Formulations for delivery of a colloidal prodrug to lower intraocular pressure (IOP) for a prolonged time

Abstract

The present disclosure relates to colloidal drug aggregates (CDAs) that are further embedded in hydrogels for sustained local delivery. Preferred drugs include beta-blocker prodrugs and ROCK inhibitors for use in treating glaucoma. In particularly preferred embodiments, the CDAs comprise prodrugs of timolol or netarsudil in combination with phospholipid stabilizers. Preferred hydrogels include hyaluronan crosslinked using an oxime bond.

Description

[0001] FORMULATIONS FOR DELIVERY OF A COLLOIDAL PRODRUG TO LOWER INTRAOCULAR PRESSURE (IOP) FOR A PROLONGED TIME

[0002] FIELD

[0003] The present disclosure relates to colloidal drug aggregates (CDAs) embedded in hydrogels for sustained local delivery, and more particularly the present disclosure relates to prodrug-derived CDAs embedded in the hydrogel, wherein the promoiety present on the prodrug enables formation of CDAs that occur when the therapeutic agent is in its prodrug form, and wherein the active drug is subsequently released from the hydrogel upon conversion of the prodrug.

[0004] BACKGROUND

[0005] In the field of ocular drug delivery, there has been significant research focused on developing sustained-release formulations to prolong the therapeutic effect of beta-blockers for reducing intraocular pressure (IOP) in patients with glaucoma (Allyn et al., 2022). Conventional eye drops containing beta-blockers, such as timolol, are widely prescribed, but they are associated with poor bioavailability and patient compliance issues due to the need for frequent administration (typically two to three times per day) (Inoue, 2014). As a result, only a small percentage of the drug reaches the target ocular tissues, while the majority is lost through drainage or systemic absorption (Dosmar et al., 2022). This has driven efforts to develop alternative delivery systems that may provide longer durations of action while minimizing the risk of systemic side effects. Several strategies have been employed to address these challenges, including the use of nanoparticle-based controlled release systems.

[0006] Liposomes, polymeric nanoparticles, and nanocrystals have been shown to improve the pharmacokinetics of encapsulated drugs, protect them from degradation, and enhance delivery to diseased ocular tissues (Dang & Shoichet, 2024). Despite these advances, many nanoparticle-based formulations are limited by low drug-loading efficiencies, with most systems achieving less than 30% drug loading (Allyn et al., 2022). Furthermore, materials commonly used for nanoparticle construction, such as tetraethyl orthosilicate and gold nanoparticles, are often unsuitable for ocular applications due to their potential toxicity and poor biocompatibility (Zhong et al., 2016).

[0007] In addition to nanoparticles, other approaches such as soft contact lenses, hydrogels, and implants have been explored as sustained-release platforms for ocular drug delivery. For instance, formulations like Nyogel® and Timoptic XE® have been developed using gellan gum and other gelling agents to extend drug release and reduce the frequency of administration (Baranowski et al., 2014). However, these formulations provide only short-term release, typically less than 24 hours (Shedden et al., 2001). Similarly, Pech et al. synthesized amphiphilic timolol prodrugs, such as palmitoyl timolol malonate, to improve corneal retention, but their efficacy is limited to a few hours, necessitating frequent re-application (Pech et al., 1996). Lipid-based nanoparticle systems, such as lipid-conjugated micelles, have shown some promise in improving ocular retention and providing sustained release of timolol for up to 14 days (Taskar et al., 2017). However, these systems often suffer from nanoparticle stability issues, which limit their long-term effectiveness. Biodegradable polymeric systems, such as poly(lactic-co-glycolic acid) (PLGA) microspheres, have also been investigated for delivering timolol over extended durations, with some studies reporting drug release for up to 90 days in animal models (Lavik et al., 2016). Despite this, the low drug-loading capacities (often less than 5%) and the potential generation of acidic degradation products that may cause ocular toxicity have raised concerns regarding the clinical translation of these systems.

[0008] Colloidal drug aggregates (CDAs) are another emerging platform for drug delivery. CDAs are self-assembled, drug-rich nanoparticles that allow for high drug-loading efficiencies (typically greater than 70%) with minimal use of excipients (Ganesh et al., 2017). Stabilization of CDAs with polymers (McLaughlin et al., 2016), proteins (Ganesh et al., 2019), lipids (Donders et al., 2023), or other small-molecule aggregators (Chen et al., 2023) has enabled their use in intravenous drug delivery. However, not all drugs are known to form CDAs, therefore limiting the use of CDAs for drug delivery. Given the limitations of current sustained-release formulations for lOP-lowering drugs, including low drug loading, toxicity concerns, and the need for frequent re-administration, there remains an unmet need for a non-invasive, long-acting delivery system with high drug-loading capacity and minimal systemic exposure.

[0009] SUMMARY

[0010] The present disclosure provides colloidal drug aggregates (CDAs) of prodrugs embedded in hydrogels for sustained local delivery, and with particular attention focused on beta-blocker prodrug CDAs and Rho-associated protein kinase (ROCK) inhibitor prodrug CDAs and beta-blocker and ROCK inhibitor colloid hydrogel formulations formulated for ophthalmic administration to the eye to lower intraocular pressure (IOP) for the treatment of ocular hypertension or glaucoma.

