Methods and agents for glaucoma

Abstract

To provide: methods and compositions for detecting, identifying and treating glaucoma diseases; and compositions and methods for affecting intraocular pressure and increasing ocular outflows in glaucoma.SOLUTION: The invention provides an aqueous pharmaceutical composition for treating a glaucoma disease, reducing intraocular pressure, and / or reducing ocular extracellular vesicle complexes, the composition comprising an active agent selected from cetylpyridinium chloride, polymyxin B sulfate, a mycin, and heparin sodium.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to methods and compositions for detecting, identifying and treating glaucoma disease. In particular, the present invention discloses compositions and methods for affecting intraocular pressure and increasing intraocular outflow in glaucoma. [Background technology]

[0002] Glaucoma disease is the leading cause of vision loss worldwide, affecting an estimated 70 million people. Glaucoma is a permanent visual impairment that is asymptomatic until the patient experiences severe vision loss. Diagnosis of glaucoma is often delayed.

[0003] Forms of glaucoma are described as open-angle or angle-closure glaucoma. Primary open-angle glaucoma (POAG) is the most prevalent, accounting for approximately 75% of cases. Narrow-angle and rarer forms account for the other 25%. In POAG, the anterior chamber angle appears normal and open, and there is elevated intraocular pressure (IOP) in the absence of underlying disease.

[0004] Risk factors for POAG include elevated IOP, increasing age, family history, African ancestry, myopia, and complications of diabetes or hypertension. The pathophysiology underlying glaucoma is related to increased resistance to aqueous humor outflow, but the direct mediator of this process remains unknown. The etiology of glaucoma is poorly understood, and factors that contribute to its progression have not been identified.

[0005] The signs and symptoms of glaucoma include degeneration of retinal ganglion cells, changes to the optic nerve, and damage to the optic nerve with corresponding visual field loss. Elevated IOP is associated with retinal ganglion cell (RGC) death and eventual visual field (VF) loss. IOP-related optic nerve damage is important in the pathogenesis of POAG. Patients with POAG and intraocular hypertension (OHT) have elevated IOP. Elevated IOP is a significant risk factor for progression from OHT to POAG. Elevated IOP is the only common clinical finding in the various secondary glaucoma types. Lowering IOP has been shown to reduce the risk of progression in NTG. In animal models, elevated IOP precedes glaucomatous nerve damage. In general, the pathogenesis of elevated IOP may be due to reduced aqueous humor outflow.

[0006] Medical treatments for glaucoma are directed at lowering IOP, which can slow disease progression in some patients. Drawbacks to current treatments include lack of efficacy and side effects of the drugs.

[0007] What is needed are effective methods and compositions for glaucoma, as well as medical devices that lower IOP and improve intraocular outflow.

[0008] There is an urgent need for methods, kits and compositions for detecting, identifying and treating glaucoma. Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention provides methods, compositions, devices, kits and reagents for detecting, identifying and treating glaucoma disease. [Means for solving the problem]

[0010] In some aspects, the present invention provides methods and compositions for lowering intraocular pressure and improving intraocular outflow in glaucoma subjects. Aspects of the invention can reduce the formation and presence of aggregate features and structures in ocular aqueous solution.

[0011] In a further aspect, the present disclosure provides a therapeutic composition for glaucoma.

[0012] Embodiments of the present invention provide devices for measuring and characterizing the coagulation characteristics of glaucoma, as well as intraocular pressure and outflow.

[0013] Further aspects of the present disclosure include diagnostic and screening medical devices for glaucoma. Further embodiments include kits and reagents for carrying out the above.

[0014] Specific examples of the present invention include:

[0015] An aqueous pharmaceutical composition for ophthalmic use comprising an active agent selected from cetylpyridinium chloride, polymyxin B sulfate, mycin, and heparin sodium. The mycin may be selected from neomycin, salinomycin, azithromycin, rapamycin, gentamicin, erythromycin, adriamycin, bleomycin, dactinomycin, mitomycin, plicamycin, dihydrostreptomycin, kanamycin, natamycin, rifamycin, and tobramycin. The active agent may comprise 0.01-2% w / v of the composition. The active agent may comprise 0.01-0.2% w / v of the composition. The composition may have a pH of about 7.3.

[0016] The composition, when administered to the eye, is capable of reducing intraocular pressure.

[0017] The composition, when administered to the eye, is capable of reducing ocular extracellular vesicle complexes.

[0018] The composition, when administered to the eye, can be effective in treating glaucoma.

[0019] The composition may further comprise one or more of a solubilizer, a surfactant, an isotonicity agent, 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, polyethylene glycol esters, polyethylene glycol esters, copolymer esters, polyoxyethylene sorbitan esters, cyclodextrins, polyvinyl alcohols, povidone, hydroxypropyl methylcellulose, poloxamers, carboxymethylcellulose, hydroxyethylcellulose, polyacrylates, and combinations thereof. The isotonicity agent may be selected from sodium chloride, trehalose, mannitol, sorbitol, dextrose, potassium chloride, and combinations thereof. Preservatives may be selected from benzalkonium chloride, polyquaternium-1, benzododecinium bromide, sorbic acid, methylparaben, propylparaben, chlorobutanol, benzyl alcohol, phenylethyl alcohol, oxychloro complexes, thimerosal, sodium perborate, disodium edetate, and combinations thereof.

[0020] The above compositions can be used in medical therapy, treatment of the human or animal body, reducing intraocular pressure in the human or animal body, reducing ocular extracellular vesicle complexes in the human or animal body, or preparing or manufacturing a medicament for preventing, ameliorating, or treating a disease or condition related to glaucoma in a subject in need thereof.

[0021] The present invention also provides a method for treating glaucoma, reducing intraocular pressure, or reducing ocular extracellular vesicle complexes in a subject in need thereof. The administration can be by injection. The ocular extracellular vesicle complexes can be aggregates of extracellular vesicles having a diameter greater than about 300 nanometers. [Brief description of the drawings]

[0022] [Figure 1]Figure 1 shows the anatomy of a normal eye (left) compared to a glaucomatous eye (right).

[0023] [Diagram 2] FIG. 2 shows an electron micrograph of a sample of trabecular meshwork (left) and an illustration of aqueous humor flow within the ocular trabecular meshwork (right).

[0024] [Diagram 3] 3 shows a schematic diagram of an embodiment of a device for detecting flow and pressure of body compositions in a channel. The channel can contain a meshwork having properties similar to the trabecular meshwork of the eye. The channel network can have a uveal meshwork portion, a corneoscleral meshwork portion, and a trabecular meshwork portion. The channel can be a microfluidic channel.

[0025] [Figure 4] FIG. 4 shows an embodiment of a series of test channels for detecting the effect of various compounds, compositions and substances on the flow and pressure of the fluid composition in the channels.

[0026] [Diagram 5] 5 shows an example of a device and system for detecting the flow and pressure of a fluid composition in a channel. The channel can contain a meshwork having properties similar to the trabecular meshwork of the eye. Alternatively, the device can utilize a reservoir for a test sample of the fluid composition. The channel can be a microfluidic channel.

[0027] [Figure 6]Figure 6 shows that imaging of extracellular vesicles with conventional fixation techniques resulted in poor EV imaging due to failure of EV adhesion. Imaging was surprisingly improved using irreversible cross-linking reagents. (a) Representative photomicrographs of isolated bovine vitreous EVs loaded at 4 million and fixed on copper grids with glutaraldehyde followed by UA and lead citrate solution show few negatively stained EVs (arrowheads) at low magnification (left) and no EVs visible in other pictures (middle and right). Negatively stained EVs are indicated by signal surrounding the periphery of the EVs (black) and low signal in the center (white or grey). (b) Schematic showing failure of EVs in solution applied to electron microscope grid surface to adhere. EVs were present in the effluent (black box) and the inventors quantified the size and number of EVs using nanoparticle tracking analysis (NTA). (c) NTA shows the size and concentration of EVs applied to the TEM grid surface. (d) NTA shows the size and concentration of EVs present in the effluent. The numbers represent the size and number of EVs that failed to adhere to the TEM grid surface. (g) Graphical representation compares the amount of EVs applied to the electron microscopy grid surface (black bars) with the size and concentration of EVs present in the effluent (grey bars, n=3). (f) Representative TEM micrographs of isolated bovine vitreous EVs after EDC-glutaraldehyde fixation, negative staining and TEM imaging reveal that substantially more EVs were visualized at low (left), medium (center) and high (right) magnification. (g) Graphical representation of the mean and ± standard deviation (log2) shows that significantly more EVs were counted per image (350x) from EDC-fixed specimens compared to Glut-fixed grids (n=3, average of 7 images counted per biological replica, *p < 0.05). (h) Representative TEM micrographs of isolated bovine aqueous humor after EDC-glutaraldehyde fixation show robust negative staining surrounding the borders of EVs. (i) TEM images from human aqueous humor without EV isolation show the presence of numerous EVs in situ. Scale bars are (a) 1000 nm (left), 600 nm (middle), 125 nm (right); (f) 600 nm (left), 125 nm (right); (h) 500 nm; and (i) 1 μm.

[0028] [Figure 7] Figure 7 shows healthy human aqueous humor, control and patient no. 1, showing the diffuse distribution of non-aggregated EVs. (a-c) Representative micrographs of diluted human healthy control aqueous humor EVs (EVs were not isolated by ultracentrifugation, but only diluted in buffered saline) fixed on copper grids with EDC-glutaraldehyde and subsequently stained with UA solution. Photographs were taken and recorded by transmission electron microscopy. These images show many negatively stained EVs of various sizes, with many EVs present independently in the fluid without aggregation. Single EV aggregates were observed and were in the minority of the samples imaged. Scale bars are indicated on the photographs.

[0029] [Figure 8] Figure 8 shows healthy human aqueous humor, control and patient 2, showing the diffuse distribution of non-aggregated EVs. (a-d) Representative micrographs of diluted human healthy control aqueous humor EVs (EVs were not isolated by ultracentrifugation, only diluted in buffered saline) fixed on copper grids with EDC-glutaraldehyde and subsequently stained with UA solution. Photographs were taken and recorded by transmission electron microscopy. The images show many negatively stained EVs of various sizes. Many EVs are present in the fluid independently, without aggregation. Single EV aggregates were observed and were in the minority of the samples imaged. No EV aggregates larger than 2 μm were observed, and no large clumps of EVs were visible. Scale bars are indicated on the photographs.

[0030] [Figure 9] Figure 9 shows POAG aqueous humor, demonstrating large glaucoma-associated EV aggregates in patient #1. (a-c) Representative transmission electron micrographs show POAG aqueous humor samples (EVs were not isolated by ultracentrifugation, only diluted in buffered saline) fixed on copper grids with EDC-glutaraldehyde and subsequently stained with UA solution. Photographs were taken and recorded by transmission electron microscopy. Dilute POAG specimens show evidence of glaucoma-associated EV aggregates present in the fluid. Images show that few, if any, were observed (a-c). Scale bars are noted on the photographs.

[0031] [Figure 10] FIG. 10 shows POAG aqueous humor, demonstrating large glaucoma-associated EV aggregates in patient #2. (a-c) Representative transmission electron micrographs from a second POAG sample (EVs were not isolated by ultracentrifugation, but only diluted in buffered saline) fixed on copper grids with EDC-glutaraldehyde and subsequently stained with UA solution. Photographs were taken and recorded by transmission electron microscopy. The second POAG aqueous humor specimen shows evidence of sizeable glaucoma-associated EV aggregates and larger free EVs. Scale bars are noted on the photographs.

[0032] [Figure 11] FIG. 11 shows that a known glaucoma drug, bimatoprost, reduces the size of POAG-associated EV complexes in the aqueous humor of glaucoma patients compared to untreated glaucoma samples. (a-b) Representative transmission electron micrographs show untreated human aqueous humor (placebo, control, buffered saline) collected from patients with POAG, showing evidence of extracellular matrix and glaucoma-associated EV complexes in the aqueous humor. The images show large collagen-like matrices in all panels, and these electron-dense structures are a few microns in size. (c-d) Representative photographs show POAG aqueous humor samples treated with the glaucoma drug, bimatoprost, showing no evidence of large extracellular matrix or glaucoma-associated EV aggregates. High-power imaging shows what appear to be larger globules that are not aggregated. Scale bars are noted in the figures.

[0033] [Figure 12] FIG. 12 shows that treatment of aqueous humor of a glaucoma patient with buffered saline has no effect on POAG-associated EV complexes in a second subject diagnosed with glaucoma. (FIG. 12) Representative transmission electron micrographs show untreated human aqueous humor (placebo, control, buffered saline) collected from a patient with POAG, showing evidence of glaucoma-associated EV complexes. Images show large collagen-like matrices in all panels. Placebo treatment has no effect on glaucoma-associated EV complexes. Scale bars are noted on the figures.

[0034] [Figure 13] FIG. 13 shows that bimatoprost reduces the size of POAG-associated EV complexes in the aqueous humor of a second subject diagnosed with glaucoma, compared to a control-treated glaucoma sample. (FIG. 13) Representative photographs show POAG aqueous humor from subject #2 sample treated with bimatoprost, showing disruption of large electron-dense glaucoma-associated EV aggregates. The glaucoma-associated EV aggregates are smaller in size, compared to the control (shown in FIG. 12). With high power imaging, not many aggregated EVs are observed. Scale bars are noted in the figures.

[0035] [Figure 14] FIG. 14 shows that aqueous humor from glaucoma patients has more electron-dense structures in the aqueous humor that are not present in healthy controls. The graphical representation depicts the size and number of unidentified substances, which the inventors termed "glaucoma-associated EV aggregates" in healthy control or POAG specimens. The inventors obtained healthy control or POAG human aqueous humor, fixed the samples with EDC, and imaged the specimens with transmission electron microscopy. The photographs were analyzed to identify and plot the number of glaucoma-associated EV aggregates for each variable. In aqueous humor obtained from a single healthy control subject, the data show that most EVs are free of glaucoma-associated EV aggregates. The data show that three subjects diagnosed with POAG have numerous glaucoma-associated EV aggregates several micrometers in size. These aggregates were not observed in healthy controls. The graph shows a substantial difference in the size of glaucoma-associated EV aggregates in POAG compared to healthy controls.

[0036] [Figure 15]FIG. 15 shows aqueous humor of glaucoma patients containing EVs that are aggregated and touch each other compared to healthy controls with fewer EVs touching each other. The graphical representation depicts the number of EVs touching each other (X-axis) and the count frequency (percent of total). The inventors obtained human aqueous humor of healthy controls or POAG, fixed the samples with EDC, and imaged the specimens with transmission electron microscopy. The photographs were analyzed and the number of EVs touching each other for each variable was determined. In aqueous humor obtained from a single healthy control subject, the data shows that most are free and not present as aggregates. The data shows that two subjects diagnosed with POAG have aqueous humor that contains EVs that touch many other EVs. The graph shows a substantial difference between the number of EVs touching each other in POAG compared to healthy controls.

[0037] [Figure 16] Figure 16 shows a curve chart of the data from Figure 15. The aqueous humor of glaucoma patients contains EVs that are aggregated and touch each other compared to healthy controls who have fewer EVs touching each other.

[0038] [Figure 17] FIG. 17 shows that extracellular vesicles are present in human aqueous humor obtained from a healthy control patient, with a predominant population between 100 and 200 nm. The graphical representation depicts the EV population in human aqueous humor obtained from a single healthy control patient. The EV count frequency is shown as a function of size. We measured EV diameter after fixing human aqueous humor from a healthy control fixed with EDC fixation and imaged with transmission electron microscopy, and healthy control human aqueous humor. The data show that healthy control human aqueous humor contains EVs. For this patient, the majority of EVs are between 100 and 200 nm in diameter. All visualized EVs were not in contact with other EVs (not aggregated).

[0039] [Figure 18]FIG. 18 shows that extracellular vesicles in the aqueous humor of subject #1 diagnosed with POAG are located within "glaucoma-associated EV aggregates," with all EVs located within the glaucoma-associated EV aggregates. (a) Graphical representation of EV population in human aqueous humor obtained from subject #1 diagnosed with POAG. The inventors fixed the aqueous humor with EDC and imaged it by transmission electron microscopy. The pictures were analyzed, and EV size and number were quantified and graphed. The inventors observed that EVs were present inside aggregates (defined as EVs that are in contact with each other or glaucoma-associated EV aggregates). The data shows the number and size of individual EVs present inside glaucoma-associated EV aggregates (gray bars) as a function of the total number of EVs counted. A significant number of EVs are between 36 nm and 300 nm in size, with a high number of EVs between 100-200 nm. In the aqueous humor of subject #1, the inventors could not observe free EVs (EVs that are not in contact with each other).

[0040] [Figure 19] FIG. 19 shows that extracellular vesicles in aqueous humor obtained from a human subject diagnosed with primary open angle glaucoma (POAG) either contacted each other to form aggregates or EVs were present as free EVs (non-aggregated). (a-b) Graphical representation of EV population in human aqueous humor obtained from a single subject diagnosed with POAG. The inventors fixed the aqueous humor with EDC fixation and imaged it by transmission electron microscopy. The pictures were analyzed to quantify EV size and number. The inventors observed EVs present within aggregates (defined as EVs that contact each other) or free EVs (defined as EVs that do not contact each other). The data shows a population of EVs that do not contact each other (non-aggregated EVs). A significant number of EVs were between 100-500 nm in size, with some larger.

[0041] [Figure 20] Figure 20 shows a graph displaying the size and frequency of EVs in contact with other EVs located within the "glaucoma-associated EV aggregates" of the patient in Figure 19. A significant amount of EVs were between 36 and 300 nm, with slightly larger free EVs observed.

