a few larger free EVs were observed.

[0044] Figure 21 Shows extracellular vesicles in aqueous humor of a single subject diagnosed with POAG located within "glaucoma-related EV aggregates", with a large number of EVs present within the glaucoma-related EV aggregates. (a) Illustration of the EV population in human aqueous humor obtained from a single subject diagnosed with POAG. We fixed the aqueous humor with EDC fixation and imaged it using transmission electron microscopy. The photographs were analyzed, and the size and number of EVs were quantified. We observed EVs present within aggregates (defined as EVs contacting each other) or free EVs (EVs not contacting each other). The data show the number and size of individual EVs present within the glaucoma-related EV aggregates (black bars) or the number and size of unaggregated EVs (free EVs, black and white striped bars) as a function of the total number of EVs counted. A large number of EVs are located within the glaucoma-related EV aggregates. A large number of EVs within the aggregates have a diameter size of 100 - 300 nm. Larger EVs have a higher population, showing a large number of free EVs (unaggregated EVs) in the larger size range.

[0045] Figure 22Extracellular vesicles from two separate glaucoma patients showed similar sizes and numbers of EVs located within "glaucoma-associated EV aggregates". (a) Schematic illustration of EV populations in human aqueous humor obtained from two subjects diagnosed with POAG. We fixed the aqueous humor of each subject with EDC and imaged the specimens using transmission electron microscopy. The photographs were analyzed, and the sizes and numbers of EVs were quantified. We observed EVs present within aggregates (defined as EVs in contact with each other). The data show the sizes and numbers of EVs present within glaucoma-associated EV aggregates for subject #1 (gray bars) or subject #2 (black bars). The data were normalized and show that the two samples had similar EV distributions present within glaucoma-associated EV aggregates.

[0046] Figure 23 The sizes and frequencies of extracellular vesicles in the aqueous humor of human subjects diagnosed with POAG were shown to be different compared to those of healthy control subjects. (a) Schematic illustration of EV populations in human aqueous humor obtained from subjects diagnosed with POAG and normal healthy controls. We fixed the aqueous humor of each subject with EDC and imaged the specimens using transmission electron microscopy. The photographs were analyzed, and the sizes and numbers of EVs were quantified. We observed that the sizes and frequencies of free EVs (non-aggregates, defined as EVs not in contact with each other) were different compared to healthy controls. The data show the sizes and numbers of free EVs from healthy controls (white bars) or POAG from subject #2 (black bars). We observed that the free EVs present in the aqueous humor of patient #2 were significantly larger EVs compared to healthy controls.

[0047] Figure 24 The sizes and frequencies of extracellular vesicle POAG aggregates were shown to be similar to those of healthy human subjects. (a) Schematic illustration of EV populations in human aqueous humor obtained from healthy controls (free EVs) or POAG subjects (aggregated EVs). We fixed the aqueous humor of each subject with EDC and imaged the specimens using transmission electron microscopy. The photographs were analyzed, and the sizes and numbers of EVs were quantified. We observed that the sizes and frequencies of free EVs (non-aggregates, defined as EVs not in contact with each other) were similar compared to EVs present within aggregates in two separate POAG subjects. The data show the sizes and numbers of free EVs from healthy controls (white bars), aggregated EVs from POAG subject #1 (gray bars), and aggregated EVs from POAG subject #2 (striped bars).

[0048] Figure 25It is shown that, compared with the control, the agent cetylpridinium chloride reduced the intraocular pressure (IOP) in a glaucoma model. The agent was tested by controlling the flow and measuring the relative IOP in a microfluidic device. The agent was compared with a placebo (buffered saline) by preparing each sample in bovine vitreous humor (BVH) and pre-incubating at 37 °C for 24 hours. The time points of injection into the device are indicated by the arrow and the letter "a". Reference Figure 25 , after injection of the placebo sample, the IOP of the placebo (dashed line) increased significantly. The IOP steadily rose to a maximum pressure of approximately 64 mmHg. In contrast, the IOP (solid line) after injection of the agent cetylpridinium chloride - BVH sample was significantly lower than that of the placebo, and this difference persisted. The results indicate that the agent cetylpridinium chloride was unexpectedly effective in reducing the IOP in a glaucoma model.

[0049] Figure 26 It is shown that, compared with the control, the agent polymyxin B reduced the intraocular pressure (IOP) in a glaucoma model. The agent was tested by controlling the flow and measuring the relative IOP in a microfluidic device. The agent was compared with a placebo (buffered saline) by preparing each sample in bovine vitreous humor (BVH) and pre-incubating at 37 °C for 24 hours. The time points of injection into the device are indicated by the arrow and the letter "a". Reference Figure 26 , after injection of the placebo sample, the IOP of the placebo (dashed line) increased significantly. The IOP steadily rose to a maximum pressure of approximately 250 mmHg. In contrast, the IOP (solid line) after injection of the agent polymyxin B was 78% lower than that of the placebo, and this difference persisted. The results indicate that the agent polymyxin B was unexpectedly effective in reducing the IOP in a glaucoma model.

[0050] Figure 27 It is shown that, compared with the control, the agent neomycin reduced the intraocular pressure (IOP) in a glaucoma model. The agent was tested by controlling the flow and measuring the relative IOP in a microfluidic device. The agent was compared with a placebo (buffered saline) by preparing each sample in bovine vitreous humor (BVH) and pre-incubating at 37 °C for 24 hours. The time points of injection into the device are indicated by the arrow and the letter "a". Reference Figure 27 , after injection of the placebo sample, the IOP of the placebo (dashed line) increased significantly. The IOP steadily rose to a maximum pressure of approximately 64 mmHg. In contrast, the IOP (solid line) after injection of the agent neomycin was 72% lower than that of the placebo, and this difference persisted. The results indicate that the agent neomycin was unexpectedly effective in reducing the IOP in a glaucoma model.

[0051] Figure 28It is shown that compared with the control, the agent sodium heparin reduces intraocular pressure (IOP) in a glaucoma model. The agent was tested by controlling the flow and measuring the relative IOP in a microfluidic device. The agent was compared with a placebo (buffered saline) by preparing each sample in bovine vitreous humor (BVH) and pre-incubating at 37 °C for 24 hours. The time points of injection into the device are indicated by the arrow and the letter "a". Reference Figure 28 , after injection of the placebo sample, the IOP of the placebo (dashed line) increased significantly. The IOP steadily rose to a maximum pressure of approximately 67 mmHg. In contrast, the IOP (solid line) after injection of the agent sodium heparin was 32% lower than that of the placebo, and this difference persisted. The results indicate that the agent sodium heparin is unexpectedly effective in reducing IOP in a glaucoma model.

[0052] Figure 29 It is shown that the compound sodium dodecyl sulfate is a negative control for intraocular pressure (IOP) in a glaucoma model. The compound was tested by controlling the flow and measuring the relative IOP in the microfluidics of a microfluidic device. The compound was compared with a placebo (buffered saline) by preparing each sample in bovine vitreous humor (BVH) and pre-incubating at 37 °C for 24 hours. The time points of injection into the device are indicated by the arrow and the letter "a". Reference Figure 29 , after injection of the placebo sample, the IOP of the placebo (dashed line) increased significantly. The IOP steadily rose to a maximum pressure of approximately 60 mmHg. However, the IOP (solid line) after injection of sodium dodecyl sulfate was significantly higher than that of the placebo. The results indicate that sodium dodecyl sulfate is a negative control that does not reduce IOP in a glaucoma model. DETAILED DESCRIPTION OF THE INVENTION

[0054] The present invention provides methods, compositions, devices, kits, and reagents for detecting, identifying, and treating glaucoma disease. Multiple embodiments of the present invention utilize the ultrastructural features of aqueous humor as a guide and marker for glaucoma treatment methods.

[0055] In some aspects, the present invention provides methods and compositions for reducing intraocular pressure and increasing aqueous humor outflow in glaucoma subjects. Multiple aspects of the present invention can reduce the formation and presence of aggregated features and structures in aqueous humor.

[0056] In other aspects, the present disclosure provides therapeutic compositions for glaucoma.

[0057] Multiple embodiments of the present invention provide devices for measuring and characterizing glaucoma aggregated features, as well as intraocular pressure and aqueous humor outflow.

[0058] Other aspects of the present disclosure include methods for diagnosing and screening glaucoma. Other embodiments include kits and reagents for performing the foregoing operations.

[0059] Multiple embodiments of the present invention can provide glaucoma diagnosis based on unique and reliable ultrastructural biomarkers identified herein. The ultrastructural components can block the trabecular meshwork, increase IOP, and over time, increase the resistance to aqueous humor outflow in the eye, leading to elevated intraocular pressure and ultimately vision loss. The ultrastructural components in the aqueous humor of patients with POAG can be reflected in EV aggregates formed together as large EV complexes. The size of the EV complexes can be several micrometers and is a glaucoma-related EV complex. The EV complexes can be present in samples from glaucoma patients and are large enough to block the trabecular meshwork.

[0060] Multiple embodiments of the present invention can provide compositions and methods for purifying and / or synthesizing EV complexes of the ultrastructural components for therapeutic and biological methods.

[0061] Multiple embodiments of the present invention can provide therapeutic agents and treatments for POAG and compositions and methods for testing aqueous humor specimens from POAG.

[0062] In some embodiments, glaucoma-related EV complexes can be reduced by dissociation and other means.

[0063] In additional embodiments, the compositions and methods of the present invention can reduce intraocular pressure and / or increase aqueous humor outflow.

[0064] Multiple embodiments of the present invention further contemplate methods for treating glaucoma.

[0065] In certain aspects, glaucoma disease can be treated by administering a surfactant that affects EV complexes. The surfactant can be used to improve, alleviate, inhibit, mitigate, delay, and / or prevent at least one symptom or condition of glaucoma.

[0066] The eye can be regarded as a closed chamber. ( Figure 1 , left) IOP can be determined by the rate of aqueous humor formation and the rate of fluid drainage. ( Figure 1 , right) Typically, reduced aqueous humor outflow in glaucoma may be associated with elevated IOP. Aqueous humor outflow may be associated with elevated IOP and glaucomatous visual damage. Aqueous humor leaves the eye through two pathways: the trabecular meshwork and, to a lesser extent, the uveoscleral outflow. Figure 2 )

[0067] Abnormal aqueous humor outflow can cause elevated IOP. The trabecular meshwork (TM) can be the main site of outflow. The TM is a filter-like tissue composed of a series of fenestrated strips that allow aqueous humor to flow through Schlemm's canal and out of the anterior chamber. The main function of the TM is to allow aqueous humor to leave the eye and establish IOP.

[0068] The juxtacanalicular tissue (JCT), or cribriform area, abuts the Schlemm's canal, which is a TM area that may be associated with establishing IOP. The site with the greatest resistance to aqueous humor outflow may be the JCT tissue, which measures approximately 2 - 20 μm. The JCT consists of a loosely arranged extracellular matrix (ECM) in which cells are embedded.

[0069] Abnormal regulation of aqueous humor flow through the TM may be associated with elevated IOP. The ECM of the TM may be a barrier that can sequester the outflow of ocular fluid.

[0070] The ultrastructural features or compositions in the aqueous humor of glaucoma patients are physically larger than the fenestrae of the JCT exit or other TM tissue fenestrae and can block the TM.

[0071] The ultrastructural features or compositions in the aqueous humor may include extracellular vesicle (EV)-based structures. EVs are transport nanovesicles associated with intercellular communication through the transfer of biomolecules (such as proteins, lipids, and nucleic acids) from one cell to another.

[0072] Typically, various cell types secrete EVs into fluids such as blood, cerebrospinal fluid, and urine. Examples include exomeres of approximately 35 nm, exosomes of approximately 40 - 100 nm, larger micro-vesicles of approximately 100 - 1000 nm, and apoptotic bodies of approximately 1 - 5 μm. EVs may be associated with the pathophysiology of diseases.

[0073] In some embodiments of the present invention, EV-based ultrastructural features and compositions are used to characterize ocular fluid.

[0074] In other embodiments, EV-based ultrastructural features and compositions can be used in a device for determining IOP. The EV-based ultrastructural features and compositions can be monitored to determine the therapeutic effect of reducing IOP. The EV-based ultrastructural features and compositions can be monitored as biomarkers for determining the therapeutic effect of reducing IOP.

[0075] In additional embodiments, EV-based ultrastructural features and compositions can be used in a device for measuring the outflow of ocular fluid. The EV-based ultrastructural features and compositions can be monitored to determine the therapeutic effect of increasing the outflow of ocular fluid. The EV-based ultrastructural features and compositions can be monitored as biomarkers for determining the therapeutic effect of increasing the outflow of ocular fluid.

[0076] In other embodiments, EV-based ultrastructural features and compositions are used to reduce the formation and presence of aggregated features, structures, and particles in ocular fluid.

[0077] In certain embodiments, EV-based ultrastructural features and compositions are used in devices for detecting ocular conditions and parameters.

[0078] In additional embodiments, EV-based ultrastructural features and compositions are used for identifying glaucoma in a subject.

[0079] In other embodiments, EV-based ultrastructural features and compositions are used in methods, kits, and reagents for glaucoma.

[0080] Without wishing to be bound by any particular theory, EV-based ultrastructural features and compositions in glaucoma can have a large structure, thereby blocking the TM and / or other outflows.

[0081] Glaucoma conditions that can be treated with the methods and compositions described herein, referred to herein as "glaucoma", include but are not limited to preglaucoma open angle with borderline findings, anatomical narrow angle primary angle closure suspect, steroid responder, ocular hypertension, primary angle closure without glaucoma damage (PAS or high IOP without optic nerve or visual field defect), unspecified open-angle glaucoma, primary open-angle glaucoma, chronic simple glaucoma, low-tension glaucoma, pigmentary glaucoma, capsular glaucoma with pseudo-exfoliation of lens, residual stage of open-angle glaucoma, unspecified primary angle-closure glaucoma, acute angle-closure glaucoma attack, chronic angle-closure glaucoma, intermittent angle-closure glaucoma, residual stage of angle-closure glaucoma, glaucoma secondary to ocular trauma, glaucoma secondary to ocular inflammation, glaucoma secondary to other ocular conditions,The other eye diseases include retinal vascular occlusion, diabetes type 1 complicated, diabetes type 2 complicated, lens diseases, intraocular lens diseases, diseases after other eye symptoms, neoplasms, benign neoplasms or malignant tumors. Also included are glaucoma secondary to drugs, glaucoma with increased episcleral venous pressure, hypersecretion glaucoma, aqueous misdirection malignant glaucoma, glaucoma in other classified diseases, congenital glaucoma, axenfeld’s anomaly, buphthalmos, glaucoma of childhood, glaucoma of newborn, hydrophthalmos, keratoglobus, congenital glaucoma macrocornea with glaucoma, macrophthalmos in congenital glaucoma, megalocornea with glaucoma, and absolute glaucoma. Also included are the adverse effects of ophthalmic drugs and preparations, acute follicular conjunctivitis, the adverse effects of carbonic anhydrase inhibitors, and the adverse effects of under-dosed ophthalmic drugs and preparations.,

[0082] In some embodiments, the compositions of the present disclosure can be administered systemically. Systemic administration can be achieved by intravenous administration, oral administration, intra-arterial administration, inhalation, intranasal administration, intraperitoneal administration, intracavitary administration, subcutaneous administration, intra-articular administration, intrathecal administration, transdural administration, transdermal administration, submucosal administration, sublingual administration, enteral administration, parenteral administration, percutaneous administration, periarticular administration, or intraventricular administration.

[0083] In other embodiments, the compositions of the present disclosure can be administered topically. The compositions can be topically administered to ocular tissue. As used herein, the term ocular tissue refers to the eye, including tissues within the sclera (e.g., the retina) and tissues outside the sclera (e.g., extraocular muscles within the orbit). Ocular tissue also includes tissues that are neurologically connected to but distinct from the eye, such as the optic nerve, the lateral geniculate nucleus, and the visual cortex. Topical administration to ocular tissue can be achieved by intravitreal administration. Intravitreal administration can be performed by intracameral administration, intravitreal injection, or subretinal injection.

[0084] In additional embodiments, topical administration to ocular tissue can be achieved by periocular administration. Periocular administration can be performed by subconjunctival injection, sub-Tenon’s injection, direct periocular injection, or depot periocular injection.

[0085] A therapeutically effective amount of the composition can be administered to a subject. A therapeutically effective amount can be an amount effective to ameliorate, relieve, inhibit, mitigate, delay, and / or prevent at least one symptom or condition of the condition being treated.

[0086] In certain embodiments, a therapeutically effective amount can be an amount effective to ameliorate the ocular condition being treated. The dosage can be determined based on various parameters, particularly the severity, age, and weight of the patient to be treated; the route of administration; and the desired regimen. A physician will be able to determine the route of administration and dosage required for any particular patient. The dosage can vary depending on the relative potency of the composition being administered and can generally be estimated based on the half-maximal effective concentration (EC50) found to be effective in in vitro and in vivo models.

[0087] Extracellular Vesicles and Aggregates in Glaucoma

[0088] Multiple embodiments of the present invention provide methods for detecting EVs in biological fluids. In certain methods, crosslinkers can be used to provide robust imaging of EV ultrastructure, for example, by electron microscopy. In other methods, glutaraldehyde-alternative cross-linkers can be used.

