A multi-functional solid nanoparticle system for treating ocular disease

Abstract

Compositions and methods are provided for glaucoma or ocular hypertension therapy. The compositions of the disclosure include solid drug nanoparticles comprised of at least two active agents: (i) a neuroprotectant, and (ii) a β adrenergic blocker. In some embodiments the neuroprotectant is maprotiline. In some embodiments the β adrenergic blocker is betaxolol. The combination of agents in nanoparticle form is shown to have synergistic benefits in the treatment of ocular disease.

Description

ATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056A MULTI-FUNCTIONAL SOLID NANOPARTICLE SYSTEM FOR TREATING OCULAR DISEASE

[0001] This application claims the benefit of U.S. Provisional Patent Application No.63 / 778,240, filed March 26, 2025, the disclosures of which application is incorporated herein by reference in their entirety.GOVERNMENT SUPPORT RESEARCH

[0002] This invention was made with Government support under contracts EY023295 and EY035088 awarded by the National Institutes of Health. The Government has certain rights in the invention.BACKGROUND

[0003] Glaucoma is a leading cause of irreversible blindness worldwide, characterized by elevated intraocular pressure (IOP) as well as the progressive loss of retinal ganglion cells (RGCs) and optic nerve damage. It affects over 76 million people as of 2020, with projections rising to 111.8 million by 2040 due to the increase in aging populations. Key risk factors of glaucoma include age, IOP, genetic predisposition, myopia, and ethnicity, with subtypes such as open-angle and angle-closure glaucoma differing in prevalence and severity. Elevated IOP is by far the only modifiable risk factor in the clinic. However, IOP reduction alone is insufficient to halt disease progression. A significant proportion of glaucoma patients still experience progressive damage to RGCs and the optic nerve even following lOP-lowering treatments (including topical medications, laser therapy, or surgery). Identifying therapeutics capable of providing neuroprotection in glaucoma therapy and incorporating them into formulations that simultaneously target IOP reduction and neuroprotection is a promising strategy but remains an unmet need in current glaucoma treatment approaches.

[0004] Often, drugs with different modes of action are combined and topically applied to obtain better control over IOP, such as Combigan (brimonidine / timolol), Cosopt (dorzolamide / timolol), and Simbrinza (brinzolamide / brimonidine), commonly prescribed as fixed combination antiglaucoma medications. However, this combination therapy is limited to the delivery of lOP-lowering drugs to the anterior segment via topical administration. To date, no topical combination therapy has been developed to target both the anterior and posterior segments. The topical route has only had limited success in delivering neuroprotective agents to the posterior segment (e.g., retina and optic nerve) due to transport challenges posed byATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056the ocular barriers (tear film barrier, corneal barrier, blood-aqueous barrier, vitreal barrier, blood-retina barrier).

[0005] Several FDA-approved drugs have demonstrated neuroprotective benefits that preserve RGCs and can potentially be repurposed for glaucoma treatment. To obtain desired benefits, they are often delivered into the eye via intravitreal injections, which is not ideal for long-term glaucoma medication due to the burden on patients and the healthcare system. In contrast, topical administration is still deemed the most convenient route of administration for glaucoma medication

[0006] Therefore, there is a strong need for novel ocular drug formulations that can enhance bioavailability, improve therapeutic outcomes, and reduce the dosing frequency to alleviate the burden on patients.SUMMARY

[0007] Compositions and methods are provided for treatment of ocular disease. The disease may be characterized by increased intraocular pressure. The disease may be glaucoma or ocular hypertension. The compositions of the disclosure include solid drug nanoparticles comprised of at least two active agents: (i) a neuroprotectant, and (ii) a p adrenergic blocker. In some embodiments the neuroprotectant is maprotiline. In some embodiments the adrenergic blocker is betaxolol. In some embodiments the active agents are present in an unionized, “free” form. The nanoparticles are uniform in size. The combination of agents in nanoparticle form is shown to have synergistic benefits in the treatment of ocular disease relative to treatment with a soluble form of the agents.

[0008] In some embodiments a dry, e.g. lyophilized, formulation of the drug nanoparticles is provided. The dry formulation may comprise an effective amount of a stabilizing agent. The stabilizing agent may be a saccharide, e.g. a disaccharide, a monosaccharide, etc. The disaccharide may be, for example and without limitation, trehalose or an analog thereof. The dry formulation may be provided in a unit dose effective for treatment of ocular disease. In some embodiments a liquid formulation of the nanoparticles is provided. The liquid formulation may comprise the dry formulation, reconstituted in an aqueous buffer, e.g. an ophthalmically acceptable diluent. The liquid formulation may be provided in a device suitable for dispensing to the eye, e.g. dispenser, pipette, dropper, etc.

[0009] In an embodiment, methods are provided for the treatment of ocular disease, e.g.glaucoma or ocular hypertension, comprising administering an effective dose of solid drug nanoparticles of the disclosure. The nanoparticles may be administered as eyedrops. The eyedrops may comprise a lyophilized formulation of the nanoparticles, reconstituted inATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056aqueous buffer. The nanoparticle eyedrops are shown herein to have a number of benefits relative to delivery ofthe component active agents in solution, which benefits include improved stability, ocular biocompatibility, tissue permeability, sustained IOP lowering and neuroprotection efficacy. The co-formulation the two agents improves the delivery of the neuroprotectant to the posterior area of the eye, thereby enhancing its efficacy.

[0010] In an embodiment, a method is provided for manufacturing uniform solid drug nanoparticles comprising (i) a neuroprotectant, e.g. maprotiline and (ii) a p-1 adrenergic blocker, e.g. betaxolol. In an embodiment, each of the active agents is dissolved in polar diluent, e.g. methanol. A stream of the active agents is mixed with an aqueous stream, e.g. in a vortex mixer, to produce uniform nanoparticles. In some embodiments the streams are mixed in a multi-inlet vortex mixer (MIVM). The nanoparticles thus formed are collected, and the diluents removed. In some embodiments the nanoparticles are admixed with an effective amount of a stabilizing agent. The stabilizing agent may be a disaccharide. The disaccharide may be, for example and without limitation, trehalose or an analog thereof. The admixture is then lyophilized, and may be stored. For use, the dry formulation is reconstituted with an aqueous buffer.

[0011] In some embodiments a kit is provided, comprising the compositions of the invention, and optionally instructions for use. A kit may comprise nanoparticles of the disclosure. The nanoparticles may be provided in a dry formulation, optionally in a unit dose. The unit dose may be provided in a device suitable for reconstitution and application. The kit can further contain a least one additional reagent, e.g. a suitable diluent. Kits may comprise an applicator device, e.g. pipette, eye-dropper, etc. Kits typically include a label indicating the intended use of the contents of the kit. The term label includes any writing, or recorded material supplied on or with the kit, or which otherwise accompanies the kit.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures.

[0013] FIGS. 1A-1 F. Preparation, cryoprotectant percentage optimization, and characterization of MAP / BX NP. (A) Scalable preparation process of MAP / BX NP. (B) Consistent size distribution of different batches. (C, D) Size distribution with different percentage of trehalose before and after freeze-drying. (E, F) Transmission electronATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056microscopy (TEM) images with insets displaying a representative single particle and size distributions of MAP / BX NP before and after freeze-drying.

[0014] FIGS. 2A-2G. Stability, in vitro drug release, and ex vivo corneal permeation of MAP / BX NP. (A, B) The MAP / BX NP appearance after freeze-drying and reconstitution in PBS. (C) Variation of hydrodynamic size and polydispersity index (PDI) of the reconstituted MAP / BX NP in PBS at 4 °C. (D, E) In vitro drug release profile of MAP / BX NP and the size change during the drug release process (F, G) Ex vivo cornea permeation profile of MAP / BX NP and the cumulative mass transport curve during permeation. The data were expressed as mean ± standard deviation (n = 3).

[0015] FIGS. 3A-3E. In vitro evaluation of cytotoxicity and ex vivo evaluation of irritation of MAP / BX NP. (A) HCE-2 cytotoxicity following 24-h incubation. The data were expressed as mean ± standard deviation (n = 6). (B) Representative photos of the chorioallantoic membrane (CAM) before and after treatment with PBS (negative control) and 0.1 M NaOH (positive control). (C, D) Representative photos of the CAM before and after treatment with different concentrations of MAP / BX free drug or MAP / BX NP. (E) Irritation evaluation table displaying different formulations, concentrations, and the irritation scores. The data were expressed as mean ± standard deviation (n = 3).

[0016] FIGS. 4A-4D. Biodistributions of MAP and BX in the eyeball after 1 daily dose and 7 successive daily dose of MAP / BX NP. (A, B) Concentrations and mass ratios of MAP and BX in different ocular tissues after 1 daily dose of MAP / BX free drug or MAP / BX NP. (C, D) Concentrations and mass ratios of MAP and BX in different ocular tissues after 7 successive daily dose of MAP / BX free drug or MAP / BX NP. The data were expressed as mean ± standard deviation (n = 5 for A and B, n = 7 for C and D). * p < 0.05, ** p < 0.01 , “* p < 0.001 , **** p < 0.0001.

[0017] FIGS. 5A-5E. High spatial resolution matrix-assisted laser desorption / ionization imaging mass spectrometry (MALDI-IMS) of MAP / BX NP in the eyeball. (A) Representative MALDI-IMS images of eyeball after MAP / BX free drug or MAP / BX NP treatment. (B, C) Summary mass spectra from MALDI-IMS images of eyeball after MAP / BX free drug or MAP / BX NP treatment. (D, E) Relative intensities of MAP and BX in different ocular areas. The data were expressed as mean ± standard deviation (n = 3). * p < 0.05, ** p < 0.01 , *** p < 0.001, ““ p < 0.0001.

[0018] FIGS. 6A-6B. In vivo evaluation of lOP-lowering effect of MAP / BX NP. (A) IOP- lowering effects of MAP / BX free drug and MAP / BX NP with single-dose in normotensive rats. (B) lOP-lowering effects of MAP / BX free drug and MAP / BX NP with multiple-dose inATTORNEY DOCKET NAME: STAN-2260WQ CLIENT REFERENCE: S25-056normotensive rats. The data were expressed as mean ± standard deviation (n = 8). * p < 0.05, “ p < 0.01 , *** p < 0.001 , **** p < 0.0001.

[0019] FIGS. 7A-7H. In vivo evaluation of neuroprotection efficacy of MAP / BX NP. (A) Schematic illustration of the timelines of the silicone oil-induced ocular hypertension (SOHU) glaucoma model development and neuroprotection evaluation. (B) Quantification of P1-N2 amplitude of pattern electroretinogram recording (PERG) at 3 weeks post initial treatment. (C) Representative optical coherence tomography (OCT) images of mouse retina in living animals. Ganglion cell complex (GCC) is indicated as double end red arrows. (D) Quantification of GCC thickness measured by OCT. (E) Representative light microscope images of semi-thin transverse sections of optic nerve with PPD staining. (F) Quantification of surviving axons in optic nerve sections. (G) Representative confocal images of flat-mounted peripheral, middle, and central retinas showing surviving RBPMS + (red) retina ganglion cells (RGCs). (H) Quantification of surviving RGC somata in flat-mounted peripheral, middle and central retinas. Violin plots display data distribution, with the dotted lines indicating the median (center) and interquartile range (top and bottom) (n > 6). * p < 0.05, “ p < 0.01 , *** p < 0.001 , **** p < 0.0001.

[0020] FIGS. 8A-8H. In vivo evaluation of biosafety of MAP / BX NP. (A) Schematic illustration of the timeline of dosing and methods used in the biosafety evaluation. (B) The representative images of corneal fluorescein staining after instillation of PBS or MAP / BX NPs for 7 days. (C) Representative optical coherence tomography (OCT) images of mouse cornea in living animals. (D) Representative OCT images of mouse retina in living animals. Ganglion cell complex (GCC) is indicated by double-ended red arrows. (E) Relative cornea and GCC thicknesses after 7-day MAP / BX NP treatment compared to PBS control. (F) Representative confocal images of flat-mounted peripheral, middle, and central retinas showing surviving RBPMS + (red) retina ganglion cells (RGCs). (G) Quantification of surviving RGC somata in flat-mounted peripheral, middle and central retinas. Violin plots display data distribution, with the dotted lines indicating the median (center) and interquartile range (top and bottom). (H) Hematoxylin and eosin (H&E) histological images of the main organs of the treated mice. No significant histopathological abnormalities were observed after 7-day MAP / BX NP treatment (n = 5).

[0021] FIG. 9. 1 H NMR spectrum of the maprotiline hydrochloride (MH) after desaltification.The1H NMR spectrum shows the desaltification of MH using TEA to obtain the free base form of MAP, without altering its chemical structure. The spectrum reveals proton peaks corresponding to the different proton environments in MAP. For example, the peaks from 1 .5 ppm to 3 ppm correspond to the protons from alkyl chain, including the methyl protons (a),ATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056methylene protons (b, c, d), and bridgehead methylene protons (g, h). The aromatic protons around 7 ppm (f) and the aromatic peri-proton around 4.25 ppm (i) appear in the expected region. All the integrations suggest the presence of the correct number of protons in different groups. The absence of new peaks or abnormal shifts support that the desaltification procedure was successful. The peak at 7.25 ppm is assigned to residual chloroform and aromatic protons at the (e) position, while the overall integration confirm that the desired product, MAP, was obtained in its free base form without alterations.

[0022] FIG. 10. The drug release profiles of MAP / BX NP fitted with five mathematical models.The data were expressed as mean ± standard deviation (n = 3).

[0023] FIG. 11. HCE-2 cytotoxicity of trehalose following 24-h incubation. The data were expressed as mean ± standard deviation (n = 6).

[0024] FIGS. 12A-12C. The IC50s of different formulations determined with four- parameter regression fitting model. (A) MAP / BX NP + Trehalose; (B) MAP / BX NP; (C) MAP / BX free drug. R2 > 0.99 for all. The data were expressed as mean + standard deviation (n = 6).

[0025] FIGS. 13A-13D. Representative rat eye images before (A, C) and after (B, D) 7 successive daily doses of MAP / BX free drug or MAP / BX NP. Neither formulation caused any irritation, such as abnormal tearing or blinking.

[0026] FIGS. 14A-14D. Biodistributions of MAP in the eyeball following topical treatment with 1 daily dose or 7 successive daily doses of MAP free drug and MAP NP, and the comparison of concentration between single and combination formulations (MAP free drug vs MAP / BX free drug, MAP NP vs MAP / BX NP). (A) Timeline of 1 daily dose experiment. (B) Concentrations of MAP in different ocular tissues after 1 daily dose. (C) Timeline of 7 successive daily dose experiment. (D) Concentrations of MAP in different ocular tissues after 7 successive daily doses. The data were expressed as mean ± standard deviation (n = 8). * p < 0.05, ** p < 0.01 , p < 0.001 , **** p < 0.0001 .