[0011] A colloidal nanoparticle, a colloidal nanoparticle hydrogel formulation, a method of producing a colloidal nanoparticle hydrogel formulation for controlled and sustained release, and a method of treating ocular hypertension in a patient are provided. Accordingly, in an aspect, there is provided a colloidal nanoparticle. The colloidal nanoparticle comprises a therapeutic agent capable of forming colloidal drug aggregates (CDAs) under conditions effective to form CDAs, the therapeutic agent being a prodrug of a parent drug that is chemically conjugated to a promoiety and lipid excipients present in an amount effective to enhance the stability and delivery of the therapeutic agent, wherein a mass ratio of the therapeutic agent to the lipid excipients is from about 1 :99 to about 99:1 (w / w). The promoiety comprises (i) one or more hydrocarbon groups selected from alkyl, aryl, and cycloalkyl carbon chains each having at least six carbons, and / or (ii) an alkyl-based or non-alkyl segment having a carbon backbone and / or ring system interrupted or substituted by one or more heteroatoms selected from oxygen, nitrogen, and sulfur, or by one or more halogens selected from fluorine, chlorine, bromine, and iodine, The parent drug is conjugated to the promoiety via a bond selected from ester, amide, carbamate, hydrazone, disulfide, boronic ester, borate ester, phosphonate, glycosidic, anhydride, thioester, phosphate, urea, thiourea, imine, oxime, acetal, ketal, peptidic, succinimide, ether, azo, lactone, lactam, carbonate, sulfonic ester, sulfonamide, siloxane, pyrophosphate, phosphoramide, phosphoramidate, thioether, orthoester, oxoester, vinyl ether, thionoester, and sulfenamide bonds. In a particular case, the promoiety comprises one or more functional groups selected from ether, thioether, amide, carbamate, urea, carbonate, and ester linkages.

[0012] In another case, the promoiety further comprises one or more segments selected from aryl, heteroaryl, cycloalkyl, heterocycloalkyl, and polyethylene glycol) (PEG) segments, or combinations thereof.

[0013] In yet another case, the parent drug is a beta-blocker drug, and the prodrug is beta-blocker prodrug or wherein the parent drug is a ROCK inhibitor drug and the pro-drug is a ROCK inhibitor prodrug.

[0014] In yet another case, the beta-blocker is timolol and the beta-blocker prodrug comprises at least one compound selected from the group consisting of timolol octanoate, timolol palmitate, timolol decylbenzoate, and timolol-abietate.

[0015] In yet another case, the beta-blocker is timolol and the beta-blocker prodrug timolol palmitate.

[0016] In yet another case, the ROCK inhibitor drug is netarsudil-M1 and the ROCK-inhibitor is netarsudil.

[0017] In yet another case, the lipid excipients comprise one or more lipids selected from the group consisting of phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines (PS), phosphatidylglycerols, phosphatidylinositols (PI), phosphatidic acids (PA), sphingolipids, cholesterol and cholesterol derivatives, triglycerides, diglycerides, cationic lipids, neutral lipids, ionizable lipids, polyethylene glycol (PEG) conjugated lipids, ceramides and derivatives, fatty acids, and fatty acid esters.

[0018] Accordingly, in an aspect, there is provided a colloidal nanoparticle hydrogel formulation. The formulation comprises the colloidal nanoparticles as described herein; and a biocompatible hydrogel matrix, wherein the biocompatible hydrogel matrix is formed by crosslinking of a biocompatible hydrogel precursor in solution and the colloidal nanoparticles are embedded within the biocompatible hydrogel matrix forming a colloid nanoparticle hydrogel formulation for controlled release of the parent drug and wherein the release of the parent drug from the colloidal nanoparticle is mediated by hydrolysis of the bonds releasing the parent drug from the promoiety.

[0019] In a particular case, the release rate of the parent drug is modulated by a factor selected from the number of carbons of the promoiety, the length of the promoiety, the degree of branching of the promoiety, the steric bulkiness of the promoiety, and any combination thereof.

[0020] In another case, the release rate of the parent drug is modulated by the number of carbons in the promoiety, with shorter alkyl chains and smaller cycloalkyl or aryl groups resulting in a faster release and longer alkyl chains and larger cyclo-alkyl or aryl groups resulting in a slower release.

[0021] In yet another case, the biocompatible hydrogel is formed from one or more polymers selected from the group consisting of natural polymers and synthetic polymers.

[0022] In yet another case, the biocompatible hydrogel is a hyaluronan-based hydrogel.

[0023] In yet another case, the biocompatible hydrogel is a polyethylene glycol- based hydrogel.

[0024] In yet another case, the biocompatible hydrogel is a mixed hydrogel comprising a hyaluronan-based hydrogel and a polyethylene glycol-based hydrogel.

[0025] In yet another case, the colloidal nanoparticle hydrogel formulation as described herein further comprises at least one additional drug.

[0026] In yet another case, the parent drug is a beta-blocker and the at least one additional drug comprises at least one non-beta-blocker drug.

[0027] In yet another case, the beta-blocker comprises at least one compound selected from the group consisting of acebutolol, atenolol, betaxolol, bisoprolol, carvedilol, labetalol, metoprolol, nebivolol, nadolol, pindolol, propranolol, sotalol, timolol, and combinations thereof, and wherein the colloidal nanoparticle hydrogel formulation is formulated for ophthalmic administration for lowering intraocular pressure.

[0028] In yet another case, the beta-blocker is timolol and the beta-blocker prodrug comprises at least one compound selected from the group consisting of timolol octanoate, timolol palmitate, timolol decylbenzoate, and timolol-abietate.

[0029] In yet another case, the beta-blocker is timolol and the beta-blocker prodrug timolol palmitate.

[0030] In yet another case, the at least one additional drug comprises at least one non-beta-blocker intraocular pressure-lowering drug. In yet another case, the at least one non-beta-blocker intraocular pressure-lowering drug comprises one or more agents selected from the group consisting of carbonic anhydrase inhibitors (CAIs), Rho kinase (ROCK) inhibitors, alpha-agonists and prostaglandin analogs.

[0031] In yet another case, the parent drug is a ROCK Inhibitor and the at least one additional drug comprises at least one non-ROCK-inhibitor drug.

[0032] In yet another case, the ROCK inhibitor is netarsudil-M1 , and wherein the colloidal nanoparticle hydrogel formulation is formulated for ophthalmic administration for lowering intraocular pressure.

[0033] In yet another case, the at least one additional drug comprises at least one non-ROCK-inhibitor intraocular pressure-lowering drug.

[0034] In yet another case, the at least one non-ROCK inhibitor intraocular pressure-lowering drug comprises one or more agents selected from the group consisting of beta blockers carbonic anhydrase inhibitors (CAIs), alpha-agonists and prostaglandin analogs.