[0089] Additional embodiments of the present invention contemplate detecting and characterizing EV complexes in glaucoma. EV complexes in glaucoma can block the TM or JCT and inhibit the aqueous humor outflow pathway of the eye.

[0090] In certain aspects, EVs in glaucoma can aggregate as EV complexes. The size or diameter of glaucoma-associated EV complexes can be up to several micrometers.

[0091] In glaucoma, EV complexes may be an ultrastructural feature of the disease. This ultrastructural feature can be a target for characterizing glaucoma. EV complexes can be used to detect treatment parameters and methods for glaucoma. In some embodiments, EV complexes can be used for the diagnosis, prognosis, and / or screening of glaucoma compositions. EV complexes can also be used in devices for determining therapeutic compositions, dosages, and regimens.

[0092] As used herein, the term diameter refers to the longest linear dimension of an irregularly shaped particle (such as an extracellular vesicle complex). For regularly shaped particles (such as spherical vesicles), the term diameter has its usual meaning, i.e., the line segment whose endpoints on the sphere pass through the center.

[0093] In some aspects, the present disclosure provides compositions of purified EV complexes from glaucoma. The purified EV complexes from glaucoma can be used in devices for assaying and detecting changes in EV ultrastructural components that may be associated with intraocular pressure and aqueous humor outflow. The present invention provides devices comprising purified EV complexes, which can be used to characterize and measure ocular obstruction and aqueous humor outflow. The purified EV complexes from glaucoma can be used in devices for screening the effects of therapeutic agents on ocular EV ultrastructural components.

[0094] In other aspects, the present disclosure provides compositions of synthetic EV complexes for characterizing glaucoma. The synthetic EV complexes for characterizing glaucoma can be used in devices for assaying and detecting changes in EV ultrastructural components that may be associated with intraocular pressure and aqueous humor outflow. The present invention provides devices comprising synthetic EV complexes, which can be used to characterize and measure ocular obstruction and aqueous humor outflow. The synthetic EV complexes from glaucoma can be used in devices for screening the effects of therapeutic agents on ocular EV ultrastructural components.

[0095] EV ultrastructural components (such as EV complexes) can consist of complexes or aggregates of extracellular vesicles. Examples of extracellular vesicles include exophers, exosomes, multivesicular bodies, intraluminal vesicles (ILVs), multivesicular endosomes (MVE), oncosomes, microvesicles, apoptotic bodies, and vesicles derived from endosomes or the plasma membrane.

[0096] The complexes or aggregates of extracellular vesicles can be protein-EV structures having a micron diameter or a diameter greater than about 1 micron.

[0097] The size of the extracellular vesicle aggregates can be from about 360 to about 21,000 nanometers (nm).

[0098] For example, the diameter of exosome particles can be about 35 nm, the diameter of exosomes can be about 40 - 100 nm, the diameter of microvesicles can be about 100 - 1000 nm, and the diameter of apoptotic bodies can be about 1 - 5 microns.

[0099] Complexes or aggregates of extracellular vesicles can contain 10, 20, 30, 40, 50, 100, 200, 500 or more extracellular vesicles.

[0100] For example, healthy subjects can have free EVs that are non - aggregated EVs with a diameter of about 100 - 200 nm, which are mainly exosomes, and some vesicles in the micron size range. The aqueous humor of healthy subjects may not have EV aggregates or EV ultrastructural features with a diameter greater than 0.4 - 20 microns.

[0101] For example, in glaucoma, the diameter of EV aggregates in a subject can be greater than 0.4 - 20 microns. Healthy subjects can have small EV aggregates of about 36 - 300 nm, which are mainly exosome particles, and some microvesicles. The amount of free EVs in glaucoma subjects may be reduced, or the remaining free EVs are few. The size of free EVs in glaucoma may be larger than that in healthy subjects. EV aggregates in glaucoma can consist of exosome particles, exosomes, microvesicles, and / or apoptotic bodies, as well as other types of vesicles or bodies.

[0102] Devices for Glaucoma Aggregation Features

[0103] The device of the present invention can be used to characterize the activity of bioactive agents against glaucoma. The device of the present invention can be used to detect or characterize eye conditions or parameters in a model system or patient pathology.

[0104] The bioactive agent may be able to provide a therapeutic benefit, especially in glaucoma. In some embodiments, the bioactive agent can be a known drug that is effective in treating eye diseases.

[0105] In some aspects, the fluid composition in the device of the present invention can be analyzed by various techniques. For example, the fluid composition can be analyzed by imaging techniques.

[0106] Examples of imaging techniques include electron microscopy, stereomicroscopy, wide - field microscopy, polarizing microscopy, phase - contrast microscopy, multiphoton microscopy, differential interference contrast microscopy, fluorescence microscopy, laser scanning confocal microscopy, multiphoton excitation microscopy, x - ray microscopy, and ultrasonic microscopy.

[0107] Examples of imaging techniques include positron emission tomography, computed tomography, and magnetic resonance imaging.

[0108] Examples of measurement techniques include colorimetric assays, chemiluminescence assays, spectrophotometry, and light scattering.

[0109] In some embodiments, the present invention can provide an apparatus for measuring the pressure and flow rate of a fluid composition.( Figure 3 ) The apparatus can have a channel that has an inlet at a first end and an outlet at a second end, wherein the inlet and the outlet are in fluid communication. The apparatus can have a reticular structure composition located in the channel to provide flow resistance. The reticular structure composition can have any one or more or all of the following parts: the uveal reticular structure, the corneoscleral reticular structure, and the juxtacanalicular reticular structure.

[0110] In some embodiments, the reticular structure composition can be composed of glass beads, microbeads, magnetic beads, gel particles, dextran particles, or polymer particles. The reticular structure composition can also be composed of glass fibers, polymer fibers, inorganic fibers, organic fibers, or metal fibers.

[0111] In certain embodiments, the fenestrae of the uveal reticular structure can be about 25 microns. The fenestrae of the corneoscleral reticular structure can be about 2 - 15 microns. The fenestrae of the juxtacanalicular reticular structure can be about 1 to 4 microns or less.

[0112] The apparatus can further include a fluid reservoir for containing the fluid composition such that the fluid reservoir is in fluid communication with the inlet of the channel to introduce the fluid composition into the inlet of the channel.

[0113] The apparatus of the present disclosure can have a pressure source for applying pressure to the fluid composition in the fluid reservoir to introduce the fluid composition into the inlet of the channel.

[0114] The apparatus of the present invention can have a flow sensor in fluid communication with the fluid composition for measuring the flow rate and pressure of the fluid composition at the inlet of the channel and transmitting the flow rate and pressure to a processor.

[0115] Signals and data from the apparatus can be received by the processor. The processor can display the flow rate and pressure. A memory or medium can store instructions or files, such as a machine-readable storage medium. The machine-readable storage medium can be non-transitory.

[0116] The processor of the present disclosure can be a general-purpose or special-purpose computer. The processor can execute instructions stored in a machine-readable storage device or medium. The processor can include an integrated circuit chip, a microprocessor, a controller, a digital signal processor, any of which can be used to receive and / or transmit data and execute the stored instructions. The processor can also transform data and / or store data in a memory, a medium, or a file. The processor can receive and execute instructions, which can include one or more steps of performing the method of the present invention. The device of the present invention can include one or more non-transitory machine-readable storage media, one or more processors, one or more storage devices, and / or one or more user interfaces. The processor can have an integrated display for displaying data or transformed data.

[0117] In some aspects, the device can have microfluidic channels. One or more channels can also be arranged in a microfluidic chip.

[0118] The device of the present disclosure can include one or more detectors for analyzing the fluid composition within the channel or at the entrance of the channel or exiting the outlet. One or more detectors can also be arranged to detect the fluid composition within the channel.

[0119] The device of the present invention can include a reticular structure composition containing extracellular vesicles or extracellular vesicle complexes. The EV complex for the reticular structure composition can be purified from glaucoma ocular fluid, aqueous humor, or vitreous humor. The ocular fluid can be of animal or clinical origin. The EV complex for the reticular structure composition can be composed of extracellular vesicles and can have a diameter of about 360 to about 21,000 nanometers.

[0120] In certain embodiments, the EV complex for the reticular structure composition can include a fixing agent, a stabilizing component, or a cross-linking component that can transform the structure into a stable and uniform composition.

[0121] Examples of stabilizing components include fixing agents as described herein, cross-linking compounds as described herein, organic solvents, polypeptides, and pharmaceutically acceptable organic salts.

[0122] Examples of salts include ammonium salts, alkali metal salts (including sodium salts, lithium salts, and potassium salts), alkaline earth metal salts (including calcium salts and magnesium salts), salts with organic bases (e.g., organic amines, such as benzathines, dicyclohexylamine, hydrabamines formed with N,N-bis(dehydroabietyl)ethylenediamine), N-methyl-D-glucamine, N-methyl-D-glucamide, tert-butylamine), and salts with amino acids (including arginine and lysine).

[0123] Examples of salts include acetate, adipate, alginate, ascorbate, aspartate, benzoate, benzenesulfonate, bisulfate, borate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, chloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, mesylate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, pectinate, persulfate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptanoate, hexanoate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, sulfonate, tartrate, thiocyanate, toluenesulfonate, and undecanoate.

[0124] The crosslinked extracellular vesicle complex can be crosslinked reversibly or irreversibly.

[0125] The crosslinked extracellular vesicle can be crosslinked reversibly or irreversibly.

[0126] In some embodiments, the device of the present invention may comprise an EV complex reticular structure composition, which can be used to identify or screen active agents for the effect of reducing IOP and / or increasing aqueous humor outflow. The EV complex used in the reticular structure composition may include a drug delivery excipient. The EV complex reticular structure composition for the device can be a synthetic EV complex or a purified EV complex.

[0127] Figure 4 An embodiment of the arrangement of the channels of the present invention is shown.

[0128] Figure 5 An embodiment of the device of the present invention is shown.

[0129] In further embodiments, the device of the present invention can be used to measure the amount or level of EV complex in a test sample. Measuring the amount or level of EV complex in a test sample can provide a diagnostic marker level for the test sample. The device of the present invention can be used to identify glaucoma or pre-glaucoma in a subject.

[0130] In other embodiments, the device of the present invention can be used to measure a pressure, which can be related to the amount or level of EV complex in a test sample. The pressure value in the channel can be directly related to the amount or level of EV complex in the test sample.

[0131] In certain embodiments, the devices of the present invention can be used to measure a determination value that can be related to the amount or level of EV complexes in a test sample. The determination value of the composition in the channel can be directly related to the amount or level of EV complexes in the test sample. For example, the determination can be a colorimetric determination, a chemiluminescent determination, a spectrophotometric determination, or a light scattering determination.

[0132] In some aspects, an aqueous humor sample from a subject can be provided and analyzed for the amount of glaucoma extracellular vesicle complexes. Based on the amount exceeding a reference value, the subject can be identified as having glaucoma or pre-glaucoma. The reference value can be the amount or level of glaucoma extracellular vesicle complexes in a reference population of healthy individuals. The subject can be diagnosed as having glaucoma or pre-glaucoma. Subsequent test samples from the subject can be used to monitor whether the amount or level of glaucoma extracellular vesicle complexes in the subsequent test sample exceeds or does not exceed that in the previous test sample, which may be related to reducing IOP and / or increasing aqueous humor outflow in the subject.

[0133] In certain embodiments, the amount or level of glaucoma extracellular vesicle complexes can include one or more of the number, size, density, morphology, and spatial distribution of the extracellular vesicle complexes.

[0134] In some embodiments, the reference value can be the amount or level of glaucoma extracellular vesicle complexes in a reference population of healthy individuals. The reference value can be the average value in a sample from the reference population.

[0135] Glaucoma can be detected in a subject, wherein a test sample from the subject contains an amount or level of glaucoma extracellular vesicle complexes that exceeds the glaucoma reference value.

[0136] In certain embodiments, the glaucoma reference value can be that the number of extracellular vesicle complexes (which contain 10 or more aggregated extracellular vesicles) in each sample is zero. In certain embodiments, the glaucoma reference value can be that the number of extracellular vesicle complexes (which contain 10 or more aggregated extracellular vesicles) in each sample is 10. In certain embodiments, the glaucoma reference value can be that the number of extracellular vesicle complexes (which contain 10 or more aggregated extracellular vesicles) in each sample is 50. In certain embodiments, the glaucoma reference value can be that the number of extracellular vesicle complexes (which contain 10 or more aggregated extracellular vesicles) in each sample is 100.

[0137] In certain embodiments, the glaucoma reference value can be zero for the number of extracellular vesicle complexes (which contain 10 or more aggregated extracellular vesicles, where the complex is greater than 360 nm) in each sample. In certain embodiments, the glaucoma reference value can be 10 for the number of extracellular vesicle complexes (which contain 10 or more aggregated extracellular vesicles, where the complex is greater than 360 nm) in each sample. In certain embodiments, the glaucoma reference value can be 50 for the number of extracellular vesicle complexes (which contain 10 or more aggregated extracellular vesicles, where the complex is greater than 360 nm) in each sample. In certain embodiments, the glaucoma reference value can be 100 for the number of extracellular vesicle complexes (which contain 10 or more aggregated extracellular vesicles, where the complex is greater than 360 nm) in each sample.

[0138] In certain embodiments, the glaucoma reference value can be 10 for the number of extracellular vesicle complexes greater than 360 nm in each sample. In certain embodiments, the glaucoma reference value can be 50 for the number of extracellular vesicle complexes greater than 360 nm in each sample. In certain embodiments, the glaucoma reference value can be 100 for the number of extracellular vesicle complexes greater than 360 nm in each sample. In certain embodiments, the glaucoma reference value can be 200 for the number of extracellular vesicle complexes greater than 360 nm in each sample.

[0139] In another aspect, the reticular structure composition in the device of the present invention can be the anterior half or part of the eye of an animal with a lens, where the TM of the eye is oriented between the inlet and outlet of the channel.

[0140] Method

[0141] The extracellular vesicles in the aqueous humor of patients with POAG can be compared with a healthy control population. The ultrastructure of EV complexes in the aqueous humor of subjects with ocular pathologies (such as glaucoma) can be compared with healthy controls (such as subjects without ocular pathologies other than cataracts). The level of EV complexes in the aqueous humor of glaucoma subjects may exceed that of healthy subjects.

[0142] Such EVs in glaucoma aqueous humor may be larger than those of healthy subjects. In some embodiments, the level of larger EV structures can be reduced to unclog the aqueous humor network structure and increase aqueous humor outflow. The EV complexes in glaucoma aqueous humor may be larger than any EVs in healthy subjects. In some embodiments, the level of EV complexes can be reduced to unclog the aqueous humor network structure and increase aqueous humor outflow.

[0143] In certain embodiments, EVs in the aqueous humor of healthy individuals can be dispersed and evenly distributed without aggregation. Healthy control aqueous humor may contain EVs that are not aggregated and are dispersed.

[0144] Glaucoma EV complexes may be larger than the EVs observed in healthy controls and may block the trabecular meshwork.

[0145] In some embodiments, purified EV complexes can be obtained from the aqueous humor in POAG. The size of the purified EV complexes can be several micrometers. Glaucoma EV complexes may be larger than the openings of the JCT (1 to 4 μm, or up to 2 to 20 μm), which can be large enough to block the juxtacanalicular tissue. The EV complexes in POAG can be used to block the trabecular meshwork and reduce aqueous humor outflow. In certain embodiments, the level of the EV complexes can be reduced to unclog the trabecular meshwork and increase aqueous humor outflow.

[0146] For example, the EV complexes can be contacted with a composition comprising an active agent such as bimatoprost. In these embodiments, the level of the EV complexes can be reduced to unclog the trabecular meshwork and increase aqueous humor outflow.

[0147] In certain aspects, the level or amount of glaucoma EV complexes in POAG subjects can be reduced by administering an active agent such as bimatoprost.

[0148] In other embodiments, the size of the purified EV complexes can be from about 360 nm to 21,000 nm. The purified EV complexes may be significantly larger than any particles found in healthy aqueous humor.

[0149] In additional embodiments, the size of the purified EV complexes can be from about 360 nm to about 21,000 nm, or 360 nm to about 10,000 nm, or 360 nm to about 5,000 nm, or 360 nm to about 3,000 nm, or 360 nm to about 2,000 nm, or 360 nm to about 1,000 nm.

[0150] In the purified EV complexes, the number of EVs in contact with each other can be from about 5 to about 300, or 10 to 300, or 10 to 200, or 10 to 100, or 10 to 50, or 10 to 40, or 10 to 20.

[0151] In the purified EV complexes, the number of EVs in contact with each other can be 20 to 300, or 30 to 300, or 40 to 300, or 50 to 300.

[0152] In the purified EV complex, the number of EVs in contact with each other can be 20 to 200, or 20 to 100, or 30 to 200, or 30 to 100, or 40 to 200, or 40 to 100, or 50 to 200, or 50 to 100.

[0153] In some embodiments, the purified EV complex can provide particles sized for the uveal meshwork. The purified EV complex for the uveal meshwork can be from about 10,000 nm to about 25,000 nm, or 15,000 nm to 25,000 nm, or 20,000 nm to 25,000 nm.