[0027] FIGS. 15. MALDI-TOF mass spectrum of CHCA matrix.

[0028] FIG. 16A-16F. In vivo evaluation of neuroprotection efficacy of BX free drug. (A) Representative OCT images of mouse retina in living animals. GCC is indicated as double end black arrows. (B) Quantification of GCC thickness measured by OCT represented as percentage of GCC thickness in the SOHU eyes compared to the sham contralateral control eyes. (C) Representative light microscope images of semi-thin transverse sections of optic nerve with PPD staining. (D) Quantification of surviving axons in optic nerve sections represented as percentage in SOHU eyes compared to the sham contralateral control eyes. (E) Representative confocal images of flat-mounted entire retina, peripheral, middle, andATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056central retinas showing surviving RBPMS + (red) RGCs. (F) Quantification of surviving RGC somata in flat-mounted peripheral, middle and central retinas represented as percentage in SOHU eyes compared to the sham contralateral control eyes. The data were expressed as mean ± standard deviation (n Y 13).

[0029] FIG. 17. Overview of method.DETAILED DESCRIPTION

[0030] Before the present methods and compositions are described, it is to be understood that this invention is not limited to particular method or composition described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0031] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0032] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. It is understood that the present disclosure supercedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0033] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "theATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056peptide" includes reference to one or more peptides and equivalents thereof, e.g. polypeptides, known to those skilled in the art, and so forth.

[0034] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0035] As used herein, compounds which are "commercially available" may be obtained from commercial sources including but not limited to Acros Organics (Pittsburgh PA), Aldrich Chemical (Milwaukee Wl, including Sigma Chemical and Fluka), Apin Chemicals Ltd. (Milton Park UK), Avocado Research (Lancashire U.K.), BDH Inc. (Toronto, Canada), Bionet (Cornwall, U.K.), Chemservice Inc. (West Chester PA), Crescent Chemical Co. (Hauppauge NY), Eastman Organic Chemicals, Eastman Kodak Company (Rochester NY), Fisher Scientific Co. (Pittsburgh PA), Fisons Chemicals (Leicestershire UK), Frontier Scientific (Logan UT), ICN Biomedicals, Inc. (Costa Mesa CA), Key Organics (Cornwall U.K.), Lancaster Synthesis (Windham NH), Maybridge Chemical Co. Ltd. (Cornwall U.K.), Parish Chemical Co. (Orem UT), Pfaltz & Bauer, Inc. (Waterbury CN), Polyorganix (Houston TX), Pierce Chemical Co. (Rockford IL), Riedel de Haen AG (Hannover, Germany), Spectrum Quality Product, Inc. (New Brunswick, NJ), TCI America (Portland OR), Trans World Chemicals, Inc. (Rockville MD), Wako Chemicals USA, Inc. (Richmond VA), Novabiochem and Argonaut Technology.

[0036] Compounds can also be made by methods known to one of ordinary skill in the art. As used herein, "methods known to one of ordinary skill in the art" may be identified though various reference books and databases. Suitable reference books and treatises that detail the synthesis of reactants useful in the preparation of compounds of the present invention, or provide references to articles that describe the preparation, include for example, "Synthetic Organic Chemistry", John Wiley & Sons, Inc., New York; S. R. Sandler et al., "Organic Functional Group Preparations," 2nd Ed., Academic Press, New York, 1983; H. O. House, "Modern Synthetic Reactions", 2nd Ed., W. A. Benjamin, Inc. Menlo Park, Calif. 1972; T. L. Gilchrist, “Heterocyclic Chemistry”, 2nd Ed., John Wiley & Sons, New York, 1992; J. March, “Advanced Organic Chemistry: Reactions, Mechanisms and Structure”, 4th Ed., Wiley-lnterscience, New York, 1992. Specific and analogous reactants may also be identified through the indices of known chemicals prepared by the Chemical Abstract Service of the American Chemical Society, which are available in most public and university libraries, as well as through on-line databases (the American Chemical Society, Washington, D.C., may be contacted for more details). Chemicals that are known but not commercially available inATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056catalogs may be prepared by custom chemical synthesis houses, where many of the standard chemical supply houses (e.g., those listed above) provide custom synthesis services.

[0037] Beta-blocker. Beta-blockers are approved for the treatment of glaucoma, by reducing fluid production in the eye and lowering intraocular pressure. Beta-blockers work by blocking beta-adrenergic receptors in the ciliary body, the area of the eye that produces aqueous humor (the fluid that circulates in the eye). By blocking these receptors, beta-blockers reduce the production of aqueous humor, which in turn lowers the pressure inside the eye. Specifically, P-1 receptors are G-protein-coupled receptors that activate cyclic AMP (cAMP) through a cascade of events. Cyclic AMP activation interacts with cAMP-dependent protein kinases, increasing calcium ion concentration. This increase in calcium leads to different physiological changes depending on the location of the receptor.

[0038] Beta-blockers approved for use in treating glaucoma include selective blockers that primarily target beta-1 receptor, e.g. Betaxolol. Non-selective beta blockers block both beta-1 and beta-2 receptors, include timolol, levobunolol, metipranolol, and carteolol. Currently available formulations are a solution of the salt of the blocker, however the use in the present disclosure is preferably an un-ionized form, i.e. not a salt.

[0039] Betaxolol. In some embodiments the beta blocker present in the nanoparticles of the disclosure is Betaxolol, which is a lipophilic p-adrenergic blocker selective to the p-1 adrenergic receptor.ATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056

[0040] Betaxolol can be administered by topical (ophthalmic) or systemic (oral) routes. Topical betaxolol is FDA-approved for ocular hypertension and chronic open-angle glaucoma. Glaucoma is an optic neuropathy defined by the death of retinal ganglion cells and characteristic changes to the optic nerve head. Intraocular pressure, as well as other factors, are factors for the loss of ganglion cells. Betaxolol’s selective p-1 adrenergic blockade decreases the likelihood of systemic and respiratory adverse effects compared to nonselective p-blockers.

[0041] Conventionally, Betaxolol is administered topically as a 0.5% betaxolol hydrochloride ophthalmic solution for ocular hypertension and open-angle glaucoma. The current guidelines recommend 1 drop of 0.5% betaxolol hydrochloride ophthalmic solution twice daily in the affected eye(s). This dosing equates to approximately 28 pg / d. The onset of action in conventional betaxolol ophthalmic solution reaches its peak effect around 2 hours postadministration. A single dose usually leads to a 12-hour reduction in intraocular pressure. A stabilization in the decrease in intraocular pressure should be observed within a few weeks.

[0042] The American Academy of Ophthalmology guidelines state that there is a 20% to 25% reduction in intraocular pressure with betaxolol. Important baseline diagnostic testing includes visual field evaluation, central corneal thickness measurement, and imaging of the optic nerve head, retinal nerve fiber layer, and macula. The response to treatment is monitored during follow-up by regular evaluation of the optic nerve appearance and quantitative measurement with visual field testing and imaging of the optic nerve head, retinal nerve fiber layer, and macula.

[0043] Timolol. In some embodiments the beta blocker present in the nanoparticles of the disclosure is Timolol, which is a nonselective p-blocker that may be administered topically and orally.ATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-0568 / "

[0044] Topical timolol is conventionally prescribed to reduce intraocular pressure in patients with open-angle glaucoma and ocular hypertension. The exact mechanism of action by which timolol reduces the intraocular pressure in patients with open-angle glaucoma is unknown. Timolol is thought to inhibit p-receptors on the ciliary epithelium, which normally functions to increase the production of aqueous humor.

[0045] A conventional dose of timolol solution is an initial 1 drop of 0.25% solution instilled in the affected eye(s) 2 times a day, which may be increased to a maximum dose of 1 drop of 0.5% solution 2 times a day. Timolol maleate ophthalmic solution is systemically absorbed, with bioavailability ranging from 60% to 78%. Peak plasma concentrations (Cmax) are typically attained within 15 minutes of ocular application. The onset of action usually occurs within 15 to 30 minutes, and the maximum reduction in intraocular pressure (IOP) is typically reached within 1 to 5 hours.

[0046] Neuroprotective agent. The term “neuroprotective” as used herein refers to the ability to protect neurons or their axons or synapses in the central or peripheral nervous system from damage or death. Many different types of insult can lead to neuronal damage or death, for example: metabolic stress caused by hypoxia, hypoglycemia, diabetes, loss of ionic homeostasis or other deleterious process, physical injury of neurons, exposure to toxic agents and numerous diseases affecting the nervous system including inherited disorders. The presence of an agent that is neuroprotective enables a neuron to remain viable upon exposure to insults that would otherwise cause a loss of functional integrity in an unprotected neuron.

[0047] In some embodiments a neuroprotective agent is maprotiline:ATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056

[0048] Maprotiline is a tetracyclic antidepressant (TeCA) that is used in the treatment of depression. In terms of its chemistry and pharmacology, maprotiline is closely related to such- other secondary-amine TCAs as nortriptyline and protriptyline and has similar effects to them. It is conventionally sold as the methanesulfonate (mesylate) salt, however the use in the present disclosure is preferably an un-ionized form, i.e. not a salt.

[0049] Stabilizing agent. The solid drug nanoparticles of the disclosure may be admixed with an effective dose of a stabilizing agent prior to lyophilization. A stabilizing agent of interest may act as a cryoprotectant. Agents of interest for this purpose may include, for example, saccharides and disaccharides. Monosaccharides of use for this purpose include fructose, galactose, inositol, xylose, raffinose, glucose, mannitol, etc. Disaccharides of use for this purpose include sucrose, maltose, sorbital, trehalose and analogs thereof, etc.

[0050] In some embodiments a stabilizing agent is trehalose or an analog thereof. Trehalose (a-D-glucopyranosyl-(1 -> 1)-a-D-glucopyranoside) is a water-soluble non-reducing disaccharide made up of two glucose subunits, joined by a 1 ,1 -glycosidic bond. Trehalose is biosynthesised by a wide variety of non-mammalian organisms as an energy source and is used in some organisms to protect against freezing and desiccation, allowing them to survive over winter or in other harsh environments. Trehalose is a non-penetrating cryoprotectant. Lactotrehalose and galactotrehalose are analogs of interest that offer similar benefits to trehalose.

[0051] By "treatment" it is meant that at least an amelioration of one or more symptoms associated with a neurodegenerative disorder afflicting the subject is achieved, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g., a symptom associated with the impairment being treated. As such, treatment also includes situations where a pathological condition, or at least symptoms associatedATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056therewith, are completely inhibited, e.g., prevented from happening, or stopped, e.g., terminated, such that the adult mammal no longer suffers from the impairment, or at least the symptoms that characterize the impairment. In some instances, "treatment", "treating" and the like refer to obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. "Treatment" may be any treatment of a disease in a mammal, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; or (c) relieving the disease, i.e., causing regression of the disease. Treatment may result in a variety of different physical manifestations, e.g., lowered IOP, maintenance or rejuvenation of the optic nerve (ON), etc. Treatment of ongoing disease, where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient, occurs in some embodiments. Such treatment may be performed prior to complete loss of function in the affected tissues. The subject therapy may be administered during the symptomatic stage of the disease, and in some cases after the symptomatic stage of the disease.

[0052] Treating may refer to any indicia of success in the treatment or amelioration or prevention of disease, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating. The treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of an examination by a physician. Accordingly, the term "treating" includes the administration of the compounds or agents of the present invention to prevent or delay, to alleviate, or to arrest or inhibit development of the symptoms or conditions associated with glaucoma. The term "therapeutic effect" refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in the subject.

[0053] The terms “recipient,” “individual,” “subject,” “host,” and “patient” are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans. "Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, camels, etc. In some embodiments, the mammal is human.ATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056

[0054] "Suitable conditions" shall have a meaning dependent on the context in which this term is used. That is, when used in connection with an antibody, the term shall mean conditions that permit an antibody to bind to its corresponding antigen. When this term is used in connection with nucleic acid hybridization, the term shall mean conditions that permit a nucleic acid of at least 15 nucleotides in length to hybridize to a nucleic acid having a sequence complementary thereto. When used in connection with contacting an agent to a cell, this term shall mean conditions that permit an agent capable of doing so to enter a cell and perform its intended function. In one embodiment, the term "suitable conditions" as used herein means physiological conditions.

[0055] The terms “co-administration” and “in combination with” include the administration of two or more therapeutic agents either simultaneously, concurrently or sequentially within no specific time limits. In one embodiment, the agents are present in the cell or in the subject's body at the same time or exert their biological or therapeutic effect at the same time. In one embodiment, the therapeutic agents are in the same composition or unit dosage form. In other embodiments, the therapeutic agents are in separate compositions or unit dosage forms. In certain embodiments, a first agent can be administered prior to (e.g., minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks) after the administration of a second therapeutic agent.

[0056] "In combination with", "combination therapy" and "combination products" refer, in certain embodiments, to the concurrent administration to a patient of a first active agent and a second active agent. When administered in combination, each component can be administered at the same time or sequentially in any order at different points in time.

[0057] "Concomitant administration" of a known therapeutic agent with a pharmaceutical composition of the present invention means administration of of a first active agent and a second active agent at such time that both will have a therapeutic effect. Such concomitant administration may involve concurrent (i.e. at the same time), prior, or subsequent administration of the drug with respect to the administration of a compound of the present invention. A person of ordinary skill in the art would have no difficulty determining theATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056appropriate timing, sequence and dosages of administration for particular drugs and compositions of the present invention.

[0058] As used herein, the term “correlates,” or “correlates with,” and like terms, refers to a statistical association between instances of two events, where events include numbers, data sets, and the like. For example, when the events involve numbers, a positive correlation (also referred to herein as a “direct correlation”) means that as one increases, the other increases as well. A negative correlation (also referred to herein as an “inverse correlation”) means that as one increases, the other decreases.

[0059] "Dosage unit" refers to physically discrete units suited as unitary dosages for the particular individual to be treated. Each unit can contain a predetermined quantity of active compound(s) calculated to produce the desired therapeutic effect(s) in association with the required pharmaceutical carrier. The specification for the dosage unit forms can be dictated by (a) the unique characteristics of the active compound(s) and the particular therapeutic effect(s) to be achieved, and (b) the limitations inherent in the art of compounding such active compound(s).

[0060] "Pharmaceutically acceptable excipient "means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients can be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.

[0061] The terms "pharmaceutically acceptable", "physiologically tolerable" and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a human without the production of undesirable physiological effects to a degree that would prohibit administration of the composition.

[0062] A "therapeutically effective amount" means the amount that, when administered to a subject for treating a disease, is sufficient to effect treatment for that disease.