[0042] [Figure 21] FIG. 21 shows that extracellular vesicles in aqueous humor of a single subject diagnosed with POAG are located within "glaucoma-associated EV aggregates" and a significant population of EVs are present within glaucoma-associated EV aggregates. (a) Graphical representation of EV population in human aqueous humor obtained from a single subject diagnosed with POAG. The inventors fixed aqueous humor with EDC fixation and imaged it by transmission electron microscopy. The pictures were analyzed to quantify EV size and number. The inventors observed EVs present within aggregates (defined as EVs that are in contact with each other) or free EVs (defined as EVs that are not in contact with each other). The data show the number and size of individual EVs present within glaucoma-associated EV aggregates (black bars) or the number and size of non-aggregated EVs (free EVs, black and white striped bars) as a function of the total number of EVs counted. A significant number of EVs were located within glaucoma-associated EV aggregates. A significant number of EVs within the aggregates show diameters from 100-300 nm in size. The larger EVs had a higher population indicating a significant number of free EVs (non-aggregated EVs) in the larger size range.

[0043] [Figure 22] FIG. 22 shows that extracellular vesicles in two separate glaucoma patients show similar size and number of EVs located in "glaucoma-associated EV aggregates." (a) Graphical representation of EV population in human aqueous humor obtained from two subjects diagnosed with POAG. The inventors fixed aqueous humor from each subject with EDC and imaged it with the specimens by transmission electron microscopy. The pictures were analyzed to quantify EV size and number. The inventors observed EVs present within the aggregates (defined as EVs that are in contact with each other). The data shows the size and number of EVs present within the glaucoma-associated EV aggregates from subject #1 (gray bars) and subject #2 (black bars). The data was normalized to show that both samples have a similar distribution of EVs present within the glaucoma-associated EV aggregates.

[0044] [Figure 23]FIG. 23 shows that extracellular vesicles in the aqueous humor of human subjects diagnosed with POAG differ in size and frequency compared to that of healthy control subjects. (a) Graphical representation of EV population in human aqueous humor obtained from subjects diagnosed with POAG and normal healthy controls. The inventors fixed the aqueous humor of each subject with EDC and imaged it with the specimens by transmission electron microscopy. The pictures were analyzed to quantify EV size and number. The inventors observed that free EVs (defined as non-aggregated, EVs that do not touch each other) differed in size and frequency compared to the healthy controls. Data shows the size and number of free EVs from POAG-derived healthy controls (white bars) or subject #2 (black bars). The inventors observed that the free EVs present in the aqueous humor of patient #2 had significantly larger EVs compared to the healthy controls.

[0045] [Figure 24] FIG. 24 shows that extracellular vesicle POAG aggregates are comparable in size and frequency to healthy human subjects. (a) Graphical representation of EV populations from aqueous humor of human subjects obtained from healthy controls (free EVs) or POAG subjects (aggregated EVs). We fixed aqueous humor from each subject with EDC and imaged it along with the specimens by transmission electron microscopy. The pictures were analyzed to quantify EV size and number. We observed that free EVs (defined as non-aggregated, EVs that do not touch each other) were comparable in size and frequency compared to EVs located within aggregates in two separate POAG subjects. Data shows size and number of free EVs from healthy control (white bars), aggregated EVs from POAG subject #1 (gray bars), and free EVs from subject #2 (striped bars).

[0046] [Diagram 25]FIG. 25 shows that the agent cetylpyridinium chloride reduced intraocular pressure (IOP) in a glaucoma model compared to the control. The agents were tested by controlling flow and using a microfluidic device to measure relative IOP. The agents were each compared to a placebo (phosphate buffer) by preparing 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 an arrow and the letter "a." Referring to FIG. 25, the IOP of the placebo (dashed line) increased significantly after injection of the placebo sample. The IOP steadily increased to a maximum pressure of about 64 mmHg. Conversely, after injection of the agent cetylpyridinium chloride-BVH sample (solid line), it was significantly lower than that for the placebo, and the difference was sustained. The results showed that the agent cetylpyridinium chloride was surprisingly effective in reducing IOP in a glaucoma model.

[0047] [Figure 26] FIG. 26 shows that the agent polymyxin B reduced intraocular pressure (IOP) in a glaucoma model compared to the control. The agents were tested by controlling flow and using a microfluidic device to measure relative IOP. The agents were each compared to a placebo (phosphate buffer) by preparing 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 an arrow and the letter "a." Referring to FIG. 26, the IOP of the placebo (dashed line) increased significantly after injection of the placebo sample. The IOP steadily increased to a maximum pressure of about 250 mmHg. Conversely, after injection of the agent polymyxin B sample (solid line), it was significantly lower than that for the placebo, and the difference was sustained. The results showed that the agent polymyxin B was surprisingly effective in reducing IOP in a glaucoma model.

[0048] [Figure 27]FIG. 27 shows that the agent neomycin reduced intraocular pressure (IOP) in a glaucoma model compared to the control. The agents were tested by controlling the flow and using a microfluidic device to measure the relative IOP. The agents were each compared to a placebo (phosphate buffer) by preparing 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 an arrow and the letter "a." Referring to FIG. 27, the IOP of the placebo (dashed line) increased significantly after injection of the placebo sample. The IOP steadily increased to a maximum pressure of about 64 mmHg. Conversely, after injection of the agent neomycin (solid line), it was 72% lower than that for the placebo, and the difference was sustained. The results showed that the agent neomycin was surprisingly effective in reducing IOP in a glaucoma model.

[0049] [Figure 28] FIG. 28 shows that the agent heparin chloride reduced intraocular pressure (IOP) in a glaucoma model compared to the control. The agents were tested by controlling flow and using a microfluidic device to measure relative IOP. The agents were each compared to a placebo (phosphate buffer) by preparing 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 an arrow and the letter "a." Referring to FIG. 28, the IOP of the placebo (dashed line) increased significantly after injection of the placebo sample. The IOP steadily increased to a maximum pressure of about 67 mmHg. Conversely, after injection of the agent heparin chloride (solid line), it was 32% lower than that for the placebo, and the difference was sustained. The results showed that the agent heparin chloride was surprisingly effective in reducing IOP in a glaucoma model.

[0050] [Figure 29]FIG. 29 shows that the agent sodium dodecyl sulfate reduced intraocular pressure (IOP) in a glaucoma model compared to the control. The agents were tested by controlling the flow and using a microfluidic device to measure the relative IOP. The agents were each compared to a placebo (phosphate buffer) by preparing 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 an arrow and the letter "a." Referring to FIG. 29, the IOP of the placebo (dashed line) increased significantly after injection of the placebo sample. The IOP steadily increased to a maximum pressure of about 60 mmHg. In contrast, after injection of the agent sodium dodecyl sulfate (solid line), it was significantly higher than that for the placebo. This result indicated that the agent sodium dodecyl sulfate is a negative control that does not reduce IOP in a glaucoma model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] The present invention provides methods, compositions, devices, kits and reagents for detecting, identifying and treating glaucoma disease. Embodiments of the present invention utilize ultrastructural characteristics of intraocular aqueous humor as guides and markers for medical devices to treat glaucoma.

[0052] In some aspects, the present invention provides methods and compositions for lowering intraocular pressure and increasing intraocular outflow in glaucoma subjects. Aspects of the invention can reduce the formation and presence of aggregate features and structures in ocular aqueous solution.

[0053] In a further aspect, the present disclosure provides a therapeutic composition for glaucoma.

[0054] Embodiments of the present invention provide devices for measuring and characterizing the coagulation characteristics of glaucoma, as well as intraocular pressure and outflow.

[0055] Further aspects of the present disclosure include diagnostic and screening medical devices for glaucoma. Further embodiments include kits and reagents for carrying out the same.

[0056] The present invention may provide a glaucoma diagnosis based on the unique and reliable ultrastructural biomarkers identified herein. The ultrastructural components may block the trabecular meshwork, elevating IOP and over time, increasing intraocular aqueous humor outflow resistance, elevating intraocular pressure and ultimately resulting in vision loss. The ultrastructural components in the aqueous humor fluid of POAG patients may be reflected in EV aggregates formed together into large EV complexes. The EV complexes may be several microns in size and are glaucoma-associated EV complexes. The EV complexes may be present in glaucoma patient samples and are large enough to block the trabecular meshwork.

[0057] Embodiments of the present invention may provide compositions and methods for purifying and / or synthesizing this ultrastructural component EV complex for use in therapeutic and biological methods.

[0058] Embodiments of the present invention may provide therapies and treatments for POAG, as well as compositions and methods for testing POAG aqueous humor specimens.

[0059] In some embodiments, glaucoma-associated EV complexes may be reduced by dissociation or other routes.

[0060] In additional embodiments, the compositions and methods of the present invention may reduce intraocular pressure and / or increase intraocular outflow.

[0061] Embodiments of the present invention further contemplate a method of treating glaucoma.

[0062] In certain aspects, glaucoma diseases can be treated by administering a surfactant that acts on the EV complex, which can be used to ameliorate, alleviate, inhibit, relieve, delay, and / or prevent at least one symptom or condition of a glaucoma disorder.

[0063] The eye can be viewed as a closed chamber (Figure 1, left). IOP can be determined by the rate of aqueous humor formation and the rate of fluid exit (Figure 1, right). In general, reduced aqueous humor outflow in glaucoma can be associated with elevated IOP. Aqueous humor outflow can be associated with elevated IOP and glaucomatous visual damage. Aqueous humor exits the eye via two pathways: the trabecular meshwork and, to a lesser extent, the uveoscleral outflow (Figure 2).

[0064] Abnormal aqueous humor outflow can cause elevated IOP. The trabecular meshwork (TM) may be the primary site of outflow. The TM is a filter-like tissue consisting of a series of fenestrated striations that allow aqueous humor to exit the anterior chamber via Schlemm's canal. The primary function of the TM is to allow aqueous humor to exit the eye and establish IOP.

[0065] The corneoscleral tissue (JCT) or cribriform region is the region of the TM that lies next to Schlemm's canal and may be involved in the establishment of IOP. The site of 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 the cells are embedded.

[0066] Abnormal regulation of aqueous humor flow through the TM would be associated with elevated IOP. The ECM of the TM may be a barrier capable of isolating intraocular fluid outflow.

[0067] Ultrastructural features or compositions in the aqueous humor of glaucoma patients that are larger than the perforations at the JCT outlet and other TM tissues may block the TM.

[0068] Ultrastructural features or compositions in aqueous humor can include structures based on extracellular vesicles (EVs), which are transport nanovesicles involved in intracellular communication by transporting biomolecules such as proteins, lipids, and nucleic acids from one cell to another.

[0069] In general, 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 microvesicular bodies of approximately 100-1000 nm, and apoptotic bodies of approximately 1-5 μm. EVs may be associated with the pathophysiology of diseases.

[0070] In some embodiments of the present invention, EV-based ultrastructural features and compositions are utilized to characterize intraocular fluids.

[0071] In further embodiments, the EV-based ultrastructural characteristics and compositions may be utilized in devices for determining IOP. The EV-based ultrastructural characteristics and compositions may be monitored to determine the efficacy of treatment in lowering IOP. The EV-based ultrastructural characteristics and compositions may be monitored as biomarkers to determine the efficacy of treatment in lowering IOP.

[0072] In additional embodiments, the EV-based ultrastructural characteristics and compositions may be utilized in devices for measuring intraocular outflow. The EV-based ultrastructural characteristics and compositions may be monitored to determine the efficacy of a treatment in increasing intraocular outflow. The EV-based ultrastructural characteristics and compositions may be monitored as biomarkers to determine the efficacy of a treatment in increasing intraocular outflow.

[0073] In further embodiments, the EV-based ultrastructural features and compositions are utilized to reduce the formation and presence of aggregate features, structures and particles in ocular water.

[0074] In certain embodiments, the EV-based ultrastructural characteristics and compositions are utilized in devices for determining intraocular conditions and parameters.

[0075] In additional embodiments, the EV-based ultrastructural characteristics and compositions are utilized to identify glaucoma in a subject.

[0076] In further embodiments, the EV-based ultrastructural characteristics and compositions are utilized in methods, kits and reagents for glaucoma.

[0077] Without being bound to any particular theory, the ultrastructural features and composition of EVs in glaucoma may have larger structures that block TM and / or other outflow.

[0078] Glaucoma disorders that may be treated with the methods and compositions described herein, referred to herein as "glaucoma", include, but are not limited to, preglaucomatous open angle with borderline findings, open angle, low risk, suspected anatomical narrow angle primary angle closure, steroid positive response, elevated intraocular pressure, primary angle closure without glaucomatous damage (PAS or high IOP without optic nerve or visual field loss), open angle glaucoma not otherwise specified, primary open angle glaucoma, chronic simplex glaucoma, low It includes intraocular pressure glaucoma, pigmentary glaucoma, capsular glaucoma with lenticular pseudoexfoliation, residual open angle glaucoma, primary angle-closure glaucoma not otherwise specified, acute angle-closure attack, chronic angle-closure glaucoma, intermittent angle-closure glaucoma, residual angle-closure glaucoma, glaucoma secondary to ocular trauma, glaucoma secondary to ocular inflammation, retinal vascular infarction, type 1 diabetes complications, type 2 diabetes complications, lens damage, intraocular lens damage, damage following other ocular conditions, neoplasms, or benign or malignant neoplasms. Also included are drug-induced glaucoma, glaucoma with elevated episcleral venous pressure, hypersecretory glaucoma, aqueous misdirection malignant glaucoma, glaucoma in diseases classified elsewhere, congenital glaucoma, Axenfeld's anomaly, buphthalmos, pediatric glaucoma, neonatal glaucoma, hydrophthalmia, keratoconus, congenital macrokeratitis with glaucoma, macrophthalmos in congenital glaucoma, macrocornea with glaucoma, absolute glaucoma. Also included are side effects of ophthalmic drugs and preparations, acute follicular conjunctivitis, side effects of carbonic anhydrase inhibitors, and side effects of dosing of ophthalmic drugs and preparations.

[0079] In some embodiments, the compositions of the present disclosure may be administered systemically, which may be accomplished by intravenous, oral, intra-arterial, inhalation, intranasal, intraperitoneal, intraabdominal, subcutaneous, intra-articular, intrathecal, transdural, transdermal, submucosal, sublingual, enteral, parenteral, percutaneous, periarticular, or intraventricular administration.

[0080] In further embodiments, the compositions of the present disclosure may be administered locally. The compositions may be administered locally to ocular tissue. As used herein, the term "ocular tissue" refers to the eye, including tissues inside the sclera, e.g., the retina, and outside the sclera, e.g., the eye muscles inside the orbit. Ocular tissue also includes tissues that are distinct from but neurologically connected to the eye, such as the optic nerve, geniculate body, and visual cortex. Local administration to ocular tissue may be achieved by intraocular administration. Intraocular administration may be performed by intracameral administration, intravitreal administration, or subretinal administration.

[0081] In additional embodiments, local administration to ocular tissues may be achieved by periocular administration, which may be performed by subconjunctival injection, sub-Tenon injection, direct intraocular injection, or depot periocular injection.

[0082] A subject can be administered a therapeutically effective amount of the composition, which can be an amount effective to ameliorate, alleviate, inhibit, relieve, delay, and / or prevent at least one symptom or condition of the disease being treated.

[0083] In certain embodiments, a therapeutically effective amount can be an amount effective to improve the ocular condition being treated. The dose can be determined according to various parameters, particularly the severity of the condition to be treated, the age and weight of the patient; the route of administration; and the required dosing regimen. For any particular patient, the route of administration and dose required can be determined. The dose can vary depending on the relative efficacy of the composition administered, and is usually estimated based on the median effective concentration (EC50) found to be effective in in vitro and in vivo models.

[0084] Extracellular vesicles and aggregates in glaucoma Embodiments of the present invention provide methods for detecting EVs in biological fluids. In certain methods, crosslinkers may be used to provide reliable imaging of EV ultrastructure, for example, by electron microscopy. In further methods, alternative crosslinkers to glutaraldehyde may be used.

[0085] An additional embodiment of the present invention contemplates detecting and characterizing EV complexes in glaucoma, which may block the TM or JCT and inhibit the aqueous humor outflow pathway in the eye.

[0086] In certain aspects, EVs in glaucoma may aggregate together in EV complexes. Glaucoma-associated EV complexes are up to several microns in size or diameter.

[0087] In glaucoma, EV complexes can be an ultrastructural feature of the disease. This ultrastructural feature can be a target for characterizing glaucoma. EV complexes can be used in detection of treatment parameters and medical devices for glaucoma. In some embodiments, EV complexes can be used for diagnosis, prognosis, and / or screening of glaucoma compositions. EV complexes can also be used in devices for determining treatment compositions, doses, and administration schedules.

[0088] As used herein, the term "diameter" refers to the longest linear dimension of an atypical particle, such as an extracellular vesicle complex. For a typical particle, such as a spherical vesicle, the term "diameter" has its usual meaning of a line segment that passes through the center and has an endpoint on a sphere.

[0089] In some aspects, the present disclosure provides a composition of purified EV complexes from glaucoma. The purified EV complexes from glaucoma can be used in a device to analyze and detect changes in EV ultrastructural components that may be related to intraocular pressure and intraocular outflow. The present invention provides a device containing purified EV complexes that can be used to characterize and measure intraocular obstruction and intraocular outflow. The purified EV complexes from glaucoma can be used in a device for screening effects of therapeutic agents on intraocular EV ultrastructural components.

[0090] In a further aspect, the present disclosure provides a composition 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 related to intraocular pressure and intraocular outflow. The present invention provides devices containing synthetic EV complexes that can be used to characterize and measure intraocular obstruction and intraocular outflow. The synthetic EV complexes from glaucoma can be used in devices for screening effects of therapeutic agents on intraocular EV ultrastructural components.

[0091] EV ultrastructural components such as EV complexes may consist of complexes or aggregates of extracellular vesicles, examples of which include exomeres, exosomes, multivesicular bodies, intraluminal vesicles (ILVs), multivesicular endosomes (MVEs), oncosomes, microvesicular bodies, apoptotic bodies, or vesicles originating from endosomes or the plasma membrane.