[0154] In other embodiments, the purified EV complex can provide particles sized for the corneoscleral meshwork. The purified EV complex for the corneoscleral meshwork can be from about 1,000 nm to about 15,000 nm, or 2,000 nm to 10,000 nm, or 2,000 nm to 5,000 nm.

[0155] In further embodiments, the purified EV complex can provide particles sized for the juxtacanalicular meshwork. The purified EV complex for the juxtacanalicular meshwork can be from about 360 nm to about 1,000 nm, or 360 nm to 2,000 nm, or 260 nm to 3,000 nm, or 1,000 nm to 3,000 nm.

[0156] The TM region, which may be associated with establishing IOP, is adjacent to Schlemm's canal and is referred to as the juxtacanalicular tissue (JCT) or cribriform area.

[0157] The EV complex can be synthesized by contacting EVs with a reagent to form a larger structure. The reagent can include a fixative, a crosslinker, and a buffer suspension. The synthesized EV complex may consist of many EVs in contact with each other to form aggregates.

[0158] In the synthesized EV complex, the number of EVs in contact with each other can be from about 5 to about 300, or 10 to 300, or 10 to 200, or 10 to 100, or 10 to 50, or 10 to 40, or 10 to 20.

[0159] In the synthesized EV complex, the number of EVs in contact with each other can be 20 to 300, or 30 to 300, or 40 to 300, or 50 to 300.

[0160] In the synthesized EV complex, the number of EVs in contact with each other can be 20 to 200, or 20 to 100, or 30 to 200, or 30 to 100, or 40 to 200, or 40 to 100, or 50 to 200, or 50 to 100.

[0161] In additional embodiments, the size of the synthetic EV complex can be from about 360 nm to about 25,000 nm, or 360 nm to 21,000 nm, or 360 nm to about 10,000 nm, or 360 nm to about 5,000 nm, or 360 nm to about 3,000 nm, or 360 nm to about 2,000 nm, or 360 nm to about 1,000 nm.

[0162] In some embodiments, the synthetic EV complex can provide particles sized for the uveal meshwork. The synthetic EV complex for the uveal meshwork can be from about 10,000 nm to about 25,000 nm, or 15,000 nm to 25,000 nm, or 20,000 nm to 25,000 nm.

[0163] In other embodiments, the synthetic EV complex can provide particles sized for the corneoscleral meshwork. The synthetic EV complex for the corneoscleral meshwork can be from about 1,000 nm to about 15,000 nm, or 2,000 nm to 10,000 nm, or 2,000 nm to 5,000 nm.

[0164] In additional embodiments, the synthetic EV complex can provide particles sized for the juxtatubular meshwork. The synthetic EV complex for the juxtatubular meshwork can be from about 360 nm to about 1,000 nm, or 360 nm to 2,000 nm, or 260 nm to 3,000 nm, or 1,000 nm to 3,000 nm.

[0165] Synthesis and Purification of Extracellular Vesicles and EV Complexes

[0166] In some embodiments, extracellular vesicles (including exosomes) and EV complexes can be synthesized, separated, and / or purified by size exclusion chromatography or gel filtration chromatography.

[0167] In certain embodiments, extracellular vesicles (including exosomes) and EV complexes can be synthesized, separated, and / or purified by centrifugation, differential centrifugation, density gradient centrifugation, or ultracentrifugation.

[0168] In additional embodiments, precipitation reagents such as polymeric precipitation reagents, protamine, sodium acetate, or organic solvents can be used to synthesize, separate, and / or purify extracellular vesicles (including exosomes) and EV complexes.

[0169] In some embodiments, immunoaffinity capture techniques can be used to synthesize, separate, and / or purify extracellular vesicles (including exosomes) and EV complexes.

[0170] In other embodiments, microfluidic devices, acoustic fluidic devices, and microfluidic chips can be used to synthesize, isolate, and / or purify extracellular vesicles (including exosomes) and EV complexes.

[0171] In additional embodiments, sequential filtration techniques can be used to synthesize, isolate, and / or purify extracellular vesicles (including exosomes) and EV complexes.

[0172] In other embodiments, extracellular vesicles, including exosomes, can be detected by resistive pulse sensing using tunable pore sensors or tunable resistive pulse sensing.

[0173] In certain embodiments, extracellular vesicles, including exosomes, can be detected by electron microscopy, optical microscopy, and flow cytometry.

[0174] In additional embodiments, extracellular vesicles, including exosomes, can be detected by dynamic light scattering and / or mass spectrometry.

[0175] In some aspects, extracellular vesicles (including exosomes) and EV complexes can be synthesized, isolated, and / or purified by first isolating vesicles from cell cultures.

[0176] In certain aspects, extracellular vesicles (including exosomes) and EV complexes can be synthesized, isolated, and / or purified by first isolating vesicles from body fluids (such as ocular fluid). The isolated vesicles can be diluted, filtered, and protected with protease inhibitors.

[0177] In other embodiments, the steps for purifying extracellular vesicles (including exosomes) and EV complexes include contacting with a fixative.

[0178] In some aspects, extracellular vesicles, including exosomes, can be synthesized by controlled biogenesis and release from in vitro grown cell lines.

[0179] Active Agent

[0180] Examples of active agents include small molecule drugs, proteins, nucleic acids, polysaccharides, biological agents, and combinations thereof.

[0181] Examples of active agents include cytokines, growth factors, proteins, peptides, antimetabolites, signal transduction modulators, antibiotics, antibodies, chemotherapeutic compounds, and combinations thereof.

[0182] Examples of active agents include anti-infective agents, anesthetics, anti-VEGF agents, anti-inflammatory agents, intraocular pressure lowering agents, and combinations thereof.

[0183] Examples of active agents include anesthetics, analgesics, and combinations thereof.

[0184] Examples of active agents include cell transport or motility inhibitors such as colchicine, vincristine, cytochalasin B, and combinations thereof.

[0185] Examples of active agents include antiglaucoma drugs.

[0186] Examples of active agents include β-blockers such as timolol, betaxolol, atenolol, prostaglandins, and combinations thereof.

[0187] Examples of active agents include lipid receptor agonists or prostaglandin analogs such as bimatoprost, travoprost, tafluprost, latanoprost, unoprostone, and combinations thereof.

[0188] Examples of active agents include α-adrenergic agonists including brimonidine, dipivefrine, and combinations thereof.

[0189] Examples of active agents include carbonic anhydrase inhibitors such as acetazolamide, methazolamide, dichlorphenamide, diamox, and combinations thereof.

[0190] Examples of active agents include neuroprotective agents such as nimodipine.

[0191] Examples of active agents include agents for dry AMD such as rapamycin, glatiramer acetate, complement C5aR blockers, ciliary neurotrophic factor, fenretinide, rheopheresis, and combinations thereof.

[0192] Examples of active agents include agents for wet AMD such as mecamylamine; aflibercept (VEGF trap eye), complement inhibitor POT-4.

[0193] Examples of active agents include kinase inhibitors such as bevacizumab, BIBW 2992, cetuximab, imatinib, trastuzumab, gefitinib, ranibizumab, pegaptanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib, lapatinib, panitumumab, vandetanib, E7080, and combinations thereof.

[0194] Examples of active agents include antibiotics such as tetracycline, chlortetracycline, bacitracin, neomycin, polymyxin, gramicidin, oxytetracycline, chloramphenicol, gentamicin, and erythromycin.

[0195] Examples of active agents include antibacterial agents such as sulfonamides, sulfacetamide, sulfamethizole, and sulfisoxazole.

[0196] Examples of active agents include antifungal agents such as fluconazole, nitrofurazone, amphotericin B, and ketoconazole.

[0197] Examples of active agents include antiviral agents such as trifluorothymidine, acyclovir, ganciclovir, DDI, AZT, foscamet, vidarabine, trifluorouridine, idoxuridine, ribavirin, protease inhibitors, and anti-cytomegalovirus agents.

[0198] Examples of active agents include anti-allergy drugs such as methapyriline; chlorpheniramine, pyrilamine, and prophenpyridamine.

[0199] Examples of active agents include anti-inflammatory drugs such as hydrocortisone, dexamethasone, fluocinolone, prednisone, prednisolone, methylprednisolone, fluorometholone, betamethasone, and triamcinolone.

[0200] Examples of active agents include decongestants such as phenylephrine, naphazoline, and tetrahydrazoline; miotics and muscarinics.

[0201] Examples of active agents include anti-cholinesterases such as pilocarpine, carbachol, di-isopropyl fluorophosphate, phospholineiodine, and demecarium bromide.

[0202] Examples of active agents include mydriatics such as atropine sulfate, cyclopentolate, homatropine, scopolamine, tropicamide, and eucatropine.

[0203] Examples of active agents include sympathomimetics such as epinephrine.

[0204] Examples of active agents include ranibizumab, bevacizumab, and triamcinolone.

[0205] Examples of active agents include anti-inflammatory agents, such as non-steroidal anti-inflammatory drugs (NSAIDs), including acetylsalicylic acid, ibuprofen, indomethacin; and COX-2 inhibitors.

[0206] Examples of active agents include immunosuppressive agents, including sirolimus.

[0207] Examples of active agents include matrix metalloproteinase (MMP) inhibitors, such as tetracycline.

[0208] Examples of active agents include anticoagulants, such as heparin, antifibrinogen, fibrinolysin, and anti-clotting activase.

[0209] Examples of active agents include anti-diabetic agents, including acetohexamide, chlorpropamide, glipizide, glyburide, tolazamide, tolbutamide, insulin, and aldose reductase inhibitors.

[0210] Examples of active agents include amines, such as Thonzonium.

[0211] Examples of active agents include detergents, such as Taurocholic acid, Glycocholic acid, Glycochenodeoxycholic Acid, Benzalkonium, Cetylpyridinium, Taurochenodeoxycholic acid, Polidocanol, and Tyloxapol.

[0212] Examples of active agents include lipids, such as sodium dodecyl sulfate.

[0213] Examples of active agents include antibacterial agents, such as polymyxin B.

[0214] Examples of active agents include amines, such as Thonzonium and related compounds Thonzylamine, mepyramine, and Piribedil.

[0215] Examples of active agents include ophthalmic drugs such as Aceclidine, Acetazolamide, Acetylcysteine, Acyclovir, Aflibercept, Alcaftadine, Alclometasone, Alteplase, Ampicillin, Anecortave, Ascorbic acid, Atropine, Azelastine, Azithromycin, Befunolol, Bendazac, Benzylpenicillin, Besifloxacin, Betamethasone, Betaxolol, Bibrocathol, Bimatoprost, Brimonidine, Brinzolamide, Bromfenac, Carbamoylcholine, Carteolol, Cefuroxime, Cenegermin, Chloramphenicol, Chlorhexidine, Chlortetracycline, Chymotrypsin, Cinchocaine, Ciprofloxacin, Clobetasone, Clonidine, Cocaine, Cortisone, Cromoglicic acid, Cyclopentolate, Cyclosporine, Cysteamine, Dapiprazole, Demecarium, Desonide, Dexamethasone, Dexpanthenol, Dibrompropamidine, Diclofenac, Diclofenamide, Dihydrostreptomycin, Dipivefrin, Dorzolamide, Echothiophate, Edetatesodium), Emedastine, Ephedrine, Epinastine, Epinephrine, Erythromycin, Ethylmorphine, Famciclovir, Fludrocortisone, Fluocinolone acetonide, Fluocortolone, Fluorescein, Fluorometholone, Flurbiprofen, Fomivirsen, Formocortal, Framycetin, Fusidic acid, Ganciclovir, Gatifloxacin, Gentamicin, Guaiazulen, Guanethidine, Heparin, Hexamidine, Homatropine, Hyaluronicacid), Hydrocortisone, Hypromellose, Ibopamine, Idoxuridine, Indometacin, Inosine, Kanamycin, Ketorolac, Ketotifen, Latanoprost, Levobunolol, Levocabastine, Levofloxacin, Lidocaine, Lifitegrast, Lodoxamide, Lomefloxacin, Loteprednol, Medrysone, Methazolamide, Methscopolamine, Methylprednisolone, Metipranolol, Micronomicin, Moxifloxacin, Nandrolone, Naphazoline, Natamycin, Nedocromil, Neomycin, Neostigmine, Nepafenac, Netarsudil, Netilmicin, Nitrofural, Norfloxacin, Ocriplasmin, Ofloxacin, Olopatadine, Oxybuprocaine, Oxymetazoline, Oxyphenbutazone, Oxytetracycline, Paraoxon, Pegaptanib, Phenylephrine, Physostigmine, Picloxydine, Pilocarpine, Piroxicam, Polymyxin B, Potassium Iodide, Povidone-iodine, Pranoprofen, Prednisolone, Procaine, Propamidine, Propanoic acid, Proparacaine, Ranibizumab, Resorcinol, Riboflavin, Rifamycin, Rimexolone, Rose bengal free acid, Salicylicacid), scopolamine, sirolimus, sodium borate, spaglumic acid, sulfacetamide, sulfadicramide, sulfamethizole, sulfaphenazole, sulfafurazole, synephrine, tafluprost, tetracaine, tetracycline, tetrahydrozoline, timolol, tobramycin, travoprost, triamcinolone, trifluridine, tropicamide, tyrothricin, unoprostone, verteporfin, vidarabine, vitamin A, and xylometazoline.

[0216] Examples of active agents include anti-cancer agents such as 5-fluorouracil, doxorubicin, asparaginase, azacitidine, azathioprine, bleomycin, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, cyclosporine, cytarabine, dacarbazine, dactinomycin, daunorubicin, doxorubicin, estramustine, etoposide, etretinate, filgrastin, floxuridine, fludarabine, fluorouracil, fluoxymesterone, flutamide, goserelin, hydroxyurea, ifosfamide, leuprolide, levamisole, lomustine, nitrogen mustard, melphalan, mercaptopurine, methotrexate, mitomycin, mitotane, pentostatin, pipobroman, plicamycin, procarbazine, sargramostin, streptozocin, tamoxifen, taxol, teniposide, thioguanine, uracil mustard, vinblastine, vincristine and vindesine.

[0217] Examples of active agents include hormones, peptides, steroids, nucleic acids, sugars, lipids, glycolipids and glycoproteins.

[0218] Examples of active agents include endocrine hormones such as pituitary, insulin, insulin-related growth factors, thyroid, and growth hormones.

[0219] Examples of active agents include heat shock proteins.

[0220] Examples of active agents include immunomodulators such as muramyl dipeptide, cyclosporins, interferons, interleukin-2, cytokines, tacrolimus, tumor necrosis factor, pentostatin, thymopentin, transforming growth factor-β2, and erythropoietin.

[0221] Examples of active agents include brain-derived neurotrophic factor (BNGF), ciliary neurotrophic factor (CNGF), and vascular endothelial growth factor (VEGF).

[0222] Examples of active agents include anticoagulants, antiproliferatives, quinoxalines, and potassium channel blockers.

[0223] Examples of active agents include guanylate cyclase inhibitors such as methylene blue, butylated hydroxyanisole, N-methylhydroxylamine, 2-(4-methylaminobutoxy)diphenylmethane, and apraclonidine.

[0224] Examples of active agents include prostaglandins such as metabolites derivatives of arachidonic acid.

[0225] Examples of active agents include sugars such as trehalose.

[0226] Examples of active ingredients include viscoelastic agents including hyaluronic acid, dimethicone, and hydroxypropyl methylcellulose.

[0227] Examples of active ingredients include ophthalmic viscoelastic surgical devices, sodium hyaluronate, chondroitin sulfate, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose, hyaluronic acid, dimethicone, and hydroxypropyl methylcellulose.

[0228] Examples of active agents include detergents, purifying agents, or cleaning agents such as salts of long-chain aliphatic bases or acids, which may have cleaning, oil-solubilizing, and / or antimicrobial effects. Examples of active agents include glycochenodeoxycholic acid, glycocholic acid, peanut oil, benzylhydroxylamine, cetylpyridinium, taurochenodeoxycholic acid, polidocanol, tyloxapol, taurocholic acid, and N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate.

[0229] Embodiments of the present invention further contemplate the use of an active agent for treating glaucoma conditions. In some aspects, glaucoma conditions can be treated by administering a surfactant for affecting EV complexes. An effective amount of the surfactant can be administered to ameliorate, relieve, inhibit, mitigate, delay, and / or prevent at least one symptom or condition of a glaucoma condition. As used herein, the term "surface active agent" broadly refers to surfactants, detergents, purifying or cleaning agents, soaps, and their modified variants.

[0230] Examples of active agents include surfactants, including Lucinactant, Calfactant, Beractant, tyloxapol, Sodium laurylsulfoacetate, tonzonium bromide, Nonoxynol-9, Cetalkonium, dimethicone, polyethylene glycol 400, Sinapultide, palmitoyl oleoyl-phosphatidylglycerol, Lapyrium, sodium lauryl sulfate (SLS), polyethylene glycol 300, Trolamine, polysorbate 80, poloxamer 407, sodium lauryl sulfate, polysorbate 20, dioctyl sulfosuccinate, Poractant alfa, Colfosceril palmitate, castor oil, benzylhydroxylamine, N-alkyl ethyl benzyl dimethyl ammonium (c12-c14), Quaternium-15, Ambroxol, Pumactant, ethanolamine, lecithin, soy, and Cocamidopropyl betaine.