[0035] In yet another case, the colloidal nanoparticle hydrogel formulation is administered by a route selected from the group consisting of subconjunctival injection, intravitreal injection, topical application, transscleral injection, intracameral injection, suprachoroidal injection, subtenon injection, subretinal injection, intravenous administration.

[0036] In yet another case, the route of administration is selected for the treatment of ocular hypertension or glaucoma.

[0037] In yet another case, the selected factor modulating the release rate of the parent drug is sufficient to maintain therapeutic levels of the drug in the eye for a period of at least 24 hours post-administration.

[0038] In yet another case, the selected factor modulating the release rate of the parent drug is the number of carbons in the promoiety.

[0039] In yet another case, wherein the period of time is greater than about 7 days and up to about 1 year.

[0040] In yet another case, the period of time is from about 7 days to about 6 months.

[0041] In yet another case, the period of time is from about 7 days to about 3 months. In yet another case, the period of time is from about 7 days to about 1 month.

[0042] In yet another case, the period of time is from about 7 days to about 14 days.

[0043] In yet another case, the promoiety comprises from about six to about hundred carbons, wherein higher numbers of carbons are associated with longer release times and lower numbers of carbons are associated with shorter release times.

[0044] In yet another case, the promoiety has from about six to about twenty- four carbons.

[0045] Accordingly, in an aspect, there is provided a method of producing a colloidal nanoparticle hydrogel formulation for controlled and sustained release of a parent drug, the formulation comprising colloidal nanoparticles that comprise a prodrug of the parent drug, the prodrug being capable of forming colloidal drug aggregates. The method comprises the steps of mixing the prodrug with lipid excipients in an organic solvent to form a mixture; suspending the mixture in an aqueous buffer solution to induce formation of prodrug colloidal nanoparticles; concentrating the prodrug colloidal nanoparticles to form a suspension of concentrated colloidal nanoparticles; mixing the suspension of concentrated colloidal nanoparticles with a biocompatible hydrogel precursor; and inducing gelation of the hydrogel precursor to embed the concentrated colloidal nanoparticles within a biocompatible hydrogel formed by crosslinking of the biocompatible hydrogel precursor, thereby forming , thereby forming the colloidal nanoparticle hydrogel formulation, wherein controlled and sustained release of the parent drug is mediated by hydrolysis of the bonds releasing the parent drug from the alkyl carbon chains, aryl groups, or cyclo-alkyl carbon chains of the promoiety.

[0046] In a particular case, the method described herein further comprises the step of synthesizing the prodrug by reacting the parent drug with promoiety comprising hydrocarbons selected from the group consisting of alkyl carbon chains, aryl groups, and cyclo-alkyl carbon chains, the alkyl carbon chains, aryl groups, or cyclo-alkyl carbon chains having at least six carbons, the parent drug being conjugated to the alkyl carbon chains, aryl groups, or cyclo-alkyl carbon chains through bonds selected from the group consisting of ester bonds, amide bonds, carbamate bonds, hydrazone bonds, disulfide bonds, boronic ester bonds, borate ester bonds, phosphonate bonds, glycosidic bonds, anhydride bonds, thioester bonds, phosphate bonds, urea bonds, thiourea bonds, imine bonds, oxime bonds, acetal bonds, ketal bonds, peptidic bonds, succinimide bonds, ether bonds, azo bonds, lactone bonds, lactam bonds, carbonate bonds, sulfonic ester bonds, sulfonamide bonds, siloxane bonds, pyrophosphate bonds, phosphoramide bonds, phosphoramidate bonds, thioether bonds, orthoester bonds, oxoester bonds, vinyl ether bonds, thionoester bonds, and sulfenamide bonds.

[0047] In another case, the promoiety has from about six to about hundred carbons.

[0048] In yet another case, the release rate of the parent drug is modulated by a factor selected from the number of carbons of thepromoiety, the length of the promoiety, the degree of branching of the promoiety, the steric bulkiness of the promoiety, and any combination thereof.

[0049] In yet another case, the release rate of the parent drug is modulated by the number of carbons of the promoiety, with shorter alkyl chains and smaller cyclo-alkyl or aryl groups resulting in a faster release and longer alkyl chains and larger cyclo-alkyl or aryl groups resulting in a slower release.

[0050] In yet another case, the biocompatible hydrogel is formed from one or more polymers selected from the group consisting of natural polymers and synthetic polymers.

[0051] In yet another case, the biocompatible hydrogel is a hyaluronan-based hydrogel.

[0052] In yet another case, the biocompatible hydrogel is a polyethylene glycol- based hydrogel.

[0053] In yet another case, the biocompatible hydrogel is a mixed hydrogel comprising a hyaluronan-based hydrogel and a polyethylene glycol-based hydrogel.

[0054] In yet another case, the lipid excipients comprise one or more lipids selected from the group consisting of phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines (PS), phosphatidylglycerols, phosphatidylinositols (PI), phosphatidic acids (PA), sphingolipids, cholesterol and cholesterol derivatives, triglycerides, diglycerides, cationic lipids, neutral lipids, ionizable lipids, polyethylene glycol (PEG) conjugated lipids, ceramides and derivatives, fatty acids, and fatty acid esters.

[0055] In yet another case, the lipid excipients comprise one or more lipids selected from the group consisting of phosphatidylcholines selected from Dipalmitoylphosphatidylcholine (DPPC), Distearoylphosphatidylcholine (DSPC), and Soybean Phosphatidylcholine (SPC); phosphatidylethanolamines selected from Distearoylphosphatidylethanolamine (DSPE) and Dimyristoylphosphatidylethanolamine (DMPE); phosphatidylglycerols selected from Dioleoylphosphatidylglycerol (DOPG); phosphatidylinositols (PI); sphingolipids selected from Sphingomyelin; cationic lipids selected from 1 ,2- dioleoyl-3-trimethylammonium-propane (DOTAP) and 1 ,2-dioleoyl-3- dimethylammonium-propane (DODAP); neutral lipids selected from Dioleoylphosphatidylcholine (DOPC); ionizable lipids selected from SM-102 and DLin-MC3-DMA; and polyethylene glycol (PEG) conjugated lipids selected from 1 ,2-dimyristoyl-rac-glycerol-PEG2000 (DMG-PEG2000) and DSPE-PEG2000.