[0092] Extracellular vesicle complexes or aggregates can be protein-EV structures with micrometer diameters, or diameters greater than about 1 micrometer.

[0093] The extracellular vesicle aggregates can have a size of about 360 to about 21,000 nanometers (nm).

[0094] For example, exomeres can be about 35 nm in diameter, exosomes can be about 40-100 nm in diameter, microvesicular bodies can be about 100-1000 nm in diameter, and apoptotic bodies can be about 1-5 micrometers in diameter.

[0095] A complex or aggregate of extracellular vesicles may contain 10, 20, 30, 40, 50, 100, 200, 500 or more extracellular vesicles.

[0096] For example, a healthy subject will have free EVs, which are non-aggregated EVs with diameters of approximately 100-200 nm, which are primarily exosomes, as well as vesicles of several micrometers in size, and will not have EV aggregates or EV ultrastructural features larger than 0.4-20 micrometers in diameter in the aqueous humor.

[0097] For example, in glaucoma, subjects may have EV aggregates larger than 0.4-20 micrometers in diameter. Healthy subjects have small EV aggregates of about 36-300 nm, which are mainly exomeres, as well as some microvesicular bodies. Glaucoma subjects may have a reduced amount of free EVs, or very few remaining free EVs. Free EVs in glaucoma are larger in size than free EVs in healthy subjects. EV aggregates in glaucoma can be composed of either exomeres, exosomes, microvesicular bodies, and / or apoptotic bodies, as well as other types of vesicles or bodies.

[0098] Devices for the aggregation characteristics of glaucoma The devices of the invention may be used to characterize the activity of biologically active agents on glaucoma.The devices of the invention may be used to detect or characterize intraocular conditions or parameters in model systems or patient pathologies.

[0099] The active agent may be capable of providing therapeutic benefit, particularly in glaucoma, hi some embodiments, the active agent may be a known drug effective in treating an eye disease.

[0100] In some aspects, the fluid composition in the device of the present invention can be analyzed by a variety of techniques. For example, the fluid composition can be analyzed by imaging techniques.

[0101] 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 ultrasound microscopy.

[0102] Examples of imaging techniques include positron emission tomography, computed tomography, and magnetic resonance imaging.

[0103] Examples of assay techniques include colorimetric assays, chemiluminescent assays, spectrophotometric methods, and light scattering methods.

[0104] In some embodiments, the present invention may provide a device (FIG. 3) for measuring pressure and flow rate of a fluid composition. The device may have a channel with an inlet at a first end and an outlet at a second end, the inlet and outlet fluidly connected. The device may have a meshwork composition that resides within the channel to provide resistance to flow. The meshwork composition may have any one or more or all of the following regions: uveal meshwork, corneoscleral meshwork, and endothelial meshwork.

[0105] In some embodiments, the meshwork composition may be comprised of glass beads, microbeads, magnetic beads, gel particles, dextran particles, or polymer particles. The meshwork composition may also be comprised of glass fibers, polymeric fibers, inorganic fibers, organic fibers, or metal fibers.

[0106] In certain embodiments, the uveal meshwork may have a fenestrations of about 25 micrometers, the corneoscleral meshwork may have a fenestrations of about 2 to 15 micrometers, and the endothelial meshwork may have a fenestrations of about 1 to less than 4 micrometers.

[0107] The device may further comprise a fluid reservoir for holding a fluid composition, the fluid reservoir being in fluid communication with the inlet of said channel for introducing said fluid composition to the inlet of said channel.

[0108] The device of the present disclosure may have a pressure source that applies pressure to a fluid composition in the fluid reservoir to introduce the fluid composition into the inlet of the channel.

[0109] The devices of the invention may have a flow sensor in fluid communication with the fluid composition to measure the flow rate and pressure of the fluid composition at the inlet of the channel and transfer the flow rate and pressure to a processor.

[0110] Signals and data from the device may be received by a processor. The processor may display flow rates and pressures. A memory or medium, such as a machine-readable storage medium, may store instructions or files. The machine-readable storage medium may be non-transitory.

[0111] The processor of the present disclosure may be a general-purpose or special-purpose computer. The processor may execute instructions stored in a machine-readable storage device or medium. The processor may include an integrated circuit chip, a microprocessor, a controller, a digital signal processor, any of which may be used to receive and / or transfer data and execute stored instructions. The processor may also convert data and / or store data in a memory, medium, or file. The processor may receive and execute instructions that may include performing one or more steps of the method of the present invention. The device of the present invention may include one or more non-transitory machine-readable storage media, one or more processors, one or more memory devices, and / or one or more user interfaces. The processor may have an integrated display for displaying data or the converted data.

[0112] In some aspects, the device can have microfluidic channels. One or more channels can be disposed in a microfluidic chip.

[0113] The devices of the present disclosure may include one or more detectors to analyze the fluid composition within the channel, or at the inlet or exiting the outlet of the channel. One or more detectors may also be positioned to detect the fluid composition within the channel.

[0114] The device of the present invention can include an omentum composition containing extracellular vesicles or extracellular vesicle complexes. The EV complexes for use in the omentum composition can be purified from glaucomatous ocular aqueous, aqueous humor, or vitreous humor. The ocular aqueous can be from an animal or clinical source. The EV complexes for use in the omentum composition can be composed of extracellular vesicles and can have a diameter of about 360 to about 21,000 nanometers.

[0115] In certain embodiments, an EV complex for use in an omentum composition can include a fixative, stabilizing component, or cross-linking component that can transform its structure into a stable and homogenous composition.

[0116] Examples of stabilizing components include fixatives described herein, cross-linking compounds described herein, inorganic solvents, polypeptides, and pharma- ceutically acceptable organic salts.

[0117] Examples of salts include alkali metal salts, including ammonium, sodium, lithium, and potassium salts; alkaline earth metal salts, including calcium and magnesium salts; salts with organic bases, such as organic amines, such as benzathine, dicyclohexylamine, N,N-bis(dehydroabietyl)ethylenediamine, N-methyl D-glucamine, N-methyl D-glucamide, hydrabamine formed with t-butylamine, and salts with amino acids, including arginine and lysine.

[0118] Examples of salts are acetate, adipate, alginate, ascorbate, aspartate, benzoate, benzenesulfonate, hydrogensulfate, borate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, shroom salt, and the like. Salts include, but are not limited to, urate, pectinate, persulfate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, sulfonate, tartrate, thiocyanate, toluenesulfonate, and undecanoate.

[0119] The crosslinked extracellular vesicle complexes may be reversibly or irreversibly crosslinked.

[0120] The crosslinked extracellular vesicles may be reversibly or irreversibly crosslinked.

[0121] In some embodiments, the device of the present invention can contain an EV complex plexus composition that can be used to identify or screen active agents for their effect on lowering IOP and / or increasing intraocular outflow. The EV complexes that can be used in the plexus composition can include drug delivery excipients. The EV complex plexus composition for the device can be a synthetic EV complex or a purified EV complex.

[0122] A specific example of the channel arrangement of the present invention is shown in FIG.

[0123] An embodiment of the device of the present invention is illustrated in FIG.

[0124] In additional embodiments, the device of the present invention can be used to measure the quantity or level of EV complexes in a test sample. Measuring the quantity or level of EV complexes in a test sample can provide a diagnostic marker for the test sample. The device of the present invention can be used to identify glaucoma or pre-glaucoma in a subject.

[0125] In a further embodiment, the device of the present invention can be used to measure pressure that can be related to the quantity or level of EV complexes in a test sample. The pressure value in the channel can be directly related to the quantity or level of EV complexes in the test sample.

[0126] In certain embodiments, the device of the present invention can be used to measure an assay value that can relate to the quantity or level of EV complexes in a test sample. The assay value of the composition in the channel can be directly related to the quantity or level of EV complexes in the test sample. For example, the assay can be a colorimetric assay, a chemiluminescent assay, a spectroscopic assay, or a light scattering assay.

[0127] In some aspects, a sample of aqueous humor from a subject can be provided and analyzed for the quantity of glaucoma extracellular vesicle complexes. The subject can be identified as having glaucoma or pre-glaucoma based on the quantity exceeding a reference value. The reference value can be the quantity 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 the quantity or level of glaucoma extracellular vesicle complexes, which may or may not exceed the previous test sample, and which can be related to lowering IOP and / or increasing intraocular outflow in the subject.

[0128] In certain embodiments, the quantity or level of glaucoma extracellular vesicle complexes can include one or more of the number, size, density, morphology, and spatial distribution of said extracellular vesicle complexes.

[0129] In some embodiments, the reference value may be the quantity or level of glaucoma extracellular vesicle complexes in a reference population of healthy individuals. The reference value may be the average value of samples from the reference population.

[0130] Glaucoma can be found in a subject, where a test sample from said subject contains a number or level of glaucoma extracellular vesicle complexes that exceeds a glaucoma reference value.

[0131] In certain embodiments, the glaucoma reference value is a number of extracellular vesicle complexes containing 10 or more aggregated extracellular vesicles of zero per sample. In certain embodiments, the glaucoma reference value is a number of extracellular vesicle complexes containing 10 or more aggregated extracellular vesicles of 10 per sample. In certain embodiments, the glaucoma reference value is a number of extracellular vesicle complexes containing 10 or more aggregated extracellular vesicles of 50 per sample. In certain embodiments, the glaucoma reference value is a number of extracellular vesicle complexes containing 10 or more aggregated extracellular vesicles of 100 per sample.

[0132] In certain embodiments, the glaucoma reference value contains 10 or more aggregated extracellular vesicles and the number of extracellular vesicle complexes larger than 360 nm is zero per sample. In certain embodiments, the glaucoma reference value contains 10 or more aggregated extracellular vesicles and the number of extracellular vesicle complexes larger than 360 nm is 10 per sample. In certain embodiments, the glaucoma reference value contains 10 or more aggregated extracellular vesicles and the number of extracellular vesicle complexes larger than 360 nm is 50 per sample. In certain embodiments, the glaucoma reference value contains 10 or more aggregated extracellular vesicles and the number of extracellular vesicle complexes larger than 360 nm is 100 per sample.

[0133] In certain embodiments, the glaucoma reference value is a number of extracellular vesicle complexes greater than 360 nm per sample of 10. In certain embodiments, the glaucoma reference value is a number of extracellular vesicle complexes greater than 360 nm per sample of 50. In certain embodiments, the glaucoma reference value is a number of extracellular vesicle complexes greater than 360 nm per sample of 100. In certain embodiments, the glaucoma reference value is a number of extracellular vesicle complexes greater than 360 nm per sample of 200.

[0134] In a further aspect, the omentum composition in the device of the invention is the anterior chamber or portion of a crystalline animal eye, where the TM of the eye faces between the entrance and exit of said channel.

[0135] method The aqueous humor extracellular vesicles of POAG patients can be compared to a population of healthy controls. The aqueous humor EV complex ultrastructure of subjects with ophthalmic pathology, such as glaucoma, can be compared to healthy controls, such as subjects with no ophthalmic pathology other than cataract. The aqueous humor EV complex levels of glaucoma subjects can exceed those of healthy subjects.

[0136] The types of EVs in the aqueous humor of glaucoma are larger than those in healthy subjects. In some embodiments, the level of the larger EV structures can be reduced to eliminate the blockage of the aqueous network and increase aqueous humor outflow. The EV complexes in the aqueous humor of glaucoma are larger than any EVs in healthy subjects. In some embodiments, the level of the EV complexes can be reduced to eliminate the blockage of the aqueous network and increase aqueous humor outflow.

[0137] In certain embodiments, EVs in healthy human aqueous humor can be diffuse and evenly distributed without aggregation. Healthy control aqueous humor can contain EVs that are non-aggregated and have a diffuse distribution.

[0138] Glaucomatous EV complexes are larger than the EVs observed in healthy controls and are capable of blocking the trabecular meshwork.

[0139] In some embodiments, purified EV complexes can be obtained from the aqueous humor in POAG. The purified EV complexes can be several microns in size. The glaucomatous EV complexes are larger than the opening of the JCT (1 to 4 μm, or 2 to 20 μm) and are large enough to block the corneoscleral 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 EV complexes can be reduced to unblock the trabecular meshwork and increase aqueous humor outflow.

[0140] For example, the EV complexes may be contacted with a composition containing an active agent, such as bimatoprost, In these embodiments, the level of the EV complexes may be reduced to unblock the trabecular meshwork and increase aqueous humor outflow.

[0141] In certain aspects, the level or quantity of glaucomatous EV complexes can be reduced in POAG subjects by administering an active agent such as bimatoprost.

[0142] In a further embodiment, the purified EV complexes can have a size of about 360 nm to 21,000 nm, which is substantially larger than any particle found in normal aqueous humor.

[0143] In additional embodiments, the purified EV complexes may have a size of 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.

[0144] In the purified 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.

[0145] In the purified 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.

[0146] In a 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.

[0147] In some embodiments, the purified EV complexes can provide particles sized for the uveal plexus. The purified EV complexes for the uveal plexus can be 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.

[0148] In further embodiments, the purified EV complexes can provide particles sized for the corneoscleral meshwork. The purified EV complexes for the corneoscleral meshwork can be from about 1,000 nm to about 15,000 nm, or from 2,000 nm to 10,000 nm, or from 2,000 nm to 5,000 nm.

[0149] In additional embodiments, the purified EV complexes can provide particles of a size for the meshwork endothelial membrane. The purified EV complexes for the meshwork endothelial membrane can be 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.

[0150] The region of the TM involved in establishing IOP is next to Schlemm's canal and is called the corneoscleral tissue (JCT) or cribriform region.

[0151] EV complexes can be synthesized by contacting EVs with reagents to form larger structures. The reagents can include fixatives, crosslinkers, and buffer suspensions. Synthetic EV complexes can be composed of many EVs that contact each other to form aggregates.

[0152] The number of EVs in contact with each other in a synthetic EV complex 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.

[0153] In a synthetic EV complex, the number of EVs in contact with each other can be between 20 and 300, or between 30 and 300, or between 40 and 300, or between 50 and 300.

[0154] The number of EVs in contact with each other in a synthetic EV complex 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.

[0155] In additional embodiments, the synthetic EV complexes may have a size of 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.

[0156] In some embodiments, the synthetic EV complexes can provide particles sized for the uveal plexus. The synthetic EV complexes for the uveal plexus can be 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.

[0157] In further embodiments, the synthetic EV complexes can provide particles sized for the corneoscleral meshwork, which can be from about 1,000 nm to about 15,000 nm, or from 2,000 nm to 10,000 nm, or from 2,000 nm to 5,000 nm.

[0158] In additional embodiments, the synthetic EV complexes can provide particles of a size for the meshwork endothelial tissue. The synthetic EV complexes for the meshwork endothelial tissue can be 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.

[0159] Synthesis and purification of extracellular vesicles and EV complexes In some embodiments, extracellular vesicles including exosomes and EV complexes may be synthesized, isolated and / or purified by size exclusion or gel filtration chromatography.

[0160] In certain embodiments, extracellular vesicles including exosomes and EV complexes may be synthesized, isolated and / or purified by centrifugation, differential centrifugation, density gradient centrifugation, or ultracentrifugation.

[0161] In additional embodiments, extracellular vesicles including exosomes and EV complexes may be synthesized, isolated and / or purified using precipitation reagents, such as polymeric precipitation reagents, protamine, sodium acetate, or organic solvents.

[0162] In some embodiments, extracellular vesicles including exosomes and EV complexes may be synthesized, isolated and / or purified using immunoaffinity capture techniques.

[0163] In further embodiments, extracellular vesicles including exosomes and EV complexes may be synthesized, isolated and / or purified using microfluidic devices, acoustofluidic devices, and microfluidic chips.

[0164] In additional embodiments, extracellular vesicles including exosomes and EV complexes may be synthesized, isolated and / or purified using sequential filtration techniques.

[0165] In a further embodiment, extracellular vesicles containing exosomes may be detected using resistive pulse sensing or tunable resistive pulse sensing with a tunable pore sensor.

[0166] In certain embodiments, extracellular vesicles, including exosomes, can be detected by electron microscopy, light microscopy, and flow cytometry.

[0167] In additional embodiments, extracellular vesicles including exosomes may be detected by dynamic light scattering and / or mass spectrometry.

[0168] In some aspects, extracellular vesicles including exosomes and EV complexes can be synthesized, isolated and / or purified by first isolating the vesicles from cell culture.

[0169] In certain aspects, extracellular vesicles including exosomes and EV complexes can be synthesized, isolated and / or purified by first isolating the vesicles from a bodily fluid, such as ocular fluid, and the isolated vesicles can be diluted, filtered and protected with protease inhibitors.

[0170] In a further embodiment, the step of purifying extracellular vesicles, including exosomes, and EV complexes comprises contacting with a fixative.

[0171] In some aspects, extracellular vesicles, including exosomes, can be synthesized by controlled biogenesis and release from in vitro grown cell lines.

[0172] Activator Examples of active agents include small molecule drugs, proteins, nucleic acids, polysaccharides, biopharmaceuticals, and combinations thereof.

[0173] Examples of active agents include cytokines, growth factors, proteins, peptides, antimetabolites, signaling modulators, antibiotics, antibodies, chemotherapeutic compounds, and combinations thereof.

[0174] Examples of active agents include anti-infective agents, anesthetic agents, anti-VEGF agents, anti-inflammatory agents, intraocular pressure reducing agents, and combinations thereof.

[0175] Examples of active agents include anesthetics, analgesics, and combinations thereof.

[0176] Examples of active agents include cell trafficking or migration inhibitors, such as colchicine, vincristine, cytochalasin B, and combinations thereof.

[0177] Examples of active agents include anti-glaucoma agents.

[0178] Examples of active agents include beta blockers such as timolol, betaxolol, atenolol, prostaglandins, and combinations thereof.

[0179] Examples of active agents include lipid receptor agonists or prostaglandin analogs such as bimatoprost, travoprost, tafluprost, latanoprost, unoprostone, and combinations thereof.