[0231] Examples of active agents include agents as described herein that can be modified for delivery or metabolic acceptability, or can be PEGylated with polyethylene glycol chains, or have attached polypropylene glycol chains. The present disclosure includes the above agents as modified active agents or PEGylated or having other chain modifications.

[0232] Examples of active agents include antibodies, antibody fragments, VEGF inhibitors, small molecules, corticosteroids, and combinations thereof.

[0233] Examples of active agents include tyrosine kinase inhibitors, monoclonal antibodies, and combinations thereof.

[0234] Examples of active agents include antibodies and antibody fragments. As used herein, the term "antibody" includes whole antibodies (e.g., two heavy chains and two light chains), antibody-binding fragments thereof (e.g., single-chain antibodies (scFv)), single-domain antibodies (e.g., nanobodies) or Fv, Fab, Fab’, F(ab’)2 and variants thereof, such as tandem scFv, Fd fragments, diabodies, triabodies. Antibody fragments can be obtained using conventional techniques known to those skilled in the art, and the fragments can be used in the same manner as the intact antibody.

[0235] With respect to the binding domain, the antibodies and antibody fragments disclosed herein can be monovalent, divalent or trivalent, and the binding domain can be monospecific, bispecific or trispecific by design in terms of binding specificity. Suitable antibodies include monoclonal antibodies or mixtures of polyclonal antibodies. Antibodies can be chimeric antibodies, CDR-grafted antibodies, humanized antibodies or any of the foregoing antigen-binding portions thereof. Therapeutic antibodies can be derived from a variety of species, including but not limited to mouse, human, camel, llama, goat, rabbit, bovine and cartilaginous fish.

[0236] In some embodiments, the antibody or its antigen-binding fragment can be used to treat eye diseases or conditions. Examples of antibodies or antigen-binding fragments include antibodies or antigen-binding fragments that bind and preferentially block or reduce integrin activity associated with the disease, such as anti-αvβ3 integrin antibodies and anti-α4β1 integrin antibodies, anti-epidermal growth factor receptor antibodies, anti-vascular endothelial growth factor (VEGF) receptor antibodies, anti-VEGF antibodies (e.g., bevacizumab, ranibizumab), anti-TNFα antibodies (e.g., infliximab and adalimumab), anti-fibroblast growth factor antibodies, anti-epidermal growth factor antibodies, anti-CD20 antibodies, anti-CD52 antibodies, anti-CD11a antibodies and anti-IL-2 antibodies.

[0237] In other embodiments, the therapeutic protein can be an antibody mimetic.

[0238] As used herein, the term "antibody mimetic" encompasses any organic compound, such as a peptide or polypeptide, that can specifically bind an antigen like an antibody and is from about 3 - 20 kDa. An antibody mimetic can comprise a scaffold that binds its target antigen through amino acids in an exposed loop similar to the CDR loops of an antibody. Antibody mimetics include adnectins, lipocalins, Kunitz domain-based adhesives, avimers, knottins, fynomers, atrimers, and cytotoxic T-lymphocyte-associated protein 4 (CTLA4)-based adhesives. Some examples are given in Weidle et al., The Emerging Role of New Protein Scaffold-based Agents for the Treatment of Cancer, Cancer Genomics & Proteomics 10:155 - 168 (2013).

[0239] Examples of active agents include agents for Parkinson's disease such as Benzatropine, Ropinirole, Tolcapone, Trihexyphenidyl, Procyclidine, Pramipexole, Entacapone, Biperiden, Amantadine, Selegiline, Bromocriptine, Levodopa, Dexetimide, Piribedil, Budipine, Melevodopa, Profenamine, Cabergoline, Lisuride, Progabide, Gabapentin, Memantine, Orphenadrine, 3,5-Dinitrocatechol, Pimavanserin, Ifenprodil, Opicapone, Benserazide, Metixene, Apomorphine, Pergolide, Rasagiline, Rotigotine, Etilevodopam, Tropatepine, Dihydroergocryptine, Phenglutarimide, Mazaticol, Etybenzatropine, Bornaprine, Etanautine, Carbidopa, Safinamide, and Dexpramipexole.

[0240] Examples of the active agent include prostaglandins, such as Epoprostenol, Dinoprost, Carboprost tromethamine, Dinoprost tromethamine, Dinoprostone, Prostaglandin D2, Prostalene, Reidispongiolide C, Unoprostone, Gemeprost, Limaprost, Iloprost, Latanoprost, Cloprostenol, Sepetaprost, Bimatoprost, Fenprostalene, Latanoprostene Bunod, Travoprost, Carboprost tromethamine, Dinoprost, Tafluprost, Cabazitaxel, Cloprostenol Sodium, Bimatoprost, (-)-Corey Lactone 4-Phenylbenzoate Alcohol, Dutasteride, Isopropyl Unoprostone, Beraprost Sodium, Prostaglandin E1, Cloprostenol, Remodulin, Trenbolone Cyclohexylmethylcarbonate, Prostaglandin F2a, Iloprost, Misoprostol Acid, Gemeprost, 9-Deoxy-9-methylene-16,16-dimethyl prostaglandin E2, Enoprostil, Ornoprostil, Epoprostenol, Sulprostone, Iloprost, Rosaprostol, (+)-Cloprostenol sodium, Carboprost tromethamine, Carboprost, Misoprostol, Prostacyclin sodium salt, Prostaglandin E2 and Limaprost.

[0241] Examples of the active agent include vitamins, such as 1-α,25-dihydroxy-20-epi-22-oxa-24,26,27-trihomovitamin D3.

[0242] Examples of the active agent include antibiotics, such as Brefeldin A, fusidic acid, Ovalicin, Narasin, and Salinomycin.

[0243] Examples of active agents include steroids such as Hydrocortisone cypionate, Hydrocortisone valerate, Hydrocortisone butyrate, Hydrocortisone probutate, Hydrocortisone aceponate, Prednisolone tebutate, Trilostane, Hydrocortisone acetate, Cholesteryl Linoleate, Methylprednisolone aceponate, Eldecalcitol, Pregnenolone acetate, Testosterone propionate, Drospirenone, Clascoterone, Norethindrone enanthate, Prednisolone hemisuccinate, Hydroxyprogesterone caproate, Trenbolone acetate, Fluprostenol, Anecortave acetate, Oleandrin, Cortisone acetate, Calcipotriol, Gestodene, Dimethyl carbate, Calcipotriol 1alpha, 24S-dihydroxyvitamin D224S-Dihydroxyvitamin D2), Ethynodiol diacetate, Nandrolone decanoate, Testosterone cypionate, Testosterone undecanoate, Carbenoxolone, progesterone-11-alpha-ol-hemisuccinate, Eplerenone, Testosterone succinate, and Boldenone undecylenate.,

[0244] Examples of active agents include statins such as Lovastatin, atorvastatin, pravastatin, rosuvastatin, fluvastatin, and simvastatin.

[0245] Examples of active agents include aggregation inhibitors such as Pentoxifylline, Argatroban, and Von Willebrand Factor Human.

[0246] Examples of active agents include prostacyclin such as Treprostinil.

[0247] Examples of active agents include amyloid targeting agents such as caprospinol and similar compounds including cholesteryl linoleate, pregnenolone acetate, and P-57AS3.

[0248] Examples of active agents include lactones such as Reidispongiolide A and Soraphen A, and Canrenone.

[0249] Examples of active agents include benzoquinones such as Antroquinonol.

[0250] Examples of active agents include Oxepanes such as Triptolide PG-701.

[0251] Examples of active agents include lipids such as gibberellin A4 and Fumagillin.

[0252] Examples of active agents include hypoglycemic agents such as Mitiglinide.

[0253] Examples of active agents include central nervous system depressants such as Glutethimide.

[0254] Examples of active agents include benzenoids such as sildenafil, udenafil, and vardenafil.

[0255] Examples of active agents include carbohydrates such as Fusicoccin and trehalose.

[0256] Examples of active agents include cholinergic agents such as biperiden, Cycrimine, procyclidine, and trihexyphenidyl.

[0257] Examples of active agents include terpenes such as cyclohexanes.

[0258] The molecules, compounds, and / or compositions of the present disclosure may be asymmetric and have one or more chiral stereocenters. Compounds containing one or more chiral centers may include substances described as "isomers", "diastereomers", "stereoisomers", "optical isomers", "enantiomers", or "racemic mixtures". Conventions for stereochemical nomenclature, such as the stereoisomer nomenclature rules of Cahn, Ingold, and Prelog, as well as methods for determining stereochemistry and separating stereoisomers are known in the art. See, for example, March’s Advanced Organic Chemistry (7th Edition, 2013). The compounds, compositions, and structures of the present disclosure are intended to cover all possible isomers, stereoisomers, diastereomers, enantiomers, and / or optical isomers of the compounds, compositions, and / or structures present, including any mixtures, racemates, or racemic or other mixtures thereof.

[0259] Compounds may exist in non-solvated and solvated forms or hydrated forms. In the present disclosure, solvated forms with pharmaceutically acceptable solvents (such as water or ethanol) will be considered equivalent to non-solvated forms. Compounds and salts or their solvates may also exist in tautomeric forms, which are considered equivalent.

[0260] The molecules, compounds, and / or compositions of the present disclosure may exist in different crystalline forms, which are intended to be covered by the present disclosure.

[0261] Compositions and Formulations

[0262] The active agents of the present disclosure may include drugs and medicaments for eye diseases, including small molecule drugs, peptides, antibodies, and protein medicaments.

[0263] The preparation of the active agent can be prepared by dissolving the composition in water to produce an aqueous solution and rendering the solution sterile.

[0264] The preparations of the present disclosure may be in the form of sterile injectable aqueous or oily suspensions. Suspensions containing dispersing or wetting agents may be formulated. The sterile injectable preparation may be a sterile injectable solution or suspension in a non-toxic pharmaceutically acceptable diluent or solvent.

[0265] Examples of solvents include water, water for injection, Ringer's solution, balanced salt solution, isotonic sodium chloride solution, 1,3-butanediol, synthetic glycerol monoesters or diesters, and fatty acids such as oleic acid.

[0266] The preparations of the present disclosure may be in the form of eye drops for topical delivery.

[0267] The ophthalmic preparation may be a solution or suspension for topical administration. The composition may be a viscous or semi-viscous gel, or other solid or semi-solid composition.

[0268] The ophthalmic preparation may be topically delivered by direct injection or by using an infusion pump.

[0269] The ophthalmic preparation may include an artificial tear carrier.

[0270] The ophthalmic preparation may include a phospholipid carrier.

[0271] The ophthalmic preparation may include surfactants, preservatives, antioxidants, tonicity-adjusting excipients, buffers, cosolvents, and viscosity-imparting excipients.

[0272] The ophthalmic preparation may include excipients to adjust the osmolality of the preparation.

[0273] The ophthalmic preparation may include viscosity-imparting excipients such as polysaccharides, hyaluronic acid, chondroitin sulfate, dextran, cellulose polymers, vinyl polymers, and acrylic polymers.

[0274] The viscosity of the ophthalmic preparation may be from 1 to 400 centipoise, or from 1 to 100 centipoise, or from 2 to 40 cps. The viscosity of the ophthalmic preparation may be about 15, 20, 25, 30, 40, or 50 centipoise.

[0275] Examples of excipients or carriers for the preparations of the present invention include ophthalmologically acceptable preservatives, viscosity enhancers, penetration enhancers, buffers, sodium chloride, sterile water, water for injection, and combinations thereof.

[0276] The dosage form of the composition of the present invention can be liquid or emulsion. The dosage form of the composition of the present invention can be solid, which can be reconstituted in a liquid before administration.

[0277] The composition of the present disclosure can also be in the form of an oil-in-water emulsion. The oil phase can be vegetable oil or mineral oil.

[0278] Examples of emulsifiers include naturally occurring gums, gum arabic, tragacanth, phospholipids, fatty acid esters, hexitols, sorbitan monooleate, and polyoxyethylene sorbitan monooleate.

[0279] Multiple embodiments of the present invention can advantageously provide effective activity of the active agent at dosage levels significantly lower than conventional dosage levels.

[0280] An effective amount of the active agent composition of the present disclosure can be an amount sufficient to ameliorate or alleviate the symptoms of the disease being treated.

[0281] The composition can be administered in a single dose or according to a regimen of repeated dosing.

[0282] An appropriate dosage level of the active agent can be determined by a person skilled in the art. In some embodiments, the active agent can be present in the composition in an amount of about 0.001% to about 40%, or about 0.01% to about 20%, or about 0.1% to 10% by weight of the total formulation.

[0283] The active agent of the present disclosure can be combined with one or more pharmaceutically acceptable carriers. The carriers can be in various forms, including fluids, viscous solutions, gels, or solubilizing particles. Examples of carriers include pharmaceutically acceptable diluents, solvents, saline, and various buffers.

[0284] Some examples of carriers, excipients, and additives are given in the following literature: U.S. Pharmacopeia National Formulary (2014); Handbook of Pharmaceutical Excipients (7th Edition, 2013); Handbook of Preservatives (2004, Synapse Information Resources); Remington: The Science and Practice of Pharmacy (22nd Edition, 2013); Remington's Pharmaceutical Sciences (Mack Publishing Co., 1990). Some examples of drugs and delivery are given in the following literature: Goodman and Gilman, The Pharmacological Basis of Therapeutics (13th Edition, 2018, McGraw Hill, NY).

[0285] In certain embodiments, the active agent can be delivered without a carrier to reduce the extracellular vesicle complex in glaucoma eye drops.

[0286] Examples of carriers include pyrogen-free water, isotonic saline, Ringer's solution, ethanol, and phosphate buffer solution.

[0287] The formulations of the present disclosure can include polymers having a molecular weight of about 0.2 to about 50 kDa, such as polyethylene glycol (PEG), polypropylene glycol, or poly(lactic-co-glycolic acid).

[0288] Examples of carrier polymers include polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone, chitosan, collagen, sodium alginate, gelatin, hyaluronic acid, polylactic acid, poly(lactic-glycolic acid) copolymer, polyhydroxybutyric acid, poly(hydroxybutyric-glycolic acid) copolymer, cellulose, hydroxymethylcellulose, hydroxypropylcellulose, fatty acid esters, and polyglycerol.

[0289] Examples of additives include sugars, sucrose, mannitol, lactose, L-arabinose, D-erythrose, D-ribose, D-xylose, D-mannose, D-galactose, lactulose, cellobiose, gentiobiose, glycerol, polyethylene glycol, N-methylpyrrolidone, low molecular weight polyvinyl alcohol, ethanol, ethylene glycol, and propylene glycol.

[0290] Examples of solubility enhancers include cyclodextrins.

[0291] The formulation may include galactose, lactose, mannitol, monosaccharides, fructose, maltose, galactose, glucose, D-mannose, sorbose, disaccharides, lactose, sucrose, trehalose, cellobiose, polysaccharides, maltodextrin, dextran, starch, mannitol or xylitol.

[0292] The ophthalmic formulation may include lipids such as dipalmitoyl ethylphosphocholine, dioleoyl phosphatidylethanolamine or 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol.

[0293] The ophthalmic formulation may include lipids such as 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine], 1,2-dioleoyl-sn-glycero-3-phosphate.

[0294] The ophthalmic formulation may include lipids such as 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, distearoyl phosphatidylcholine, diarachidonoyl phosphatidylcholine, dipalmitoyl phosphatidylethanolamine.

[0295] The ophthalmic formulation may include fatty acids, oleic acid, myristoleic acid or arachidonic acid.

[0296] The ophthalmic formulation may include phospholipids such as phosphatidylcholine, lecithin, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine and phosphatidylethanolamine.

[0297] The ophthalmic formulation may include polymers such as polyvinylpyrrolidone, hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl starch, cyclodextrin, 2-hydroxypropyl-β-cyclodextrin, sulfobutyl ether-β-cyclodextrin, polyethylene glycol, pectin, poly(lactide-co-glycolide), polylactide, polyethyleneimine or poly-L-lysine.

[0298] In some embodiments, the ophthalmic formulation may include one or more of a pH adjusting excipient, a buffering excipient, an isotonicity excipient, a viscosity excipient or a wetting excipient. In certain embodiments, the ophthalmic formulation may include an acidifying excipient, a preservative, an antioxidant, a solubilizing excipient, a wetting agent or a suspending excipient.

[0299] The ophthalmic formulation may include additives, diluents, delivery vehicles or carrier materials such as polymers, polyethylene glycol, dextran, diethylaminoethyl dextran, cyclodextrin or carboxymethyl cellulose.

[0300] Examples of excipients include sodium chloride, sodium dihydrogen phosphate monohydrate and disodium hydrogen phosphate anhydrous.

[0301] Examples of formulation additives include vegetable oils, olive oil, sesame oil, coconut oil, mineral oil and paraffin.

[0302] Examples of dispersants or wetting agents include lecithin, polyoxyethylene stearate, heptadecaethyleneoxyhexadecanol, polyoxyethylene sorbitan monooleate, and polyvinyl dehydrated sorbitan monooleate.