[0056] In yet another case, the organic solvent comprises one or more solvents selected from the group consisting of ethanol, methanol, isopropanol, butanol, dichloromethane (DCM), chloroform, dimethyl sulfoxide (DMSO), acetone, acetonitrile, tetrahydrofuran (THF), dimethylformamide (DMF), and toluene.

[0057] In yet another case, the aqueous buffer solution comprises one or more buffers selected from the group consisting of phosphate-buffered saline (PBS), Tris-buffered saline (TBS), HEPES buffer, citrate buffer, acetate buffer, borate buffer, sodium bicarbonate buffer, MES buffer, and malate buffer.

[0058] In yet another case, concentrating the colloidal nanoparticles to form a suspension of concentrated colloidal nanoparticles comprises using one or more of centrifugal filters, dialysis bags, or tangential flow filtration to remove excess solvent and unincorporated materials, thereby concentrating the nanoparticle suspension while maintaining the stability and integrity of the colloids.

[0059] In yet another case, the parent drug is a beta-blocker drug, and the prodrug is beta-blocker prodrug.

[0060] In yet another case, the beta-blocker comprises at least one compound selected from the group consisting of acebutolol, atenolol, betaxolol, bisoprolol, carvedilol, labetalol, metoprolol, nebivolol, nadolol, pindolol, propranolol, sotalol, timolol, and combinations thereof.

[0061] In yet another case, the beta-blocker is timolol and the beta-blocker prodrug comprises at least one compound selected from the group consisting of timolol octanoate, timolol palmitate, timolol decylbenzoate, and timolol-abietate.

[0062] In yet another case, the beta-blocker is timolol and the beta-blocker prodrug timolol palmitate.

[0063] In yet another case, the method described herein further comprises the step of adding at least one non-beta-blocker intraocular pressure-lowering drug.

[0064] In yet another case, the at least one non-beta-blocker intraocular pressure-lowering drug is added during the step of mixing the suspension of concentrated colloidal nanoparticles with the biocompatible hydrogel precursor.

[0065] In yet another case, the at least one non-beta-blocker intraocular pressure-lowering drug comprises one or more agents selected from the group consisting of carbonic anhydrase inhibitors (CAIs), Rho kinase (ROCK) inhibitors, alpha-agonists and prostaglandin analogs.

[0066] In yet another case, the parent drug is a ROCK inhibitor drug, and the pro-drug is a ROCK inhibitor prodrug.

[0067] In yet another case, the ROCK inhibitor is netarsudil-M1 .

[0068] In yet another case, the method described herein comprises the step of adding at least one non-ROCK-inhibitor intraocular pressure-lowering drug.

[0069] In yet another case, the at least one non-ROCK-inhibitor intraocular pressure-lowering drug is added during the step of mixing the suspension of concentrated colloidal nanoparticles with the biocompatible hydrogel precursor.

[0070] In yet another case, the at least one non-ROCK inhibitor intraocular pressure-lowering drug comprises one or more agents selected from the group consisting of beta blockers carbonic anhydrase inhibitors (CAIs), alpha-agonists and prostaglandin analogs.

[0071] Accordingly, in an aspect, there is provided a colloidal nanoparticle hydrogel formulation produced according to the method described herein.

[0072] In a particular case, the parent drug is a beta-blocker drug, and the prodrug is beta-blocker prodrug or wherein the parent drug is a ROCK inhibitor, and the prodrug is ROCK-inhibitor prodrug. Accordingly, in an aspect, there is provided a method of treating ocular hypertension in a patient. The method comprises administering to an eye of the patient the colloidal nanoparticle hydrogel formulation described herein.

[0073] In a particular case, the colloidal nanoparticle hydrogel formulation is administered by subconjunctival, intravitreal, topical, transscleral, intracameral, suprachoroidal, subtenon, or subretinal injection for the treatment of ocular hypertension or glaucoma.

[0074] In another case, the number of carbons in the hydrocarbons of the promoiety is sufficient to maintain therapeutic levels of the drug in the eye for a period of at least 24 hours post-administration.

[0075] In yet another case, the period of time is greater than about 7 days and up to about 1 year.

[0076] In yet another case, the period of time is from about 7 days to about 6 months.

[0077] In yet another case, the period of time is from about 7 days to about 3 months.

[0078] In yet another case, the period of time is from about 7 days to about 1 month.

[0079] In yet another case, the period of time is from about 7 days to about 14 days.

[0080] A further understanding of the functional and advantageous aspects of the disclosure may be realized by reference to the following detailed description and drawings.

[0081] BRIEF DESCRIPTION OF THE DRAWINGS

[0082] Embodiments will now be described, by way of example only, with reference to the drawings, in which:

[0083] Figure 1 illustrates scattering intensity versus concentration for timolol. Linear regression fit shows that timolol does not form colloid drug aggregates (n = 4, mean ± standard deviation).

[0084] Figure 2 shows timolol prodrug colloids, dispersed in hyaluronan-oxime hydrogel, are tested for in vitro release and in vivo efficacy for local and sustained delivery of timolol. Timolol, a p-blocker used in the treatment of glaucoma, was chemically modified into hydrophobic hydrolysable ester prodrugs, thereby forming timolol prodrugs. Hydrophobically-modified timolol self-assembles into drug-rich colloidal drug aggregates. Timolol release from the colloid-loaded hydrogel was evaluated first in vitro and then in vivo by measuring the IOP after subconjunctival injection in rats.