[0063] The phrase “determining the treatment efficacy” and variants thereof can include any methods for determining that a treatment is providing a benefit to a subject. The term “treatment efficacy” and variants thereof are generally indicated by alleviation of one or more signs or symptoms associated with the disease and can be readily determined by one skilled in the art. “Treatment efficacy” may also refer to the prevention or amelioration of signs and symptoms of toxicities typically associated with standard or non-standard treatments of a disease. Determination of treatment efficacy is usually indication and disease specific and canATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056include any methods known or available in the art for determining that a treatment is providing a beneficial effect to a patient. For example, evidence of treatment efficacy can include but is not limited to remission of the disease or indication. Further, treatment efficacy can also include general improvements in the overall health of the subject, such as but not limited to enhancement of patient life quality, increase in predicted subject survival rate, decrease in depression or decrease in rate of recurrence of the indication (increase in remission time). (See, e.g., Physicians' Desk Reference (2010).)

[0064] Glaucomas are a group of eye disorders characterized by progressive optic nerve damage in which an important part is a relative increase in intraocular pressure (IOP) that can lead to irreversible loss of vision. Glaucomas are categorized as open-angle glaucoma or angle-closure glaucoma. The “angle” refers to the angle formed by the junction of the iris and cornea at the periphery of the anterior chamber. The angle is where > 98% of the aqueous humor exits the eye via either the trabecular meshwork and the Schlemm canal or the ciliary body face and choroidal vasculature. Glaucomas are further subdivided into primary (cause of outflow resistance or angle closure is unknown) and secondary (outflow resistance results from a known disorder), accounting for > 20 adult types. Another group of glaucoma patients does not have IOP elevation, which in general is called normal tension glaucoma (NTG). NTG is also associated with progressive optic nerve degeneration and RGC death.

[0065] Axons of retinal ganglion cells travel through the optic nerve carrying visual information from the eye to the brain. Damage to these axons causes ganglion cell death with resultant optic nerve atrophy and patchy vision loss. Elevated intraocular pressure (IOP; in unaffected eyes, the average range is 11 to 21 mm Hg) plays a role in axonal damage, either by direct nerve compression or diminution of blood flow. However, the relationship between externally measured pressure and nerve damage is complicated. Of people with IOP > 21 mm Hg (ie, ocular hypertension), only about 1 to 2% / year (about 10% over 5 years) develop glaucoma. Additionally, about one third of patients with glaucoma do not have IOP > 21 mm Hg (known as low-tension glaucoma or normal-tension glaucoma).

[0066] IOP is determined by the balance of aqueous secretion and drainage. Elevated IOP is caused by inhibited or obstructed outflow, not oversecretion; a combination of factors in the trabecular meshwork (eg, dysregulation of extracellular matrix, cytoskeletal abnormalities) appear to be involved. In open-angle glaucoma, IOP is elevated because outflow is inadequate despite an angle that appears unobstructed. In angle-closure glaucoma, IOP is elevated when a physical distortion of the peripheral iris mechanically blocks outflow.ATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056

[0067] Symptoms and signs of glaucoma vary with the type of glaucoma, but the defining characteristic is optic nerve damage as evidenced by an abnormal optic disk and certain types of visual field deficits. Glaucoma is diagnosed when characteristic findings of optic nerve damage are present and other causes have been excluded. Elevated IOP makes the diagnosis more likely, but elevated IOP can occur in the absence of glaucoma and is not essential for making the diagnosis.

[0068] Ocular hypertension is a condition that occurs when pressure within the eye increases without affecting a person’s vision or damaging their eye anatomy. It arises when fluid doesn’t drain out of the eye at its regular rate, resulting in a buildup that increases intraocular (within the eye) pressure. Ocular hypertension may be an early sign of glaucoma, an eye disease that can cause irreversible vision loss over time if it is not treated. Intraocular pressure levels greater than 21 mm Hg confirm a diagnosis of ocular hypertension. Hypertension may be measured, for example, by tonometry, goniscopy, etc.

[0069] Older adults are more likely than those under age 40 to have ocular hypertension, which is more likely to develop as people age. An estimated 4% to 10% of Americans aged 40 and older have ocular hypertension.

[0070] Treatment of ocular hypertension can reduce the risk of developing glaucoma. For example, incidence of glaucomatous damage in people with ocular hypertension is about 2.6- 3% for intraocular pressures of 21-25 mmHg, 12-26% for intraocular pressures of 26-30 mmHg, and approximately 42% for those higher than 30 mmHg. Individuals with intraocular pressure of greater than 21 mmHg, greater than 25 mmHg, greater 30 mmHg may be selected for treatment with the compositions of the disclosure.Compositions

[0071] A pharmaceutical composition comprising solid drug nanoparticles is provided. The compositions include solid drug nanoparticles comprised of at least two active agents: (i) a neuroprotectant, and (ii) a p-1 adrenergic blocker. In some embodiments the neuroprotectant is maprotiline. In some embodiments the p-1 adrenergic blocker is betaxolol.

[0072] In some embodiments the active agents are present in the un-ionized form, i.e. the agents are in a free form, not a salt.

[0073] The ratio of : (i) a neuroprotectant, and (ii) a p-1 adrenergic blocker is pre-determined, and may be (mass / mass) a ratio of about 1 :20; 1 :15; 1 :12.5; 1 :10; 1 :7.5; 1 :5; 1 :2.5; 1 :1 ; 2.5:1 ; 5:1 ; 7.5:1 ; 10:1 ; 12.5:1 ; 15:1 ; 20:1, etc.ATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056

[0074] In some embodiments the average hydrodynamic diameter of the nanoparticles is at least about 50 nm, at least about 100 nm, at least about 125 nm, at least about 150 nm, at least about 175 nm, at least about 200 nm, at least about 225 nm, at least about 250 nm. In some embodiments the average hydrodynamic diameter of the nanoparticles is not more than about 1000 nm, not more than about 900 nm, not more than about 800 nm, not more than about 700 nm, not more than about 600 nm, not more than about 500 nm, not more than about 400 nm, not more than about 300 nm, not more than about 250 nm. For example, the population of nanoparticles may have an average hydrodynamic diameter of from about 50 to about 500 nm; of from about 100 to about 300 nm; of from about 150 to about 250 nm.

[0075] The population of nanoparticles may be relatively uniform in size. The uniformity helps ensure consistent drug release and stability, as well as controlling the properties and performance of the nanoparticles. Uniformity may be expressed, for example, by Polydispersity Index (PDI), which is a parameter that describes the width or spread of particle size distribution. It is calculated using the square of the standard deviation divided by the mean particle size. In some embodiments the PDI of a population of solid drug nanoparticles of the disclosure is less than about 0.5, less than about 0.4, less than about 0.2, less than about 0.15, less than about 0.125, less than about 0.11.

[0076] In a dry formulation, the nanoparticles may be admixed with a stabilizing agent, e.g.trehalose or an equivalent. The dry formulation may be lyophilized. The dry formulation may be packaged for use, e.g. in a sterile container suitable for reconstitution. An effective amount of the stabilizing agent may be at least about 0.25% (mass / volume) of the volume of nanoparticles, at least about 0.5%, at least about 0.75%, at least about 1%, at least about 1 .25%, at least about 1.5%, at least about 2%, at least about 2.5%, at least about 3%, and may be less than about 5%, less than about 4.5%, less than about 4%, less than about 3.5%. In some embodiments the amount is from about 0.5% to about 2.5%, from about 0.5% to about 1 .5%, and may be around about 1%.

[0077] The dry formulation may be reconstituted with an aqueous diluent for topical administration to the eye. Any suitable aqueous diluent may be used, for example normal saline (0.9% NaCI), phosphate-buffered saline, a balanced salt solution, etc. For example a balance salt solution may be a sterile balanced salt solution, each mL containing sodium chloride (NaCI) 0.64%, potassium chloride (KCI) 0.075%, calcium chloride dihydrate (CaCI2'2H2O) 0.048%, magnesium chloride hexahydrate (MgCI26-H2O) 0.03%, sodium acetate trihydrate (C2H3NaO2-3H2O) 0.39%, sodium citrate dihydrate (C6H5Na3O7-2H2O) 0.17%, sodium hydroxide and / or hydrochloric acid (to adjust pH), and water for injection. The pH is approximately 7.5. The osmolality is approximately 300 mOsm / Kg.ATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056

[0078] The reconstituted formulation may comprise at least about 0.5% nanoparticles (w / v);at least about 0.75% nanoparticles (w / v); at least about 0.1% nanoparticles (w / v); at least about 0.15% nanoparticles (w / v); at least about 0.2% nanoparticles (w / v); at least about 0.25% nanoparticles (w / v); at least about 0.3% nanoparticles (w / v); at least about 0.35 nanoparticles (w / v); at least about 0.4% nanoparticles (w / v); at least about 0.45% nanoparticles (w / v); at least about 0.5% nanoparticles (w / v); at least about 0.6% nanoparticles (w / v); at least about 0.7% nanoparticles (w / v); at least about 0.8% nanoparticles (w / v); at least about 0.9% nanoparticles (w / v); at least about 1% nanoparticles (w / v); at least about 1 .5% nanoparticles (w / v); at least about 2% nanoparticles (w / v); at least about 2.5% nanoparticles (w / v); and up about 10% nanoparticles, up to about 7.5%, up to about 5%, up to about 2.5%.

[0079] A typical dose is from 1 to 2 drops per eye, where a drop may be from about 5 mL;from about 10 mL; from about 15 mL; from about 20 mL; from about 25 mL; from about 30 mL; up to about 50 mL; up to about 40 mL.

[0080] The effective dose of the active agents may be at least about 0.1 mg / eye; at least about 0.25 mg / eye; at least about 0.5 mg / eye; at least about 0.75 mg / eye; at least about 1 mg / eye; at least about 1.5 mg / eye; at least about 2 mg / eye; at least about 2.5 mg / eye; at least about 3 mg / eye; at least about 3.5 mg / eye; at least about 4 mg / eye; at least about 4.5 mg / eye; at least about 5 mg / eye; and up to about 50 mg / eye; and up to about 30 mg / eye; and up to about 25 mg / eye; and up to about 20 mg / eye; and up to about 25 mg / eye; and up to about 15 mg / eye; and up to about 10 mg / eye.

[0081] Formulations for pharmaceutical compositions are well known in the art. For example, Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 19th Edition, 1995, describes exemplary formulations (and components thereof) suitable for pharmaceutical delivery of disclosed compounds. Pharmaceutical compositions comprising at least one of the subject compounds can be formulated for use in human or veterinary medicine. Particular formulations of a disclosed pharmaceutical composition may depend, for example, on the mode of administration and / or on the location of the infection to be treated. In some embodiments, formulations include a pharmaceutically acceptable carrier in addition to at least one active ingredient, such as a subject compound. In other embodiments, other medicinal or pharmaceutical agents, for example, with similar, related or complementary effects on the affliction being treated can also be included as active ingredients in a pharmaceutical composition.

[0082] The term "unit dosage form," as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of a subject compound calculated in an amount sufficient to produce the desired effectATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for a subject compound depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

[0083] Certain embodiments of the pharmaceutical compositions comprising a subject compound may be formulated in unit dosage form suitable for individual administration of precise dosages. The amount of active ingredient administered will depend on the subject being treated, the severity of the affliction, and the manner of administration, and is known to those skilled in the art. Within these bounds, the formulation to be administered will contain a quantity of the extracts or compounds disclosed herein in an amount effective to achieve the desired effect in the subject being treated.Methods of manufacture

[0084] The solid drug nanoparticles of the disclosure are preferably formed by a process that provides for uniform particle size. In an embodiment, a method is provided for manufacturing uniform solid drug nanoparticles comprising (i) a neuroprotectant, e.g. maprotiline and (ii) a 0- 1 adrenergic blocker, e.g. betaxolol. In an embodiment, each of the active agents is dissolved in polar diluent, e.g. methanol. Each active agent may be separately or jointly dissolved at a concentration of from about 0.1 mg / mL; from about 0.25 mg / mL; from about 0.5 mg / mL; from about 0.75 mg / mL; from about 1 mg / mL; from about 1.5 mg / mL; from about 2 mg / mL; from about 2.5 mg / mL; from about 5 mg / mL; from about; from about 7.5 mg / mL; from about 10 mg / mL; from about 20 mg / mL; from about 30 mg / mL; from about 40 mg / mL; from about 50 mg / mL; up to about 100 mg / mL, up to about 75 mg / mL, up to about 50 mg / mL. The two active agents are combined or jointly dissolved at a pre-determined ratio.

[0085] A stream containing the solution of active agents is mixed with an aqueous stream, such as ultra-pure water, etc. e.g. in a vortex mixer, to produce uniform nanoparticles. In some embodiments the streams are mixed in a multi-inlet vortex mixer (MIVM).

[0086] A four-stream MIVM allows control of both the supersaturation and the final solvent quality by varying stream velocities. The design also enables the separation of reactive components prior to mixing. Finally, the design enables mixing of streams of unequal volumetric flows. The process utilizes flash nano-precipitation, which requires fast mixing of two or more streams to create supersaturation. A dissolved solute and stabilizing amphiphilic polymer are rapidly mixed with an anti-solvent to create high supersaturation over a time scale shorter than the characteristic nucleation and growth time scales for the nanoparticles. This rapid growth from a uniform concentration field and uniform “poisoning” of the nanoparticle surfaces by adsorbed amphiphilic polymer uniformly stops the growth to produce narrowATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056particle size distributions. The process can also provide the capability to coat particles and create composite multi-functional particles.

[0087] The nanoparticles thus formed are collected, and the diluents removed by any suitable method, e.g. rotary evaporation. In some embodiments the nanoparticles are admixed with an effective amount of a stabilizing agent. The stabilizing agent may be a disaccharide. The disaccharide may be, for example and without limitation, trehalose or an analog thereof. The admixture is then lyophilized, and may be stored. For use, the dry formulation is reconstituted with an aqueous buffer.

[0088] As an example, shown in FIG. 1 A, there can be five main steps to synthesize this solid nanoparticle: Dissolve the agents in methanol. Introduce the methanol solution into one inlet of MIVM and water into another three inlets. The four streams are mixed in MIVM. The injection volumes of these four inlets are the same. The nanoparticle suspension is collected from the outlet of MIVM and methanol removed by rotary evaporation. Trehalose is mixed into nanoparticle suspension and dissolved completely. The admixture is snap frozen with liquid nitrogen and then lyophilized. The dry formulation is reconstituted with an aqueous diluent before use.Methods of T reatment

[0089] Aspects of the instant disclosure include methods of treating a subject for glaucoma, e.g. open-angle glaucoma or angle-closure glaucoma, for optic hypertension, etc. In some embodiments, provided herein is a method of treating glaucoma in a mammalian subject in need thereof, comprising administering an effective dose of a nanoparticle formation, e.g. by administration of eye drops. In an example, 1 or 2 eyedrops are instilled in an affected eye. Drops may be administered one or a plurality of days, and in some embodiments is administered twice daily, daily, every two days, semi-weekly, weekly, etc. for a period of from about 1, about 2, about 3, about 4, about 5, about 6, about 7 or more weeks, up to a chronic maintenance level of dosing. Therapeutic entities of the present invention are usually administered on multiple occasions.