[0180] Examples of active agents include alpha adrenergic agonists, including brimonidine, dipivefrin, and combinations thereof.

[0181] Examples of active agents include carbonic anhydrase inhibitors such as acetazolamide, methazolamide, diclophenamide, diamox, and combinations thereof.

[0182] Examples of active agents include neuroprotective agents such as nimodipine.

[0183] 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.

[0184] Examples of active agents include agents for wet AMD such as mecamylamine; VEGF Trap-Eye, the complement inhibitor POT-4.

[0185] 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.

[0186] Examples of active agents include antibiotics such as tetracycline, chlortetracycline, bacitracin, neomycin, polymyxin, gramicidin, oxytetracycline, chloramphenicol, gentamicin, and erythromycin.

[0187] Examples of active agents include antibacterial agents such as sulfonamides, sulfacetamide, sulfamethizole and sulfafurazole.

[0188] Examples of active agents include antifungals such as fluconazole, nitrofurazone, amphotericin B, and ketoconazole.

[0189] Examples of active agents include trifluridine, acyclovir, ganciclovir, DDI, AZT, foscarnet, vidarabine, trifluorouridine, idoxuridine, ribavirin, protease inhibitors, and antiviral agents such as anti-cytomegalovirus agents.

[0190] Examples of active agents include metapyrelin; anti-allergy agents such as chlorpheniramine, nepyramine and profenpyridamine.

[0191] Examples of active agents include anti-inflammatory agents such as hydrocortisone, dexamethasone, fluorocinolone, prednisone, prednisolone, methylprednisolone, fluorometholone, betamethasone and triamcinolone.

[0192] Examples of active agents include decongestants such as phenylephrine, naphazoline, and tetrahydrozoline; miotics, and muscarinics.

[0193] Examples of active agents include anticholinesterase agents such as pilocarpine, carbachol, disopropylfluorophosphate, phosphonate iodide, and derma potassium bromide.

[0194] Examples of active agents include mydriatics such as atropine sulfate, cyclopentalate, homatropine, scopolamine, tropicamide, eucatropine.

[0195] Examples of active agents include sympathomimetics such as epinephrine.

[0196] Examples of active agents include ranibizumab, bevacizamab, and triamcinolone.

[0197] Examples of active agents include non-steroidal anti-inflammatory drugs (NSAIDs), including acetylsalicylic acid, ibuprofen, indomethacin; and anti-inflammatory drugs such as COX-2 inhibitors.

[0198] Examples of active agents include immunosuppressants including sirolimus.

[0199] Examples of active agents include matrix metalloproteinase (MMP) inhibitors such as tetracycline.

[0200] Examples of activators include anticoagulants such as heparin, antifibrinogen, fibrinolysin, and the anticoagulant activase.

[0201] Examples of active agents include acetohexamide, chlorpropamide, glipizide, glibenclamide, tolazamide, tolbutamide, insulin, and antidiabetic agents including aldose reductase inhibitors.

[0202] Examples of activators include amines such as thonzonium.

[0203] Examples of active agents include detergents such as taurocholic acid, glycocholic acid, glycochenodeoxycholic acid, benzalkonium, cetylpyridinium, taurochenodeoxycholic acid, polidocanol, and tyloxalol.

[0204] Examples of active agents include lipids such as sodium lauryl sulfate.

[0205] Examples of active agents include antibacterial agents such as polymyxin B.

[0206] Examples of active agents include amines such as thonzonium and the related compounds thonzylamine, mepyramine and piribedil.

[0207] Examples of active agents include aceclidine, acetazolamide, acetylcysteine, acyclovir, aflibercept, alcaftadine, alclometasone, alteplase, ampicillin, anecortave, ascorbic acid, atropine, azelastine, azithromycin, befunolol, bendazac, benzylpenicillin, besifloxacin, betamethasone, betaxolol, vibrocatol, bimatoprost, brimonidine, brinzolamide, bromfenac, carbamoylcholine, carteolol, ceruloxime, senegelmine, chloramphenicol, cloram ... hexidine, chlortetracycline, chymotrypsin, cinchocaine, ciprofloxacin, clobetasone, clonidine, cocaine, cortisone, cromoglycate, cyclopentalate, cyclosporine, cysteamine, dapiprazole, demecarium, desonide, dexamethasone, dexpanthenol dibromopropamidine, diclofenac, diclofenamide, dihydrostreptomycin, dipivefrin, dorzolamide, echothiophate, sodium edetate, emedastine, ephedrine, epinastine, epinephrine, erythromycin , ethylmorphine, famciclovir, fludrocortisone, fluorocinolone acetonide, fluocortolone, fluorescein, fluorometholone, flurbiprofen, fomivirsen, formocortal, framycetin, fusidic acid, ganciclovir, gatifloxacin, gentamicin, guaiazulene, guanethidine, heparin, hexamidine, homatropine, hyaluronic acid, hydrocortisone, hypromellose, ibopamine, idoxuridine, indomethacin, inosine, kanamycin, ketorolac, ketotifen, latanoprost, Levobunolol, levocabastine, levofloxacin, lidocaine, lifitegrast, lodoxamide, lomefloxacin, loteprednol, medrysone, methazolamide, methascopolamine, methylprednisolone, metipranolol, micronomycin, moxifloxacin, nandrolone, naphazoline, natamycin, nedocromil, neomycin, neostigmine, nepafenac, netarsudil, netilmicin, nitrofurazone, norfloxacin, ocriplasmin, ofloxacin, olopatadine, oxybuprocaine, oxymetazoline,Oxyphenbutazone, oxytetracycline, paraoxon, pegaptanib, phenylephrine, physostigmine, picloxyzine, pilocarpine, piroxicam, polymyxin B, potassium iodide, povidone iodine, pranoprofen, prednisolone, procaine, propamidine, propionic acid, proparacaine, ranibizumab, resorcinol, riboflavin, rifamycin, rimexolone, rose bengal free acid, salicylic acid, scopolamine, ciloli These include ophthalmic agents such as mus, sodium borate, spabulumic acid, sulfacetamide, sulfadiclamide, sulfamethizole, sulfaphenazole, sufafurazole, synephrine, tafluprost, tetracaine, tetracycline, tetrizoline, timolol, tobramycin, travoprost, triamcinolone, triflidine, tropicamide, tyrothricin, unoprostone, verteporfin, vidarabine, vitamin A, and xylometazoline.

[0208] Examples of active agents include 5-fluorouracil, adriamycin, asparginase, azacitidine, azathioprine, bleomycin, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, cyclosporine, cytarabine, dacarbazine, dactinomycin, daunorubicin, doxorubicin, estramustine, etoposide, etretinate, filgrastim, floxuridine, fludarabine, fluorouracil, fluoxymethamphetamine, fluoxetine ... These include anti-cancer drugs such as luteamide, 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.

[0209] Examples of active agents include hormones, peptides, steroids, nucleic acids, saccharides, lipids, glycolipids, and glycoproteins.

[0210] Examples of active agents include endocrine hormones such as pituitary, insulin, insulin-related growth factor, thyroid, and growth hormone.

[0211] Examples of active agents include heat shock proteins.

[0212] Examples of active agents include immune response modifiers such as muramyl dipeptide, cyclosporine, interferon, interleukin-2, cytokines, tacrolimus, tumor necrosis factor, pentostatin, thymopentin, transforming factor beta 2, and erythropoietin.

[0213] Examples of active agents include brain nerve growth factor (BNGF), ciliary nerve growth factor (CNGF), and vascular endothelial growth factor (VEGF).

[0214] Examples of active agents include anticoagulants, antiproliferative agents, quinoxalines, and potassium channel blockers.

[0215] Examples of active agents include methylene blue, butylated hydroxyanisole, N-methylhydroxylamine, 2-(4-methylaminobutoxy)diphenylmethane, and guanylate cyclase inhibitors such as apraclonidine.

[0216] Examples of active agents include prostaglandins, such as metabolite derivatives of arachidonic acid.

[0217] Examples of active agents include sugars such as trehalose.

[0218] Examples of active ingredients include viscoelastic agents including hyaluronic acid, dimethicone, and hypromellose.

[0219] Examples of active ingredients include ophthalmic viscosurgical devices, sodium hyaluronate, chondroitin sulfate, hydroxypropyl methylcellulose, hydroxy-propyl-methylcellulose, hyaluronic acid, dimethicone, and hypromellose.

[0220] Examples of active agents include detergents, purifying or cleaning agents, such as salts of long chain fatty bases or acids, which may have cleansing, oil-soluble and / or antibacterial effects. Examples of active agents include glycochenodeoxycholic acid, glycocholic acid, peanut oil, benzalkonium, cetylpyridinium, taurochenodeoxycholic acid, polidocanol, tyloxalol, taurocholic acid, and N-dodecyl-N,N-dimethyl 3-ammonio-1-propanesulfonate.

[0221] The embodiment of the present invention further contemplates the use of the active agent to treat glaucoma disorders. In some aspects, glaucoma disorders can be treated by administering a surfactant that acts on the EV complex. An effective amount of the surfactant can be administered to improve, alleviate, inhibit, relieve, delay, and / or prevent at least one symptom or condition of glaucoma disorders. As used herein, the term "surfactant" broadly refers to surfactants, detergents, cleaning or cleaning agents, soaps, and modified versions thereof.

[0222] Examples of active agents include surfactants including lucinactant, calfactant, beractant, tyloxalol, sodium lauryl sulfate, thonzonium, nonoxynol-9 cetalkonium, dimethicone, polyethylene glycol 400, sinaplutide, palmitoyl oleoyl-phosphatidylglycerol, lapyrium, sodium lauryl sulfate (SLS), polyethylene glycol 300, trolamine, polysorbate 80, poloxamer 407, sodium tetradecyl sulfate, polysorbate 20, docusate, polactant alpha, colfosseryl palmitate, castor oil, benzalkonium, N-alkylethylbenzyldimethylammonium salts (c12-c14), quaternium-15, ambroxol, pumactant, ethanolamine, soy lecithin, and cocamidopropyl betaine.

[0223] Examples of active agents are described herein and include agents that can be modified for delivery or metabolic availability, or that can be PEGylated with a polyethylene glycol chain or have a polypropylene glycol chain attached. The present disclosure includes modified active agents, as described above, that have PEGylation or other chain modifications.

[0224] Examples of active agents include antibodies, antibody fragments, VEGF inhibitors, small molecules, corticosteroids, and combinations thereof.

[0225] Examples of active agents include tyrosine kinase inhibitors, monoclonal antibodies, and combinations thereof.

[0226] Examples of active agents include antibodies and antibody fragments. The term "antibody" as used herein includes whole antibodies, e.g., dual heavy and dual light chains, and 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, e.g., tandem scFv, Fd fragments, diabodies, triabodies. Antibody fragments can be obtained by conventional techniques known to those skilled in the art, and the fragments can be used in the same manner as intact antibodies.

[0227] The antibodies and antibody fragments disclosed herein may be monovalent, bivalent, or trivalent with respect to the binding domains, which may be monospecific, bispecific, or trispecific in design in binding specificity. Suitable antibodies include monoclonal or polyclonal antibody mixtures. The antibodies may be chimeric, CDR-grafted, humanized, or any of the antigen-binding sites described above. Therapeutic antibodies may be derived from a variety of species, including, without limitation, mouse, human, camel, llama, goat, rabbit, bovine, and cartilaginous fish.

[0228] In some embodiments, the antibodies or antigen-binding fragments may be used to treat ocular diseases or conditions. Examples of antibodies or antigen-binding fragments include those that bind to and preferentially block or reduce the activity of disease-associated integrins, 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.

[0229] In a further embodiment, the therapeutic protein can be an antibody mimetic.

[0230] As used herein, the term "antibody mimetic" refers to any organic compound, e.g., a peptide or polypeptide, that can specifically bind to an antigen like an antibody and is about 3-20 kDa. An antibody mimetic can include a scaffold that binds to its target antigen via amino acids in exposed loops similar to the CDR loops of an antibody. Antibody mimetics include adnectins, lipocalins, Kunitz domain-based binders, avimers, knottins, finomers, atrimers, and cytotoxic T-lymphocyte-associated protein-4 (CTLA4)-based binders. Some examples are provided 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).

[0231] Examples of active agents include benztropine, ropinirole, tolcapone, trihexyphenidyl, procyclidine, pramipexole, entacapone, biperiden, amantadine, selegiline, bromocriptine, levodopa, dextimide, piribedil, budipine, merevodopa, profenamine, cabergoline, lisuride, progabide, gabapentin, memantine, orphenadrine, 3,5-dinitrocatechol. , pimavanserin, ifenprodil, opicapone, benserazide, methixene, apomorphine, pergolide, rasagiline, rotigotine, etilepodopam, tropatepine, dihydroergocryptine, phenglutarimide, mazaticol, ethibenztropine, bornaprine, ethanautine, carbidopa, safinamide, and dexpramipexole.

[0232] Examples of active agents include epoprostenol dinoprostone, carboprost tromethamine, dinoprostone tromethamine, dinoprostonone, prostaglandin D2, prostalene, radispongiolide C, unoprostone, gemeprost, limaprost, iloprost, latanoprost, cloprostenol, cepetaprost, bimatoprost, fenprostalene, latanoprost bunod, travoprost, carboprost tromethamine, dinoprostone, tafluprost, cabazitaxel, cloprostenol sodium, bimatoprost, (-)-Corey lactone 4-phenylbenzoate alcohol, dutasteride, isopropyl unoprostone prostaglandins such as sulprostone, 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.

[0233] Examples of active agents include vitamins such as 1-alpha, 25-dihydroxyl-20-epi-22-oxa-24, 26,27-trihomovitamin D3.

[0234] Examples of active agents include antibiotics such as brefeldin A, fusidic acid, ovalicin, narasin, and salinomycin.

[0235] Examples of active agents are 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 norethisterone enanthate, prednisolone hemisuccinate, hydroxyprogesterone capronate, trembolo These include steroids such as benzodiazepine acetate, fluprostenol anecortave acetate, oleandrin cortisone acetate, calcipotriol gestodene dimethyl carbate, calcipotriol 1-alpha, 24S-dihydroxyvitamin D2, ethylenodiol diacetate, nandrolone decanoate, testosterone cypionate, testosterone undecanoate, carbenoxolone, progesterone-11-alpha-ol-hemisuccinate, eplerenone, testosterone succinate, and boldenone undecylenate.

[0236] Examples of active agents include statins such as lovastatin, atorvastatin, pravastatin, rosuvastatin, fluvastatin, and simvastatin.

[0237] Examples of activators include aggregation inhibitors such as pentoxyphylline, argatroban, and von Willebrand factor human.

[0238] Examples of active agents include prostacyclins, such as treprostinil.

[0239] Examples of active agents include amyloid targeting agents such as caprospinol and similar compounds including cholesteryl linoleate, pregnenolone acetate, and P-57AS3.

[0240] Examples of active agents include leidyspongiolide A and soraphen A, and lactones such as canrenone.

[0241] Examples of activators include benzoquinones such as antroquinolol.

[0242] Examples of activators include oxepanes such as triptolide PG-701.

[0243] Examples of active agents include lipids such as gibberellin A4 and fumagillin.

[0244] Examples of active agents include blood glucose lowering agents such as mitiglinide.

[0245] Examples of active agents include central nervous system depressants such as glutethimide.

[0246] Examples of active agents include benzenoids such as sildenafil, udenafil, and vardenafi.

[0247] Examples of active agents include fusicoccin and carbohydrates such as trehalose.

[0248] Examples of active agents include cholinergic agents such as biperiden, cyclimine, procyclidine, and trihexyphenidyl.

[0249] Examples of activators include terpenes such as cyclohexane.

[0250] The molecules, compounds and / or compositions of the present disclosure are 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 "racemates." Conventions for stereochemical nomenclature, such as the Cahn, Ingold, and Prelog stereoisomer naming conventions, and methods for determining stereochemistry and separating stereoisomers, are known in the art. See, for example, March's Advanced Organic Chemistry (7th ed., 2013). The compounds, compositions, and structures of the present disclosure are intended to include and incorporate all possible isomers, stereoisomers, diastereomers, enantiomers, and / or optical isomers present in the compounds, compositions, and / or structures, including any mixtures thereof, racemates, or mixtures thereof, racemic or otherwise.

[0251] Compounds can exist in unsolvated and solvated forms, or in hydrated forms.In this disclosure, solvated forms with pharma-ceutically acceptable solvents such as water or ethanol are considered equivalent to unsolvated forms.Compounds and salts, or their solvates, can also exist as tautomers, which are considered equivalent.

[0252] The molecules, compounds and / or compositions of the present disclosure may be found in various crystalline forms, which are intended to be incorporated into the present disclosure.

[0253] Compositions and Formulations Active agents of the present disclosure may include drugs and agents for ocular diseases, including small molecule drugs, peptide, antibody and protein agents.

[0254] A formulation of the active agent can be prepared by dissolving the composition in water to produce an aqueous solution, and sterilizing the solution.

[0255] The preparations of the present disclosure may be in the form of a sterile injectable aqueous or oleaginous suspension. The suspension may be formulated to contain a dispersing or wetting agent. The sterile injectable preparation may be a sterile injectable solution or suspension in a non-toxic, pharma- ceutically acceptable diluent or solvent.

[0256] Examples of solvents include water, water for injection, Ringer's solution, balanced saline, isotonic sodium chloride solution, 1,3-butanediol, synthetic mono- or diglycerides, and fatty acids such as oleic acid.

[0257] The formulations of the present disclosure may be in the form of eye drops for topical delivery.

[0258] The ophthalmic formulation may be a solution or a suspension for topical application. The composition may be a viscous or semi-viscous gel, or other solid or semi-solid composition.

[0259] Ophthalmic formulations may be delivered locally by direct injection or by use of an infusion pump.

[0260] The ophthalmic formulation may include an artificial tear carrier.