[0085] Figures 3A to 3E show chemical modification of timolol with hydrophobic moieties results in colloidal drug aggregates, in which:

[0086] Figure 3A shows the synthesis of timolol prodrugs. Timolol maleate salt (1) was treated with 2 M NaOH to enhance the basicity of the hydroxyl group. Following extraction, the resultant timolol (2) reacted with hydrophobic acyl chloride groups, leading to the formation of timolol prodrugs (3a, 3b, 3c, and 3d);

[0087] Figure 3B shows plots of scattering intensity versus concentration for timolol prodrugs with linear regression fits were used to determine critical aggregation concentration (CAC) values (n = 4, mean ± standard deviation);

[0088] Figure 3C shows representative images of cryo-TEM for timolol prodrugs 3b, 3c and 3d (scale bar = 100 nm);

[0089] Figure 3D is a histogram of hydrodynamic diameter (nm) for three (3) timolol prodrugs 3b, 3c and 3d; and

[0090] Figure 3E shows polydispersity index of lipid-stabilized timolol prodrug CDAs (n = 3, mean ± standard deviation).

[0091] Figures 4A to 4B show timolol maleate (1) treated with 2 M NaOH to form timolol (2), in which:

[0092] Figure 4A is a 1 H-NMR (500 MHz, DMSO-d6) with peaks at 5 ppm 5.02 (s, 1 H), 4.39 (dd, J = 10.6, 4.1 Hz, 1 H), 4.29 (dd, J = 10.6, 6.1 Hz, 1 H), 3.81 (t, J = 5.6 Hz, 1 H), 3.72 - 3.66 (m, 4H), 3.47 - 3.42 (m, 6H), 2.56 (dd, J = 6.0, 2.2 Hz, 2H), 1.00 (s, 9H); and

[0093] Figure 4b shows mass spectrometry with the main peak at 317.16 m / z corresponding to the expected mass of timolol (2).

[0094] Figures 5A to 5B show characterization of timolol butanoate (3a), in which: Figure 5A is a 1 H-NMR (500 MHz, DMSO-d6) with peaks at 5 ppm 5.12 (qd, J = 6.3, 3.1 Hz, 1 H), 4.52 - 4.48 (m, 1 H), 4.37 (dd, J = 11 .5, 6.6 Hz, 1 H), 3.65 - 3.56 (m, 4H), 3.39 - 3.27 (m, 4H), 2.17 (dq, J = 14.8, 7.6 Hz, 2H), 1.48 (p, J = 7.4 Hz, 1 H), 0.95 (s, 9H), 0.79 (td, J = 7.4, 2.6 Hz, 3H); and

[0095] Figure 5B shows mass spectrometry with the main peak at 317.16 m / z corresponding to the expected mass of timolol (2). Mass spectrum of 3a. The main peak at 387.21 m / z corresponds to the expected mass of 3a.

[0096] Figures 6A to 6B show characterization of timolol octanoate (3b), in which:

[0097] Figure 6A shows the analysis by 1 H-NMR (500 MHz, DMSO-d6) with peaks at 5 ppm 5.37 (d, J = 8.5 Hz, 1 H), 4.65 - 4.54 (m, 1 H), 4.44 (dd, J = 11 .7, 5.7 Hz, 1 H), 3.82 - 3.73 (m, 1 H), 3.68 (d, J = 4.8 Hz, 4H), 3.61 (d, J = 16.6 Hz, 1 H), 3.43 - 3.37 (m, 4H), 2.37 (q, J = 8.3 Hz, 2H), 2.23 (dt, J = 14.6, 7.4 Hz, 2H), 1.47 (q, J = 7.7, 7.2 Hz, 4H), 1.34 (s, 9H), 1.20 (s, 6H), 0.84 (d, J = 8.6 Hz, 3H); and

[0098] Figure 6B shows the mass spectrum of 3b. The main peak at 443.36 m / z corresponds to the expected mass of 3b.

[0099] Figures 7A to 7B show characterization of timolol palmitate (3c), in which:

[0100] Figure 7A shows the analysis by 1 H-NMR (500 MHz, DMSO-d6) of 3c revealed peaks at 5 ppm 5.36 (ddt, J = 11 .6, 5.8, 3.1 Hz, 1 H), 4.59 (dd, J = 11 .6, 3.1 Hz, 1 H), 4.43 (dd, J = 11 .7, 5.6 Hz, 1 H), 3.68 (t, J = 4.9 Hz, 4H), 3.45 - 3.35 (m, 5H), 2.43 - 2.32 (m, 2H), 2.17 (t, J = 7.4 Hz, 12H), 1.51 - 1.41 (m, 14H), 1.34 (s, 10H), 1.12 (d, J = 6.6 Hz, 3H), 0.87 (s, 3H); and

[0101] Figure 7B shows the mass spectrum of 3c. The main peak at 555.40 m / z corresponds to the expected mass of 3c.

[0102] Figures 8A to 8B show characterization of timolol decylbenzoate (3d), in which: Figure 8A shows the analysis by 1 H-NMR (500 MHz, DMSO-d6) of 3d revealed peaks at 5 ppm 7.14 (d, J = 12.2 Hz, 4H), 5.30 (d, J = 4.8 Hz, 1 H), 4.06 (dd, J = 10.4, 4.1 Hz, 1 H), 3.98 (q, J = 4.2, 3.5 Hz, 1 H), 3.61 (s, 4H), 3.31 - 3.21 (m, 4H), 2.55 (dd, J = 8.6, 6.6 Hz, 2H), 1.56 - 1 .52 (m, 2H), 1 .49 (s, 9H), 1.25 - 1.21 (m, 16H), 0.85 (s, 3H); and

[0103] Figure 8B shows the mass spectrum of 3d. The main peak at 561 .34 m / z corresponds to the expected mass of 3d.