[0303] Examples of antioxidants include ascorbic acid, cysteine hydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, lecithin, propyl gallate, α-tocopherol, citric acid, ethylenediaminetetraacetic acid, sorbitol, tartaric acid, and phosphoric acid.

[0304] Examples of formulation additives include thickeners such as beeswax, paraffin wax, or cetyl alcohol.

[0305] Examples of formulation excipients include suspending excipients, sodium carboxymethyl cellulose, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum, or gum arabic.

[0306] Ophthalmic formulations may include carriers or cosolvents such as polysorbate 20, 60, or 80, Pluronic F-68, F-84, or P-103, tyloxapol, Cremophor, sodium dodecyl sulfate, glycerol, PEG 400, propylene glycol, cyclodextrin, and combinations thereof. The carrier or cosolvent may be used at a concentration of about 0.01% to about 2% by weight.

[0307] Ophthalmic formulations may include gel excipients such as gellan gum, xanthan gum, and combinations thereof.

[0308] Ophthalmic formulations may include viscosity enhancers such as polyvinyl alcohol, methylcellulose, hydroxypropyl carboxymethyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, methylcellulose, polyvinylpyrrolidone, and combinations thereof. The viscosity enhancer may be used at a concentration of about 0.01% to about 2% by weight.

[0309] Ophthalmic formulations may include preservatives such as benzalkonium chloride, chlorobutanol, dodecyldimethylbenzylammonium bromide, methyl paraben, propyl paraben, phenethyl alcohol, disodium edetate, sorbic acid, onamer, polyquaternium-1, parabens, sodium benzoate, phenol, cresol, parachlorometacresol, benzyl alcohol, thimerosal, sorbic acid, benzethonium chloride, and combinations thereof. The preservative may be used at a concentration of about 0.001% to about 1.0% by weight.

[0310] Unit dose compositions may be sterile but may not contain a preservative.

[0311] Ophthalmic preparations may include pH-adjusting excipients such as citrate buffers, acetate buffers, succinate buffers, malate buffers, and gluconate buffers.

[0312] Ophthalmic preparations may include additional acids (such as hydrochloric acid) or additional bases (such as sodium hydroxide) to adjust the pH.

[0313] Examples of pH control agents include arginine, sodium hydroxide, glycine, hydrochloric acid, and citric acid.

[0314] Ophthalmic preparations may include buffers such as citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, phthalic acid, tris, tromethamine, and phosphate buffers.

[0315] Ophthalmic preparations may include surfactants.

[0316] Examples of surfactants include nonionic surfactants, polysorbate-80, polysorbate-20, polysorbates, sorbitan esters, lipids, phospholipids, lecithin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, fatty acids, fatty esters, cholesterol.

[0317] Examples of surfactants include oleic acid, sorbitan trioleate, and long-chain diglycerides.

[0318] Examples of surfactants include beractant, poractant alfa, and calfactant.

[0319] Ophthalmic preparations may include tonicity agents, tonicity-adjusting excipients.

[0320] Examples of tonicity-adjusting excipients, isotonic excipients include sodium chloride, mannitol, and sorbitol.

[0321] Examples of tonicity-adjusting excipients include sugars, polyols, amino acids, and organic and inorganic salts.

[0322] Multiple embodiments of the present invention include kits that contain any reagents, pharmaceutical excipients, active agents, and instructions for use.

[0323] The kit may include a container or preparation that contains one or more active agents formulated in a pharmaceutical product for delivery. The ophthalmic preparation kit may be in a multi-dose form.

[0324] The kit may include a dispenser or dropper device for topical delivery and use.

[0325] The kit may include one or more unit doses of the composition for delivery. The unit doses may be hermetically sealed to maintain sterility.

[0326] Sample Preparation and Detection of Ultrastructural Targets

[0327] Describes the preparation and processing of samples. Aqueous humor or vitreous humor specimens collected for EV isolation are immediately processed without fixation. EVs are isolated from bovine vitreous humor or aqueous humor; or 4T1 cells using the ultracentrifugation protocol described below. EVs are isolated using the method described. Patient aqueous humor was imaged without isolation and without treatment. Dilute the sample as described.

[0328] Describes the isolation and purification of extracellular vesicles. We employed a method for isolating extracellular vesicles from fluids. For bovine vitreous humor or aqueous humor, approximately 8 ml of vitreous humor (or 100 μl of aqueous humor) was placed in a 15 ml tube and centrifuged at 2000 g (2500 rpm) for 30 minutes at 4 °C in a Sorvall legend RT swinging bucket (Sorvall). The supernatant was then transferred to a new 15 ml tube. The centrifugation step was then repeated. The supernatant was then transferred to a new tube and centrifuged at 10,000 g for 30 minutes at 4 °C in a Sorvall RC-58 centrifuge (Sorvall) using an SS-34 rotor (DuPont). The supernatant was then transferred and the step was repeated. The sample was transferred to an ultracentrifuge tube (Beckman) and placed in a swinging bucket rotor (SW-41, Beckman) and centrifuged at 100,000 g for 1 hour at 4 °C in an L7-55 ultracentrifuge (Beckman). The supernatant was transferred to a new tube. The step was repeated. The sample was resuspended in 50 μl of sterile tris-buffered saline (TBS, pH 8) and placed in a silanized tube. Samples for imaging were processed immediately and the remaining samples were frozen at -80 °C.

[0329] Describes nanoparticle tracking analysis. Nanoparticle tracking analysis was performed using a NanoSight NS300 system (Malvern) to characterize particles in solution from 30 - 800 nm. Extracellular vesicles isolated from vitreous humor, aqueous humor, or 4T1 cells were resuspended in 100 μl of tris-buffered saline (TBS, pH 7.0). The particles were loaded, the camera was focused, and 5 videos were captured for 60 seconds each. The videos were recorded and then analyzed using NanoSight software (version 3.0) to determine the size distribution and particle concentration of the EVs. Graphs were created. The Brownian motion of each particle was tracked between frames and the size was ultimately calculated by applying the Stokes-Einstein equation.

[0330] Describes the conventional glutaraldehyde-only fixation of liquid samples for electron microscopy. The EV solution treated with the conventional TEM fixation method is called "glutaraldehyde-only" or "Glut-only". As described above, EVs were obtained and resuspended in a buffer solution. We obtained Formvar / carbon-coated EM grids (Electron Microscopy Sciences) and coated the surface with a poly-L-lysine solution (%, Sigma Aldrich). We applied approximately 15 μl of poly-L-lysine to the Formvar / carbon-coated surface of the EM grid and incubated the sample in a humid chamber at room temperature for 15 minutes. We removed the poly-L-lysine solution with a pipette. We allowed the grid to dry at room temperature for 10 minutes.

[0331] Next, 5 μL of the EV-containing solution was pipetted onto the poly-L-lysine-Formvar / carbon-coated EM grid and incubated in a humid chamber at room temperature for 30 minutes. Next, the EV solution was removed with a pipette. The sample was fixed in a "glutaraldehyde fixation solution"; it consisted of 2.5% glutaraldehyde, 4% paraformaldehyde, and 0.02% picric acid in 0.1 M sodium cacodylate buffer. We pipetted 15 μl of the glutaraldehyde solution onto the EM grid and incubated the sample at room temperature for 15 minutes 19. After that, the glutaraldehyde fixation solution was removed with a pipette. The grid was washed with 15 μl of double-distilled water at room temperature for 5 minutes. The sample was washed 2 times at room temperature, 5 minutes each time. As described below, the sample was dried at room temperature and observed on a JEM 1400 electron microscope (JEOL, USA, Inc). The EDC-formalin-fixed specimens were further processed as described below.

[0332] Describes the preparation of the EDC-ETT solution. The method for EDC solution fixation was modified from previous reports 17, 18. We prepared a 0.1 M 1-methylimidazole buffer solution (0.1 M 1-methylimidazole, 300 mM NaCl, pH adjusted to 8.0 with 12 N NaOH) and stored the solution at room temperature for up to 3 months. Next, we freshly prepared the EDC solution for each experiment. We measured 0.96 ml of the 0.1 M 1-methylimidazole buffer solution and added 13 mg of 5-(ethylthio)-1H-tetrazole (ETT, Sigma Aldrich, final concentration 0.1 M). The pH was adjusted to 8.0 with 12 N NaOH. Next, we added 19.2 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (Sigma Aldrich, final concentration 0.10 M), and then readjusted the pH to 8.0 using 12 M HCl. The EDC-ETT solution was placed on ice until use.

[0333] Describes the EDC-glutaraldehyde fixation of liquid samples on an electron microscope. All isolated EVs were resuspended in 20 μl of TBS (pH 8.0) and kept at 4 °C. We obtained Formvar / carbon-coated EM grids (ElectronMicroscopy Sciences) and coated the surface with poly-L-lysine solution (%, Sigma Aldrich). We applied approximately 15 μl of poly-L-lysine onto the Formvar / carbon-coated surface of the EM grid and incubated the sample in a humid chamber at room temperature for 15 minutes. We removed the poly-L-lysine solution with a pipette and placed the grid in the humid chamber until it was ready for use. Next, we combined aliquots of freshly prepared EDC / ETT solution and EV solution by adding 5 μl of ice-cold EDC / ETT solution and 5 μl of ice-cold EVs suspended in TBS (pH 8.0) into a 1.5 ml pre-cooled silanized tube. The sample was incubated on ice for 30 minutes. Then, we applied 10 μl of the EDC / ETT-EV solution onto the surface of the Formvar / carbon-coated EM grid and incubated the sample at 4 °C in a humid chamber for 30 minutes. To activate the cross-linking ability of the EDC reagent, we placed the sample in the humid chamber of an incubator at 50 °C for 3 hours. The sample was removed from the incubator and the EDC solution was removed using a pipette. The sample was fixed by secondary fixation using a glutaraldehyde-based cross-linking solution (containing 2.5% glutaraldehyde, 4% paraformaldehyde, 0.02% picric acid in 0.1 M sodium cacodylate buffer) and incubated at room temperature for 15 minutes. The glutaraldehyde solution was removed by pipetting out the bubbles from the EM grid. The grid was washed by placing 15 μl of double-distilled water onto the grid and incubating at room temperature for 5 minutes. The water was removed and a second wash was performed. Finally, the sample was negatively stained or stained for DNA, RNA, and proteins as described below. For negative staining, the sample was contrasted successively in 2% uranyl acetate (pH 7) and 2% methylcellulose / 0.4% uranyl acetate (pH 4). After staining with the corresponding dyes, the EM grid was mounted for imaging on an electron microscope as described below.

[0334] Describes the transmission electron microscopy imaging. All EM grids were observed on a JEM 1400 electron microscope (JEOL, USA, Inc) operating at 100 kV. Digital images were captured on a Veleta 2K x 2K CCD camera (Olympus-SIS). Electron microscope images were recorded and the size and frequency of EVs were analyzed using ImageJ software.

[0335] Describes the transmission electron microscopy of vitreous humor and eye tissues. Human or bovine vitreous tissues were obtained as described above. Cells in the samples were removed by low-speed centrifugation, and the whole-mount specimens were tested by H and E staining and imaging as described below. For vitreous humor, 2 μL was pipetted onto the module and fixed in a 0.1 M sodium cacodylate buffer solution of 2.5% glutaraldehyde, 4% paraformaldehyde, and 0.02% picric acid, and incubated at room temperature for 60 minutes 19. The specimens were washed with an excess volume of buffer (pH 7.3) at room temperature for 5 minutes each time. The samples were post-fixed with 1% OsO 4 -1.5% potassium ferricyanide (aqueous solution) at room temperature for 60 minutes 20. The samples were washed with buffer 3 times at room temperature for 5 minutes each time. The samples were placed whole and stained with 1.5% uranyl acetate at room temperature for 60 minutes. The samples were dehydrated through a series of gradient ethanol and transitioned through acetonitrile. The samples were infiltrated and embedded in Embed 812 resin (Electron Microscopy Sciences). Tissue sections were cut at 60-65 nm using a Diatome diamond knife (Diatome) on a Leica Ultracut T ultramicrotome (Leica Microsystems). The sections were contrasted with lead citrate 21 and observed on a JEM 1400 electron microscope (JEOL, USA, Inc) operating at 100 kV. Digital images were captured on a Veleta 2K×2K CCD camera (Olympus-SIS). Electron microscope images were recorded, and the size and frequency of EVs were analyzed using ImageJ software. For TEM staining of nucleic acids, we incubated the acridine orange staining solution (Exo-Red exosome RNA fluorescent labeling, System Biosciences) with 5 μl of ultracentrifugation-purified EVs at 25 °C for 30 minutes. For EVs stained with ethidium bromide (EtBr), we mixed a 5 μg / ml EtBr solution with 5 μl of ultracentrifugation-purified EVs at 25 °C for 30 minutes. For protein staining on TEM, we mixed 500 μM CFSE diluted in TBS (pH 7.4) with 5 μl of ultracentrifugation-purified EVs at 25 °C for 30 minutes. Then all the above samples were fixed, mounted, and imaged with TEM as above.

[0336] Describes the statistical analysis. Excel (version 2011, Microsoft) was used for graph visualization and calculations. Unless otherwise stated, all experiments were performed with n≥3. For nanoparticle tracking analysis, we used the Stokes-Einstein equation to calculate particle size, concentration, and distribution. Using SPSS software, unpaired Student's t-tests were performed for statistical analysis, and a p-value < 0.05 was considered significant.

[0337] Describes a method for imaging healthy control aqueous humor samples and POAG aqueous humor samples. The samples are shown in Table 1.

[0338] Table 1: Reagents / Samples

[0339]

[0340] Method:

[0341] For 1 ml of EDC / ETT solution in 0.1 M 1-methylimidazole buffer (pH 8.0):

[0342] 1. EDC-HCl powder should be stored at -20 °C under argon. To avoid moisture condensation, open the bottle only after it has reached room temperature, i.e., remove the bottle from the freezer 1 hour before weighing the sample.

[0343] 2. The EDC / 5-ETT solution should be prepared no earlier than 1 hour before use.

[0344] 3. Pipette 5.76 ml of 0.1 M 1-methylimidazole buffer (pH 8.0) into a 15 ml tube and vortex vigorously.

[0345] 4. Carefully spoon out 0.078 g of ETT found in group "G" onto a piece of paper. Carefully pour this 0.078 g into the tube.

[0346] 5. Use pH test paper and pipette to check if the pH is 8.0

[0347] 6. Use weighing paper to measure out 0.12 g of EDC. Carefully pour it into the tube. Vortex vigorously.

[0348] 7. Discard the unused solution.

[0349] 8. Use pH test paper and pipette to check if the pH is 8.0

[0350] Poly-L-lysine grids, EDC fixation, Glut fixation, UA staining

[0351] 1. Use Formvar-coated grids.

[0352] 2. Place them, with the Formvar side down, onto a drop of polylysine and then let stand for 5 - 10 min.

[0353] 3. Then you will withdraw the poly-L-lysine

[0354] 4. Fix a piece of parchment paper down to a 100 mm dish with tape or a heavy object. Place the grids down on the parchment paper.

[0355] Mix the EDC solution and EV

[0356] 1. Mix 10 μl (cold) EV with 10 μl (cold) EDC solution in a 1.5 ml silanized tube.

[0357] 2. In a cold room, apply 10 μl of the sample (aqueous humor) to the grid, wait for 30 minutes to dry, and allow the EV to settle to the bottom of the grid.

[0358] 3. Place in a humid chamber and incubate at 50 °C for at least 3 hours.

[0359] Secondary fixation with Glut

[0360] 1. Use a pipette to remove the EDC-EV solution from the sample.

[0361] 2. Fix with Glut, add 2 - 5 μl of Glut fixative, and then wait for 5 minutes

[0362] 3. Use a pipette or lens paper to remove Glut

[0363] Rinse Glut and EDC

[0364] 1. Apply a water bubble to the grid for 5 minutes

[0365] 2. Use a pipette to remove the water bubble

[0366] 3. Add 5 μl of uranyl acetate to the grid (4 drops, blotting between each drop)

[0367] 4. Allow the grid to dry, then place it in a grid box

[0368] 5. Use a pipette to remove the stain

[0369] Image with TEM. Image at 25k (approx. 50 pictures) and very rarely at 50K magnification shortly after completion.

[0370] Method of treating POAG aqueous humor samples with bimatoprost and fixing fluid with EDC, negative staining, and TEM imaging. We attempted to determine the effect of bimatoprost on EV in the aqueous humor of glaucoma patients. For this purpose, we diluted POAG aqueous humor samples with tris-buffered saline (1:10 dilution) and added 1:1 volume of the diluted POAG sample and tris-buffered saline (control) or bimatoprost.

[0371] Lumigan is obtained directly from the dispensing bottle and is undiluted. Mix 10 ul of the POAG sample with 10 ul of TBS or mix 10 ul of the POAG sample with 10 ul of undiluted Lumigan. Then incubate the tube in a thermal cycler PCR machine and let it stand at 37 °C for 72 hours. Then dilute the sample (1:10). Next, we use the following protocol to immobilize EDC on the electron microscopy grid.