[0090] Various subjects may be treated in the methods of the present disclosure. In some instances, treated subjects may be mammals, including but not limited to e.g., rodents (e.g., rats, mice, etc.), non-human primates (e.g., macaques, marmosets, tamarins, spider monkeys, owl monkeys, vervet monkeys, squirrel monkeys, baboons, chimpanzees, etc.), humans, and the like. In some instances, a treated subject may be an animal model (e.g., a rodent model, a non-human primate model, etc.) of glaucoma.ATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056

[0091] In some instances, a treated subject is a human subject, including but not limited to e.g., a human subject having an optic neuropathy such as glaucoma, a human subject at increased risk of developing an optic neuropathy, a human subject of advanced age (e.g., at least 60 years of age, at least 65 years of age, at least 70 years of age, at least 75 years of age, at least 80 years of age, at least 85 years of age, at least 90 years of age, etc.), or an individual having multiple such risk factors. Treated subjects may or may not be symptomatic, e.g., a subject may or may not display or have previously displayed one or more symptoms of an optic neuropathy.

[0092] Administration of an agent to a subject, as described herein, may be performed employing various routes of administration, preferably in a topical form. The route of administration may be selected according to a variety of factors including, but not necessarily limited to, the condition to be treated, the formulation and / or device used, the patient to be treated, and the like. Routes of administration that are useful in the disclosed methods include, but are not limited to, topical eye drops. Formulations for these dosage forms are described herein.

[0093] Those of skill in the art will readily appreciate that dose levels can vary as a function of the specific compound, the nature of the delivery vehicle, and the like. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means.

[0094] In those embodiments where an effective amount of an active agent is administered to the subject, the amount or dosage is effective when administered for a suitable period of time, such as daily, twice daily, semi-weekly, etc., so as to evidence a reduction in the disorder, e.g., a reduction in a symptom of the disorder or in a marker of disease pathology. For example, an effective dose is the dose that, when administered for a suitable period of time, such as at least about one week, and maybe about two weeks, or more, up to a period of about 3 weeks, 4 weeks, 8 weeks, or longer, will reduce a symptom of the disorder, for example, by about 10% or more, by about 20% or more, e.g., by 30% or more, by 40% or more, or by 50% or more, in some instances by 60% or more, by 70% or more, by 80% or more, or by 90% or more, for example, and will halt progression of the disorder in the subject. In some instances, an effective amount or dose of active agent will not only slow or halt the progression of the disease condition but will also induce the reversal of the condition, i.e., will cause an improvement in the neurological health of the subject. For example, in some instances, an effective amount is the amount that when administered for a suitable period of time, for example, at least about one week, and / or about two weeks, or more, up to a period of about 3 weeks, 4 weeks, 8 weeks, or longer will improve, stabilize, or at least reduce the progression of a disorder in subject, for example 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, in someATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056instances 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or more relative to the subject’s condition prior to administration.

[0095] In some instances, in those embodiments where an effective amount of an active agent is administered to the subject, the amount or dosage is effective when administered for a suitable period of time to result in a reduction in RGC degeneration in the subject. Such a reduction may manifest in various ways, including but not limited to e.g., an increase in the number, size or length of RGCs, or a reduction in the amount of degeneration of RGCs, or their axons or soma, or the like. In some instances, methods of the present disclosure may result in at least a 5%, e.g., at least a 10%, at least a 15%, at least a 20%, at least a 25%, at least a 30%, at least a 35%, at least a 40%, at least a 45%, at least a 50%, at least a 55%, at least a 60%, at least a 65%, at least a 70% at least a 75%, at least a 80%, e.g., reduction in RGC degeneration. In some instances, methods of the present disclosure may result in at least a 5%, e.g., at least a 10%, at least a 15%, at least a 20%, at least a 25%, at least a 30%, at least a 35%, at least a 40%, at least a 45%, at least a 50%, at least a 55%, at least a 60%, at least a 65%, at least a 70% at least a 75%, at least a 80%, e.g., increase in RGC number, size or length of RGC axons or somata. Various methods of assessing the amount of RGC degeneration or increase in number, size or length of RGC axons or somata may be employed, including invasive and non-invasive techniques, such as electrophysiology measurement for RGC neuronal function, visual acuity, OCT imaging, fundus imaging, histology studies of RGC somata and axons morphology.

[0096] An effective amount of a subject formulation will depend, at least, on the particular method of use, the subject being treated, the severity of the affliction, and the manner of administration of the therapeutic composition. A "therapeutically effective amount" of a composition is a quantity of a specified compound sufficient to achieve a desired effect in a subject (host) being treated. Therapeutically effective doses of a subject compound or pharmaceutical composition can be determined by one of skill in the art, with a goal of achieving local (e.g., tissue) concentrations that are at least as high as the IC50 of an applicable compound disclosed herein. The specific dose level and frequency of dosage for any particular subject may be varied and will depend upon a variety of factors, including the activity of the subject compound, the metabolic stability and length of action of that compound, the age, body weight, general health, sex and diet of the subject, mode and time of administration, rate of excretion, drug combination, and severity of the condition of the host undergoing therapy.

[0097] Conversion of an animal dose to human equivalent doses (HED) may, in some instances, be performed using the conversion table and / or algorithm provided by the U.S.ATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (ODER) in, e.g., Guidance for Industry: Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers (2005) Food and Drug Administration, 5600 Fishers Lane, Rockville, MD 20857, the disclosure of which is incorporated herein by reference).

[0098] In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated.

[0099] In other embodiments, for therapeutic applications, therapeutic entities of the present invention are administered to a patient suspected of, or already suffering from such a disease in an amount sufficient to cure, or at least partially arrest, the symptoms of the disease (biochemical, histologic and / or behavioral), including its complications and intermediate pathological phenotypes in development of the disease. An amount adequate to accomplish therapeutic or prophylactic treatment is defined as a therapeutically- or prophylactically- effective dose. In both prophylactic and therapeutic regimes, agents are usually administered in several dosages until a sufficient response has been achieved.

[0100] The pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration. Preferably, a therapeutically effective dose will provide therapeutic benefit without causing substantial toxicity.

[0101] Toxicity of the nanoparticles described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD50 (the dose lethal to 50% of the population) or the LD100 (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index. The data obtained from these cell culture assays and animal studies can be used in formulating a dosage range that is not toxic for use in human. The dosage of the proteins described herein lies preferably within a range of circulating concentrations that include the effective dose with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See, e.g., Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, Ch.1).ATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056

[0102] Also within the scope of the invention are kits comprising the compositions of the invention and instructions for use. The kit can further contain a least one additional reagent, e.g. a suitable diluent. Kits may comprise an applicator device, e.g. pipette, eye-dropper, etc. Kits typically include a label indicating the intended use of the contents of the kit. The term label includes any writing, or recorded material supplied on or with the kit, or which otherwise accompanies the kit.

[0103] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible. It is also understood that the terminology used herein is for the purposes of describing particular embodiments

[0104] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or only and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[0105] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the appended claims.EXPERIMENTAL

[0106] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of howto make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.ATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056Example 1NeuProlO: A Dual-Action Solid Drug Nanoparticle Formulation for Simultaneous Intraocular Pressure Reduction and Neuroprotection in Glaucoma

[0107] Glaucoma is a leading cause of irreversible blindness worldwide, primarily due to elevated intraocular pressure (IOP) that damages the optic nerve. Most current treatments focus solely on lowering IOP without directly protecting or regenerating the optic nerve, as drug delivery to the posterior eye remains a significant challenge. In this study, we developed NeuProlO, a dual-action solid drug nanoparticle (NP) eyedrop formulation designed for both IOP reduction and neuroprotection. Using a flash nanoprecipitation method, we successfully fabricated the first NeuProlO solid NPs incorporating the repurposed neuroprotective agent maprotiline (MAP) and the lOP-lowering agent betaxolol (BX), forming MAP / BX NPs for synergistic glaucoma treatment. This formulation offers significant advantages over conventional eyedrops, including improved drug stability, ocular biocompatibility, tissue permeability, and sustained therapeutic efficacy. Notably, BX not only lowers IOP but also enhances MAP delivery to the posterior eye, maximizing its neuroprotective potential. The MAP / BX NP formulation represents a promising advancement in glaucoma management by integrating sustained IOP control with targeted neuroprotection. Furthermore, the use of a multiple-inlet vortex mixer ensures reproducibility, scalability, and precise formulation control. NeuProlO can redefine glaucoma treatment, offering a more effective and long-lasting solution to preserving vision and improving patient outcomes.

[0108] The development of nanotechnology has significantly advanced the formulation of hydrophobic drugs by enhancing their permeability across lipophilic membranes. Two main strategies are employed in nanoparticle (NP) formulations: using nanocarriers like polymeric micelles and liposomes or creating solid drug NP composed solely of the drug. Solid drug NPs offer higher drug loading and improved permeability across biological barriers such as the cornea. However, traditional nanoprecipitation methods can result in inconsistent particle sizes and poor batch-to-batch consistency, posing challenges for industrial production. Innovative methods like the multi-inlet vortex mixer (MIVM) have been developed to produce uniform NPs in a continuous process, offering a promising solution for scaling up production. Additionally, it is crucial to prepare these solid NPs in a dry form without compromising their structure and function. This allows for easy storage and reconstitution in aqueous buffer right before use, ensuring the NPs retain their therapeutic effectiveness while offering better bioavailability and longer action time for ocular drug delivery.ATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056

[0109] To address the urgent need for an effective glaucoma treatment that combines neuroprotection with IOP reduction, while also overcoming the challenges associated with drug formulation scalability, we repurposed the antidepressant MAP for its neuroprotective properties and combined it with IOP reduction drug BX in a solid NP formulation— NeuProlO. Leveraging the MIVM, we developed a continuous and scalable method to produce this MAP / BX solid drug NP (MAP / BX NP) system. Our in vitro and in vivo studies demonstrate that NeuProlO offers extended efficacy in treating glaucoma by simultaneously reducing IOP and providing neuroprotection. This innovation not only sheds new light on the potential of drug repurposing in glaucoma treatment but also presents a scalable solution for the industrial production of advanced ocular drug formulations, ultimately improving patient outcomes and compliance.Experiment section

[0110] Materials. Maprotiline hydrochloride (MH) was purchased from Sigma-Aldrich (St.Louis, MO, USA). BX and betaxolol hydrochloride (BH) were purchased from MedChem Express LLC (Monmouth Junction, NJ, USA). Trehalose was purchased from TCI America (Portland, OR, USA). Cell proliferation reagent CCK-8 was purchased from Apexbio Technology LLC (Boston, MA, USA). White Leghorn chicken eggs (SPF fertile) were acquired from AVS Bio (Norwich, CT, USA). Fresh rabbit whole eyes were purchased from Pel-Freeze Biologicals (Rogers, AR, USA). Keratinocyte serum-free medium for human corneal epithelial cells (HCE-2, ATCC, Manassas, VA, USA) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Acetonitrile (ACN), triethylamine (TEA) and phosphate buffered saline (PBS) were purchased from Fisher Scientific (Pittsburgh, PA, USA). Ultrapure water (18.2 MO cm) was produced by a Millipore water purification system (Milli-Q IQ 7000, Millipore Sigma, St. Louis, MO, USA).

[0111] Cell line. HCE-2 cells were cultured in keratinocyte serum-free medium supplemented with EGF human recombinant and Bovine Pituitary Extract in a 5% CO2 incubator at 37 °C. All the plates for HCE-2 cell were pre-coated with coating mixture containing fibronectin (0.01 mg / mL), bovine collagen type I (0.03 mg / mL) and bovine serum albumin (0.01 mg / mL).

[0112] Animals. Normotensive Wistar rat (8-16 weeks old, male and female) was used for in vivo IOP reduction and biodistribution studies. C57BL / 6J WT (#000664) mice (7-9 weeks old, male) with silicone oil-induced ocular hypertension (SOHU) glaucoma model were used for neuroprotection efficacy assessment. All the animals were housed in standard cages under a 12-h light-dark cycle, with room temperature maintained at 25 + 2°C and humidity levelsATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056between 40% and 60%. All experimental procedures were conducted following the animal protocol (#19222 and #32093) approved by the Institutional Animal Care and Use Committee (IACUC) at Missouri University of Science and Technology and Stanford University School of Medicine.

[0113] Preparation of MAP and MAP / BX NP. To prepare MAP, MH (2 mg / mL) was dissolved in ultrapure water and then 50 pL of TEA was added to remove HCI group in MH. Centrifugation was conducted and then the MAP precipitate was washed three times with ultrapure water. Finally, MAP was collected after lyophilization and characterized.

[0114] To prepare MAP / BX NP, MAP (5 mg) and BX (5 mg) were dissolved in methanol (5 mL). This drug solution was then introduced to one inlet of MIVM and ultrapure water (3 x 5 mL) was introduced to the remaining three inlets. The four streams were mixed in the MIVM with a flow rate of 40 mL / min. After removal of methanol by rotary evaporation, the MAP / BX NP suspension was obtained. Subsequently, 1% w / v trehalose was added to the NP suspension as the cryoprotectant and then dissolved completely by vortex mixing. The liquid nitrogen was utilized to conduct snap freezing and frozen NP suspension was lyophilized. The yielded white powder was stored at room temperature. The PBS was added to reconstitute it before use.Characterization

[0115] Proton nuclear magnetic resonance (1H NMR) spectroscopy. 1 H NMR spectrum of MAP was obtained on a Bruker 400 MHz spectrometer using CDCI3 as solvent .

[0116] Transmission Electron Microscopy (TEM). TEM images were captured using a JEM- 1400 microscope (JEOL, Japan) operated at 120 kV after negative staining with sodium phosphotungstate. A 2 pL aliquot of the different NP suspensions was placed onto a carbon- coated copper grid (300 mesh, Fisher Scientific, Pittsburgh, PA, USA). Excess liquid was removed by wicking, and the grids were immediately transferred onto droplets of freshly prepared, filtered 2% w / v aqueous sodium phosphotungstate. After a 2-min staining, excess stain was wicked off, and the grids were thoroughly dried before imaging.