[0104] Figures 9A to 9D show the stability study of timolol prodrug colloids with and without lipid excipients, in which:

[0105] Figure 9A illustrates hydrodynamic diameter of bare timolol prodrug colloids over 28 d (n = 4, mean ± standard deviation);

[0106] Figure 9B shows scattering intensity of bare timolol prodrug colloids over 28 d (n = 4, mean ± standard deviation);

[0107] Figure 9C illustrates hydrodynamic diameter of timolol prodrug colloids with lipid excipients over 28 d (n = 3, mean ± standard deviation); and

[0108] Figure 9D shows scattering intensity of timolol prodrug colloids with lipid excipients over 28 d (n = 3, mean ± standard deviation);

[0109] Figures 10A to 10B show 1 H-NMR of HA-modified ketone (HAK) and aldehyde (HAA), in which:

[0110] Figure 10A shows the 1 H-NMR of HAK. The ketone substitution onto HA was determined to be approximately 38%. The N-acetyl group of HA was used as a reference to calculate ketone degree of substitution; and

[0111] Figure 10B shows the 1 H-NMR of HA-modified tertbutyl carbazate. The aldehyde substitution onto HA was determined to be approximately 48% using the integration of the tert-butyl peak as a proxy.

[0112] Figure 11 shows the 1 H-NMR of PEG(NH2)4, PEG(ONHBoc)4and PEGOA4. All 1 H-NMR were acquired at 500 MHz with 512 scans in CDCh. PEG(NH2)4(bottom) underwent a reaction with 2- ((tertbutoxycarbonyl)amino)oxy)acetic acid, resulting in PEG(ONHBoc)4 (middle). Subsequent deprotection with acid yielded PEGOA4 (top).

[0113] Figures 12A to 12E show that CDA-loaded HA-oxime hydrogel is injectable and non-swelling, in which:

[0114] Figure 12A illustrates the impact of CDAs on mechanical properties of the HA-oxime gel was investigated;

[0115] Figure 12B shows that CDA-loaded HA-oxime gels display similar time sweep profiles of G’ and G” moduli compared to HA-oxime gel alone;

[0116] Figure 12C shows that gel only vs. CDA-loaded gel are injectable by hand through a 30-gauge needle over 15 min (n=3, mean ± standard deviation);

[0117] Figure 12D shows the compressive modulus of CDA-loaded HA-oxime hydrogel compared to gel alone (n = 4, mean ± standard deviation, Student’s t- test); and

[0118] Figure 12E shows that swelling of CDA-loaded HA-oxime gel over 28 d in PBS is minimal as measured by change in mass relative to initial hydrogel mass (n = 3, mean ± standard deviation).

[0119] Figures 13A to 13F show that in vitro release kinetics of timolol from a CDA-loaded HA-oxime hydrogel is tuned via ester hydrolysis, in which:

[0120] Figure 13A illustrates the in vitro release kinetic studies of timolol prodrug CDA-loaded HA-oxime gels in 2-mL Eppendorf tubes. Aliquots were taken over time to quantify the amount of timolol released;

[0121] Figure 13B shows the cumulative timolol release from timolol prodrug CDA-loaded hydrogels. All gels (100 pL) were loaded with 0.05% w / v timolol, and PBS release medium (900 pL) was sampled and replenished over a period of 7 d to quantify the amount of timolol released via UV-UPLC (n = 3, mean ± standard deviation, two-way ANOVA with Tukey’s post-hoc analysis, *p < 0.05, **p < 0.01 , ***p < 0.001 , ****p < 0.0001); Figure 13C shows the diffusion constants of timolol from different gel formulations. Slopes (k) from B) were extended until R2 > 0.97 (n = 3, mean ± standard deviation);

[0122] Figure 13D shows diffusivity calculated from slope (k) of diffusion constants of timolol from prodrug CDA-loaded hydrogels (n = 3, mean ± standard deviation, one-way ANOVA with Tukey’s post-hoc analysis, ***p < 0.001 , ****p < 0.0001 );

[0123] Figure 13E shows the cumulative timolol release from either timolol palmitate 3c, timolol palmitamide 3c’ CDA- vs. timolol maleate-loaded hydrogel (n = 3, mean ± standard deviation, two-way ANOVA with Tukey’s post-hoc analysis, ***p < 0.001 , ****p < 0.0001); and

[0124] Figure 13F shows the diffusivity of timolol from timolol palmitate 3c, timolol palmitamide 3c’ CDA- vs. timolol maleate-loaded hydrogel (n = 3, mean ± standard deviation, one-way ANOVA with Tukey’s post-hoc analysis, ***p < 0.001 , ****p < 0.0001 ).

[0125] Figure 14 shows recovery of timolol from HA-oxime hydrogels after 28 d of drug release. Crosslinked hydrogels were enzymatically degraded after 28 d, at the terminal timepoint of the release studies to measure any remaining timolol. All gels were initially loaded an equivalence of 50 pg timolol. The remaining drug was quantified by UV-UPLC (n = 3, mean ± standard deviation).

[0126] Figure 15 shows the baseline IOP measurements in healthy Long-Evans rats prior to treatment. IOP was measured on days -2, -1 , and 0, at the same time of day within a 4 h period, prior to subconjunctival injection, ensuring a defined initial IOP for each animal and a standardized assessment protocol (n = 24 eyes, mean ± standard deviation, one-way ANOVA with Tukey’s post-hoc analysis).

[0127] Figures 16A to 16G show 3c CDA-loaded HA-oxime hydrogel exhibits long-acting lOP-lowering effect in rats, in which: Figure 16A illustrates the experimental design of the efficacy study in healthy 6-week-old Long-Evans rats. At 0 d, rats received a 30-pL subconjunctival injection of either saline, HA-oxime gel, timolol maleate (1 mg / mL timolol) or 3c CDA-loaded gel (1 mg / mL timolol). IOP was measured to monitor the lOP-lowering effect and blood was collected from the tail vein for the PK study over 56 d;

[0128] Figure 16B shows the representative H&E images of the cornea from Long-Evans rats at 33 d post-treatment;

[0129] Figure 16C shows the total corneal thickness measured via Imaged which displayed no difference across all experimental groups at 33 d postinjection (n = 3, mean ± standard deviation, one-way ANOVA with Tukey’s post- hoc analysis);

[0130] Figure 16D shows the corneal epithelium (CE) thickness measured via Imaged which displayed no difference across all experimental groups at 33 d post-injection (n = 3, mean ± standard deviation, one-way ANOVA with Tukey’s post-hoc analysis);

[0131] Figure 16E shows the plasma levels of timolol at 6-h post-injection (n = 3, mean ± standard deviation, Student’s t-test, **** p < 0.0001);

[0132] Figure 16F shows the IOP (mmHg) over the first 24 h post-injection in rats (n = 8, mean ± standard deviation, two-way ANOVA with Tukey’s post-hoc analysis, **p < 0.01 , **** p < 0.0001 ); and

[0133] Figure 16G shows the normalized IOP over 56 d post-injection in rats (n = 8, mean ± standard deviation, two-way ANOVA with Tukey’s post-hoc analysis, **p < 0.01 , **** p < 0.0001 ).