[0261] The ophthalmic formulation may include a phospholipid carrier.

[0262] Ophthalmic formulations may include surfactants, preservatives, antioxidants, tonicity adjusting excipients, buffers, co-solvents, and viscosity excipients.

[0263] Ophthalmic formulations can include excipients to adjust the osmolality of the formulation.

[0264] Ophthalmic formulations may include viscosity excipients such as polysaccharides, hyaluronic acid, chondroitin sulfate, dextran, cellulosic polymers, vinyl-based polymers, and acrylic-based polymers.

[0265] The ophthalmic formulation may have a viscosity of 1 to 400 centipoise, or 1 to 100 centipoise, or 2 to 40 cps. The ophthalmic formulation may have a viscosity of about 15, 20, 25, 30, 40, or 50 centipoise.

[0266] Illustrative examples of excipients or carriers for the formulations of the present invention include ophthalmically acceptable preservatives, viscosity enhancing agents, penetration enhancers, buffers, sodium chloride, sterile water, water for injection, and combinations thereof.

[0267] The dosage form of the composition of the present invention may be a liquid or emulsion. The dosage form of the composition of the present invention may be a solid, which can be reconstituted into a liquid prior to administration.

[0268] The compositions of the present disclosure may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil or a mineral oil.

[0269] Examples of emulsifying agents include naturally occurring gums, gum acacia, gum tragacanth, phosphatides, esters of fatty acids, hexitols, sorbitan monooleate, and polyoxyethylene sorbitan monooleate.

[0270] Embodiments of the present invention may advantageously provide effective activity of active agents at dosage levels significantly lower than conventional dosage levels.

[0271] An effective amount of an active agent composition of the present disclosure can be an amount sufficient to ameliorate or reduce the symptoms of the disease being treated.

[0272] The compositions may be administered in a single dose or may be administered in a multiple dose regimen.

[0273] Appropriate dosage levels of the active agent can be determined by one of skill in the art. In some embodiments, the active agent may 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.

[0274] The active agent of the present disclosure may be combined with one or more pharma- ceutically acceptable carriers.Carriers may be in various forms, including fluid, viscous solution, gel, or solubilized particles.Examples of carriers include pharma-ceutically acceptable diluents, solvents, saline, and various buffers.

[0275] Some examples of carriers, excipients and additives are given in US Pharmacopeia National Formulary (2014); Handbook of Pharmaceutical Excipients (7th ed., 2013); Handbook of Preservatives (2004, Synapse Information Resources); Remington: The Science and Practice of Pharmacy (22nd ed. 2013); Remington's Pharmaceutical Sciences (Mack Publishing Co. 1990). Some examples of drugs and delivery are given in Goodman and Gilman, The Pharmacological Basis of Therapeutics (13th ed. 2018, McGraw Hill, NY).

[0276] In certain embodiments, the active agent can be delivered without a carrier to reduce extracellular vesicle complexes in glaucomatous ocular aqueous solution.

[0277] Examples of the carrier include pyrogen-free water; isotonic saline, Ringer's solution, ethyl alcohol, and phosphate buffer solution.

[0278] Polymers such as polyethylene glycol (PEG), polypropylene glycol, or poly(lactic-co-glycolic acid) having a molecular weight of about 0.2 to about 50 kDa can be included.

[0279] Examples of carrier polymers include polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone, chitosan, collagen, sodium alginate, gelatin, hyaluronic acid, polylactic acid, poly(lactic acid-glycolic acid) copolymer, polyhydroxybutyric acid, poly(hydroxybutyric acid-glycolic acid) copolymer, cellulose, hydroxymethylcellulose, hydroxypropylcellulose, fatty acid esters, and polyglycerin.

[0280] Examples of additives include sugars, sucrose, mannitol, lactose, L-arabinose, D-erythrose, D-ribose, D-xylose, D-mannose, D-galactose, lactulose, cellobiose, gentibiose, glycerin, polyethylene glycol, N-methylpyrrolidone, oligovinyl alcohol, ethanol, ethylene glycol, and propylene glycol.

[0281] Examples of solubility enhancers include cyclodextrins.

[0282] 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.

[0283] Ophthalmic formulations can include lipids such as dipalmitoylethylphosphocholine, dioleoylphosphatidylethanolamine, or 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol.

[0284] Ophthalmic formulations can include lipids such as 1,2-dioleoyl-sn-glycero-3-[phospho-ol-serine], 1,2-dioleoyl-sn-glycero-3-phosphate.

[0285] Ophthalmic formulations can include lipids such as 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, distearoylphosphatidylcholine, diarachidoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine.

[0286] The ophthalmic formulations can include the fatty acids oleic acid, myristoleic acid, or arachidonic acid.

[0287] Ophthalmic formulations can include phospholipids such as phosphatidylcholine, lecithin, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, and phosphatidylethanolamine.

[0288] Ophthalmic formulations can include polymers such as polyvinylpyrrolidone, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, hydroxyethylstarch, cyclodextrin, 2-hydroxypropyl-β-cyclodextrin, sulfobutylether-β-cyclodextrin, polyethylene glycol, pectin, poly(lactide-co-glycolide), polylactide, polyethyleneimine, or poly-L-lysine.

[0289] In some embodiments, the ophthalmic formulation can include one or more of a pH adjusting excipient, a buffering excipient, an isotonic excipient, a viscous excipient, or a wetting excipient. In certain embodiments, the ophthalmic formulation can include an acidifying excipient, a preservative, an antioxidant, a solubilizing excipient, a humectant, or a suspending excipient.

[0290] The ophthalmic formulation can include an excipient, diluent, delivery vehicle, or carrier material, such as a polymer, polyethylene glycol, dextran, diethylaminoethyl dextran, cyclodextrin, or carboxymethylcellulose.

[0291] Examples of excipients include sodium chloride, sodium dihydrogen phosphate monohydrate, and disodium hydrogen phosphate anhydrous.

[0292] Examples of formulation additives include vegetable oils, olive oil, sesame oil, coconut oil, mineral oil, and paraffin.

[0293] Examples of dispersing or wetting agents include lecithin, polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyethylene sorbitan monooleate.

[0294] Examples of antioxidants include ascorbic acid, cysteine ​​hydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, lecithin, propyl gallate, alpha-tocopherol, citric acid, ethylenediaminetetraacetic acid, sorbitol, tartaric acid, and phosphoric acid.

[0295] Examples of formulation additives include thickeners, such as beeswax, paraffin, or cetyl alcohol.

[0296] Examples of excipients include suspending excipients, sodium carboxymethylcellulose, methylcellulose, hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, or gum acacia.

[0297] Ophthalmic formulations may include a carrier or co-solvent, 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 co-solvent may be used at a concentration of about 0.01% to about 2% by weight.

[0298] The ophthalmic formulation may include gel excipients such as gellan, xanthan gum, and combinations thereof.

[0299] The ophthalmic formulations can include thickening agents such as polyvinyl alcohol, methylcellulose, hydroxypropyl carboxymethylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, methylcellulose, polyvinylpyrrolidone, and combinations thereof. The thickening agents can be used at a concentration of about 0.01% to about 2% by weight.

[0300] Ophthalmic formulations can include preservatives such as benzalkonium chloride, chlorobutanol, benzododecinium bromide, methylparaben, propylparaben, phenylethyl alcohol, disodium edetate, sorbic acid, onamer, polyquaternium-1, hydroxybenzoic acid, sodium benzoate, phenol, cresol, p-chloro-m-cresol, benzyl alcohol, thimerosal, sorbic acid, benzethonium chloride, and combinations thereof. Preservatives can be used at a concentration of about 0.001% to about 1.0% by weight.

[0301] The unit dosage compositions can be sterile but may contain no preservatives.

[0302] Ophthalmic formulations can include pH adjusting excipients such as citrate buffers, acetate buffers, succinate buffers, malate buffers, and gluconate buffers.

[0303] The ophthalmic formulations can include additional acids, such as hydrochloric acid, or / and additional bases, such as sodium hydroxide, for pH adjustment.

[0304] Examples of pH control agents include arginine, sodium hydroxide, glycine, hydrochloric acid, and citric acid.

[0305] Ophthalmic formulations can include buffers such as citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, phthalic acid, Tris, tromethamine hydrochloride, and phosphate buffers.

[0306] The ophthalmic formulation may include a surfactant.

[0307] Examples of surfactants can include non-ionic surfactants, polysorbate 80, polysorbate 20, polysorbates, sorbitan esters, lipids, phospholipids, lecithin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, fatty acids, fatty acid esters, cholesterol.

[0308] Examples of surfactants include oleic acid, sorbitan trioleate, and long chain diglycerides.

[0309] Examples of surfactants include beractant, poractant alpha, and calfactant.

[0310] Ophthalmic formulations can include tonicity adjusting excipients.

[0311] Examples of isotonicity adjusting excipients include sodium chloride, mannitol, and sorbitol.

[0312] Examples of isotonicity adjusting excipients include sugars, polyols, amino acids, and organic and inorganic salts.

[0313] Embodiments of the invention include kits containing any of the reagents, pharmaceutical excipients, active agents, and instructions for use.

[0314] The kit can include a container or formulation containing one or more active agents that are formulated into a pharmaceutical preparation for delivery. The ophthalmic formulation kit can be in multi-dose form.

[0315] The kit can include a dispenser or dropping device for topical delivery and use.

[0316] The kit may include one or more unit dosage forms of the composition for delivery. The unit dosage forms may be sealed to maintain sterility.

[0317] Sample preparation and detection of ultrastructural targets Sample preparation and processing are described. Aqueous humor or vitreous humor specimens collected for EV isolation were processed immediately without fixation. EVs were isolated from bovine vitreous or aqueous humor; or from 4T1 cells using the ultracentrifugation protocol described below. EVs were isolated using the methods described below. Patient aqueous humor was imaged without isolation or processing.

[0318] Extracellular vesicle isolation and purification is described. We adopted a method to isolate extracellular vesicles from fluids. For bovine vitreous or aqueous humor, approximately 8 ml of vitreous (or 100 μL of aqueous humor) was placed in a 15 ml tube and centrifuged at 2,000 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 a SS-34 rotor (DuPont). The supernatant was then transferred and the step was repeated. The samples were transferred to ultracentrifuge tubes (Beckman) in a swinging bucket rotor (SW-41, Beckman) and spun at 100,000 g in an L7-55 ultracentrifuge (Beckman) for 1 hour at 4°C. The supernatant was transferred to a new tube. The step was repeated. Samples were resuspended in 50 μL of sterile Tris-buffered saline (TB, pH 8) and placed in siliconized tubes. Samples for imaging were processed immediately and the remaining samples were frozen at -80°C.

[0319] Nanoparticle tracking analysis is described. Nanoparticle tracking analysis was performed using a NanoSight NS300 system (Malvern) to characterize particles between 30 and 800 nm in solution. We resuspended extracellular vesicles isolated from vitreous humor, aqueous humor, or 4T1 cells in 100 μL of Tris-buffered saline (TB, pH 7.0). Particles were loaded and the camera was focused to capture five videos of 60 seconds each. Videos were recorded and then analyzed using NanoSight software (Version 3.0) to determine the size distribution of EVs and particle concentration. Graphs were generated. The Brownian motion of each particle was tracked from frame to frame, and finally, the Stokes-Einstein equation was applied to allow for the calculation of size.

[0320] Conventional glutaraldehyde-only fixation of liquid samples for electron microscopy is described. EV solutions processed by conventional TEM fixation methods are referred to as "glutaraldehyde-only" or "Glut-only". EVs were obtained and resuspended in a buffered solution as described above. We obtained formvar / carbon-coated EM grids (Electron Microscopy Sciences) and coated their surfaces with poly-L-lysine solution (%, Sigma Aldrich). We applied approximately 15 μL of poly-L-lysine to the formvar / carbon-coated surface of the EM grids and incubated the samples in a humidified chamber for 15 minutes at room temperature. We removed the poly-L-lysine solution with a pipette. We allowed the grids to dry for 10 minutes at room temperature.

[0321] Next, 5 μL of the EV-containing solution was pipetted onto a poly-L-lysine-formvar / carbon-coated EM grid and incubated in a humidified chamber for 30 min at room temperature. The EV solution was then removed with a pipette. The samples were fixed in a “glutaraldehyde fixative solution” consisting of 2.5% glutaraldehyde, 4% paraformaldehyde, and 0.02% picric acid in 0.1 M sodium cacodylate buffer. We pipetted 15 μL of glutaraldehyde solution onto the EM grid and incubated the samples for 15 min at room temperature. The glutaraldehyde fixative solution was then pipetted away. The grid was washed with 15 μL of double-distilled water for 5 min at room temperature. The samples were washed twice, 5 min each time at room temperature. The samples were dried at room temperature and observed under a JEM 1400 electron microscope (JEOL, USA, Inc) as described below. EDC-formalin fixed specimens were further processed as described below.

[0322] The EDC-ETT solution preparation is described. The method of EDC solution immobilization was adopted from previous reports17,18. We prepared 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. Then, we prepared fresh EDC solution for each experiment. We weighed out 0.96 ml of 0.1 M 1-methylimidazole buffer solution and added 13 mg of 5-(ethylthio)-1H-tetrazole (ETT, Sigma Aldrich, final concentration was 0.1 M). The pH was adjusted to 8.0 with 12 N NaOH. We then 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 with 12 M HCl. The EDC-ETT solution was kept on ice until use.

[0323] EDC-glutaraldehyde fixation of liquid samples for electron microscopy is described. 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 (Electron Microscopy Sciences) and coated their surfaces with poly-L-lysine solution (%, Sigma Aldrich). We applied approximately 15 μL of poly-L-lysine to the formvar / carbon-coated surface of the EM grids and incubated the samples in a humidified chamber for 5 minutes at room temperature. We removed the poly-L-lysine solution with a pipette and set the grids aside in the humidified chamber until ready to use. We then combined equal volumes of freshly made EDC / ETT solution and the EV solution by adding 5 μL of ice-cold EDC / ETT solution with 5 μL of ice-cold EV suspended in TBS (pH 8.0) to a 1.5 ml pre-chilled siliconized tube. The sample was incubated on ice for 30 minutes. We then applied 10 μL of EDC / ETT-EV solution to the surface of a formvar / carbon-coated EM grid and incubated the sample for 30 minutes at 4° C. in a humid chamber. To initiate the cross-linking ability of the EDC reagent, we placed the sample in a humid chamber in 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 samples were fixed with a secondary fixation using a glutaraldehyde-based crosslinking solution containing 2.5% glutaraldehyde, 4% paraformaldehyde, 0.02% picric acid in 0.1 M sodium cacodylate buffer and incubated for 15 minutes at room temperature. The glutaraldehyde solution was removed from the EM grid by pipetting air bubbles. The grid was washed by placing 15 μL double distilled water on the grid and incubating for 5 minutes at room temperature. The water was removed and a second wash was performed. Finally, the samples were negatively stained or stained for DNA, RNA and protein as described below.For negative staining, the samples were successively counterstained in 2% uranyl acetate, pH 7, and in 2% methylcellulose / 0.4% uranyl acetate, pH 4. After staining with the stains, the EM grids were then mounted for imaging in an electron microscope, as described below.

[0324] Transmission electron microscopy imaging is described. All EM grids were observed with a JEM 1400 electron microscope (JEOL, USA, Inc) operating at 100 kV. Digital images were captured with a Veleta 2K x 2K CCD camera (Olympus-SIS). Electron microscopy images were recorded and the size and frequency of EVs were analyzed using ImageJ software.

[0325] Transmission electron microscopy of vitreous humor and ocular tissues is described. Human or bovine vitreous tissue was obtained as described above. Samples were decellularized by low speed centrifugation and whole mount preparations were examined for H and E staining as described below. For vitreous, 2 μL was pipetted onto a block and fixed in a solution of 2.5% glutaraldehyde, 4% paraformaldehyde, 0.02% picric acid in 0.1 M sodium cacodylate buffer and incubated for 60 minutes at room temperature19. The specimens were washed with an excess volume of buffer (pH 7.3) for 5 minutes each at room temperature. Samples were post-fixed with 1% OsO4-1.5% K-ferricyanide (aqueous) for 60 minutes at room temperature20. Samples were washed with buffer three times for 5 minutes each at room temperature. Samples were set together and stained with 1.5% uranyl acetate for 60 minutes at room temperature. Samples were dehydrated through a graded ethanol series and transferred through acetonitrile. Samples were infiltrated and embedded in Embed 812 resin (Electron Microscopy Sciences). Tissue sections were cut to 60-65 nm using a Diatome diamond knife (Diatome) on a Leica Ultracut T ultramicrotome (Leica Microsystems). Sections were contrasted with lead citrate 21 and viewed under a JEM 1400 electron microscope (JEOL, USA, Inc) operating at 100 kV. Digital images were captured with a Veleta 2K x 2K CCD camera (Olympus-SIS). Electron microscopy images were recorded and analyzed for size and frequency of EVs using ImageJ software. For TEM staining of nucleic acids, we incubated 5 μL of acridine orange staining solution (Exo-Red Exosome RNA Fluorescent Label, System Biosciences) with centrifugation-purified EVs for 30 min at 25 °C. For ethidium bromide (EtBr) staining EVs, we mixed 5 μg / ml EtBr solution with 5 μL of ultracentrifugation-purified EVs for 30 min at 25°C.For protein staining on the TEM, we mixed 500 μm CFSE diluted in TBS (pH 7.4) with 5 μL of ultracentrifugally purified EVs for 30 min at 25° C. All samples were then fixed, mounted, and imaged on a TEM as described above.

[0326] Statistical analysis is described. Graphical presentation and calculations were performed using Excel (version 2011, Microsoft). All experiments were performed with n ≧ 3 unless otherwise stated. For nanoparticle tracking analysis, we used the Stokes-Einstein equation to calculate particle size, concentration and distribution. Statistical analysis was performed using Student's t-test for two independent groups using SPSS software, and a p-value < 0.05 was assumed to be a significant difference.