[0372] For 1 ml of EDC / ETT solution in 0.1 M 1-methylimidazole buffer (pH 8.0):

[0373] 1. EDC-HCl powder should be stored at -20 °C under argon. To avoid moisture condensation, only open the bottle after it has reached room temperature, i.e., take the bottle out of the freezer 1 hour before weighing the sample.

[0374] 2. The EDC / 5-ETT solution should be prepared no earlier than 1 hour before use.

[0375] 3. Pipette 5.76 ml of 0.1 M 1-methylimidazole buffer (pH 8.0) into a 15 ml tube and vortex vigorously.

[0376] 4. Carefully pour 0.078 g of ETT found in group "G" onto a piece of paper using a spatula. Carefully pour this 0.078 g into the tube.

[0377] 5. Use pH test paper and pipette to check if the pH is 8.0

[0378] 6. Use weighing paper to measure out 0.12 g of EDC. Carefully pour it into the tube. Vortex vigorously.

[0379] 7. Discard the unused solution.

[0380] 8. Use pH test paper and pipette to check if the pH is 8.0

[0381] Poly-L-lysine grid, EDC immobilization, Glut immobilization, UA staining

[0382] 1. Use Formvar-coated grids.

[0383] 2. Place them face down with the Formvar side onto the polylysine drop and then let them stand for 5 - 10 min.

[0384] 3. Then you will withdraw the poly-L-lysine

[0385] 4. Fix a piece of parchment paper down to a 100 mm dish with tape or a heavy object. Place the grid down on the parchment paper.

[0386] Mix the EDC solution and EV

[0387] 1. Mix 2.5 μl (cold) EV with 5 μl (cold) EDC solution in a 1.5 ml silanized tube.

[0388] 2. Apply 5 μl of the sample (aqueous humor) to the grid in the cold room, wait for 15 minutes for it to dry, and allow the EV to sediment to the bottom of the grid.

[0389] 3. Place in a humidified chamber and incubate at 50 °C for at least 3 hours.

[0390] Secondary fixation with Glut

[0391] 1. Use a pipette to remove the EDC-EV solution from the sample.

[0392] 2. Fix with Glut, add 2 - 5 μl of Glut fixative, and then wait for 5 minutes

[0393] 3. Remove the Glut with a pipette or lens paper

[0394] Rinse Glut and EDC

[0395] 1. Apply a water bubble to the grid for 5 minutes

[0396] 2. Remove the water bubble with a pipette

[0397] 3. Add 5 μl of uranyl acetate to the grid (1 drop, and blot after the drop)

[0398] 4. Allow the grid to dry and place it in a grid box

[0399] 5. Remove the stain with a pipette

[0400] Then image the sample with TEM.

[0401] Additional embodiments of the present invention are as follows.

[0402] A method for identifying glaucoma or pre-glaucoma in a subject, the method comprising: providing an aqueous humor sample from a subject suspected of having glaucoma; analyzing glaucoma-related extracellular vesicle complexes in the sample, which complexes are: aggregates of extracellular vesicles; or individual extracellular vesicles having a diameter greater than 300 nm, or 300 to 3,000 nm, or 300 to 5,000 nm, or 300 to 10,000 nm; and identifying, based on the analysis, that the subject has glaucoma or pre-glaucoma.

[0403] The glaucoma-related extracellular vesicle complex is a complex of extracellular vesicles selected from exophers, exosomes, multivesicular bodies, intraluminal vesicles (ILVs), multivesicular endosomes (MVE), oncosomes, microvesicles, apoptotic bodies, and vesicles derived from endosomes or the plasma membrane.

[0404] The glaucoma-related extracellular vesicle complex is a complex of individual extracellular vesicles, wherein the diameter of the complex can be from 360 to 21,000 nanometers.

[0405] The glaucoma-related extracellular vesicle complex can be an aggregate of 10, 20, 30, 40 or more extracellular vesicles.

[0406] The glaucoma-related extracellular vesicle complex can be an aggregate of 50, 100 or 200 or more extracellular vesicles.

[0407] The above method further comprises: before the analysis, fixing the glaucoma-related extracellular vesicles in the sample. Fixing the glaucoma-related extracellular vesicles comprises: contacting the sample with an irreversible cross-linking agent; and before, after or simultaneously with the contacting of the sample with the irreversible cross-linking agent, contacting the sample with an aldehyde-containing fixative to fix the glaucoma-related extracellular vesicle complex.

[0408] The above method, wherein the irreversible cross-linking agent is selected from water-soluble carbodiimides, cyanogen halides and mixtures thereof.

[0409] The above method, wherein the irreversible cross-linking agent is 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide.

[0410] The above method, wherein the irreversible cross-linking agent is a cyanogen halide selected from cyanogen bromide, cyanogen fluoride, cyanogen chloride and cyanogen iodide.

[0411] The above method further includes: independently of the contacting with the irreversible crosslinking agent and the contacting with the aldehyde-containing fixative, and before, after or simultaneously with the contacting with the irreversible crosslinking agent and the contacting with the aldehyde-containing fixative, contacting the sample with other crosslinking agents selected from ethylene glycol di(meth)acrylate, ethylene glycol diacrylate, di(ethylene glycol) diacrylate, tetra(ethylene glycol) diacrylate, ethylene glycol dimethacrylate, di(ethylene glycol) dimethacrylate, tri(ethylene glycol) dimethacrylate, derivatives of methylene bisacrylamide, N,N'-methylenebisacrylamide, N,N-methylenebisacrylamide, N,N-(1,2-dihydroxyethylene) bisacrylamide, formaldehyde-free crosslinking agents, N-(1-hydroxy-2,2-dimethoxyethyl) acrylamide, divinylbenzene, formalin fixative, formal calcium, formal saline, zinc formalin (unbuffered), Zenker fixative, Helly fixative, B-5 fixative, Bouin solution, Hollande solution, Gendre solution, Clarke solution, Camey solution, methacam, alcoholic formalin, and formol aceticalcohol.

[0412] The above method, wherein the analysis includes: imaging the fixed glaucoma-related extracellular vesicle complex.

[0413] The above method, wherein the imaging is performed by the following techniques: transmission electron microscopy, scanning electron microscopy, cryo-electron microscopy, binocular stereomicroscopy, wide-field microscopy, polarization microscopy, phase-contrast microscopy, multiphoton microscopy, differential interference contrast microscopy, fluorescence microscopy, laser scanning confocal microscopy, multiphoton excitation microscopy, X-ray microscopy, ultrasonic microscopy, colorimetry, chemiluminescence assay, spectrophotometry, positron emission tomography, computed tomography, and magnetic resonance imaging.

[0414] The above method further includes: based on the imaging, detecting the glaucoma-related extracellular vesicle complex in the aqueous humor sample.

[0415] The above method, wherein the identification is based on the detection.

[0416] The above method, wherein the identification comprises: providing a standard image of a clinical aqueous humor sample from a subject with glaucoma, the clinical aqueous humor sample comprising a glaucoma-related extracellular vesicle complex fixed with an irreversible cross-linking agent and an aldehyde-containing fixative; comparing an image of the subject's clinical sample with the standard image with respect to the size, density, morphology, or spatial distribution of the fixed glaucoma-related extracellular vesicle complex; and determining based on the comparison whether the subject has glaucoma or pre-glaucoma.

[0417] The above method, further comprising: administering a therapeutic agent to the subject based on the determination.

[0418] The above method, wherein the therapeutic agent reduces the size of the glaucoma-related extracellular vesicle complex by disrupting two or more extracellular vesicles in contact with each other.

[0419] The above method, wherein the identification comprises monitoring the progression or regression of glaucoma and comprises: providing a previous image of a clinical aqueous humor sample of the subject, which comprises a glaucoma-related extracellular vesicle complex fixed with an irreversible cross-linking agent and an aldehyde-containing fixative; comparing an image of the subject's clinical aqueous humor sample comprising a glaucoma-related extracellular vesicle complex fixed with an irreversible cross-linking agent and an aldehyde-containing fixative with the previous image with respect to the size, density, morphology, or spatial distribution of the fixed glaucoma-related extracellular vesicle complex; and determining based on the comparison whether glaucoma is progressing or regressing.

[0420] The above method, wherein the identification of glaucoma or pre-glaucoma is performed before the subject from whom the aqueous humor sample is provided experiences any vision loss.

[0421] A method of screening the ability of a compound to treat glaucoma, the method comprising: providing a candidate agent that may be useful for treating glaucoma; providing a sample comprising a glaucoma-related extracellular vesicle complex, wherein the extracellular vesicles are aggregates of extracellular vesicles or individual extracellular vesicles having a diameter greater than 300 nanometers; contacting the candidate agent with the glaucoma-related extracellular vesicle complex; and identifying, based on the contact, a candidate compound that effectively reduces the size of the glaucoma-related extracellular vesicle complex.

[0422] The above method, wherein the glaucoma-related extracellular vesicle complex is in the aqueous humor.

[0423] The above method, further comprising: contacting a second sample containing a glaucoma-related extracellular vesicle complex with a placebo and comparing the reduction in the size of the glaucoma-related extracellular vesicle complex in the sample contacted with the placebo with the reduction achieved by the candidate agent to identify a candidate compound that effectively reduces the size of the glaucoma-related extracellular vesicle complex.

[0424] A reagent for detecting glaucoma, comprising: an isolated sample containing an extracellular vesicle complex related to glaucoma, said complex being one of the following: an aggregate of extracellular vesicles, or an individual extracellular vesicle with a diameter greater than 300 nanometers.

[0425] The above reagent, wherein the extracellular vesicle complex related to glaucoma is in the aqueous humor.

[0426] Multiple embodiments of the present invention further contemplate a composition for ophthalmic use.

[0427] In some embodiments, an aqueous pharmaceutical composition for ophthalmic use may contain an active agent selected from cetylpyridinium chloride, polymyxin B sulfate, neomycin sulfate, and sodium heparin. The active agent may account for 0.01 - 2% w / v of the composition. In some embodiments, the active agent may be 0.01 - 0.2% w / v of the composition.

[0428] For example, sodium hydroxide and hydrochloric acid can be used to adjust the pH of the composition. The composition may have a pH of 6.8 to 7.9 or about 7.3.

[0429] In some embodiments, the composition can reduce intraocular pressure when administered to the eye.

[0430] In additional embodiments, the composition can reduce the extracellular vesicle complex of the eye when administered to the eye.

[0431] In other embodiments, the composition can effectively treat glaucoma disease when administered to the eye.

[0432] The composition of the present invention may further contain one or more of a solubilizer, a surfactant, a tonicity agent, and a preservative.

[0433] Examples of solubilizers include phosphates, citric acid monohydrate, trisodium citrate, and combinations thereof.

[0434] Examples of phosphates include sodium dihydrogen phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and combinations thereof.

[0435] Examples of surfactants include phospholipids, polyglycerol esters, propylene glycol esters, polyethylene glycol esters, copolymer esters, polyoxyethylene sorbitan esters, cyclodextrins, polyvinyl alcohol, polyvinylpyrrolidone, hydroxypropyl methylcellulose, poloxamers, carboxymethylcellulose, hydroxyethylcellulose, polyacrylates, and combinations thereof.

[0436] Examples of tonicity agents include sodium chloride, trehalose, mannitol, sorbitol, dextrose, potassium chloride, and combinations thereof.

[0437] Examples of preservatives include benzalkonium chloride, polyquaternium-1, dodecyl dimethyl benzyl ammonium bromide, sorbic acid, methyl paraben, propyl paraben, chlorobutanol, benzyl alcohol, phenethyl alcohol, chloro-oxo-complex, thimerosal, sodium perborate, disodium edetate, and combinations thereof.

[0438] In the case of ophthalmic compositions, solubilizers can also function as buffers, or as stabilizers, or as thickeners.

[0439] In the case of ophthalmic compositions, surfactants can also function as solubilizers.

[0440] The compositions of the present invention can consist of only an active agent and a carrier.

[0441] For example, the composition can be an aqueous solution of an active agent.

[0442] Examples of carriers include water, sterile water, water for injection, water for irrigation, phosphate buffer solution, and combinations thereof.

[0443] The compositions of the present invention can be used for medical treatment.

[0444] The compositions of the present invention can be used to treat the human or animal body.

[0445] The compositions of the present invention can be used to reduce intraocular pressure in the human or animal body.

[0446] The compositions of the present invention can be used to reduce the extracellular vesicle complex in the eye of the human or animal body.

[0447] The compositions of the present invention can be used to prepare or manufacture a medicament for preventing, ameliorating, or treating glaucoma-related diseases or conditions in a subject in need thereof.

[0448] Multiple embodiments of the present invention also contemplate a method of treating glaucoma disease, reducing intraocular pressure, or reducing the extracellular vesicle complex in the eye in a subject in need thereof by administering the compositions of the present disclosure to the eye of the subject.

[0449] Administration can be by injection.

[0450] The extracellular vesicle complex in the eye can be an aggregate of extracellular vesicles having a diameter greater than about 300 nanometers.

[0451] For all purposes, all publications cited in this specification, including patents, patent application publications, and non-patent publications, are hereby expressly incorporated by reference in their entirety.

[0452] Although the foregoing disclosure has been described in detail by way of example for purposes of clear understanding, it will be apparent to those skilled in the art that certain changes and modifications are encompassed within the present disclosure and can be practiced within the scope of the appended claims without undue experimentation, which are presented by way of illustration and not limitation. The present invention includes all such additional embodiments, equivalents, and modifications. The present invention includes any combination or mixture of the features, materials, elements, or limitations of the various exemplary components, examples, and claimed embodiments.

[0453] The terms "a / an", "the", and similar terms as used in the description of the present invention and the claims are to be construed as including both the singular and the plural. Example

[0454] Example 1. Use of improved fixation to enhance imaging of extracellular vesicles in biological fluids. The EV ultrastructure in fluids can be detected by transmission electron microscopy (TEM) combined with negative staining. However, in our laboratory, we found that this technique led to inconsistent or often negative results. When examining a known amount of EVs applied to the solution, we observed a significant difference between the high number of EVs applied and the fewer EVs imaged finally. Usually, the results were inconsistent and technical replicates would vary. Therefore, there were flaws in the method and it hindered efficient, consistent, and representative EV imaging in solution. Thus, we evaluated each step of the EV imaging procedure and tried to identify points where EVs might be lost. We found that the conventional TEM procedure led to inefficient binding of EVs to the surface of the electron microscopy grid and that most EVs could not attach. To bind EVs more efficiently, we cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), which retained the EVs and enabled robust TEM imaging. Finally, we demonstrated that this method could be used to image EVs in various biological fluids (including: blood (plasma); cerebrospinal fluid; nipple aspirate fluid; aqueous humor) or isolated EVs suspended in buffer.

[0455] Example 2. Due to the significant loss of vesicles into the discarded solution, the yield of imaging EVs suspended in liquid using conventional methods is low. To improve the established TEM and negative staining procedures, we used EVs isolated from bovine vitreous humor (a gel-like matrix located between the lens and retina of the eye) and aqueous humor as a model system. First, we dissected the vitreous humor from the posterior chamber of the eye, homogenized the sample, isolated the EVs using ultracentrifugation, and resuspended the sample in buffered saline. Next, we quantified the number and size of the EVs using nanoparticle tracking analysis (NTA; 3.98 × 108 EVs / ml). To visualize the ultrastructure of vitreous EVs suspended in fluid, we followed a conventional glutaraldehyde-based TEM imaging protocol ( Figure 6 , first row). We applied approximately 4 x 106 EVs to an electron microscopy grid and followed the standard protocol for glutaraldehyde fixation and negative staining with uranyl acetate solution. We subsequently imaged the specimen using TEM, and the results showed the presence of very few EVs (0.033 ± 0.182 EVs were observed per high-power microscopic field at 25,000x magnification, n = 3 biological replicates, and 10 photographs were captured), which was an unexpectedly low number and inconsistent with loading 4 million EVs. At lower magnification, we observed sparse and spaced-out EVs ( Figure 6 , first row, left), and in most photographic frames, we were unable to clearly identify the negatively stained EVs ( Figure 6 , first row, middle and right). These results suggest that the EVs were disrupted during specimen processing, failed to attach to the surface of the TEM grid, or were lost during the protocol. To verify the presence of EVs in the vitreous specimen, we imaged the vitreous basal lamina (the tightly adherent part of the gel-like matrix attached to the retina), sectioned the sample, and prepared it for TEM. Indeed, we observed many electron-dense signals in the shape of EVs in the vitreous tissue sections (data not shown), confirming the presence of EVs in the samples we prepared for TEM. Therefore, we hypothesized that millions of EVs applied to the surface of the electron microscopy grid were discarded in the discarded solution and did not attach to the grid surface ( Figure 6 , second row, left panel). To determine whether a large number of EVs were present in the discarded solution, we measured and compared the number and size of EVs isolated from the 4T1 breast cancer cell line and resuspended in buffered saline. We found that the concentration of 4T1 EVs was 8.63 x 108 particles / ml (± 4.96 x 108 particles / ml), and we applied 8.63 x 106 EVs to the grid surface ( Figure 6, second line, middle and Table 2). Next, we measured the amount of EVs present in the discard solution using nanoparticle tracking analysis (NTA) and found that in 3 separate experiments, at least 8.16 x 10^8 particles / ml (±2.43 x 10^7 particles / ml) or 8.16 x 10^6 particles were lost to the wash buffer ( Figure 6 , second line, right, Figure 6 , third line, left and Table 2). These data indicate that most of the EVs applied to the grid surface failed to attach to the electron microscopy grid surface and were lost to the discard solution ( Figure 6 , 3rd line, left). Therefore, we infer that imaging EVs suspended in liquid using conventional fixation, negative staining, and electron microscopy is severely hampered by poor crosslinking of EVs to the grid surface.