[0134] Figures 17A to 17F show in vivo biocompatibility study via H&E staining, in which:

[0135] Figure 17A illustrates the representative H&E images of the retina from Long-Evans rats at day 33 post-injection. (Scale bar = 50 pm).; Figure 17B shows the quantification of retina thickness via Imaged, which displayed no difference across all treatment groups at day 33 postinjection (n = 3, mean ± standard deviation, one-way ANOVA with Tukey’s post- hoc analysis;

[0136] Figure 17C shows the quantification of outer nuclear layer (ONL) thickness via Imaged, which displayed no difference across all treatment groups at day 33 post-injection (n = 3, mean ± standard deviation, one-way ANOVA with Tukey’s post-hoc analysis;

[0137] Figure 17D shows the quantification of inner nuclear layer (INL) thickness via Imaged, which displayed no difference across all treatment groups at day 33 post-injection (n = 3, mean ± standard deviation, one-way ANOVA with Tukey’s post-hoc analysis;

[0138] Figure 17E shows the quantification of ganglion cell layer (GCL) thickness via Imaged, which displayed no difference across all treatment groups at day 33 post-injection (n = 3, mean ± standard deviation, one-way ANOVA with Tukey’s post-hoc analysis; and

[0139] Figure 17F shows the representative H&E images of the trabecular meshwork from Long-Evans rats at day 33 post-injection. (Scale bar = 50 pm).

[0140] Figure 18 shows the plasma levels of timolol over 56 d in Long-Evans healthy rats. Plasma levels of timolol from 3c CDA-loaded hydrogel group were undetectable after 14 d post-injection (n = 3, mean ± standard deviation).

[0141] Figure 19 shows plots of scattering intensity versus concentration for netarsudil with linear regression fits were used to determine CAC value (n = 4, mean ± standard deviation).

[0142] Figures 20A to 20B show the stability of netarsudil colloids (100 pM) at 37 °C following the addition of lipid excipients. Stability was assessed at 0, 1 , 3, and 6 hours, and at 1 , 3, 7, 14, and 21 days, in which:

[0143] Figure 20A shows the changes in scattering intensity (n = 4, mean ± standard deviation); and Figure 20B shows the hydrodynamic diameters which were measured using DLS (n = 4, mean ± standard deviation).

[0144] Figures 21 A to 21 C show the release behavior of the hydrogel-colloid composite (HAK:HAA 1 :0, 0.7 wt% PEGOA4, 300 pM netarsudil), which demonstrated a prolonged release profile of netarsudil over 28 days, in which:

[0145] Figure 21 A shows a schematic representation of colloid release from the hydrogel into the release buffer;

[0146] Figure 21 B shows the cumulative release profile of netarsudil over 28 days, wherein butyrylcholinesterase-containing buffer was added on days 14, 15, and 21 (arrows); and

[0147] Figure 21 C shows the mechanism of hydrolysis of netarsudil to its M1 metabolite in the presence of butyrylcholinesterase.

[0148] DETAILED DESCRIPTION

[0149] A detailed description is provided below to facilitate a thorough understanding of the disclosed embodiments and connections thereof. The description is not limited to any particular example included herein.

[0150] Various embodiments and aspects of the disclosure will be described with reference to the details discussed below. The following description and drawings are illustrative of the disclosure and are not to be construed as limiting the disclosure. Numerous specific details are described to provide a thorough understanding of various embodiments of the present disclosure. The Figures are not to scale. Further, in certain instances, well-known or conventional details are not described in order to provide a concise discussion of embodiments of the present disclosure.

[0151] As used herein, the terms, “comprises” and “comprising” are to be construed as being inclusive and open ended, and not exclusive. Specifically, when used in the specification and claims, the terms, “comprises” and “comprising” and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps or components. As used herein, the term “exemplary” means “serving as an example, instance, or illustration,” and should not be construed as preferred or advantageous over other configurations disclosed herein.

[0152] As used herein, the terms “about” and “approximately”, when used in conjunction with ranges of dimensions of particles, compositions of mixtures or other physical properties or characteristics, are meant to cover slight variations that may exist in the upper and lower limits of the ranges of dimensions so as to not exclude embodiments where on average most of the dimensions are satisfied but where statistically dimensions may exist outside this region. It is not the intention to exclude embodiments such as these from the present disclosure. Unless otherwise specified, the terms “about” and “approximately” mean plus or minus 25 percent or less.

[0153] It is to be understood that unless otherwise specified, any specified range or group is as a shorthand way of referring to each and every member of a range or group individually, as well as each and every possible sub-range or sub-group encompassed therein and similarly with respect to any sub-ranges or sub-groups therein. Unless otherwise specified, the present disclosure relates to and explicitly incorporates each and every specific member and combination of sub-ranges or sub-groups.

[0154] As used herein, the term "on the order of", when used in conjunction with a quantity or parameter, refers to a range spanning approximately one tenth to ten times the stated quantity or parameter.

[0155] As used herein, the term “hydrolysis” refers to the cleavage of one or more covalent bonds by reaction with water, and includes both enzymatic hydrolysis (e.g., mediated by esterases or other hydrolases) and non-enzymatic hydrolysis (including acid-catalyzed, base-catalyzed, or spontaneous hydrolysis under physiological or formulation conditions).