[0327] Methods for imaging healthy control and POAG aqueous humor samples are described. The samples are shown in Table 1.

[0328] [Table 1]

[0329] method:

[0330] For 1 ml of EDC / ETT solution in 0.1 M 1-methylimidazole buffer (pH 8.0): 1. EDC-HCl powder should be stored under argon at -20° C. To avoid moisture condensation, only bottles that had reached room temperature were opened, i.e., removed from the refrigerator 1 hour before drawing samples. 2. The EDC / 5-ETT solution should be made up to 1 hour before use. 3. Pipette 5.76 ml of 0.1 M 1-methylimidazole buffer (pH 8.0) into the 15 ml tube and vortex vigorously. 4. Carefully remove 0.078 g of the ETT found in group "G" into a spoonful of paper. Carefully pour the 0.078 g into the tube. 5. Using pH paper and a pipette, check that the pH is 8.0. 6. Using paper, measure out 0.12 g of EDC. Carefully pour into the tube. Vortex vigorously. 7. Discard any unused solution. 8. Using pH paper and a pipette, check that the pH is 8.0.

[0331] Poly-L-lysine grid, EDC fixation, Glut fixation, UA staining 1. Use Formvar-Court Grid. 2. Place them formvar side down towards the poly-lysine drop and leave for 5-10 minutes. 3. The poly-L-lysine is then withdrawn. 4. Secure the parchment paper to the 100mm pan with tape or weights. Place the grid on top of the parchment paper.

[0332] Mixing EDC solution with EVs In a 1.5 ml siliconized tube, 10 μL (cold) EVs are mixed with 10 μL (cold) EDC solution. 2. In the cold room, apply a 10 μL sample (aqueous humor) to the grid and leave for 30 minutes to dry and allow the EVs to settle to the bottom of the grid. 3. Place in a humid chamber and incubate at 50°C for at least 3 hours.

[0333] Secondary fixation with Glut 1. Using a pipette, remove the EDC-EV solution from the sample. 2. Fix with Glut: add 2-5 μL of glut fixative and wait 5 minutes. 3. Remove the Glut with a pipette or lens paper.

[0334] Glut and EDC Rinse 1. Place the blisters on the grid for 5 minutes. 2. Remove the bubbles with a pipette. Add 3.5 μL of uranyl acetate onto the grid (4 drops, blotting between each drop). 4. Dry the grid and place it in the grid box. 5. Remove the stain with a pipette.

[0335] Imaging with a TEM. Immediately after completion, images are taken at 25k (approximately 50 photos) at approximately 50K magnification.

[0336] Methods for treating POAG aqueous humor samples with bimatoprost, as well as fixation, negative staining and TEM imaging of the fluid with EDC. We sought to determine the effect of bimatoprost on EVs in the aqueous humor of glaucoma patients. To do this, we diluted POAG aqueous humor samples with Tris-buffered saline (1:10 dilution) and added POAG samples diluted with either 1:1 volume of Tris-buffered saline (control) or bimatoprost.

[0337] Lumigan was taken directly from the dispense bottle and was not diluted. 10 μL of POAG sample was mixed with 10 μL of TBS or 10 μL of POAG sample was mixed with 10 μL of undiluted Lumigan. The tubes were then incubated in a thermocycler PCR machine and left at 37° C. for 72 hours. The samples were then diluted (1:10). We then mounted with EDC on electron microscopy grids using the following protocol.

[0338] 1 ml of EDC / ETT solution in 0.1 M 1-methylimidazole buffer (pH 8.0) 1. EDC-HCl powder should be stored under argon at -20° C. To avoid humidity condensation, only open bottles that have reached room temperature, i.e., removed from the refrigerator 1 hour before drawing samples. 2. The EDC / 5-ETT solution should be made up to 1 hour before use. 3. Pipette 5.76 ml of 0.1 M 1-methylimidazole buffer (pH 8.0) into the 15 ml tube and vortex vigorously. 4. Carefully remove 0.078 g of the ETT found in group "G" into a spoonful of paper. Carefully pour the 0.078 g into the tube. 5. Using pH paper and a pipette, check that the pH is 8.0. 6. Using paper, measure out 0.12 g of EDC. Carefully pour into the tube. Vortex vigorously. 7. Discard any unused solution. 8. Using pH paper and a pipette, check that the pH is 8.0.

[0339] Poly-L-lysine grid, EDC fixation, Glut fixation, UA staining 1. Use Formvar-Court Grid. 2. Place them formvar side down towards the poly-lysine drop and leave for 5-10 minutes. 3. The poly-L-lysine is then withdrawn. 4. Secure the parchment paper to the 100mm pan with tape or weights. Place the grid on top of the parchment paper.

[0340] Mixing EDC solution with EVs In a 1.5 ml siliconized tube, 2.5 μL of (cold) EVs are mixed with 5 μL of (cold) EDC solution. 2. In the cold room, apply a 5 μL sample (aqueous humor) to the grid and leave for 15 minutes to dry and allow the EVs to settle to the bottom of the grid. 3. Place in a humid chamber and incubate at 50°C for at least 3 hours.

[0341] Secondary fixation with Glut 1. Using a pipette, remove the EDC-EV solution from the sample. 2. Fix with Glut: add 2-5 μL of glut fixative and wait 5 minutes. 3. Remove the Glut with a pipette or lens paper.

[0342] Glut and EDC Rinse 1. Place the blisters on the grid for 5 minutes. 2. Remove the bubbles with a pipette. Add 3.5 μL of uranyl acetate onto the grid (one drop, blot after application). 4. Dry the grid and place it in the grid box. 5. Remove the stain with a pipette.

[0343] The samples were imaged by TEM.

[0344] Additional illustrative examples of the invention are as follows.

[0345] A method for identifying glaucoma or pre-glaucoma in a subject, the method comprising: providing an aqueous humor sample from a subject potentially having glaucoma; analyzing the sample for glaucoma-associated extracellular vesicle complexes, either aggregates of extracellular vesicles or individual extracellular vesicles having a diameter greater than 300 nanometers, or between 300 and 3,000 nm, or between 300 and 5,000 nm, or between 300 and 10,000 nm; and identifying the subject as having glaucoma or pre-glaucoma based on the analysis.

[0346] The glaucoma-associated extracellular vesicle complex is an extracellular vesicle complex selected from the group consisting of exomeres, exosomes, multivesicular bodies, intraluminal vesicles (ILVs), multivesicular endosomes (MVEs), oncosomes, microvesicular bodies, apoptotic bodies, or vesicles originating from endosomes or plasma membranes.

[0347] The glaucoma-associated extracellular vesicle complexes may be complexes of individual extracellular vesicles, wherein the complexes may have a diameter of from 360 to 21,000 nanometers.

[0348] The glaucoma-associated extracellular vesicle complexes may be aggregates of 10, 20, 30, 40 or more extracellular vesicles.

[0349] The glaucoma-associated extracellular vesicle complex may be an aggregate of 50, 100, or 200 or more extracellular vesicles.

[0350] The method further comprises fixing the glaucoma-associated extracellular vesicles in the sample prior to analysis. Fixing the glaucoma-associated extracellular vesicles comprises contacting the sample with a non-reversible cross-linking agent; and contacting the sample with an aldehyde-containing fixative before, during or simultaneously with contacting the sample with the non-reversible cross-linking agent to fix the glaucoma-associated extracellular vesicle complexes.

[0351] The method according to claim 1, wherein the irreversible crosslinking agent is selected from the group consisting of water-soluble carbodiimides, cyanogen halides, and mixtures thereof.

[0352] The above method, wherein the irreversible crosslinking agent is 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide.

[0353] The method according to claim 1, wherein the irreversible cross-linking agent is a cyanogen halide selected from the group consisting of cyanogen bromide, cyanogen fluoride, cyanogen chloride, and cyanogen iodide.

[0354] and independently, before, after or simultaneously with the contacting with the irreversible crosslinker and the contacting with the aldehyde-containing fixative, contacting the sample with an additional crosslinker, the additional crosslinker being selected from the group consisting of 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-methylene bisacrylamide, N,N-methylene bisacrylamide, N,N-(1,2-dihydroxyethylene)bisacrylamide, formaldehyde-free crosslinkers, N-(1-hydroxy-2,2-dimethoxyethyl)acrylamide, divinylbenzene, formalin fixative, calcium formal, formal saline, zinc formalin (unbuffered), Zenker fixative, Helly fixative, B-5 ​​fixative, Bouin's solution, Hollande's solution, Gendre's solution, Clarke's solution, Carnoy's solution, metacam, alcohol formalin, and formol acetate alcohol.

[0355] The method, wherein said analyzing comprises imaging the immobilized glaucoma-associated extracellular vesicle complexes.

[0356] The above methods, wherein the imaging is performed by transmission electron microscopy, scanning electron microscopy, cryo-electron microscopy, binocular stereo microscopy, 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, ultrasound microscopy, colorimetric assay, chemiluminescence assay, spectrocolorimetry, positron emission tomography, computed tomography, and magnetic resonance imaging.

[0357] The method further comprising detecting the glaucoma-associated extracellular vesicle complexes in the aqueous humor sample based on the imaging.

[0358] The method above, wherein said identifying is based on said detecting.

[0359] The above method, wherein the identifying step comprises: providing a standard image of a clinical aqueous humor sample from a subject having glaucoma, the aqueous humor sample containing the glaucoma-associated extracellular vesicle complexes fixed with an aldehyde containing irreversible crosslinker and fixative; comparing an image of the subject's clinical sample with the standard image in terms of size, density, morphology, or spatial distribution of the fixed glaucoma-associated extracellular vesicle complexes; and determining whether the subject has glaucoma or pre-glaucoma based on the comparison.

[0360] The above method, further comprising administering a therapeutic agent to the subject based on said determining.

[0361] The method, wherein the therapeutic agent reduces the size of glaucoma-associated extracellular vesicle complexes by disrupting two or more extracellular vesicles that are in contact with one another.

[0362] The above method, wherein the identifying step involves monitoring the progression or reversal of glaucoma, and includes providing a previous image of a clinical aqueous humor sample from the subject containing glaucoma-related extracellular vesicle complexes fixed with an aldehyde containing a non-reversible crosslinking agent and a fixative; comparing the image of the clinical aqueous humor sample from the subject containing the glaucoma-related extracellular vesicle complexes fixed with an aldehyde containing a non-reversible crosslinking agent and a fixative with the previous image in terms of size, density, morphology, or spatial distribution of the fixed glaucoma-related extracellular vesicle complexes; and determining whether the glaucoma is progressing or reversing based on the comparison.

[0363] The above methods, wherein identifying glaucoma or pre-glaucoma is performed before the subject providing the aqueous humor sample experiences any vision loss.

[0364] A method for screening compounds for their ability to treat glaucoma, the method comprising: providing a candidate agent potentially effective in treating glaucoma; providing a sample containing glaucoma-associated extracellular vesicle complexes, wherein the extracellular vesicles are either aggregates of extracellular vesicles or individual extracellular vesicles having a diameter greater than 300 nanometers; contacting the candidate agent with the glaucoma-associated extracellular vesicle complexes; and identifying a candidate compound effective in reducing the size of the glaucoma-associated extracellular vesicle complexes based on the contacting.

[0365] The above method, wherein the glaucoma-associated extracellular vesicle complexes are aqueous humor.

[0366] The above method further comprises contacting a second sample containing glaucoma-associated extracellular vesicle complexes with a placebo; and comparing the reduction in size of the glaucoma-associated extracellular vesicle complexes in the sample contacted with the placebo to that achieved with the candidate agent to identify a candidate compound effective in reducing the size of glaucoma-associated extracellular vesicle complexes.

[0367] A reagent for detecting glaucoma, comprising an isolated sample containing glaucoma-associated extracellular vesicle complexes, which are either extracellular vesicles of aggregates of extracellular vesicles or individual extracellular vesicles having a diameter of more than 300 nanometers.

[0368] The above reagent, wherein the glaucoma-associated extracellular vesicle complex is aqueous humor.

[0369] Embodiments of the present invention further contemplate ophthalmic compositions.

[0370] In some embodiments, the ophthalmic aqueous pharmaceutical composition may include an active agent selected from cetylpyridinium chloride, polymyxin B sulfate, neomycin sulfate, and heparin sodium. The active agent may comprise 0.01-2% w / v of the composition. In some embodiments, the active agent may comprise 0.01-0.2% w / v of the composition.

[0371] The pH of the composition can be adjusted, for example, using sodium hydroxide and hydrochloric acid. The composition can have a pH of 6.8 to 7.9, or about 7.3.

[0372] In some embodiments, the composition is capable of reducing intraocular pressure when administered to the eye.

[0373] In additional embodiments, the composition, when administered to the eye, can reduce extracellular vesicle complexes in the eye.

[0374] In a further embodiment, the compositions, when administered to the eye, may be effective to treat glaucoma diseases.

[0375] The compositions of the present invention may further comprise one or more of a solubilizing agent, a surfactant, an isotonicity agent, and a preservative.

[0376] Examples of solubilizing agents include phosphates, citric acid monohydrate, trisodium citrate, and combinations thereof.

[0377] Examples of phosphates include monosodium dihydrogen phosphate, disodium monohydrogen phosphate, dipotassium monohydrogen phosphate, monopotassium phosphate, and combinations thereof.

[0378] Examples of surfactants include phospholipids, polyglycerol esters, polyethylene glycol esters, polyethylene glycol esters, copolymer esters, polyoxyethylene sorbitan esters, cyclodextrins, polyvinyl alcohol, povidone, hydroxypropyl methylcellulose, poloxamer, carboxymethyl, cellulose, hydroxyethyl cellulose, polyacrylates, and combinations thereof.

[0379] Examples of isotonic agents include sodium chloride, trehalose, mannitol, sorbitol, dextrose, potassium chloride, and combinations thereof.

[0380] Examples of preservatives include benzalkonium chloride, polyquaternium-1, benzododecinium bromide, sorbic acid, methylparaben, propylparaben, chlorobutanol, benzyl alcohol, phenylethyl alcohol, oxychloro complexes, thimerosal, sodium perborate, disodium edetate, and combinations thereof.

[0381] In the context of ophthalmic compositions, the solubilizer may also act as a buffering agent, or as a stabilizer, or as a viscosity enhancer.

[0382] In the context of ophthalmic compositions, surfactants may also have the effect of acting as solubilizing agents.

[0383] The compositions of the present invention may consist only of the active agent and the carrier.

[0384] For example, the composition can be an aqueous solution of the active agent.

[0385] Examples of the carrier include water, sterile water, water for injection, water for irrigation, phosphate buffer, and combinations thereof.

[0386] The compositions of the present invention can be used in medical therapy.

[0387] The compositions of the invention can be used in the treatment of the human or animal body.

[0388] The compositions of the present invention can be used to reduce intraocular pressure in the human or animal body.

[0389] The compositions of the present invention can be used to reduce ocular extracellular vesicle complexes in the human or animal body.

[0390] The compositions of the present invention can be used in preparing or manufacturing a medicament for preventing, ameliorating, or treating a disease or condition related to glaucoma in a subject in need thereof.

[0391] Embodiments of the present invention also contemplate methods of treating glaucoma disease, reducing intraocular pressure, or reducing ocular extracellular vesicle complexes in a subject in need thereof by administering a composition of the present disclosure to the eye of the subject.

[0392] Administration may be by injection.

[0393] Ocular extracellular vesicle complexes can be aggregates of extracellular vesicles having diameters greater than about 300 nanometers.

[0394] All patents, published patent applications, and non-patent publications cited in this description are each expressly incorporated herein by reference in their entirety for all purposes.

[0395] Although the above disclosure has been described in detail by way of example for purposes of clarity of understanding, it will be apparent to those skilled in the art that certain changes and modifications can be understood by the disclosure and made without undue experimentation within the scope of the appended claims, as set forth in the non-limiting drawings. The present invention includes all such attendant embodiments, equivalents, and modifications. The present invention includes all combinations and mixtures of the various exemplary components, examples, and features, materials, elements, or limitations of the claimed embodiments.

[0396] The terms "a," "an," "the," and similar terms used to describe and claim the invention should be interpreted as encompassing both the singular and the plural. EXAMPLES

[0397] Example 1 Use of factory immobilization to accelerate imaging of extracellular vesicles in biological fluids EV ultrastructure can be detected by transmission electron microscopy (TEM) in combination with negative staining. However, in our laboratory, we found this technique to be inconsistent and often yield negative results. When examining known amounts of EVs applied to solution, we found substantial discrepancies between the large number of EVs applied and the very few EVs ultimately imaged. Often, the results were inconsistent and varied with technical reproduction. Hence, methodological gaps exist, preventing efficient, consistent, and representative EV imaging in solution. Therefore, we evaluated each step of the EV imaging protocol and attempted to identify points at which EVs would be lost. We found that conventional TEM protocols lead to poor binding of EVs to the electron microscope grid surface and that most EVs do not adhere. To attach EVs more efficiently, we cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), which retains EVs and allows reliable TEM imaging. Finally, we demonstrated that this method can be used to image EVs in a variety of biological fluids, including blood (plasma); cerebrospinal fluid; nipple aspirate fluid; aqueous humor, or isolated EVs suspended in buffer.