[0117] Dynamic Light Scattering (DLS). The NP hydrodynamic size distribution, polydispersity index (PDI), and zeta potential were evaluated using a Lab Red Zetasizer (Malvern, UK) with a 632.8 nm laser, set at a 90° scattering angle. For the size analysis, samples were diluted 4- fold with ultrapure water, while a 20-fold dilution was used for the zeta potential measurement. Each sample underwent triplicate testing, and data was processed through ZS XPLORER software version 1.02.ATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056

[0118] Liquid Chromatography-Tandem Mass Spectrometry (LC-MS / MS). MAP and BX concentrations were quantified using a Shimadzu UFLC system coupled to a 4000-QTRAP tandem mass spectrometer (AB SCI EX, Concord, ON, Canada). A Synergi Hrdro-RP 80 A C18 column (250 mm x 2 mm, 4 pm particle size, Phenomenex, Torrance, CA, USA) was used for separation. The analytes were eluted at a flow rate of 0.3 mL / min using a gradient elution program. Ultrapure water containing 0.01% formic acid (FA) was used as mobile phase A and ACN with 0.01% FA was used as mobile phase B. The gradient program started with 20% mobile phase B, followed by a linear increase to 95% mobile phase B over 4 min. This condition was held for 2 min, then the gradient was returned to 20% mobile phase B over 0.01 min, and the system was equilibrated at 20% mobile phase B for 4 min before the next injection. The mass spectrometer detection conditions were optimized at 600 °C of ion source temperature, 3,000 V of ion spray voltage, 20 psi of curtain gas pressure, 40 psi of ion source gas 1, and 40 psi of ion source gas 2. The specific mass transitions and additional optimized conditions are detailed in Table 1. The HPLC-MS / MS method was validated for performance in eyeball grinding samples before being applied to the analysis of different ocular tissue samples. Verapamil was used as internal standard for all samples.Table 1.Optimized parameters for HPLC-MS / MS method. The upper ion pairs were used for quantification while the lower ion pairs were used for confirmation.Compound Ion Pairs Declustering Collision Energy (CE, V) Collision Cell Exit Potential (DP, V) Potential (CXP,V) Maprotiline 278.131 / 250.2 46 27 16278.131 / 191.1 46 49 12 Betaxolol 308.237 / 116.2 61 27 6308.237 / 72.1 61 37 12 Verapamil 455.248 / 165.2 61 39 10

[0119] MALDI-IMS was conducted using a Shimadzu iMScope QT IMS coupled to a Shimadzu 9030 Q-ToF MS system (Shimadzu, Kyoto, Japan), equipped with a nitrogen laser. The detailed parameters were listed in Table 2. Ten milligram per milliliter of a-cyano-4- hydroxycinnamic acid (CHCA) with 0.1% FA dissolved in 50% ACN was used as the matrix, applied to the slides via a Shimadzu iMLayer Aero matrix sprayer. The ions at m / z 278.191734 ([MAP + H]+) and m / z 308.089318 ([BX + H]+) were selected to test MAP and BX, respectively.ATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056Table 2. Optimized parameters for MALDI IMS. Instrument setup ValueNumber of laser shoots 200Repetition rate 2,000 HzLaser diameter 2Laser intensity 60Detector voltage 2.2 kVPitch 20 x 20 pm

[0120] In vitro drug release: MAP / BX NP or MAP / BX free drug (i.e., MH and BH) with the identical MAP and BX concentrations was prepared in PBS first. Pur-A-Lyzer dialysis tube (MWCO 6,000 Da) was immersed in PBS for 30 min to equilibrate it, then 100 pL of NP suspension or MH / BH solution was added into the tube, which was immersed in 30 mL of PBS at 37 °C. The sample was withdrawn from the 30-mL PBS at 0.5 h, 1 h, 2 h, 3 h and 4 h (n = 3). The PBS with the same volume was supplemented immediately after sampling to keep sink condition. Further sample dilution was conducted with mobile phase mixture (50% A / 50% B) before LC-MS / MS analysis. To elucidate the release kinetics, the in vitro drug release profiles of MAP and BX from MAP / BX NP were fitted to five common kinetic models (Zeroorder, First-order, Higuchi, Korsmeyer-Peppas, and Weibull models) as follows (Equations 1-5):(Zero-order model) :Mt= kox t Equation 1where Mt is the cumulative amount of drug released at time t, k0is the zero-order release rate constant.(First-order model): Mt= Mmx (1 - e_fciXt) Equation 2where M« is the maximum cumulative release, and kt is the first-order release constant. (Higuchi model): Mt= kHx Vt Equation swhere kHis the Higuchi release constant, assuming Fickian diffusion from a homogeneous matrix.(Korsmeyer-Peppas model):= kKx tnEquation 4where kKis the kinetic constant and n is the diffusion exponent indicative of the release mechanism.(Weibull model): = 1 - Equation 5where a is the scale parameter (related to the time constant of the system), and p isATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056the shape parameter. A p < 0.75 is associated with Fickian diffusion, 0.75-1 .0 with combined diffusion and erosion, and > 1.0 with complex or relaxation-controlled release.

[0121] Nonlinear regression was performed using GraphPad Prism, and the adjusted coefficient of determination (adjusted R2) was used to evaluate the goodness of fitting. The model with the highest adjusted R2was considered the most appropriate for describing the release behavior.

[0122] The hydrodynamic size was also tested during the drug release process and a similar procedure was followed. Briefly, MAP / BX NP was prepared in PBS and tested by DLS to determine the size distribution and PDI value before the drug release. The concentrations of MAP and BX were the same as the above drug release setting. Pur-A-Lyzer dialysis tube (MWCO 6,000 Da) was immersed in PBS for 30 min to equilibrate it, then 100 pL of NP suspension was added into the tube, which was immersed into 30 mL of PBS at 37 °C. Ten microliters of the MAP / BX NP sample were withdrawn from that 100 pL of NP suspension at 0.5 h, 1 h, 2 h, 3 h and 4 h (n = 3). The hydrodynamic size and PDI value were then tested by DLS.

[0123] Ex vivo corneal permeation: MAP / BX NP or MAP / BX free drug with the identical MAP and BX concentrations was prepared in PBS first. The cornea from fresh rabbit eyeball was isolated and mounted between donor chamber and receiver chamber of Franz cell. Three milliliters of PBS was added into receptor chamber and 100 pL PBS was added into donor chamber to do equilibration for 30 min at 37° C. Then the 100 pL of PBS was replaced by 100 pL of MAP / BX NP or MAP / BX free drug in donor chamber. The sample was withdrawn from that receiver chamber at 0.5, 1 , 2, 3 and 4 h (n = 3). The PBS with the same volume was supplemented immediately after sampling to keep sink condition. Further sample dilution was conducted with mobile phase mixture (50% A / 50% B) before LC-MS / MS analysis. The apparent permeability coefficient (Papp) was calculated based on Equation 6, in which the dQ / dt reflects the slope of a cumulative mass transport curve (Figure 3G), C is the drug concentration in the donor chamber, and A is effective cross-sectional area (0.2 cm2) available for diffusion. The hydration level (H) of experimental cornea was also tested afterwards following Equation 7 to evaluate its intactness.Apprent permeability coefficient (Papp~) — ~~~ Equation 6 Hydration level (H) =y 10Q%Equation 7wet weight

[0124] Cytotoxicity assay: HCE-2 cell was used to evaluate the cytotoxicity of MAP / BX NPs.HCE-2 cells were first seeded to a96-well plate with a density of 10,000 cells / well (n = 6), thenATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056incubated overnight. Afterwards, 100 pL of fresh Keratinocyte SFM medium (supplemented with EGF human recombinant and bovine pituitary extract) containing trehalose, MAP / BX NP, MAP / BX NP + trehalose, or MAP / BX free drug with the total drug concentrations from 2 pg / mL (1 pg / mL MAP + 1 pg / mL BX) to 1 ,000 pg / mL (500 pg / mL MAP + 500 pg / mL BX) was added into each well. Notably, the concentrations of trehalose group were equivalent to the trehalose concentrations in MAP / BX NP + trehalose group. After 24 h incubation, the old medium in each well was replaced with 100 pL of fresh medium and 10 pL of CCK-8. After 4 h incubation, the absorbance of each well at 450 nm was measured using a microplate reader. The relative cell viability was determined by comparing the absorbance of treated cells with that of control cells. The IC50 was calculated following a four- parameter logistic regression fitting model.

[0125] Irritation assessment by the HET-CAM Assay: The ocular tolerability and irritation potential of the MAP / BX NPs were evaluated by HET-CAM. The White Leghorn chicken eggs (specific pathogen free) were incubated at 37.5° C with constant humidity for 3 days. After incubation, the eggs were cracked open, and the embryos were transferred into sterilized weigh boats, with a piece of eggshell added for normal development. Weigh boats with chick embryos were placed into individual humidity chambers. Keep it in incubator at 37.5° C and allow to develop for another 7 days. The formulation was added to chick CAMs on day 10. A sterilized 10-mm diameter poly-band ring was placed in an area on the chick embryo with no major vessels. The 20 pL of MAP / BX NP or MAP / BX free drug with the total drug concentrations from 1 mg / mL (0.5 mg / mL MAP + 0.5 mg / mL BX) to 10 mg / mL (5 mg / mL MAP + 5 mg / mL BX) was administered inside the poly-band rings for 5 min. The concentration setting was based on the clinical dosage of BX and also to explore the proper dosing concentration for the in vivo studies. Following this, the membrane underwent careful examination for signs of vascular damage, with the time to injury onset, if any, being recorded. Three samples were placed on each egg. Normal PBS and 0.1 M sodium hydroxide were used as negative and positive controls, respectively. After the administration, imaging of each sample was conducted at 1 x magnification using a microscope to capture an overall view. The irritation evaluation is based on the calculation of an irritation score (IS). The first appearance of the three endpoints (H: time of appearance of haemorrhage; L: time of appearance of vascular lysis; C: time of appearance of coagulation) is recorded in seconds and the IS was calculated using the following Equation 8 and evaluated according to Table 3:is = (301 - H) x 5 / 300 + (301 - L) x 7 / 300 + (301 - C) x 9 / 300 Equation 8Table 3.The HET-CAM irritation score range and the corresponding irritation level.ATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056HET-CAM irritation score range Irritation category0-0.9 Non-irritant1 -4.9 Weak or slight irritation5-8.9 or 5-9.9 Moderate irritation9-21 or 10-21 Strong or severe irritation

[0126] In vivo biodistribution on normotensive rats: Wistar rat (female and male, ~8 weeks old, n 2s 7) was used to evaluate biodistribution of MAP and BX after different treatments. The right and left eyes were treated with MAP / BX NPs (reconstituted in PBS, 2 x 5 ]iL per eye, 0.2% w / v MAP and 0.2% w / v BX) and MAP / BX free drug (dissolved in PBS, 2 x 5 pL per eye, with equivalent MAP and BX concentration), respectively. The treatment was applied at 9:30 am for 1 daily dose experiment or 7 successive daily dose experiment. To unravel the boosting effect of BX for the biodistribution of MAP in the eyeball, the MAP NP and MAP free drug were used to treat the right and left eyes of normotensive rat, respectively, following the same procedure above. After 4 h of the last treatment, the rats were euthanized. The eyeballs were collected, rinsed with PBS, and subjected to procedure either for LC-MS / MS or MALDI-IMS analysis.

[0127] For the LC-MS / MS analysis, the rinsed eyeballs were dissected into different parts (cornea, aqueous humor, lens, retina, and optic nerve). The tissue samples were weighed with an analytical balance and rinsed with PBS. The aqueous humor samples were measured by a volumetric glass needle. Afterwards, all the samples were snap-frozen by liquid nitrogen immediately and then stored in -800C. Prior to LC-MS / MS analysis, each tissue sample was thawed and then immersed in 80% ACN containing 200 pg / L of internal standard (verapamil) in a safe lock tube. One spoon of zirconium oxide beads was added to this tube and thorough homogenization was conducted in a bullet blender (NEXT ADVANCE, NY, USA) at 4° C (speed 8, 10 min). Afterwards, the sample was centrifuged at 10,000 g for 15 mins. The supernatant was withdrawn and diluted further for LC-MS / MS analysis. Aqueous humor samples were thawed and mixed with 80% ACN containing 200 pg / L of the internal standard in the tube. The centrifugation was conducted immediately, and the supernatant was taken and diluted for LC-MS / MS analysis.

[0128] For the MALDI-IMS analysis, the collected eyeballs were frozen in liquid nitrogen. The frozen eyeballs were embedded in 1% w / v carboxymethylcellulose, frozen again in liquid nitrogen, and stored at -80 °C. For cryo-sectioning, the frozen blocks were equilibrated at -20 °C in a cryostat and cut into 15-pm-thick slices. The tissue sections were mounted on 1.1 mmATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056indium-tin oxide-coated glass slides, coated with matrix, and subsequently analyzed by using MALDI-IMS.

[0129] In vivo IOP reduction evaluation on normotensive rats: For single-dose experiment, Wistar rat (female and male, ~16 weeks old, n = 8) was used to evaluate IOP reduction of MAP / BX NP and MAP / BX free drug. The right and left eyes were treated with MAP / BX NPs (reconstituted in PBS, 2 x 5 pL per eye, 0.2% w / v MAP and 0.2% w / v BX) and MAP / BX free drug (dissolved in PBS, 2 x 5 pL per eye, with equivalent MAP and BX concentration), respectively. The treatment was applied at 9:30 am. Before treatment, the IOP was measured for 4 successive days and the average value was used as baseline. The lOPs were measured at 1 , 2, 4, 6, 24, 30, 48, 72, 96, 120, 144, and 168 h postdosing using ICare TONOLAB tonometer (ICare, Helsinki, Finland).

[0130] For multiple-dose experiment, Wistar rat (female and male, ~16 weeks old, n = 8) was used to evaluate IOP reduction of MAP / BX NP and MAP / BX free drug. The right and left eyes were treated with MAP / BX NPs (reconstituted in PBS, 2 x 5 pL per eye, 0.2% w / v MAP and 0.2% w / v BX) and MAP / BX free drug (dissolved in PBS, 2 x 5 pL per eye, with equivalent MAP and BX concentration), respectively. The treatment was applied at 9:30 am every 2 days, 3 times in total. Before treatment, the IOP was measured for 3 successive days and the average value was used as the baseline. IOP was measured at 9:00 am and 15:00 pm daily for the first 7 days postdosing and at 9 am for the remaining 3 days until it returned to baseline.

[0131] In vivo neuroprotection efficacy evaluation in SOHU mice: SOHU glaucoma model.The SOHU mouse modeling has been performed as the previous studies reported. In brief, mice were anesthetized by intraperitoneal injection of Avertin (about 0.3 mg / g) and received the silicon oil (Alcon Laboratories, 1 ,000 mPa s) injection at 9 weeks of age. Before injection, 0.5% proparacaine hydrochloride (Akorn, Somerset, New Jersey) was applied to the cornea as topical anesthesia. A 32G needle was tunneled through the layers of the cornea close to the limbus into the anterior chamber with no injury to the lens or iris. After that, about 2 pL silicone oil was injected slowly into the anterior chamber using a homemade sterile glass micropipette, until the oil droplet expanded to cover most surface of the iris (diameter about 1.8-2.2 mm). After the injection, veterinary antibiotic ointment (BNP ophthalmic ointment, Vetropolycin, Dechra, Overland Park, Kansas) was applied to the surface of the injected eye. Throughout the procedure, artificial tears (Systane Ultra Lubricant Eye Drops, Alcon Laboratories, Fort Worth, Texas) were applied to keep the cornea moist.