[0456] Table 2 shows poor attachment of extracellular vesicles to the electron microscopy grid. We determined the size and concentration of EVs applied to the grid surface and compared this value with the EVs present in the discard fraction. We isolated EVs from the mouse mammary tumor cell line 4T1 cell culture medium using ultracentrifugation and measured them using nanoparticle tracking analysis (NTA) with the same settings for all variables in NTA 2.3 build 17 software. The mean size, mode size, and mean concentration of EVs applied to the TEM grid surface, as well as the EVs present in the discard fraction (n = 3), are shown.

[0457] Table 2: Extracellular Vesicles and Electron Microscopy Grid

[0458]

[0459]

[0460] Samples were analyzed using NTA 3.1 build 54 software (Malvern)

[0461] Example 3. Using formalin-EDC fixation preserves EVs and allows robust imaging of EVs suspended in liquid. We hypothesized poor binding of EVs to the TEM grid and attempted to permanently attach EVs suspended in liquid to the grid by adding the heat-stable fixative EDC, a carbodiimide, to generate irreversible crosslinks between the positively charged amino side chains and carboxyl groups of proteins. To test our hypothesis, we combined 4 million isolated bovine vitreous EVs with the EDC fixation solution, applied it to the surface of a poly-L-lysine-coated Formvar TEM grid, and activated the EDC solution by heating (50°C) for 3 hr ( Figure 6, line 3, middle). After crosslinking, we removed the EDC solution, applied glutaraldehyde fixation solution, washed the samples, and performed negative staining. The images showed a large number of EVs in each photographic frame, with 16.5 EVs (±16.9) per 25,000x high magnification field under the matching conditions of conventional negative staining and TEM imaging. Additionally, when compared to glutaraldehyde alone, we identified at least 357-fold more EVs in the EDC-fixed samples ( Figure 6 , line 4, left panel, p < 0.05, n = 3). To expand the scope of this technique to other fluids, we visualized EVs isolated from bovine aqueous humor, which showed robust EV detection with electron-dense negative staining around the EV perimeter ( Figure 6 , line 4, middle). We also imaged native aqueous humor fluid from healthy patients to visualize EVs in situ. We defined in situ imaging as "imaging the solution without EV isolation and visualizing the contents of the biological fluid in situ". This is a method of visualizing EVs in fluid that is different from previous methods used to image EVs because we did not concentrate the EVs. Here, the images showed a heterogeneous population of EVs with well-defined negative staining ( Figure 6 , line 4, right). Overall, we found that crosslinking of EVs suspended in various fluids with EDC led to a substantial improvement in transmission electron microscopy imaging. These data indicate that, for imaging EVs in fluid, EDC fixation significantly increased the number of observed EVs compared to conventional aldehyde fixation.

[0462] Example 4. Comparing extracellular vesicles in the aqueous humor of healthy controls and POAG patients. Aqueous humor is a clear, watery biological fluid similar to plasma but containing 98% water as well as amino acids, electrolytes, ascorbic acid, glutathione, and immunoglobulins. Aqueous humor has been shown to contain EVs. In these studies, EVs were isolated using an ultracentrifugation protocol, and no glaucoma-related EV aggregates were observed. We hypothesized that there would be differences in the ultrastructure of EVs in the aqueous humor of subjects with ocular pathologies (such as glaucoma) compared to healthy controls (subjects without other ocular pathologies except cataract). We chose to study aqueous humor because it is typically lost during cataract surgery, and obtaining samples does not pose any additional risk to the subjects. To observe the most natural state of aqueous humor, we did not use ultracentrifugation or other EV-related protocols to isolate the EVs. We collected aqueous humor from a group of healthy patients and a group clinically diagnosed with POAG. We predicted that the EVs in the aqueous humor of glaucoma patients would be larger, enabling these structures to block the outflow of aqueous humor. To complete this study, we obtained aqueous humor specimens from 1) healthy control patients undergoing elective cataract surgery (control group) or 2) patients diagnosed with glaucoma (POAG group). The study design is listed below.

[0463] Example 5. Study design; obtaining aqueous humor from healthy and glaucoma patients. To determine the ultrastructure of EVs in aqueous humor donated by healthy controls or glaucoma patients, we conducted a prospective clinical trial. Control samples were those diagnosed with cataract but without other ocular comorbidities or other systemic comorbidities. All patients in the control group were undergoing elective outpatient, same-day cataract surgery and were generally healthy. At the start of cataract surgery, we collected samples (50 to 100 μl) of a small amount of aqueous humor that would otherwise be lost as medical waste. We then fixed the samples with an EDC crosslinker, negatively stained the samples, and performed transmission electron microscopy. Study samples would be those patients with POAG in addition to cataract and without the following conditions: diabetic retinopathy or age-related macular degeneration. The glaucoma patient pool would be selected from patients scheduled for cataract extraction and lens implantation surgery. Inclusion criteria included the following; outpatient cataract surgery patients, 18 years of age or older, without other ocular comorbidities other than cataract, or patients with a clinical diagnosis record of POAG in addition. Exclusion criteria included other ocular comorbidities in addition to cataract. The institutional review board approved the protocol for collecting samples and completing these studies.

[0464] Example 6. Obtaining aqueous humor samples from patients with and without glaucoma: During cataract surgery, an incision was made into the anterior chamber and approaching the cataract lens. Aqueous humor flowed out of the eye during the surgery and was replaced with irrigation fluid infused through the instrument. At the start of cataract surgery, we collected a small amount of aqueous humor samples that would otherwise be lost as medical waste. A numeric study ID code was assigned to the samples to de-identify them, and they were immediately transferred from the OR to the laboratory for TEM imaging analysis. For the purposes of this study, patients were not surgically treated or otherwise intervened upon, but rather those individuals who were scheduled for cataract surgery for therapeutic purposes. Cataract surgery schedules were reviewed to identify those patients who met the above inclusion or exclusion criteria to serve as controls or the POAG group. Informed consent was obtained from each patient prior to inclusion in the study. Then, with the following exceptions, patients underwent standard cataract surgery. At the start of cataract surgery, a 30-gauge needle on a TB syringe was inserted through the clear cornea to aspirate 0.05 - 0.1 cc of aqueous humor. The wound was self-sealing. Subsequently, a paracentesis incision was made in the cornea by the surgeon according to standard cataract surgery. We collected the samples and transported the samples on ice to the laboratory. Study samples were identified with a random study ID code to ensure they were de-identified. For all experiments, we did not use ultracentrifugation to isolate EVs, but rather performed in situ imaging of the biological fluid.

[0465] Example 7. Ultrastructural in situ imaging of healthy control aqueous humor shows a diffuse distribution of EVs: To understand the morphology of EVs under normal physiological conditions, we performed in situ imaging of aqueous humor from healthy patients. No previous glaucoma studies have used the EDC fixation technique to visualize EVs in situ in aqueous humor. To understand the ultrastructural content of aqueous humor, we diluted the samples 1:10 with buffered saline (Note: We did not isolate EVs), fixed the samples with EDC and then glutaraldehyde, negatively stained the specimens, and imaged them with TEM. The data indicate that, according to multiple control human subjects, healthy human aqueous humor contains a large number of EVs ( Figures 7 - 8 ). These studies show that EVs are present in the aqueous humor of healthy individuals and are diffusely distributed, with no evidence of EV aggregation. In addition, most EVs are not connected to other EVs and are relatively evenly distributed. Thus, healthy control aqueous humor contains non-aggregated and diffusely distributed EVs.

[0466] Example 8. Ultrastructural in situ imaging of POAG aqueous humor shows considerably larger glaucoma-associated EV complexes than those observed in the healthy control group. We hypothesized that there is an as-yet-unidentified substance in the aqueous humor of POAG patients that is responsible for blocking the trabecular meshwork. We hypothesized that this unidentified substance has not been described because it is below the detection threshold using current imaging techniques. Therefore, we applied EDC fixation, negative staining, and transmission electron microscopy imaging to observe the EV morphology in the aqueous humor of POAG patients. Under the same conditions used for the healthy controls, in situ imaging of the imaged aqueous humor from two different POAG patients was performed without treatment (no EV isolation). Unexpectedly, the aqueous humor of POAG patients showed multiple groups of EVs aggregated into large EV complexes that were several micrometers in size, which we termed "glaucoma-associated EV complexes" ( Figure 9 and Figure 10 ). Glaucoma-associated EV complexes were present in two different patient samples and were larger than the open pores of the JCT (1 to 4 μm, or up to 2 to 20 μm), and the complex was large enough to block the juxtacanalicular tissue. These data indicate that there is ultrastructural material in the aqueous humor of POAG patients that may be responsible for blocking the trabecular meshwork, reducing aqueous humor outflow, and potentially causing vision loss. We propose that glaucoma-associated EV complexes are the unidentified substance responsible for glaucoma pathology.

[0467] Example 9. Treatment of large EV complexes with bimatoprost disrupts the EV complexes when compared to controls. To determine whether the glaucoma-associated EV aggregates visualized using the EDC fixation method are indeed potential mediators of POAG, we hypothesized that treatment with a known glaucoma drug would alter the morphology of the glaucoma-associated EV aggregates. Therefore, we chose to use bimatoprost (Lumigan), an ocular hypotensive agent that reduces IOP in normal eyes, eyes with ocular hypertension, and glaucomatous eyes. Additionally, bimatoprost is known to enhance aqueous humor outflow by remodeling the extracellular matrix, by regulating matrix metalloproteinases, and by remodeling the extracellular matrix. Here, we hypothesized that the size of the glaucoma-associated EV complexes would be reduced by the addition of bimatoprost. To test this, we incubated aqueous humor from POAG patients with bimatoprost or buffered saline (placebo or control condition) at 37 °C for 72 hours and imaged the EV ultrastructure using EDC fixation, negative staining, and transmission electron microscopy ( Figures 11 - 13 ). After treatment with bimatoprost, we noticed a substantial decrease in the number of glaucoma-associated EV complexes in the POAG-bimatoprost samples compared to the POAG samples treated with placebo ( Figure 11 ). In a second human subject diagnosed with glaucoma, the experiment was repeated and POAG aqueous humor was mixed with placebo (buffered saline), and the samples were incubated at 37 °C for 72 hours. We observed numerous glaucoma-associated EV complexes ( Figure 12 ). However, under the same conditions, except that we used bimatoprost, we observed a substantial decrease in the number of glaucoma-associated EV complexes ( Figure 13 ). These data suggest that a known POAG treatment reduces the size of glaucoma-associated EV complexes and alters their morphology, indicating that the complex may be a potential pathological mediator of POAG.

[0468] Example 10. Aqueous humor from glaucoma patients contains larger electron-dense structures that are not present in the aqueous humor of healthy controls. Electron microscopy images show large electron-dense structures in the aqueous humor of POAG specimens that are not present in healthy controls. We hypothesized that the size of the glaucoma-associated EV aggregates would be larger and present in higher numbers when compared to healthy controls. Therefore, we obtained electron micrographs from healthy controls or POAG samples and measured the number and size of the glaucoma-associated EV aggregates. The data indicate that POAG aqueous humor has significantly larger glaucoma-associated EV aggregates, with sizes ranging from 361 nm to 20,214 nm ( Figure 14 ). Additionally, we observed that significantly more glaucoma-associated EV aggregates were present in the POAG samples when compared to healthy controls. These data suggest that POAG aqueous humor EVs contain large ultrastructural complexes that are not present in healthy controls (Figure 14 )。In addition, glaucoma-related EV aggregates are large enough to block the eye's drainage system, i.e., the trabecular meshwork. It is hypothesized that abnormal aqueous humor outflow leads to elevated IOP, which is the main risk factor for glaucoma. The TM region associated with establishing IOP is adjacent to Schlemm's canal and is called juxtacanalicular tissue (JCT) or cribriform area. The site of maximum resistance to aqueous humor outflow is the JCT, which has dimensions of approximately 2 - 20 μm (J. Ocular Biology 2013 June 1(1):3), with fenestrae of 1 - 4 μm or greater. The JCT consists of loosely arranged extracellular matrix (ECM) in which cells are embedded. The ECM of the JCT has been considered a barrier that can sequester aqueous humor outflow. Therefore, we hypothesized that this substance (glaucoma-related EV aggregates) in the aqueous humor of glaucoma patients is physically larger than the diameter of the JCT exit, potentially blocking aqueous humor outflow and being associated with glaucoma pathology.

[0469] Example 11. The aqueous humor of glaucoma patients contains EVs that contact each other and form larger structures called "glaucoma-related EV aggregates", which are not present in healthy control aqueous humor. To determine the composition of glaucoma-related EV aggregates, we analyzed TEM images and found that these ultrastructures consist of many EVs that contact each other to form larger aggregates. To quantify the number of EVs found in glaucoma-related EV aggregates, we counted the total number of EVs present in the images, quantified the number of EVs in each aggregate, or calculated the number of EVs that were not in contact with other EVs (free EVs). We classified the number of EVs in contact with each other as 0 (free EVs, no contact), less than 5 EVs in contact with each other, 5 to 10 EVs in contact with each other, 10 to 50 EVs in contact with each other, 50 to 100 EVs in contact with each other, or 100 to 300 EVs in contact with each other. The data showed that in healthy controls, few EVs were found in aggregates ( Figure 15 and Figure 16 ), while most EVs contacted fewer than 5 other EVs. The data indicate that in healthy controls, most EVs are "free EVs". Interestingly, for glaucoma patients, a large number of EVs contacted 50 to 300 other EVs. The data suggest that most EVs in glaucoma are within glaucoma-related EV aggregates, more than in healthy control samples ( Figure 15 and Figure 16 ). These data indicate that EVs in glaucoma patients contact each other, in sharp contrast to healthy controls, in which most EVs are free.

[0470] Example 12. Extracellular vesicles in the aqueous humor of healthy control subjects exist as "free EVs", and most EVs are between 100 and 200 nm in size. We sought to identify and quantify the EV population in healthy control aqueous humor. To determine the size distribution of EVs in healthy control aqueous humor, we characterized the EV population in human aqueous humor obtained from a single healthy control patient ( Figure 17 ). We fixed healthy control human aqueous humor with EDC, negatively stained the samples and imaged them with TEM. We analyzed the photographs and measured the diameters of the EVs. The data showed that for this patient, healthy control human aqueous humor contained EVs, and most of the EVs had diameters between 100 - 200 nm ( Figure 17 ). In addition, we observed that most EVs in the control group were not in contact with other EVs. This data indicates that healthy control aqueous humor contains exosomes and some microvesicles. We did not observe larger apoptotic bodies in these samples, nor did we observe glaucoma-related EV aggregates.

[0471] Example 13. Extracellular vesicles in the aqueous humor of subjects diagnosed with POAG are located within glaucoma-related EV aggregates, and their size is similar to that of the "free EVs" found in healthy controls. To study the EV population in the aqueous humor of human subject #1 diagnosed with POAG, we obtained aqueous humor, fixed the samples with EDC, negatively stained the specimens and imaged them with TEM. We analyzed the photographs and measured the EV size and counting frequency of those EVs located within glaucoma-related EV aggregates, or we counted the free EVs. In POAG specimen #1, we did not observe "free EVs". Then, we counted and measured the number and size of the EVs located within glaucoma-related EV aggregates ( Figure 18 ). The data showed that a large number of EVs located within glaucoma-related EV aggregates were between 36 nm and 300 nm in size, and the largest number of EVs were 100 - 200 nm in size ( Figure 18 ). In a second human subject diagnosed with POAG, we again found that POAG aqueous humor had some glaucoma-related EV aggregates. We observed EVs present within the aggregates (defined as EVs in contact with each other) or free EVs (EVs not in contact with each other, Figures 19 - 20 ). In addition, we found that a large number of EVs located within glaucoma-related EV aggregates were 36 - 300 nm in size ( Figure 21 ). Next, we compared the number and size of EVs present in glaucoma-related EV aggregates, and the EV populations in the two samples were similar ( Figure 22 ). These data indicate that the EV size population in POAG patients is located within glaucoma-related EV aggregates, and the data is consistent between samples.

[0472] Example 14. Free EVs in the aqueous humor of human subjects diagnosed with POAG differ in size and frequency when compared to the aqueous humor of healthy control subjects. To determine whether there are differences in free EVs in the aqueous humor of glaucoma patients and healthy controls, we compared the size and counting frequency of glaucoma and control conditions. We found that the size and frequency of EVs differed between POAG patients and healthy controls ( Figure 23 ). These data indicate that EVs that do not come into contact with other EVs are larger in POAG patients relative to healthy controls. These data may imply the function of free EVs.