[0398] Example 2 Conventional imaging of EVs suspended in fluid is low yielding due to the extensive loss of vesicles to waste fluid. To improve on 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 vitreous humor from the posterior chamber of the eye, homogenized the samples, isolated EVs using ultracentrifugation, and resuspended the samples in buffered saline. Next, we quantified the number and size of EVs using nanoparticle-tracking analysis (NTA; 3.98 x 108 EVs per ml). To visualize the ultrastructure of vitreous EVs suspended in fluid, we followed a conventional glutaraldehyde-based TEM imaging protocol (Figure 6, top). We applied approximately 4 x 106 EVs to an electron microscope grid and followed standard protocols for glutaraldehyde fixation and negative staining with uranyl acetate solution. We subsequently imaged the specimens using TEM, and the results showed that there were almost no EVs (0.033 ± 0.182 EVs observed per 25,000x high-power microscope field, n = 3 biological repeats, and 10 photographs were captured), an unexpectedly low number that is not consistent with a loading of 4 million EVs. At low magnification, we observed sparsely spaced EVs (Figure 6, top row, left), and in most photographic frames, we could not clearly identify negatively stained EVs (Figure 6, top row, center and right). These results suggest that EVs were either destroyed during specimen processing, failed to adhere to the surface of the TEM grid, or were lost during the protocol. To confirm that EVs are present in the vitreous specimen, we imaged the vitreous base, a gel-like matrix attached to the retina, sectioned the sample, and prepared it for TEM. Indeed, we observed many EV-type electron-dense signals (data not shown) in the vitreous tissue sections, confirming their presence in the samples prepared for TEM.Therefore, we found that millions of EVs applied to the surface of electron microscopy grids were discarded in the discarded solution and did not adhere to the grid surface (Figure 6, second row, left). To determine that there was a significant amount of EVs present in the effluent, we measured and compared the number and size of EVs isolated from 4T1 and 4T2 breast cancer cell lines and resuspended in buffered saline. We found that the concentration of 4T1 EVs was 8.63 x 108 particles per ml (± 4.96 x 108 particles per ml), and we applied 8.63 x 106 EVs to the grid surface (Figure 6, second row, center and Table 2). Next, we used nanoparticle tracking analysis (NTA) to measure the amount of EVs present in the relinquishment solution and found that at least 8.16 x 108 particles per ml (± 2.43 x 107 particles per ml) or 8.16 x 106 particles were lost to the wash buffer in three independent trials (Figure 6, second row, right; Figure 6, third row, left and Table 2). These data indicate that the majority of EVs applied to the surface of the grid are unable to adhere to the electron microscopy grid surface and are lost in the relinquishment solution (Figure 6, third row, left). Therefore, we found that imaging EVs suspended in liquid using conventional fixation methods, negative staining and electron microscopy is greatly hindered by poor cross-linking to the grid surface.

[0399] Table 2 shows that very few extracellular vesicles adhere to the electron microscopy grid. We determined the size and concentration of EVs applied to the entire grid surface and compared this value to the fraction present in the discarded EVs. We isolated EVs from a mouse mammalian tumor cell line, 4T1 cell media, using ultracentrifugation and measurements were performed using Nanoparticle Tracking Analysis (NTA) using the same settings for all variables in NTA 2.3 build 17 software. The average size, mode size and average concentration of EVs applied to the entire TEM grid surface and EVs present in the discarded fraction are shown (n = 3).

[0400] [Table 2]

[0401] Example 3 The use of formalin-EDC fixation preserves EVs and allows reliable imaging of EVs suspended in liquid. We hypothesized that EVs do not bind well to TEM grids and attempted to permanently attach EVs suspended in liquid to the grids by adding a heat-stable fixative, EDC, and a carbodiimide that creates irreversible crosslinks between positively charged amino side chains and carboxyl groups of proteins. To test our hypothesis, we combined 4 million isolated bovine vitreous EVs and an EDC fixation solution, applied it to the surface of a poly-l-lysine-coated formvar TEM grid, and activated the EDC solution by applying heat (50°C) for 3 hours (Figure 6, third row, center). After crosslinking, we removed the EDC solution, applied a glutaraldehyde fixation solution, washed the sample, and performed negative staining. The images show abundant EVs in each frame, with 16.5 EVs (± 16.9) per 25,000x high-power field, consistent with conventional negative staining and TEM. Moreover, we identified at least 357 times more EVs in EDC-fixed samples compared to glutaraldehyde alone (Fig. 6, fourth row, left; p < 0.05, n = 3). To extend the scope of this technique to other fluids, we visualized EVs isolated from bovine aqueous humor, which shows robust detection of EVs with electron-dense negative staining surrounding the EV periphery (Fig. 6, fourth row, center). We imaged native aqueous humor fluid from a healthy patent to visualize EVs in situ. We define in situ imaging as imaging a non-isolated solution and visualizing the contents of that biological fluid in situ. Our method for visualizing EVs in fluids differs from conventional methods for imaging EVs because we do not concentrate the EVs. Here, the images show a heterogeneous population with clear negative staining (Figure 6, fourth row, right). Ultimately, we found that crosslinking EVs suspended in multiple fluids with EDC resulted in substantially improved transmission electron microscopy imaging. These data show that for imaging EVs in fluids, EDC fixation significantly improves the number of EVs observed compared to conventional aldehyde fixation.

[0402] Example 4 Comparison of extracellular vesicles in aqueous humor from healthy controls and POAG patients. The aqueous humor is a clear watery biological fluid similar to plasma, but contains 98% water, as well as amino acids, electrolytes, ascorbic acid, glutathione, and immunoglobin. Aqueous humor has been shown to contain EVs. In these studies, EVs were isolated using an ultracentrifugation protocol, in which no glaucoma-associated EV aggregates were observed. We hypothesized that there would be differences in the EV ultrastructure of aqueous humor in subjects with ophthalmic pathology (such as glaucoma) compared to healthy controls (subjects with no ophthalmic pathology other than cataract). We chose to study the aqueous humor, which is normally lost during cataract surgery, and obtaining a sample did not pose any additional risk to the subject. In order to observe aqueous humor in its most natural state, we did not isolate EVs using ultracentrifugation or other EV-related protocols. We collected aqueous humor from a cohort of healthy patients and a cohort of patients with a clinical diagnosis of POAG. We found that EVs in the aqueous humor of glaucoma patients are larger, thus making these structures block aqueous humor outflow. To complete this study, we obtained aqueous humor specimens from 1) healthy control patients undergoing elective cataract surgery (control cohort) or 2) patients with a diagnosis of glaucoma (POAG cohort). The experimental design is listed below.

[0403] Example 5 Experimental design; from healthy and glaucoma patients. We performed a prospective clinical trial to determine the aqueous humor of aqueous humor-derived EVs donated from healthy controls or glaucoma patients with a diagnosis of cataract but without other ocular or other systemic complications. All patients in the control cohort were elective outpatient, ambulatory cataract surgery patients and were generally healthy. We collected a small sample (50 to 100 μL) of aqueous humor (usually lost as medical waste) at the start of cataract surgery. We fixed the sample with EDC crosslinker, negatively stained the sample, and performed transmission electron microscopy. The experimental sample had POAG in addition to cataract and did not have any of the following conditions: diabetic retinopathy or age-related macular degeneration. The pool of glaucoma patients is selected from patients who will have cataract extraction and lens implant surgery. Inclusion criteria included: outpatient cataract surgery patients aged 18 years or older who had no other ocular complications besides cataract or who also had a clinical diagnosis of POAG. Exclusion criteria included ocular complications other than cataract. The Institutional Review Board approved the protocols for collecting samples and completing these experiments.

[0404] Example 6 Obtaining aqueous humor samples from patients with and without glaucoma: During cataract surgery, an incision is made into the anterior chamber to reach the cataractous lens. During surgery, aqueous humor leaves the eye and is replaced by irrigation fluid injected through the instrument. We collected a small sample of aqueous humor that is normally lost as medical waste at the beginning of cataract surgery. The sample was assigned a numeric study ID code for anonymity and immediately transported from the OR to the laboratory for TEM imaging analysis. Patients were not undergoing surgery or concomitant interventions for the purposes of this study, but instead were individuals scheduled to undergo therapeutic cataract surgery. Scheduled cataract surgery was reviewed to identify patients who met the inclusion and exclusion criteria above for the control or POAG cohorts. Informed consent was obtained from each patient prior to inclusion in the study. Patients then underwent standard cataract surgery with the following exceptions. At the beginning of the cataract surgery, a 30-gauge needle attached to a TB syringe was inserted into the clear cornea to aspirate 0.05–0.1 cc of aqueous humor. The wound self-sealed. Subsequently, a corneal puncture incision was made according to standard cataract procedure by the surgeon. We collected the samples and transported them to the laboratory on ice. Experimental samples were identified with random experiment ID codes to ensure anonymity. For all experiments, we did not isolate EVs using ultracentrifugation, but rather imaged their biofluids in situ.

[0405] Example 7 Imaging healthy control aqueous humor ultrastructure in situ shows diffuse distribution of EVs: To understand the morphology of EVs in normal physiological conditions, we imaged aqueous humor from healthy patients in situ. No pre-glaucoma experiments have visualized EVs in aqueous humor in situ using EDC fixation. To understand the ultrastructure of aqueous humor, we diluted the samples 1:10 in buffered saline (note: we did not isolate EVs), fixed the samples with EDC and then glutaraldehyde, negatively stained the specimens, and imaged them by TEM. The data showed that healthy human aqueous humor contains abundant quantities of EVs from multiple control human subjects (Figures 7-8). These experiments suggest that EVs in healthy human aqueous humor were present and diffusely distributed, with no evidence of EV aggregation. Moreover, most EVs were not attached to one another and were relatively uniformly distributed. Therefore, healthy control aqueous humor contains non-aggregated and diffusely distributed EVs.

[0406] Example 8 Imaging the ultrastructure of POAG aqueous humor in situ shows significantly larger glaucoma-associated EV complexes, larger than those observed in healthy controls. We hypothesized that POAG patients have an unidentified substance present in the aqueous humor that is responsible for obstructing the trabecular meshwork. We hypothesized that this unidentified substance has not yet been described because it is below the threshold of detection using current imaging techniques. Therefore, we applied EDC fixation, negative staining, and transmission electron microscopy imaging to observe the morphology of EVs in the aqueous humor of POAG patients. Aqueous humor imaged from two different POAG patients was imaged in situ without processing (no EV isolation) and under identical conditions to those used for healthy controls. Strikingly, the aqueous humor of POAG patients showed numerous groups of EVs aggregated together in large EV complexes, several microns in size, which we called "glaucoma-associated EV complexes" (Figures 9 and 10). Glaucoma-associated EV complexes were present in two different patient samples and were measured to be larger than the JCT opening (1 to 4 μm, or 2 to 20 μm), which is large enough to occlude corneoscleral tissue. These data suggest that POAG patients have ultrastructural material present in the aqueous humor that is responsible for occluding the trabecular meshwork, reducing aqueous humor outflow, and potentially causing vision loss. We propose that glaucoma-associated EV complexes are an unidentified substance that contributes to the pathogenesis of glaucoma.

[0407] Example 9 Compared to controls, treatment of large EV complexes with bimatoprost disrupts the EV complexes. To determine whether the glaucoma-associated EV aggregates visualized using the EDC fixation method are indeed possible mediators of POAG, we hypothesized that treatment with known glaucoma drugs would alter the morphology of glaucoma-associated EV aggregates. Therefore, we chose to use bimatoprost (Lumigan), an intraocular pressure-lowering drug that lowers IOP in normal, hypertensive, and glaucomatous eyes. Moreover, bimatoprost is known to promote aqueous humor outflow by regulating matrix metalloproteinases and remodeling the extracellular matrix. Here, we hypothesized that the glaucoma-associated EV complexes would be reduced in size by the addition of bimatoprost. To test this, we incubated aqueous humor from POAG patients with bimatoprost or buffered saline (placebo or control conditions) for 72 hours at 37°C and imaged the EV ultrastructure using EDC fixation, negative staining and transmission electron microscopy (Figures 11-13). After bimatoprost treatment, we noted that the number of glaucoma-associated EV complexes in POAG-bimatoprost samples was substantially reduced compared to placebo-treated POAG samples (Figure 11). In a second human subject diagnosed with glaucoma, we repeated the experiment, mixing POAG aqueous humor with placebo (buffered saline) and incubating the samples at 37°C for 72 hours, and we observed many glaucoma-associated EV complexes (Figure 12). However, under the same conditions, except with bimatoprost, we observed a substantial reduction in the number of glaucoma-associated EV complexes (Figure 13). These data suggest that known treatments for POAG reduce the size and alter the morphology of the glaucoma-associated EV complex, suggesting that this complex may be a possible pathogenic mediator of POAG.

[0408] Example 10 Aqueous humor from glaucoma patients contains more electron-dense structures in the aqueous humor that are not present in healthy controls. Electron microscopy images showed large electron-dense structures in the aqueous humor of POAG specimens that are not present in healthy controls. We hypothesized that POAG glaucoma-associated EV aggregates are larger in size and more abundant compared to healthy controls. Therefore, we obtained electron microscopy pictures from healthy controls or POAG samples and measured the number and size of glaucoma-associated EV aggregates. The data showed that POAG aqueous humor contained substantially larger glaucoma-associated EV aggregates, measuring from 361 nm to 20,214 nm (Figure 14). Moreover, we observed substantially more glaucoma-associated EV aggregates in POAG samples compared to healthy controls. These data suggested that POAG aqueous humor EVs contained large ultrastructural complexes that were not present in healthy controls (Figure 14). Moreover, the glaucoma-associated EV aggregates were large enough to block the eye's drainage system, the trabecular meshwork. It was hypothesized that abnormal aqueous humor outflow causes elevated IOP, which is a major risk factor for glaucoma. The region of the TM involved in the establishment of IOP is next to Schlemm's canal and is called the corneoscleral tissue (JCT) or cribriform region. The site of greatest resistance to aqueous humor outflow is the JCT, which measures approximately 2-20 μm (J. Ocular Biology 2013 June 1(1):3) with perforations of 1 to 4 μm or more. The JCT is composed of a loosely arranged extracellular matrix (ECM) in which cells are embedded. The ECM of the JCT has been implicated as a barrier that can isolate ocular fluid outflow. Therefore, we hypothesized that this material (glaucoma-associated EV aggregates) in the aqueous humor of patients with glaucoma is physically larger than the diameter of the JCT outlet, obstructing aqueous humor outflow and may be related to glaucoma pathogenesis.

[0409] Example 11 The aqueous humor of glaucoma patients contains EVs that contact each other to form larger structures called "glaucoma-associated EV aggregates" that are not present in healthy control aqueous humor. To determine the composition of glaucoma-associated EV aggregates, we analyzed TEM images and found that these ultrastructures are composed of many EVs that contact each other to form larger aggregates. To quantify the number of EVs found within glaucoma-associated EV aggregates, we counted the total number of EVs present in the image and quantified the number of EVs in each aggregate, or counted the number of EVs that were not in contact with other EVs (free EVs). We categorized the number of EVs in contact with each other as 0 (free EVs, no contact), less than 5 EVs in contact with each other, 5-10 EVs in contact with each other, 10-50 EVs in contact with each other, 50-100 EVs in contact with each other, or 100-300 EVs in contact with each other. The data show that in healthy controls, very few EVs are present in aggregates (Figures 15 and 16), with most EVs in contact with fewer than 5 other EVs. The data suggested that in healthy controls, most EVs are "free EVs". Interestingly, for glaucoma patients, a substantial number of EVs were in contact with between 50 and 300 other EVs. The data suggest that in glaucoma, more than in healthy control samples, most EVs are found within glaucoma-associated EV aggregates (Figures 15 and 16). These data suggest that glaucoma patients have EVs in contact with each other, in stark contrast to healthy controls, where most EVs are free.

[0410] Example 12 Extracellular vesicles in the aqueous humor of healthy control subjects exist as "free EVs," with most EVs between 100 and 200 nm in size. We sought to determine 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 a TEM. We analyzed the photos and measured EV diameters. The data show that for this patient, healthy control human aqueous humor contains EVs, with most EVs between 100 and 200 nm in diameter (Figure 17). Furthermore, we found that most EVs in the control cohort were not in contact with other EVs. This data suggests that healthy control aqueous humor contains exosomes and some microvesicular bodies. We did not observe larger apoptotic bodies or glaucoma-associated EV aggregates in these samples.

[0411] Example 13 Extracellular vesicles in the aqueous humor of subjects diagnosed with POAG are present in glaucoma-associated EV aggregates and are similar in size to "free EVs" found in healthy controls. To study the EV population in the aqueous humor of human subject #1 with a diagnosis of POAG, we obtained aqueous humor, fixed the samples with EDC, negatively stained the specimens, and imaged them with a TEM. We analyzed the pictures and measured the EV size and count frequency of EVs located within glaucoma-associated EV aggregates, or we counted free EVs. In POAG specimen #1, we did not observe "free EVs." We then counted and measured the number and size of EVs located in glaucoma-associated EV aggregates (Figure 18). The data show that a significant number of EVs are located within glaucoma-associated EV aggregates, measuring between 36 nm and 300 nm in size, with a peak number of EVs measuring between 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-associated EV aggregates. We observed EVs present within aggregates (defined as EVs that contact each other) or free EVs (not contacting each other, Figs. 19-20). Furthermore, we found that a significant number of EVs located within glaucoma-associated EV aggregates were 36-300 nm in size (Fig. 21). We then compared the number and size of EVs present in glaucoma-associated EV aggregates, and the two sample EV populations were similar (Fig. 22). These data suggest that the EV size population in POAG patients is located within glaucoma-associated EV aggregates, and the data is consistent between samples.

[0412] Example 14 Free EVs in the aqueous humor of human subjects diagnosed with POAG differ in size and frequency compared to those 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 count frequency of glaucoma and control conditions. We found that EVs differ in size and frequency between POAG patients and healthy controls (Figure 23). These data suggest that EVs that do not contact other EVs are larger in POAG patients compared to healthy controls. These data suggest that free EVs may have implications.

[0413] Example 15 Extracellular vesicles from POAG aqueous humor present within glaucoma-associated EV aggregates are similar in size and frequency to free EVs obtained from healthy human subjects. We hypothesized that EVs present in glaucoma-associated EV aggregates are similar in size and count frequency to healthy control EVs. To test this, we compared the count frequency and size of EVs from POAG and healthy control specimens. POAG aqueous humor-derived EVs located within glaucoma-associated EV aggregates are similar in size and frequency to non-aggregated EVs in healthy human subjects (Figure 24). These data suggest that the normal population of EVs from healthy controls may be blocked from functioning because EVs in POAG are trapped in glaucoma-associated EV aggregates.