[0132] After the development of SOHU glaucoma model, the right eyes of mice were treated with MAP free drug or MAP / BX free drug (designated as SOHU MAP free drug group or SOHU MAP / BX free drug group, dissolved in PBS, 5 pL per eye, 0.2% w / v MAP and 0.2% w / v BX),ATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056and the left eyes of mice were treated with MAP NP (prepared following same procedure with MAP / BX NP preparation, including MAP dissolution in MeOH, MIVM mixing, rotary evaporation, lyophilization with 1% trehalose, and resuspension in PBS) or MAP / BX NP (designated as SOHU MAP NP group or SOHU MAP / BX NP group, reconstituted in PBS, 5 uL per eye, 0.2% w / v MAP and 0.2% w / v BX). The treatment was applied daily for 3 weeks (n 2? 6). For the control groups, the left eyes of mice were used for the SOHU glaucoma mouse model and the contralateral right eyes were injected with 2 pl of normal saline in the anterior chamber. Each mouse received a 5 pL PBS drop for both eyes daily for 3 weeks (designated as SOHU PBS group or naive group, n 5= 6). In addition, to confirm if BX has neuroprotection efficacy, BX free drug was also dosed daily for 3 weeks (designated as SOHU BX free drug group, dissolved in PBS, 5 pL per eye, 0.2% w / v BX, n 2s 13). Afterwards, the following tests were conducted for each mouse.

[0133] Pattern electroretinogram (PERG) recording. After anesthetization (0.01 mg xylazine / g + 0.08 mg ketamine / g) and pupil dilation, PERG of both eyes was recorded simultaneously with the Miami PERG system (Intelligent Hearing Systems, Miami, Florida) according to manufacturer’s instructions. The pattern remained at a contrast of 100% and a luminance of 800 cd / m2, and it consisted of four cycles of black-gray elements, with a spatial frequency of 0.052 c / d. Two consecutive recordings of 200 traces were averaged to achieve one readout; each trace recorded up to 1 ,020 ms. The first positive peak in the waveform was designated as P1 and the second negative peak as N2. The amplitude was measured from P1 to N2.

[0134] Spectral-domain optical coherence tomography imaging. After anesthetization (0.01 mg xylazine / g + 0.08 mg ketamine / g) and pupil dilation, the retina fundus images were captured with the Heidelberg Spectralis optical coherence tomography (OCT) imaging system (Heidelberg Engineering, Germany). The mouse retina was scanned with the glaucoma mode (ring scan) centered by the optic nerve head under high-resolution mode (each B-scan consisted of 1 ,536 A-scans). The ganglion cell complex (GCC) includes retinal nerve fiber layer (RNFL), ganglion cell layer (GCL), and inner plexiform layer (IPL). The average thickness of GCC around the optic nerve head was measured manually with the Heidelberg software. The investigators who measured the thickness of GCC were masked to the treatment of the samples.

[0135] Immunohistochemistry of whole mounts of retina and counting of RGCs. After perfusion fixation with 4% PFA in PBS, mice eyeballs and optic nerves were dissected out and post-fixed with 4% PFA for 2 h at room temperature. The primary antibodies used for immunostaining were as follows: anti-RBPMS at 1 :4,000 (Custom made at ProSci), SecondaryATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056antibodies were then applied (1 :200; Jackson ImmunoResearch, West Grove, Pennsylvania) and incubated for 1 h at room temperature before mounting. For whole-retina RGC counting in the SOHU glaucoma model, the entire retina was imaged with the 20 x objective lens of a Keyence fluorescence microscope. Eight circles drawn by Concentric Circle plugin of NIH Imaged were used to define the peripheral, middle, and central areas of the retina. Multiple 250 x 250 pm counting frames were applied by Fiji / lmageJ and the number of surviving RGCs was counted by RGCode software. For SOHU MAP free drug, SOHU MAP / BX free drug, MAPNP or MAP / BX NP treatment groups, RGC survival was calculated as the density of surviving RGC numbers / mm2retina. For BX free drug treatment, the percentage of RGC survival was calculated as the ratio of surviving RGC numbers in treated eyes compared to contralateral control eyes. The investigators who counted the cells were masked to the treatment of the samples.

[0136] Optic nerve semi-thin sections and quantification of surviving axons. Briefly, optic nerves were post-fixed in situ with 2% glutaraldehyde and 2% PFA. Semi-thin (1-pm) crosssections of the optic nerves (2 mm distal to the eye) were collected. After paraphenylenediamine (PPD) staining, each optic nerve was imaged by a 100 x oil objective lens of a Keyence bright field microscope to cover the entire area of the optic nerves without overlap. Multiple 10 x 10 pm counting frames were applied automatically by AxonCounter plugin of Imaged to sample about 10% of each optic nerve. The number of surviving axons automatically counted by AxoNet plugin of Imaged. For SOHU MAP free drug, SOHU MAP / BX free drug, MAPNP or MAP / BX NP treatment groups, axon survival was calculated as the density of surviving axon numbers / pm2axon. For BX free drug treatment, surviving axon number was calculated for each ON and compared to that in the contralateral control ON to yield a percentage of axon survival value. The investigators who counted the axons were masked to the treatment of the samples.

[0137] In vivo biosafety assessment in mice: C57BL / 6J WT mice (7-9 weeks old, male and female, n = 5) were used to evaluate the biosafety of MAP / BX NP formulation. The right and left eyes of each mouse were treated topically with PBS and MAP / BX NP (reconstituted in PBS, 5 pL per eye, 0.2% w / v MAP and 0.2% w / v BX, once daily, 7 days in total), respectively. After 7 days, fluorescein staining method was applied to evaluate ocular irritation response of MAP / BX NPs. Specifically, two microliters of fluorescein sodium solution (1%, w / v) were dropped on ocular surface, which was then observed with slit lamp microscopy (Microclear Medical Instruments, Suzhou, Jiangsu, China) under cobalt blue light on the 7th day of treatment. The cornea thickness and GCC thickness were then tested using OCT. Next, the mice were euthanized and immunohistochemistry test of whole mounts of retina and countingATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056of RGCs were conducted after perfusion fixation with 4% PEA in PBS. To evaluate the systemic response of MAP / BX NPs, hematoxylin and eosin (H&E) staining for organs was conducted. Specifically, the organs including heart, liver, spleen, lung, and kidney were harvested and fixed in PFA at 4° C for 24 h. The organs were subsequently immersed in 15% sucrose for 6 h and 30% sucrose for 12 h, then embedded in optimal cutting temperature compound, and frozen at -80°C until sectioning. Cryosections of 10 pm thickness were obtained using a cryostat (Leica CM1860, Wetzlar, Germany) and mounted on Superfrost microscope slides. Slides were fixed in 95% ethanol, stained with H&E using standard protocols, and visualized under a brightfield microscope (Leica) at 10x magnification

[0138] Statistical analysis: Data analysis was performed using statistical software Microsoft Excel 365 (Microsoft Corporation). Data analysis figures were drawn by Origin Pro 2021 and GraphPad Prism 10. Student’s t-test or one-way ANNOVA was conducted to compare the statistical difference.Results

[0139] Scalable and reproducible preparation of MAP / BX NP. The preparation for NeuProlO MAP / BX NP was streamlined, and the measured hydrodynamic diameters were consistent across production batches. After vigorous mixing inside MIVM and removal of methanol by rotary evaporation, the MAP / BX NP suspension was formed (Figure 2A). The average hydrodynamic diameter was 217.9 nm for 3 batches of samples. The relative standard deviation (%RSD) of hydrodynamic diameter was 6.7%, meaning it was stable and reproducible. The average PDI was 0.1 for these 3 batches, indicating the size distribution was very uniform (Figure 2B). The average zeta potential was -11 .3 mV in ultrapure water.

[0140] Trehalose preserves the integrity of MAP / BX NP during lyophilization. To extend and simplify the storage of MAP / BX NP, the dried NP form after lyophilization was preferred. We introduced trehalose as the cryoprotectant and utilized snap freezing method to minimize the formation of ice crystal and therefore protected the original MAP / BX NP structure. The suitable mass percentage of trehalose was explored (Figures 2C&D). After lyophilization, the NP with trehalose formed white powder. The dried MAP / BX NPs were resuspended into ultrapure water and evaluated by DLS. It was found that the NPs with 1% and 2% trehalose remained intact after lyophilization, and the hydrodynamic size increased from -220 nm to -300 nm and the zeta potential changed from -11.3 mV to -17.9 mV before and after lyophilization. The increase in hydrodynamic size is attributed to the formation of a structured hydration layer and surface adsorption of trehalose, which enlarges the effective particle diameter measured by DLS. This is consistent with the observed shift in zeta potential, indicating enhanced surfaceATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056charge stabilization and altered interfacial properties. The PDI values were all lower than 0.2, indicating that there was no precipitate formed after lyophilization. Considering the cost of scalable production, 1% trehalose was applied as cryoprotectant for the other experiments. The TEM images in Figures 2E&F revealed that the MAP / BX NPs had a round morphology before and after lyophilization, further indicating that 1% trehalose could protect the structure of the MAP / BX NPs. The diameter of NP was then measured using ImageJ and size distribution histogram was created by counting around 80 particles (Figures 2E&F). The average diameters of NPs were comparable before and after lyophilization (127 nm and 132 nm, before and after lyophilization). Previous studies also reported that trehalose displayed more effective cryoprotection compared to sorbitol, mannitol, lactose, and sucrose, but the applied concentration was higher (5%- 10%). They conducted a 24 - hour pre-freezing process at -60 °C or -80 °C, during which gradual crystallization might occur, and higher trehalose levels were required to preserve NP integrity. In contrast, our snap-freezing method minimized solvent crystallization, allowing 1% trehalose to sufficiently protect the formulation.

[0141] Improved colloidal stability of MAP / BX NP. The colloidal suspension of MAP / BX NPs maintained stability for at least 7 days following reconstitution in PBS under 4°C (2 mg / mL of MAP and 2 mg / mL of BX). The MAP / BX NP dried powder was white and a pronounced Tyndall effect was observed in NP suspension upon illumination with a green laser beam (532 nm of laser wavelength, Figures 3A&B). The variation of size was neglectable within the 7 days assessed (Figure 30). The PDI value increased slightly after the first day and then remained stable (~0.2).

[0142] Enhanced drug release of MAP / BX NP and size change during the release. To evaluate the drug release behavior of MAP / BX NP in ocular fluids, we next conducted in vitro drug release study. MAP / BX NP exhibited sustained-release characteristics for both MAP and BX compared to the MAP / BX free drug, which is critical for maintaining effective intraocular drug concentrations over an extended period. The MAP released from NP and free drug formulations were 82% and 100%, respectively, within 1 h and reached > 90% from NP after 4 h. The BX released from NP and free drug were 63% and 87%, respectively, within 1 h and reached 89% and 100% after 4 h (Figure 3D). The drug release kinetics of MAP and BX from MAP / BX NP were evaluated using various kinetic models, including zero-order, first-order, Higuchi, Korsmeyer-Peppas, and Weibull models (Table 4 & Figure 2). Among these, the Weibull model provided the best overall fit for both MAP and BX, as evidenced by the highest Adjusted R2values (MAP: 0.91 ; BX: 0.94) and the lowest Akaike Information Criterion (AIC) values (MAP: 90.80; BX: 82.56). The Weibull shape parameters (P) were 0.74 for both MAPATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056and BX, indicating that drug release predominantly follows Fickian diffusion. This suggests that drug molecules are primarily released by passive diffusion through the intact NR matrix rather than by erosion of the NR platform.ATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056Table 4.Parameters estimated by fitting drug release data to five mathematical models.Mathematical ModelsZero-order First-order Higuchi Korsmeyer-Peppas WeibullDrug Formulation ko R2ki Adj. R2AIC kH Adj R2AIC kK n Adj. R2AIC a P Adj. R2AIC MAP / BX free20.46 0.49 1.81 0.83 10660 61.61 0.66 119.30 78.09 0.24 0.76 11470 0.56 4.55 0.87 103.80 MAP drugMAP / BX NP 18.85 0.59 1.36 0.91 89.05 55.26 0.77 105.50 69.50 0.25 0.89 94.89 0.73 0.74 0.91 90.80 MAP / BX free23.40 0.61 1.24 0.84 10600 58.62 0.77 113.40 65.60 0.38 0.77 11470 0.78 2.85 0.91 98.11 BX drugMAP / BX NP 19.57 0.72 0.85 0.92 84.24 50.34 0.89 90.92 57.96 0.35 0.92 86.71 1.16 0.74 0.94 82.56ATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056

[0143] The hydrodynamic size of NP mainly underwent a 3-stage variation during the release process. The size was relatively stable within 0.5 h and increased dramatically from 0.5 h to 2 h due to the swelling of MAP / BX NPs. Afterwards, the NP underwent a complete dissolution and undetectable by DLS (Figure 3E). The PDI initially remained low (< 0.3), indicating a uniform size distribution, but showed a significant elevation corresponding to the increase in particle size, indicating higher heterogeneity in particle size during the drug release progress. The initial burst release observed within the first 0.5 h likely from surface-associated or weakly entrapped MAP and BX molecules. These drug fractions are readily accessible and rapidly diffuse into the release medium due to a steep concentration gradient. MAP / BX NPs exhibit a transient increase in hydrodynamic size around 2 h, suggesting matrix swelling, but this structural change does not correlate with a burst release. Instead, the continuous and stable release profile after 1 h supports a diffusion-dominated mechanism, where swelling facilitates drug diffusion without leading to particle rupture.

[0144] Enhanced corneal permeability of MAP / BX NP. To evaluate the permeability of MAP / BX NP on cornea, we conducted ex vivo cornea permeation study using Franz cell device. A more strikingly enhanced drug permeability was enabled by MAP / BX NP (Figure 3F), proving that the lipophilicity of NP formulation facilitated better penetration through the lipophilic layers of the cornea. The Papp of MAP was 1.89 x 10'7cm / s in free drug group but increased 22-fold in NP group (4.24 x 10-6cm / s). Similarly, the Papp of BX was also elevated remarkably in NP group (1 .87 x 10'6cm / s for free drug and 1.20 x 10’5cm / s for NP). In addition, the cumulative transport of BX was always higher than MAP, no matter if NP or free drug formulation. Once penetrated the cornea epithelium, BX can cross the hydrophilic middle layers of the cornea more effectively due to the higher hydrophilicity than MAP. The H value ranged from 77% to 80%, within the healthy range (76%-80%) based on the previous report. These results demonstrated that the MAP / BX NP can provide a better cornea permeation compared to free drug, without causing any damage to the corneal tissue.