[0473] Example 15. Extracellular vesicles from POAG aqueous humor present in glaucoma-related EV aggregates are similar in size and frequency to free EVs obtained from healthy human subjects. We hypothesized that EVs present in glaucoma-related EV aggregates are similar to healthy control EVs in both size and counting frequency. To test this, we compared the counting frequency and size of EVs from POAG and healthy control specimens. The size and frequency of EVs from POAG aqueous humor located within glaucoma-related EV aggregates were similar to unaggregated EVs in healthy human subjects ( Figure 24 ). These data suggest that due to EVs in POAG being trapped in glaucoma-related EV aggregates, the normal EV population from healthy controls may be prevented from functioning.

[0474] Example 16. The active agent for treating glaucoma can be cetylpyridinium of formula XI, which can be used in prodrug form or pharmaceutically acceptable salt form.

[0475]

[0476] It is 1-cetylpyridin-1-ium.

[0477] A solution of the pharmaceutical compound was prepared by weighing the compound in a microcentrifuge tube and dissolving the solid material in 1x PBS buffer at pH 7.2. For compounds with low solubility in water, a stock solution was prepared in ethanol or DMSO and then diluted 10-fold with 10% ethanol or DMSO vehicle to reach the final concentration. The solution of the compound was heated (37 °C) and vortexed to facilitate dissolution.

[0478] The concentration of cetylpyridinium chloride was 43 mg / ml.

[0479] To determine the effect of the compound on intraocular pressure (IOP), the compound was tested in bovine vitreous humor (BVH) in a microfluidic chip device.

[0480] A solution of 25% homogenized bovine vitreous humor (BVH) was prepared by diluting 100% homogenized BVH with PBS buffer. 50 μL of BVH was aliquoted into 0.5 mL PCR tubes. 50 μL of the compound solution was added to the BVH to bring the total concentration of BVH to 12.5%. The samples were vortexed briefly and then incubated overnight at 37 °C. For the control experiment, 50 μL of PBS buffer or PBS containing 10% ethanol or DMSO was prepared and incubated with 25% BVH under the same conditions.

[0481] The test BVH solution was introduced into the reservoir of the device. The fluid probe was connected to the inlet of the microfluidic chip and a flow rate of 2 μL / min was established using PBS as the source fluid. Once the fluid started to flow out of the outlet of the chip and reached a steady flow of 2 μL / min, the flow rate and pressure changes inside the microfluidic chip were recorded. The baseline flow rate and pressure readings were recorded for 5 minutes, and then 7 μL of the test BVH solution was injected into the chip through a sample syringe. The flow rate and pressure changes were continued to be recorded for an additional 50 minutes after the sample injection. Recording was stopped after 55 minutes. The relative change in chip pressure throughout the experiment was plotted on a graph.

[0482] Figure 25 It was shown that the agent cetylpyridinium chloride reduced intraocular pressure (IOP) in a glaucoma model compared to the control. The agent was tested by controlling the flow and measuring the relative IOP in a microfluidic device. The agent was compared with a placebo (buffered saline) by preparing each sample in bovine vitreous humor (BVH) and pre-incubating at 37 °C for 24 hours. The time point of injection into the device is indicated by the arrow and the letter "a". Refer to Figure 25 , after injection of the placebo sample, the IOP of the placebo (dashed line) increased significantly. The IOP steadily rose to a maximum pressure of approximately 64 mmHg. In contrast, the IOP (solid line) after injection of the agent cetylpyridinium chloride - BVH sample was significantly lower than that of the placebo, and this difference persisted. The results indicate that the agent cetylpyridinium chloride was unexpectedly effective in reducing IOP in a glaucoma model.

[0483] Example 17. The active agent for treating glaucoma can be polymyxin B of formula XXI, which can be used in the prodrug form or in the form of a pharmaceutically acceptable salt.

[0484]

[0485] A solution of the pharmaceutical compound was prepared by weighing the compound in a microcentrifuge tube and dissolving the solid material in 1x PBS buffer at pH 7.2. For compounds with low solubility in water, a stock solution was prepared in ethanol or DMSO and then diluted 10-fold with 10% ethanol or DMSO vehicle to achieve the final concentration. The solution of the compound was heated (37 °C) and vortexed to facilitate dissolution.

[0486] The concentration of polymyxin B sulfate was 10 mg / ml.

[0487] To determine the effect of the compound on intraocular pressure (IOP), the compound was tested in bovine vitreous humor (BVH) in a microfluidic chip device.

[0488] A solution of 25% homogenized bovine vitreous humor (BVH) was prepared by diluting 100% homogenized BVH with PBS buffer. 50 μL of BVH was aliquoted into 0.5 mL PCR tubes. 50 μL of the compound solution was added to the BVH to bring the total concentration of BVH to 12.5%. The samples were vortexed briefly and then incubated overnight at 37 °C. For the control experiment, 50 μL of PBS buffer or PBS containing 10% ethanol or DMSO was prepared and incubated with 25% BVH under the same conditions.

[0489] The test BVH solution was introduced into the reservoir of the device. A fluid probe was connected to the inlet of the microfluidic chip and a flow rate of 2 μl / min was established using PBS as the source fluid. Once the fluid started to flow out of the outlet of the chip and reached a steady flow of 2 μl / min, the flow rate and pressure changes inside the microfluidic chip were recorded. The baseline flow rate and pressure readings were recorded for 5 minutes, and then 7 μl of the test BVH solution was injected into the chip through a sample syringe. The flow rate and pressure changes were continued to be recorded for an additional 50 minutes after the sample injection. Recording was stopped after 55 minutes. The relative change in chip pressure throughout the experiment was plotted on a graph.

[0490] Figure 26 It was shown that the pharmaceutical polymyxin B reduced intraocular pressure (IOP) in a glaucoma model compared to the control. The pharmaceutical was tested by controlling the flow and measuring the relative IOP in a microfluidic device. The pharmaceutical was compared to a placebo (buffered saline) by preparing each sample in bovine vitreous humor (BVH) and pre-incubating at 37 °C for 24 hours. The time point of injection into the device is indicated by the arrow and the letter "a". Reference Figure 26, after injecting the placebo sample, the IOP of the placebo (dashed line) increased significantly. The IOP steadily rose to a maximum pressure of approximately 250 mmHg. In contrast, the IOP (solid line) after injecting the agent polymyxin B was 78% lower than that of the placebo, and this difference persisted. The results indicate that the agent polymyxin B is unexpectedly effective in reducing IOP in the glaucoma model.

[0491] Example 18. The active agent for treating glaucoma can be neomycin of formula XI, which can be used in the form of a prodrug or a pharmaceutically acceptable salt.

[0492]

[0493] A solution of the pharmaceutical compound was prepared by weighing the compound in a microcentrifuge tube and dissolving the solid material in 1x PBS buffer at pH 7.2. For compounds with low solubility in water, a stock solution was prepared in ethanol or DMSO and then diluted 10-fold with 10% ethanol or DMSO vehicle to achieve the final concentration. The solution of the compound was heated (37 °C) and vortexed to facilitate dissolution.

[0494] The concentration of neomycin sulfate was 35 mg / ml.

[0495] To determine the effect of the compound on intraocular pressure (IOP), the compound was tested in bovine vitreous humor (BVH) in a microfluidic chip device.

[0496] A solution of 25% homogenized bovine vitreous humor (BVH) was prepared by diluting 100% homogenized BVH with PBS buffer. 50 uL of BVH was aliquoted into 0.5 mL PCR tubes. 50 uL of the compound solution was added to the BVH to bring the total concentration of BVH to 12.5%. The sample was briefly vortexed and then incubated overnight at 37 °C. For the control experiment, 50 uL of PBS buffer or PBS containing 10% ethanol or DMSO was prepared and incubated with 25% BVH under the same conditions.

[0497] The test BVH solution was introduced into the reservoir of the device. A fluid probe was connected to the inlet of the microfluidic chip and a flow rate of 2 ul / min was established using PBS as the source fluid. Once the fluid started to flow out of the outlet of the chip and reached a stable flow of 2 ul / min, the flow rate and pressure changes inside the microfluidic chip were recorded. The baseline flow rate and pressure readings were recorded for 5 minutes, and then 7 ul of the test BVH solution was injected into the chip through a sample syringe. The flow rate and pressure changes were continued to be recorded for an additional 50 minutes after the sample injection. Recording was stopped after 55 minutes. The relative change in chip pressure throughout the experiment was plotted on a graph.

[0498] Figure 27It was shown that, compared with the control, the agent neomycin reduced intraocular pressure (IOP) in the glaucoma model. The agent was tested by controlling the flow and measuring the relative IOP in a microfluidic device. The agent was compared with a placebo (buffered saline) by preparing each sample in bovine vitreous humor (BVH) and pre-incubating at 37 °C for 24 hours. The time points of injection into the device are indicated by the arrows and the letter "a". Reference Figure 27 , after injection of the placebo sample, the IOP of the placebo (dashed line) increased significantly. The IOP steadily rose to a maximum pressure of approximately 64 mmHg. In contrast, the IOP (solid line) after injection of the agent neomycin was 72% lower than that of the placebo, and this difference persisted. The results indicate that the agent neomycin is unexpectedly effective in reducing IOP in the glaucoma model.

[0499] Example 19. The active agent for treating glaucoma can be heparin, which can be used in the form of a prodrug or a pharmaceutically acceptable salt.

[0500] A solution of the agent compound was prepared by weighing the compound in a microcentrifuge tube and dissolving the solid material in 1x PBS buffer at pH 7.2. For compounds with low solubility in water, a stock solution was prepared in ethanol or DMSO and then diluted 10-fold with a 10% ethanol or DMSO vehicle to reach the final concentration. The solution of the compound was heated (37 °C) and vortexed to facilitate dissolution.

[0501] The concentration of sodium heparin was 10 mg / ml.

[0502] To determine the effect of the compound on intraocular pressure (IOP), the compound was tested in bovine vitreous humor (BVH) in a microfluidic chip device.

[0503] A solution of 25% homogenized bovine vitreous humor (BVH) was prepared by diluting 100% homogenized BVH with PBS buffer. 50 uL of BVH was aliquoted into 0.5 mL PCR tubes. 50 uL of the compound solution was added to the BVH to bring the total concentration of BVH to 12.5%. The samples were briefly vortexed and then incubated overnight at 37 °C. For the control experiment, 50 uL of PBS buffer or PBS containing 10% ethanol or DMSO was prepared and incubated with 25% BVH under the same conditions.

[0504] Introduce the test BVH solution into the reservoir of the device. Connect the fluid probe to the inlet of the microfluidic chip and establish a flow rate of 2 μl / min using PBS as the source fluid. Once the fluid starts to flow out of the outlet of the chip and reaches a stable flow of 2 μl / min, record the flow rate and pressure changes inside the microfluidic chip. Record the baseline flow rate and pressure readings for 5 minutes, then inject 7 μl of the test BVH solution into the chip through the sample syringe. Continue to record the flow rate and pressure changes for an additional 50 minutes after the sample injection. Stop recording after 55 minutes. Plot the relative change in chip pressure throughout the experiment on a graph.

[0505] Figure 28 Shown that, compared to the control, the agent sodium heparin reduced intraocular pressure (IOP) in the glaucoma model. The agent was tested by controlling flow and measuring relative IOP in a microfluidic device. The agent was compared to a placebo (buffered saline) by preparing each sample in bovine vitreous humor (BVH) and pre-incubating at 37 °C for 24 hours. The time point of injection into the device is indicated by the arrow and the letter "a". Refer Figure 28 , after injecting the placebo sample, the IOP of the placebo (dashed line) increased significantly. The IOP steadily rose to a maximum pressure of approximately 67 mmHg. In contrast, the IOP (solid line) after injecting the agent sodium heparin was 32% lower than that of the placebo, and this difference persisted. The results indicate that the agent sodium heparin was unexpectedly effective in reducing IOP in the glaucoma model.

[0506] Example 20. Sodium dodecyl sulfate is a negative control for intraocular pressure (IOP) in the glaucoma model.

[0507] Prepare the solution by weighing the compound in a microcentrifuge tube and dissolving the solid material in 1x PBS buffer at pH 7.2. For compounds with low solubility in water, prepare a stock solution in ethanol or DMSO and then dilute 10-fold with 10% ethanol or DMSO vehicle to reach the final concentration. Heat (37 °C) and vortex the compound solution to facilitate dissolution.

[0508] The concentration of sodium dodecyl sulfate is 24 mg / ml.

[0509] To determine the effect of the compound on intraocular pressure (IOP), the compound was tested in bovine vitreous humor (BVH) in a microfluidic chip device.

[0510] A 25% solution of homogenized bovine vitreous humor (BVH) was prepared by diluting 100% homogenized BVH with PBS buffer. 50 μL of BVH was aliquoted into 0.5 mL PCR tubes. 50 μL of the compound solution was added to the BVH to bring the total BVH concentration to 12.5%. The samples were vortexed briefly and then incubated overnight at 37 °C. For the control experiment, 50 μL of PBS buffer or PBS containing 10% ethanol or DMSO was prepared and incubated with 25% BVH under the same conditions.

[0511] The test BVH solution was introduced into the reservoir of the device. The fluid probe was connected to the inlet of the microfluidic chip and a flow rate of 2 μL / min was established using PBS as the source fluid. Once the fluid started to flow out of the outlet of the chip and reached a steady flow of 2 μL / min, the flow rate and pressure changes inside the microfluidic chip were recorded. The baseline flow rate and pressure readings were recorded for 5 minutes, and then 7 μL of the test BVH solution was injected into the chip through the sample syringe. The flow rate and pressure changes were continued to be recorded for an additional 50 minutes after the sample injection. Recording was stopped after 55 minutes. The relative change in chip pressure during the entire experiment was plotted on a graph.

[0512] Figure 29 Shown, the compound sodium dodecyl sulfate is a negative control for intraocular pressure (IOP) in a glaucoma model. The compound was tested by controlling the flow and measuring the relative IOP in a microfluidic device. The compound was compared to a placebo (buffered saline) by preparing each sample in bovine vitreous humor (BVH) and pre-incubating at 37 °C for 24 hours. The time point of injection into the device is indicated by the arrow and the letter "a". Refer Figure 29 , after injecting the placebo sample, the IOP of the placebo (dashed line) increased significantly. The IOP steadily rose to a maximum pressure of approximately 60 mmHg. However, the IOP after injecting sodium dodecyl sulfate (solid line) was significantly higher than that of the placebo. This result indicates that sodium dodecyl sulfate is a negative control that does not reduce IOP in a glaucoma model.

[0513] Generally, intraocular administration can be performed by intracameral administration, intravitreal administration, or subretinal administration. Periocular administration can be performed by subconjunctival injection, subtenon injection, direct periocular injection, or depot periocular injection. Systemic administration can be performed by intravenous administration, oral administration, intraarterial administration, inhalation, intranasal administration, intraperitoneal administration, intraperitoneal administration, subcutaneous administration, intra-articular administration, intrathecal administration, transdural administration, transdermal administration, submucosal administration, sublingual administration, enteral administration, parenteral administration, transdermal administration, periaricular administration, or intraventricular administration.

[0514] Ophthalmic formulations can be delivered locally by eye drops, by direct injection, or by using an infusion pump. Intraocular administration can be carried out by intracameral administration, intravitreal administration, or subretinal administration.

Claims

1. Use of an aqueous pharmaceutical composition in the preparation of a medicament for reducing extracellular vesicle complexes in the eye of a human or animal body, said composition comprising the active agent polymyxin B sulfate, wherein the active agent accounts for 0.1-10% by weight of the composition, and wherein the extracellular vesicle complex in the eye is an aggregate of extracellular vesicles with a diameter greater than 300 nanometers.

2. The use according to claim 1, wherein the composition has a pH of 7.

3.

3. The use according to claim 1, wherein the composition further comprises one or more of a solubilizer, a surfactant, a tonicity agent, and a preservative.

4. The use according to claim 3, wherein the solubilizer is selected from phosphates, citric acid monohydrate, trisodium citrate, and combinations thereof.

5. The use according to claim 3, wherein the surfactant is selected from phospholipids, polyglycerol esters, propylene glycol esters, polyethylene glycol esters, copolymer esters, polyoxyethylene sorbitan esters, cyclodextrins, polyvinyl alcohol, polyvinylpyrrolidone, hydroxypropylmethylcellulose, poloxamer, carboxymethylcellulose, hydroxyethylcellulose, polyacrylates, and combinations thereof.

6. The use according to claim 3, wherein the tonicity agent is selected from sodium chloride, trehalose, mannitol, sorbitol, dextrose, potassium chloride, and combinations thereof.

7. The use according to claim 3, wherein the preservative is selected from benzalkonium chloride, polyquaternium-1, dodecyldimethylbenzylammonium bromide, sorbic acid, methyl paraben, propyl paraben, chlorobutanol, benzyl alcohol, phenethyl alcohol, chloramine-T, thimerosal, sodium perborate, disodium edetate, and combinations thereof.

8. The use according to claim 1, wherein the composition is administered to the eye of the subject by intracameral injection or periocular injection.

9. The use according to claim 8, wherein the composition is administered to the eye of the subject by intracameral injection, intravitreal injection, subretinal injection, subconjunctival injection, subfascial injection, direct periocular injection, or depot periocular injection.

Images