[0414] Example 16 The active agent for use in the treatment of glaucoma may be cetylpyridinium of formula XI, which may be used in a prodrug form or in a pharma- ceutically acceptable salt form.

[0415] [ka] It is 1-hexadecylpyridin-1-ium.

[0416] Solutions of drug compounds were prepared by weighing the compound in a microcentrifuge tube and dissolving the solid material in 1x PBS buffer at pH 7.2. For compounds that were poorly soluble in water, stock solutions were prepared in ethanol or DMSO and diluted 10-fold to achieve final concentrations in 10% ethanol or DMSO vehicle. Heat (37°C) and vortex mixing were applied to the compound solutions to facilitate dissolution.

[0417] The concentration of cetylpyridinium chloride was 43 mg / ml.

[0418] To determine the effect of the compounds on intraocular pressure (IOP), the compounds were tested in bovine vitreous humor (BVH) in a microfluidic chip device.

[0419] A solution of 25% homogenized bovine vitreous humor (BVH) was prepared by diluting 100% homogenized BVH with PBS buffer. 50 uL of BVH was dispensed into a 0.5 mL PCR tube. 50 μL of compound solution was added to the BVH to make the total concentration of BVH 12.5%. Samples were vortexed briefly and then incubated overnight at 37°C. In control experiments, 50 μL of either PBS buffer or PBS with 10% ethanol or PBS containing DMSO was prepared and incubated with 25% BVH under the same conditions.

[0420] The test BVH solution was introduced into the reservoir of the device. A fluid probe was attached to the inlet of the microfluidic chip and a flow rate of 2 ul / min was established with PBS as the source fluid. Once fluid started to exit the outlet of the chip and a steady flow of 2 ul / min was achieved, the flow rate and pressure changes within the microfluidic chip were recorded. After recording baseline flow rate and pressure readings for 5 minutes, 7 ul of the test BVH solution was injected into the chip through the sample injector. Recording of flow rate and pressure changes continued for an additional 50 minutes after sample injection. Recording was stopped after 55 minutes. The relative changes in chip pressure over the entire course of the experiment were plotted on a graph.

[0421] FIG. 25 shows that the agent cetylpyridinium chloride reduced intraocular pressure (IOP) in a glaucoma model compared to the control. The agents were tested by controlling flow and using a microfluidic device to measure relative IOP. The agents were each compared to a placebo (phosphate buffer) by preparing 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 an arrow and the letter "a." Referring to FIG. 25, the IOP of the placebo (dashed line) increased significantly after injection of the placebo sample. The IOP steadily increased to a maximum pressure of about 64 mmHg. Conversely, after injection of the agent cetylpyridinium chloride-BVH sample (solid line), it was significantly lower than that for the placebo, and the difference was sustained. The results showed that the agent cetylpyridinium chloride was surprisingly effective in reducing IOP in a glaucoma model.

[0422] Example 17 The active agent for use in the treatment of glaucoma may be polymyxin B of formula XXI, which may be used in a prodrug form or in a glaucoma-acceptable salt form.

[0423] [ka]

[0424] Solutions of drug compounds were prepared by weighing the compound in a microcentrifuge tube and dissolving the solid material in 1x PBS buffer at pH 7.2. For compounds that were poorly soluble in water, stock solutions were prepared in ethanol or DMSO and diluted 10-fold to achieve final concentrations in 10% ethanol or DMSO vehicle. Heat (37°C) and vortex mixing were applied to the compound solutions to facilitate dissolution.

[0425] The concentration of polymyxin B was 10 mg / ml.

[0426] To determine the effect of the compounds on intraocular pressure (IOP), the compounds were tested in bovine vitreous humor (BVH) in a microfluidic chip device.

[0427] A solution of 25% homogenized bovine vitreous humor (BVH) was prepared by diluting 100% homogenized BVH with PBS buffer. 50 uL of BVH was dispensed into a 0.5 mL PCR tube. 50 μL of compound solution was added to the BVH to make the total concentration of BVH 12.5%. Samples were vortexed briefly and then incubated overnight at 37°C. In control experiments, 50 μL of either PBS buffer or PBS with 10% ethanol or PBS containing DMSO was prepared and incubated with 25% BVH under the same conditions.

[0428] The test BVH solution was introduced into the reservoir of the device. A fluid probe was attached to the inlet of the microfluidic chip and a flow rate of 2 ul / min was established with PBS as the source fluid. Once fluid started to exit the outlet of the chip and a steady flow of 2 ul / min was achieved, the flow rate and pressure changes within the microfluidic chip were recorded. After recording baseline flow rate and pressure readings for 5 minutes, 7 ul of the test BVH solution was injected into the chip through the sample injector. Recording of flow rate and pressure changes continued for an additional 50 minutes after sample injection. Recording was stopped after 55 minutes. The relative changes in chip pressure over the entire course of the experiment were plotted on a graph.

[0429] FIG. 26 shows that the agent polymyxin B reduced intraocular pressure (IOP) in a glaucoma model compared to the control. The agents were tested by controlling flow and using a microfluidic device to measure relative IOP. The agents were each compared to a placebo (phosphate buffer) by preparing 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 an arrow and the letter "a." Referring to FIG. 26, the IOP of the placebo (dashed line) increased significantly after injection of the placebo sample. The IOP steadily increased to a maximum pressure of about 250 mmHg. Conversely, after injection of the agent polymyxin B sample (solid line), it was significantly lower than that for the placebo, and the difference was sustained. The results showed that the agent polymyxin B was surprisingly effective in reducing IOP in a glaucoma model.

[0430] Example 18 The active agent for use in treating glaucoma may be neomycin of formula XI, which may be used in a prodrug form or in a glaucoma-acceptable salt form.

[0431] [ka]

[0432] Solutions of drug compounds were prepared by weighing the compound in a microcentrifuge tube and dissolving the solid material in 1x PBS buffer at pH 7.2. For compounds that were poorly soluble in water, stock solutions were prepared in ethanol or DMSO and diluted 10-fold to achieve final concentrations in 10% ethanol or DMSO vehicle. Heat (37°C) and vortex mixing were applied to the compound solutions to facilitate dissolution.

[0433] The concentration of neomycin was 35 mg / ml.

[0434] To determine the effect of the compounds on intraocular pressure (IOP), the compounds were tested in bovine vitreous humor (BVH) in a microfluidic chip device.

[0435] A solution of 25% homogenized bovine vitreous humor (BVH) was prepared by diluting 100% homogenized BVH with PBS buffer. 50 uL of BVH was dispensed into a 0.5 mL PCR tube. 50 μL of compound solution was added to the BVH to make the total concentration of BVH 12.5%. Samples were vortexed briefly and then incubated overnight at 37°C. In control experiments, 50 μL of either PBS buffer or PBS with 10% ethanol or PBS containing DMSO was prepared and incubated with 25% BVH under the same conditions.

[0436] The test BVH solution was introduced into the reservoir of the device. A fluid probe was attached to the inlet of the microfluidic chip and a flow rate of 2 ul / min was established with PBS as the source fluid. Once fluid started to exit the outlet of the chip and a steady flow of 2 ul / min was achieved, the flow rate and pressure changes within the microfluidic chip were recorded. After recording baseline flow rate and pressure readings for 5 minutes, 7 ul of the test BVH solution was injected into the chip through the sample injector. Recording of flow rate and pressure changes continued for an additional 50 minutes after sample injection. Recording was stopped after 55 minutes. The relative changes in chip pressure over the entire course of the experiment were plotted on a graph.

[0437] FIG. 27 shows that the agent neomycin reduced intraocular pressure (IOP) in a glaucoma model compared to the control. The agents were tested by controlling the flow and using a microfluidic device to measure the relative IOP. The agents were each compared to a placebo (phosphate buffer) by preparing 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 an arrow and the letter "a." Referring to FIG. 27, the IOP of the placebo (dashed line) increased significantly after injection of the placebo sample. The IOP steadily increased to a maximum pressure of about 64 mmHg. Conversely, after injection of the agent neomycin (solid line), it was 72% lower than that for the placebo, and the difference was sustained. The results showed that the agent neomycin was surprisingly effective in reducing IOP in a glaucoma model.

[0438] Example 19 The active agent for use in treating glaucoma may be heparin, which may be used in a prodrug form or in a glaucoma-acceptable salt form.

[0439] Solutions of drug compounds were prepared by weighing the compound in a microcentrifuge tube and dissolving the solid material in 1x PBS buffer at pH 7.2. For compounds that were poorly soluble in water, stock solutions were prepared in ethanol or DMSO and diluted 10-fold to achieve final concentrations in 10% ethanol or DMSO vehicle. Heat (37°C) and vortex mixing were applied to the compound solutions to facilitate dissolution.

[0440] The concentration of heparin was 10 mg / ml.

[0441] To determine the effect of the compounds on intraocular pressure (IOP), the compounds were tested in bovine vitreous humor (BVH) in a microfluidic chip device.

[0442] A solution of 25% homogenized bovine vitreous humor (BVH) was prepared by diluting 100% homogenized BVH with PBS buffer. 50 uL of BVH was dispensed into a 0.5 mL PCR tube. 50 μL of compound solution was added to the BVH to make the total concentration of BVH 12.5%. Samples were vortexed briefly and then incubated overnight at 37°C. In control experiments, 50 μL of either PBS buffer or PBS with 10% ethanol or PBS containing DMSO was prepared and incubated with 25% BVH under the same conditions.

[0443] The test BVH solution was introduced into the reservoir of the device. A fluid probe was attached to the inlet of the microfluidic chip and a flow rate of 2 ul / min was established with PBS as the source fluid. Once fluid started to exit the outlet of the chip and a steady flow of 2 ul / min was achieved, the flow rate and pressure changes within the microfluidic chip were recorded. After recording baseline flow rate and pressure readings for 5 minutes, 7 ul of the test BVH solution was injected into the chip through the sample injector. Recording of flow rate and pressure changes continued for an additional 50 minutes after sample injection. Recording was stopped after 55 minutes. The relative changes in chip pressure over the entire course of the experiment were plotted on a graph.

[0444] FIG. 28 shows that the agent heparin chloride reduced intraocular pressure (IOP) in a glaucoma model compared to the control. The agents were tested by controlling flow and using a microfluidic device to measure relative IOP. The agents were each compared to a placebo (phosphate buffer) by preparing 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 an arrow and the letter "a." Referring to FIG. 28, the IOP of the placebo (dashed line) increased significantly after injection of the placebo sample. The IOP steadily increased to a maximum pressure of about 67 mmHg. Conversely, after injection of the agent heparin chloride (solid line), it was 32% lower than that for the placebo, and the difference was sustained. The results showed that the agent heparin chloride was surprisingly effective in reducing IOP in a glaucoma model.

[0445] Example 20 Sodium dodecyl sulfate was the negative control for intraocular pressure (IOP) in glaucoma.

[0446] Solutions were prepared by weighing the compounds in microcentrifuge tubes and dissolving the solid material in 1x PBS buffer at pH 7.2. For compounds that were poorly soluble in water, stock solutions were prepared in ethanol or DMSO and diluted 10-fold to achieve final concentrations in 10% ethanol or DMSO vehicle. Heat (37°C) and vortex mixing were applied to the compound solutions to facilitate dissolution.

[0447] The concentration of sodium dodecyl sulfate was 24 mg / ml.

[0448] To determine the effect of the compounds on intraocular pressure (IOP), the compounds were tested in bovine vitreous humor (BVH) in a microfluidic chip device.

[0449] A solution of 25% homogenized bovine vitreous humor (BVH) was prepared by diluting 100% homogenized BVH with PBS buffer. 50 uL of BVH was dispensed into a 0.5 mL PCR tube. 50 μL of compound solution was added to the BVH to make the total concentration of BVH 12.5%. Samples were vortexed briefly and then incubated overnight at 37°C. In control experiments, 50 μL of either PBS buffer or PBS with 10% ethanol or PBS containing DMSO was prepared and incubated with 25% BVH under the same conditions.

[0450] The test BVH solution was introduced into the reservoir of the device. A fluid probe was attached to the inlet of the microfluidic chip and a flow rate of 2 ul / min was established with PBS as the source fluid. Once fluid started to exit the outlet of the chip and a steady flow of 2 ul / min was achieved, the flow rate and pressure changes within the microfluidic chip were recorded. After recording baseline flow rate and pressure readings for 5 minutes, 7 ul of the test BVH solution was injected into the chip through the sample injector. Recording of flow rate and pressure changes continued for an additional 50 minutes after sample injection. Recording was stopped after 55 minutes. The relative changes in chip pressure over the entire course of the experiment were plotted on a graph.

[0451] FIG. 29 shows that the agent sodium dodecyl sulfate reduced intraocular pressure (IOP) in a glaucoma model compared to the control. The agents were tested by controlling the flow and using a microfluidic device to measure the relative IOP. The agents were each compared to a placebo (phosphate buffer) by preparing 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 an arrow and the letter "a." Referring to FIG. 29, the IOP of the placebo (dashed line) increased significantly after injection of the placebo sample. The IOP steadily increased to a maximum pressure of about 60 mmHg. In contrast, after injection of the agent sodium dodecyl sulfate (solid line), it was significantly higher than that for the placebo. This result indicated that the agent sodium dodecyl sulfate is a negative control that does not reduce IOP in a glaucoma model.

[0452] In general, intraocular administration may be performed by intracameral, intravitreal, or subretinal administration. Periocular administration may be performed by subconjunctival, sub-Tenon, direct intraocular, or depot periocular injection. Systemic administration may be achieved by intravenous, oral, intra-arterial, inhalation, intranasal, intraperitoneal, intraabdominal, subcutaneous, intra-articular, intrathecal, transdural, transdermal, submucosal, sublingual, enteral, parenteral, percutaneous, periarticular, or intraventricular administration.

[0453] Ophthalmic formulations may be delivered locally by eye drops, by direct injection, or by use of an infusion pump. Intraocular administration may be performed by intracameral, intravitreal, or subretinal administration.

Claims

1. An aqueous pharmaceutical composition for ophthalmic use, comprising an active agent selected from cetylpyridinium chloride, polymyxin B sulfate, mycin, and heparin sodium.

2. 2. The composition of claim 1, wherein the mycin is selected from neomycin, salinomycin, azithromycin, rapamycin, gentamicin, erythromycin, adriamycin, bleomycin, dactinomycin, mitomycin, plicamycin, dihydrostreptomycin, kanamycin, natamycin, rifamycin, and tobramycin.

3. The composition of claim 1, wherein the active agent comprises 0.01-2% w / v of the composition.

4. The composition of claim 1, wherein the active agent comprises 0.01-0.2% w / v of the composition.

5. 10. The composition of claim 1 having a pH of about 7.

3.

6. The composition of claim 1, which when administered to the eye reduces intraocular pressure.

7. The composition of claim 1, which reduces extracellular vesicle complexes in the eye when administered to the eye or systemically.

8. The composition of claim 1, which is effective to treat glaucoma or ocular hypertension when administered ocularly or systemically.

9. 10. The composition of claim 1, further comprising one or more of a solubilizing agent, a surfactant, a tonicity agent, and a preservative.

10. 2. The composition of claim 1, wherein the solubilizing agent is selected from phosphates, citric acid monohydrate, trisodium citrate, and combinations thereof.

11. 2. The composition of claim 1, wherein the surfactant is selected from phospholipids, polyglycerol esters, polyethylene glycol esters, polyethylene glycol esters, copolymer esters, polyoxyethylene sorbitan esters, cyclodextrins, polyvinyl alcohols, povidone, hydroxypropyl methylcellulose, poloxamers, carboxymethylcellulose, hydroxyethylcellulose, polyacrylates, and combinations thereof.

12. 2. The composition of claim 1, wherein the tonicity agent is selected from sodium chloride, trehalose, mannitol, sorbitol, dextrose, potassium chloride, and combinations thereof.

13. 2. The composition of claim 1, wherein the preservative is selected from benzalkonium chloride, polyquaternium-1, benzododecinium bromide, sorbic acid, methylparaben, propylparaben, chlorobutanol, benzyl alcohol, phenylethyl alcohol, oxychloro complexes, thimerosal, sodium perborate, disodium edetate, and combinations thereof.

14. A composition according to any one of claims 1 to 13 for use in medical therapy.

15. A composition according to any one of claims 1 to 13 for use in the treatment of the human or animal body.

16. The composition according to any one of claims 1 to 13, which reduces intraocular pressure in the human or animal body.

17. A composition according to any one of claims 1 to 13 for use in reducing ocular extracellular vesicle complexes in the human or animal body.

18. 14. The composition of any one of claims 1 to 13 for use in preparing or manufacturing a medicament for preventing, ameliorating, or treating a disease or condition related to glaucoma in a subject in need thereof.

19. A method of treating glaucoma disease, reducing intraocular pressure, or reducing ocular extracellular vesicle complexes in a subject in need thereof, comprising administering a composition according to any one of claims 1 to 13.

20. 20. The method of claim 19, wherein administration is by injection, and wherein intraocular administration is intracameral, intravitreal, or subretinal administration, and periocular administration is subconjunctival, sub-Tenon, direct intraocular, or depot periocular injection.

21. 20. The method of claim 19, wherein the administration is systemic, performed by intravenous, oral, intra-arterial, inhalation, intranasal, intraperitoneal, intraabdominal, subcutaneous, intra-articular, intrathecal, transdural, transdermal, submucosal, sublingual, enteral, parenteral, percutaneous, periarticular, or intraventricular administration.

22. 20. The method of claim 19, wherein the ocular extracellular vesicle complexes are aggregates of extracellular vesicles having diameters greater than about 300 nanometers.

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