[0145] MAP / BX NP minimizes cytotoxicity of HCE-2 cell line. Developing an ocular drug formulation with minimal toxicity is imperative. Accordingly, the potential toxic effects of the prepared formulations were assessed using an in vitro assay. The trehalose had no cytotoxicity, with the relative cell viability always higher than 90% at different concentrations. The other 3 formulations, however, exhibited an apparent dose-dependent tendency (Figure 4A). Specifically, when the total drug concentration =£ 10 pg / mL, there was no cytotoxicity for all 3 formulations. Elevating the concentration further, the cell viability of MAP / BX free drugATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056decreased dramatically, reaching less than 10% at 40 pg / mL. Conversely, the cell viability dropped mildly for MAP / BX NP and MAP / BX NP + trehalose with the increase of dosing concentration, which may be due to the slow drug release of NPs. The IC50 was then determined based on the cell viability data, and it was 35.59 pg / mL, 33.61 pg / mL, and 20.49 pg / mL for MAP / BX NP + trehalose, MAP / NX NP, and MAP / BX free drug, respectively, with MAP / BX NP + trehalose showing the lowest cytotoxicity. The improved cytotoxicity of the MAP / BX NP + trehalose compared to MAP / BX NP may be attributed to the addition of trehalose, a non-toxic, biologically compatible sugar that stabilizes membranes and protects cells from osmotic stress.

[0146] MAP / BX NP lowers irritation level in HET-CAM test. Ocular formulations must be nonirritating because the eye’s delicate tissues, such as the conjunctiva and cornea, are sensitive, and even minor irritation can lead to inflammation, discomfort, and reduce patient adherence to treatment. The HET-CAM test is a reliable model for assessing ocular irritation as its highly vascularized CAM mimics the inflammatory response of the human conjunctiva. The normal PBS and 0.1 M of NaOH were used as negative and positive control, respectively. Hemorrhage, vascular lysis and coagulation were observed on CAM surface in 10 s after NaOH treatment and the irritation score was 20.4, severe irritation level. In contrast, PBS did not cause any irritation after 5-min treatment, suggesting non-irritation (Figure 4B). When treated with MAP / BX free drug or MAP / BX NPs at different drug concentrations (Figures 4C&D), it was found that both formulations had no irritation at 1 mg / mL and then exhibited different irritation levels with the increase of dosing concentration. Specifically, 2 mg / mL of free drug resulted in moderate irritation and severe irritation was observed when further elevated to 4 mg / mL and 10 mg / mL. Compared to free drug, the MAP / BX NP displayed a lower irritation level, with slight irritation showing at 2 mg / mL and 4 mg / mL. The severe irritation also existed with 10 mg / mL of MAP / BX NP. Thus, we applied 4 mg / mL of total drug concentration (2 mg / mL MAP + 2 mg / mL BX) for the rest in vivo experiments.

[0147] Enhanced biodistributions of MAP and BX in the eyeball of normotensive rats. To clarify the biodistribution and accumulation of MAP and BX in the eyeball after NP or free drug treatment, we tested the drug concentration in different ocular tissues after 1 daily dose and 7 successive daily dose experiments.

[0148] After 1 daily dose treatment, the average MAP concentration of NP group was slightly lower than that of free drug group in cornea, whereas significantly higher in aqueous humor, retina, and optic nerve and insignificantly higher in lens (Figure 5A). Regarding BX concentration, the obvious difference was not observed between NP group and free drug group in the eyeball, except lens. Specifically, the average BX concentration of NP group wasATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056significantly higher in cornea and lens and was comparable in aqueous humor, retina, and optic nerve between these 2 groups. In addition, the corresponding mass ratios of MAP and BX were displayed in Figure 5B. Compared to free drug treatment, the mass ratios of MAP and BX were quite lower in cornea after NP treatment (MAP: 58.3% and 41.2% after free drug and NP treatment, respectively; BX: 43% and 29.3% after free drug and NP treatment, respectively), further proving that NP can penetrate the corneal barrier more effectively than free drug. Correspondingly, compared to free drug group, the mass ratios of MAP and BX in lens were obviously higher in NP group (MAP: 31.8% and 42.8% after free drug and NP treatment, respectively; BX: 47.2% and 65.6% after free drug and NP treatment, respectively), indicating the MAP and BX from NP crossed the cornea and reached the deeper area of the eyeball more efficiently. The total mass ratio of MAP in the back of the eye (retina and optic nerve) could affect its neuroprotection effect. In NP group, this total mass ratio was 7.5%, higher than that in free drug group (5.1 %), suggesting the NP had a better permeation property and potentially more competent neuroprotection effect.

[0149] Figure 5C demonstrates the accumulation of MAP and BX in the different areas of eyeball after daily dose for 7 successive days. Like the profile of 1 daily dose experiment, much more MAP of free drug group was detected in cornea than in NP group, and the MAP concentrations from NP group were significantly higher in the aqueous humor, lens, retina and optic nerve. BX concentrations of NP group were significantly higher in aqueous humor and lens and were comparable to free drug group in cornea, retina, and optic nerve. About 90% MAP was retained in the cornea in the free drug group and only 1 .8% MAP reached retina and optic nerve (Figure 5D). Conversely, after NP treatment, over 5% MAP was delivered to the back of the eye and 64.2% MAP was tested in the cornea. In terms of BX, a more even drug mass distribution was found in these 2 groups. In the free drug group, the highest and lowest mass ratios appeared in lens (32.3%) and aqueous humor (10.6%). In NP group, these 2 values showed up in lens (36.7%) and optic nerve (5.6%). The eyeball status was also monitored during the experiment. After 7-day treatments, the MAP / BX free drug and MAP / BX NP did not cause any irritation, including abnormal tearing and blinking.

[0150] The penetration of MAP to the posterior area of the eyeball is essential for the neuroprotection of glaucoma eye. To investigate if there was any boosting effect of BX for MAP biodistribution, the MAP NP and MAP free drug were also utilized to carry out 1 daily dose and 7 successive daily dose experiments following the same procedure above and MAP concentration in different ocular tissues was tested by LC-MS / MS. After 1 daily dose and 7 successive daily dose experiments, there was no obvious tendency between MAP and MAP / BX groups in cornea, aqueous humor, and lens. However, the MAP concentration wasATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056always significantly higher in the back of the eye (retina and optic nerve) in MAP / BX group compared to the corresponding MAP group in both free drug and NP formulations. For example, after 1 daily dose, the MAP concentrations of MAP / BX free drug groups were 12- and 4-fold higher than MAP free drug group in retina and optic nerve, respectively. The MAP concentrations of MAP / BX NP group were 5- and 2-fold higher than MAP NP group in retina and optic nerve, respectively. A comparable trend was also noticed after 7 successive daily dose experiment. These results indicated that MAP could penetrate the ocular tissue more effectively by incorporating BX in the formulation.

[0151] MALDI-IMS was conducted to further visualize the spatial distribution of MAP and BX in the eyeball. After free drug treatment, most of MAP was retained in the anterior segment of the eyeball. After NP treatment, however, MAP was observed in the posterior segment clearly, including retina and optic nerve (Figure 6A). As expected, BX from both groups was captured in both anterior and posterior segments, with the major distribution displaying around the lens area. Figures 6B&C display the summary mass spectra of the whole eye section. Under positive mode, both MAP and BX were detected successfully, with the [MAP+H]+ and [BX+H]+ at m / z 278.191734 and 308.089318, respectively. Other peaks with relatively high intensities (e.g., m / z 291) were mainly derived from the matrix, as confirmed by comparison with the MALDI-TOF mass spectrum of CHCA. In these 2 groups, BX displayed the most abundant signal within m / z ranging from 275 to 315. In the NP group, the MAP intensity was 40.8% lower than that in the free drug group, while BX intensity was 7.5% higher than free drug group. The relative signal intensities of MAP and BX in different ocular tissues after MAP / BX free drug or MAP / BX NP treatment were then evaluated (Figures 6D&E). For MAP, the intensity of NP group is significantly lower than free drug group in cornea, but higher in vitreous body and optic nerve. In terms of BX, after NP treatment, its intensity is significantly lower than free drug treatment in lens but higher in all the rest target areas. This finding is comparable to biodistribution study assessed by LC-MS / MS, further proving that NP formulation could penetrate the ocular tissue more effectively than free drug.

[0152] Sustained lOP-lowering effect of MAP / BX NP in normotensive rats. To investigate the action time of the MAP / BX NP, the IOP of normotensive rats was monitored after single-dose treatment and MAP / BX free drug with the identical drug concentration was dosed simultaneously as a control group. A sustained IOP reduction was observed following a single topical administration of MAP / BX NPs, with an effective IOP control (> 15% IOP reduction) maintained for up to 72 h post-dosing (Figure 7A). Specifically, following administration of either the NP formulation or the free drug, an immediate reduction in IOP was observed. However, in the free drug group, IOP rebounded at 4 h post-treatment and gradually returnedATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056to baseline by 6 h. Afterwards, there was no pronounced IOP fluctuation from 6 h to 168 h. In contrast, the MAP / BX NP exhibited sustained lOP-lowering effect. After the treatment, no significant difference was observed in the first 4 h. Impressively, the AIOP of the NP group was significantly lower than that of the free drug group at 6 h, 30 h, 48 h, and 72 h, indicating that the lOP-lowering effect of NP can last longer. The amount of free drug formulation crossing the cornea may be limited, which could be cleared via the aqueous outflow pathway quickly. The AIOP of NP group displayed a sustained decreasing trend within 48 h, then elevated gradually and returned to normal IOP after 168 h, with the lowest value showing up at 48 h.

[0153] Based on the above single-dose experiment, the 48 h of time interval was selected for the multiple-dose experiment to explore whether the MAP / BX NP could maintain therapeutic effect with reduced dose frequency. We started dosing at 9 am and dosed the formulation again every 48 h. The IOP was monitored daily at 9 am and 3 pm for the first 7 days and at 9 am from day 8 to day 10. Consistent with the single-dose experiment, a pronounced reduction in IOP was observed immediately following treatment. However, the free drug group’s IOP rapidly returned to baseline, whereas the NP group maintained a relatively low and stable IOP until the subsequent dosing (Figure 7B). In these 10 days, the average AIOP of NP group was -4.11 mmHg, which was around 6-fold lower than that of free drug group (-0.69 mmHg). Moreover, statistically significant differences in AIOP were observed at nearly all time points, further substantiating the enhanced and sustained lOP-lowering efficacy of MAP / BX NP.

[0154] Synergistic neuroprotective effect in the SOHU glaucomatous mice in MAP / BX combination groups. The SOHU mouse glaucoma model was selected to evaluate the effect of MAP / BX NP on glaucomatous neurodegeneration as it effectively models human secondary glaucoma, characterized by severe degeneration of RGCs and the optic nerve. To evaluate visual function after administration, the PERG test was conducted after 3-week treatment (Figure 8B). In SOHU PBS group, the P1-N2 amplitude decreased 43.3% compared to naive group, indicating visual function impairment of SOHU mice. In MAP free drug and MAP NP groups, the average P1-N2 amplitudes are all lower than SOHU PBS group, suggesting no visual function restoration. In contrast, although there is no significant difference, both MAP / BX NP and MAP / BX free drug treatments depict elevating trend of the amplitude over SOHU PBS treatment (MAP / BX NP, 48.9% increase; MAP / BX free drug, 76.5% increase), displaying visual function preservation. The significant thinning of GOG in SOHU PBS group was observed in OCT retinal images due to severe neurodegeneration. The MAP NP and MAP free drug did not show neuroprotection efficacy, with similar GCC thickness evaluated under OCT, compared to SOHU PBS group. Whereas, both MAP / BX NP and MAP / BX free drugATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056groups displayed neuroprotective effects, with the GCC thickness increased by 21 .3% and 39.7%, respectively, compared to SOHU PBS group (Figures 8C&D). Similarly, histological analysis of semithin optic nerve sections demonstrated significantly greater axon survival in the MAP / BX NP and MAP / BX free drug groups, ~8- and 9-fold higher than SOHU PBS group, respectively, but there was no alleviation after MAP NP or MAP free drug treatment (Figures 8E&F). The confocal images of flat-mounted peripheral, middle, and inner retinas showing surviving RBPMS + (red) RGCs (Figures 8G&H) exhibited pronounced decrease of RGCs in SOHU PBS group compared to naive group and the RGC survival of SOHU PBS group was significantly lower than naive group. After treatment with MAPNP or MAP free drug, there was no obvious elevation of RGC survival. In contrast, both MAP / BX NP and MAP / BX free drug formulations significantly increased it, with average survivals ~4- and 7-fold greater than SOHU PBS group, respectively. To confirm the better GCC thickness and RGC and axon survivals were attributed to MAP, the BX free drug was used to dose SOHU glaucoma mice following the same procedure. The results revealed no significant differences in GCC thickness, RGC, or axonal survival between the SOHU BX free drug and SOHU PBS groups, indicating that BX lacks neuroprotective efficacy.

[0155] Satisfactory biosafety of MAP / BX NP in mice. T o evaluate the translational potential of MAP / BX NP, we conducted a comprehensive investigation of its ocular irritation, corneal thickness, RGC viability, and systemic response using non-model mice (Figure 9). Based on our prior efficacy results, MAP / BX NP with the same drug concentrations was used for safety assessment. Ocular irritation was evaluated over 7 days using the fluorescein staining method. Slit lamp examination revealed no signs of corneal opacity or epithelial defects after 7-day treatment, indicating excellent ocular surface tolerance (Figure 9B). OCT images further confirmed that corneal thickness and GCC thickness in the MAP / BX NP group were comparable to the PBS control (Figures 9C-E), indicating MAP / BX NP had no damage to the cornea or retina after 7-day treatment. To further evaluate the biosafety of MAP / BX NP to RGCs, we conducted an immunohistochemistry test of whole mounts of retina and counted RGCs, which reflects RGC viability directly. As shown in Figures 9F&G, RGC densities in peripheral, middle, and central areas of retina in the MAP / BX NP group are all comparable to the PBS group, suggesting a great biocompatibility of MAP / BX NP. In addition, we also assessed the systemic response of MAP / BX NP (Figure 9H). Following 7 days of daily instillation, H&E staining showed no pathological alterations in the structure of the main organs (heart, liver, spleen, lung, and kidney). Collectively, these findings demonstrate that MAP / BX NP exhibits no ocular or systemic toxicity and holds promise for clinical application.ATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056

[0156] Since glaucoma is a multifactorial disease, a single drug treatment is usually insufficient. A study shows that nearly 40% of glaucoma patients require concomitant use of two or more antiglaucoma drugs. Thus, it is significant to fabricate a binary drug platform integrating the neuroprotectant with lOP-lowering agent to achieve IOP reduction and neuroprotection simultaneously. Inspired by the recent study that MAP, an antidepression drug, prevents neurodegeneration in glaucoma mouse model, we engineered the NeuProlO solid drug NP eye drop combining MAP and BX using flash nanoprecipitation method in MIVM. Solid drug NPs represent a transformative advance in drug delivery, characterized by the selfassembly of pure drug molecules into nanoscale structures without the extensive use of carrier excipients. Due to nanoscale size and lipophilic nature, solid drug NPs can cross the ocular barriers by endocytosis and direct permeation, reaching the deeper ocular areas more efficiently. Compared to conventional NP systems such as polymeric NPs, liposome, lipid NPs, micelles, etc., solid drug NPs offer several distinct advantages, including remarkably high drug loading, reduced immunogenicity and toxicity due to carrier absence, enhanced physicochemical stability, simplified synthesis and regulatory pathways. MIVM enables rapid mixing on the scale of milliseconds to microseconds by significantly reducing the diffusion distance between the solvent and antisolvent, due to its microscale geometry, leading to effective flash nanoprecipitation. Our results verified the MlVM’s effectiveness, enabling the preparation of MAP / BX NP with scalability, precise control, and high reproducibility.

[0157] The major obstacle to eye drop efficacy is the corneal barrier. In our study, the MAP / BX NP exhibited better corneal permeability than free drug formulation, indicating a superior drug transport through transcorneal route. It has been well-documented that NP can pass through the cell membrane by endocytosis. Conversely, due to the poor affinity between the hydrophilic free drug and lipophilic cell membrane, the MAP / BX free drug cannot cross the corneal barrier effectively. The cumulative transport percentage of MAP and BX in the same group displayed significant difference, with BX always showing higher permeability in both formulations. The cornea is composed of five layers, including epithelium, Bowman's layer, stroma, Descemet’s membrane, and endothelium from the outermost to the innermost. The epithelium and endothelium are lipophilic and other layers are all hydrophilic. After passing through the epithelium, the transport of MAP / BX NP will be hindered due to the existence of three hydrophilic layers in the middle and the drug release also happened during this process. It has been documented that the water solubilities of MAP and BX are 0.15 mg / L and 29.8 mg / L, respectively. Thus, we speculated that the released BX crossing the layer structure of cornea more effectively is attributed to its relatively higher hydrophilicity. There are another two possible routes of NP transport to the posterior eye besides the transcorneal route:ATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056conjunctival-scleral route and uveoscleral route. Conjunctival-scleral route is a more permeable alternative, exploiting the larger pore sizes and loose junctional structures of conjunctival tissue. This route facilitates efficient NP diffusion into sclera and further into the choroidal region, subsequently accessing the retina and optic nerve. Conjunctival-scleral route bypasses anterior clearance mechanisms and may account for substantial drug accumulation in posterior tissues. Additionally, evidence suggests a minor yet potentially impactful route, uveoscleral route, might also support NP distribution to posterior ocular tissues. In future studies, we can try to label NPs with near-infrared fluorescent dyes and use in vivo imaging to track their transport across ocular tissues after topical administration.

[0158] The pharmacokinetics of MAP and BX in the eyeball after NP treatment are important data for translation. It also helps establish the relationship between the administered dose and the concentration of the drug in target tissues. The elevated MAP and BX concentrations in the back of the eye after NP treatment further proved the better permeability of MAP / BX NP formulation. According to our recent study, MAP inhibits HRH1 to block ER Ca2+release, thereby restoring ER homeostasis, preventing harmful intracellular Ca2+influx, and ultimately protecting injured or diseased RGCs and optic nerves. Therefore, a higher MAP concentration in the retina and optic nerve in NP group is a promising signal, prompting us to utilize the SOHU glaucoma model to confirm its neuroprotection efficacy.

[0159] BX is a beta-1 adrenergic receptor antagonist (beta-blocker). In the ciliary body, there are beta-adrenergic receptors that regulate the production of aqueous humor. By blocking these beta-1 receptors, BX reduces the production of aqueous humor, leading to a decrease in IOP. Even though we did not test BX concentrations in the ciliary body, the concentration of BX in the aqueous humor is an indirect indicator. If BX is present in the aqueous humor, it is reasonable to infer that it has penetrated to the ciliary body and exerted its effect. Consequently, the higher concentration of BX in the aqueous humor after NP treatment is also very inspiring for our IOP in vivo study. With MALDI-IMS results, most of MAP was retained in the cornea area in free drug group but could reach the deeper area of the eyeball after NP treatment, consistent with our LC-MS / MS data. Regarding the BX, the highest intensity was observed in lens and vitreous body areas in both two groups, which also agreed well with the drug mass ratio analysis. After comparison of biodistribution between MAP NP and MAP / BX NP, MAP free drug and MAP / BX free drug groups, it was speculated that the BX might assist in the delivery of MAP to the posterior area of the eye, no matter in MAP / BX NP or MAP / BX free drug formulation. BX is a selective beta-1 adrenergic receptor antagonist known for reducing IOP. However, it has been reported to increase ocular blood flow by dilating blood vessels in the retina and optic nerve head, particularly in glaucoma patients. This improvedATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056perfusion may enhance the delivery of topically administered MAP to the deeper structures of the eye, including the retina and optic nerve. Improved blood flow facilitates better drug distribution and retention in the optic nerve and retina, potentially enhancing MAP’s neuroprotective effects. In addition, lower IOP reduces compression on the RGCs and optic nerve head, making it easier for MAP to penetrate and exert its therapeutic effects.

[0160] The IOP hypertension of SOHU glaucoma model was accomplished by injecting excessive silicone oil into the anterior chamber of the eyeball, until the oil droplet expanded to cover most areas of the iris. Given that the IOP reduction mechanism of BX is achieved by hindering the aqueous humor production, it is not suitable to directly apply the SOHU glaucoma model for lOP-lowering effect investigation, thus the normotensive rat was utilized in this study. Consistent with our hypothesis based on the biodistribution study, the sustained IOP reduction efficacy was proven after MAP / BX NP treatment, with eye drops administered every other day. This prolonged effect minimizes the frequency of dosing, enhancing patient compliance and reducing the burden of strict medication schedules, which is a common issue with conventional treatments requiring frequent application. Considering the nocturnal rhythm of rats, a 24-h IOP dynamics should be monitored in future studies to fully characterize the chronopharmacological behavior of this formulation. The SOHU glaucoma mice displayed a dramatically lower P1-N2 amplitude, GCC thickness, RGC and axon survivals than naive group, making it ideal for assessment of neuroprotection efficacy. All the MAP / BX NP and MAP / BX free drug groups exhibited greater P1-N2 amplitude of PERG, GCC thickness, RGC and axon survivals. In contrast, the MAP NP and MAP free drug did not show neuroprotective effect. The BX free drug was then utilized to confirm if it has any neuroprotective effect and the results showed that all the GCC thickness and RGC and axon survivals were lower than those in SOHU PBS group, indicating the neuroprotective effect in MAP / BX combination groups are attributed to the MAP. Recalling the biodistribution results, the BX could significantly increase the MAP delivery to the back of eyeball, especially retina and optic nerve, which provide robust evidence to elucidate this phenomenon. The BX in combination groups could facilitate the MAP delivery to the retina and optic nerve, but the MAP in single drug groups could not penetrate the ocular barriers effectively, leading to the poorly neuroprotective effect. This study further validated the synergistic therapeutic benefit of MAP / BX combination groups. Regarding biosafety evaluation in mice, the MAP / BX NP demonstrated great ocular surface tolerance, RGC safety, and no systemic toxicity after 7-day treatment. Given the better tissue permeability, biocompatibility, and sustained lOP-lowering capability, the MAP / BX NP formulation is a more promising pharmacological approach for glaucoma management.ATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056

[0161] Future studies may focus on optimizing MAP and BX dosing, elucidating the MAP / BX NP transport pathways in eyeball, particularly through advanced imaging techniques and in vivo models, assessing systemic drug exposure, investigating long-term formulation efficacy and safety. A more thorough pharmacokinetics study tracking drug concentration at different time points in the eyeball will be conducted by using larger animals, such as rabbit. The sensitivity of conjunctiva and cornea will be monitored regularly under slit lamp and OCT postadministration. The Chou-Talalay combination index method will be applied to further confirm the synergistic effect of MAP / BX group. The hypertensive glaucoma animal model suitable for IOP monitoring will also be utilized in our expanded studies in the future. In addition, reduced dosing frequency enhances patient compliance but necessitates greater precision in dosing and delivery. The delivery device that simplifies self-administration while ensuring accurate dosing is highly warranted.

[0162] In summary, this study engineered NeuProlO, a dual-action MAP / BX NP eye drop formulation, for noninvasive and efficient synergistic glaucoma treatment. The preparation process is controllable, scalable, and reproducible using a MIVM. NeuProlO significantly improved corneal permeation due to its lipophilic nature, enhancing drug delivery to deeper ocular tissues. Biocompatibility tests confirmed MAP / BX NP’s low cytotoxicity and minimal irritation. Biodistribution studies demonstrated higher MAP concentrations in the posterior eye, suggesting strong tissue permeability. Furthermore, in vivo studies showed that NeuProlO provided sustained IOP reduction, while histological analysis confirmed its neuroprotective efficacy, verifying its synergistic therapeutic benefits. Our biosafety evaluation revealed that NeuProlO exhibited superior ocular surface tolerance and RGC safety with minimal systemic response, underscoring its favorable biocompatibility profile. These findings highlight NeuProlO’s potential to advance ophthalmic therapeutics, offering an effective and noninvasive approach for glaucoma treatment and other ocular diseases.References[1] K. Allison, D. Patel, O. Alabi, Epidemiology of glaucoma: the past, present, and predictions for the future, Cureus 12(11) (2020).[2] H. Jayaram, M. Kolko, D.S. Friedman, G. Gazzard, Glaucoma: now and beyond, The Lancet 402(10414) (2023) 1788-1801.[3] D. Wang, W. Huang, Y. Li, Y. Zheng, P.J. Foster, N. Congdon, M. He, Intraocular pressure, central corneal thickness, and glaucoma in Chinese adults: the liwan eye study, American journal of ophthalmology 152(3) (2011) 454-462. e1.ATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056[4] P. Wostyn, M. Nedergaard, Should poor sleep be added to the list of risk factors for normaltension glaucoma in the future?, Eye (2025) 1-2.[5] V. Chrysostomou, F. Rezania, I. A. Trounce, J.G. Crowston, Oxidative stress and mitochondrial dysfunction in glaucoma, Current opinion in pharmacology 13(1) (2013) 12-15.[6] D.J. Calkins, Critical pathogenic events underlying progression of neurodegeneration in glaucoma, Progress in retinal and eye research 31 (6) (2012) 702-719.[7] C. Baudouin, M. Kolko, S. Melik-Parsadaniantz, E.M. Messmer, Inflammation in Glaucoma: From the back to the front of the eye, and beyond, Progress in retinal and eye research 83 (2021) 100916.[8] T. Wang, L. Cao, Q. Jiang, T. Zhang, Topical medication therapy for glaucoma and ocular hypertension, Frontiers in Pharmacology 12 (2021) 749858.[9] K. Pei, M. Georgi, D. Hill, C.F.J. Lam, W. Wei, M.F. Cordeiro, Neuroprotective Nanocarriers in Glaucoma, Pharmaceuticals 17(9) (2024) 1190.

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[0163]

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[0164] The preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention,ATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied by the appended claims.

Claims

1. ATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-056WHAT IS CLAIMED IS:1 . A composition of uniform solid drug nanoparticles comprising of at least two active agents: (i) a neuroprotectant, and (ii) a p adrenergic blocker.

2. The composition of claim 1 , wherein the neuroprotectant is maprotiline.

3. The composition of claim 1 or claim 2, wherein the adrenergic blocker is betaxolol.

4. The composition of any of the previous claims, wherein one or both of the active agents are present in an un-ionized form.

5. The composition of any of the previous claims wherein the ratio of (i) : (ii) is from about 10:1 to about 1 :10 (mass:mass).

6. The composition of any of the previous claims wherein the ratio of (i) : (ii) is about 1 :1.

7. The composition of any of the previous claims in a lyophilized formulation.

8. The composition of claim 7, wherein the lyophilized formulation comprises an effective amount of a stabilizing agent.

9. The composition of claim 8, wherein the stabilizing agent is trehalose or an analog thereof.

10. The composition of claim 8 or claim 9, wherein the stabilizing agent is present at a concentration of from about 0.5 to 2% w / v.11 . The composition of any of claims 1 -10 in an aqueous diluent.

12. The composition of claim 11 wherein the aqueous diluent is ophthalmically acceptable.ATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-05613. The composition of claim 11 or claim 12, wherein the solid drug nanoparticles are present in the solution at a concentration of from about 0.5% nanoparticles (w / v) to about 10% nanoparticles (w / v).

14. The composition of any of the previous claims, wherein the nanoparticles have an average hydrodynamic diameter of from about 50 to about 500 nm.

15. The composition of any of the previous claims, wherein the nanoparticles have an average hydrodynamic diameter of from about 100 to about 300 nm.

16. The composition of any of the previous claims, wherein the population of nanoparticles has a polydispersity index (PDI) of less than about 0.2.

17. A method for treatment of ocular disease, the method comprising administering topically to the eye of a subject an effective dose of a composition of any of claims 11-16.

18. The method of claim 17, wherein the ocular disease is glaucoma.

19. The method of claim 18, wherein the glaucoma is open-angle glaucoma.

20. The method of claim 18, wherein the glaucoma is angle-closure glaucoma.21 . The method of claim 17, wherein the ocular disease is ocular hypertension.

22. The method of any of claims 17-21 , wherein the effective dose is 1-2 drops per affected eye, where a drop is from about 5 ml to about 50 ml.

23. The method of any of claims 17-22, wherein the effective dose comprises from about 0.1 mg active agents to about 50 mg active agents.

24. The method of any of claims 17-23, comprising daily or semi-weekly administration.

25. A kit comprising a nanoparticle composition of any of claims 1-16, and instructions for use.ATTORNEY DOCKET NAME: STAN-2260WO CLIENT REFERENCE: S25-05626. The kit of claim 25, wherein the nanoparticle composition is provided in a unit dose.

27. The kit of claim 25, wherein the nanoparticle composition is provided in a device suitable for aqueous reconstitution and application.

28. A method for manufacture of the composition of any of claims 1-16, the method comprising:dissolving each of (i) a neuroprotectant and (ii) a p-1 adrenergic blocker in a polar diluent to generate an active agent solution;mixing a stream of the active agent solution with an aqueous stream in a vortex mixer to produce uniform nanoparticles;collecting the uniform nanoparticles; andremoving the polar diluent.

29. The method of claim 28, wherein the streams are mixed in a multi-inlet vortex mixer (MIVM).

30. The method of claim 28 or claim 29, wherein the uniform nanoparticles are admixed with a stabilizing agent.31 . The method of claim 30 wherein the stabilizing agent is trehalose or an analog thereof.

32. The method of claim 30 or claim 31 , further comprising lyophilizing the admixture.

33. The method of any claims 28-32, wherein the neuroprotectant is maprotiline.

34. The method of any claims 28-33, wherein the adrenergic blocker is betaxolol.

35. The method of any claims 28-34, wherein one or both of the active agents are present in an un-ionized form.

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