[0156] . As used herein, natural polymers suitable for forming biocompatible hydrogels include, without limitation, any one or combination of glycosaminoglycans such as hyaluronan (or hyaluronic acid), chondroitin sulfate, and heparin; protein-based biopolymers such as collagen, gelatin, fibrin, silk fibroin, elastin, keratin, and laminin; marine or microbial polysaccharides such as alginate, chitosan, agarose, xanthan gum, gellan gum, and carrageenan; plant polysaccharides such as cellulose and cellulose derivatives (e.g., carboxymethylcellulose, hydroxypropylcellulose), starch and modified starches, and pectin; and microbial or fungal polysaccharides such as dextran, dextrin, and pullulan.

[0157] As used herein, Synthetic polymers suitable for forming biocompatible hydrogels include, without limitation, any one or combination of hydrophilic, hydrogel-forming polymers such as polyethylene glycol (PEG), polyethylene glycol) diacrylate (PEGDA), poly (vinyl alcohol) (PVA), poly(acrylic acid) (PAA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(N-isopropylacrylamide) (PNIPAAm), polyurethanes, polyethylene oxide) (PEG), and poly(propylene glycol) (PPG), as well as biodegradable and / or hydrophobic polymers that form hydrogels when incorporated into suitable copolymer or block copolymer architectures, such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), and poly(dimethylsiloxane) (PDMS), and copolymers or block copolymers thereof.

[0158] The present disclosure relates to formulations of colloidal drug aggregates (CDAs) embedded in hydrogels for sustained local delivery. Because many drugs do not inherently form CDAs, a hydrophobic promoiety is added to the drug to enable CDA formation. In exemplary embodiments, the CDA formulations comprise prodrugs that form CDAs embedded in hydrogels for sustained local delivery. In one such embodiment, beta-blocker prodrug CDAs are embedded in hydrogels formulated for ophthalmic administration to the eye to lower IOP for the treatment of ocular hypertension or glaucoma. In another embodiment, ROCK inhibitors prodrug CDAs are embedded in hydrogels formulated for ophthalmic administration to the eye to lower IOP for the treatment of ocular hypertension or glaucoma.

[0159] According to an embodiment of the present disclosure, the prodrug colloid formulations disclosed herein comprise colloidal nanoparticles which in turn comprise a prodrug formed by a drug chemically conjugated to promoiety of hydrocarbons. The hydrocarbons may be alkyl carbon chains, aryl groups, cyclo-alkyl carbon chains, or any combination of these, wherein the hydrocarbons contain a minimum of six carbons. The drug is conjugated to the alkyl carbon chains, aryl groups, or cyclo-alkyl carbon chains. Conjugation is achieved through chemical bonds which may include ester bonds, amide bonds, carbamate bonds, hydrazone bonds, disulfide bonds, boronic ester bonds, borate ester bonds, phosphonate bonds, glycosidic bonds, anhydride bonds, thioester bonds, phosphate bonds, urea bonds, thiourea bonds, imine bonds, oxime bonds, acetal bonds, ketal bonds, peptidic bonds, succinimide bonds, ether bonds, azo bonds, lactone bonds, lactam bonds, carbonate bonds, sulfonic ester bonds, sulfonamide bonds, siloxane bonds, pyrophosphate bonds, phosphoramide bonds, phosphoramidate bonds, thioether bonds, orthoester bonds, oxoester bonds, vinyl ether bonds, thionoester bonds, and sulfenamide bonds. Those skilled in the art of organic and medicinal chemistry will readily understand how to form these bonds, using well-established methods and reaction conditions known in the literature. For instance, esterification or amidation may be used to conjugate drugs to alkyl chains, while hydrazone and disulfide bonds may be formed using hydrazine derivatives or thiol-based chemistry, respectively. Similarly, oxime bonds may be achieved through the reaction of aldehydes or ketones with hydroxylamines under mild conditions. The conjugation approach may be tailored depending on the desired bond type, which is informed by considerations such as the prodrug’s hydrolysis rate, stability, and pharmacokinetics.

[0160] In terms of utility, the colloidal nanoparticles formed from these prodrugs may be used in a variety of medical applications. These nanoparticles provide enhanced stability and solubility of the prodrug, leading to prolonged drug release and improved bioavailability. Additionally, they may enhance targeted delivery to tissues, improving therapeutic outcomes. The colloidal formulation is also beneficial for minimizing systemic absorption and side effects. Furthermore, these nanoparticles may be administered via various routes, including subconjunctival injection, topical application, or even intravenous administration, depending on the clinical indication.

[0161] The prodrug is mixed with lipid excipients in an organic solvent to form a mixture which is suspended in an aqueous buffer solution to induce formation of prodrug colloidal nanoparticles. The lipid excipients enhance the stability and delivery of the prodrug, and a mass ratio of the prodrug to lipid excipients is in a range from 1 to 99% w / w to 99 to 1 % w / w. Non-limiting examples of organic solvents include, but are not limited to, any one or combination of ethanol, methanol, isopropanol, butanol, dichloromethane (DCM), chloroform, dimethyl sulfoxide (DMSO), acetone, acetonitrile, tetrahydrofuran (THF), dimethylformamide (DMF), and toluene.

[0162] Non-limiting examples of aqueous buffer solutions include, but are not limited to, any one or combination of phosphate-buffered saline (PBS), Trisbuffered saline (TBS), HEPES buffer, citrate buffer, acetate buffer, borate buffer, sodium bicarbonate buffer, MES buffer, and malate buffer.

[0163] Non-limiting examples of lipid excipients include any one or combination of phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines (PS), phosphatidylglycerols, phosphatidylinositols (PI), phosphatidic acids (PA), sphingolipids, cholesterol and cholesterol derivatives, triglycerides and diglycerides, cationic lipids, neutral lipids, ionizable lipids, polyethylene glycol (PEG) conjugated lipids, ceramides and derivatives, fatty acids, and fatty acid esters.

[0164] Non-limiting examples of the phosphatidylcholines include dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), soybean phosphatidylcholine (SPC); non-limiting examples of the phosphatidylethanolamines include distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE); non-limiting examples of the phosphatidylglycerols include dioleoylphosphatidylglycerol (DOPG); phosphatidylinositols (PI); non-limiting examples of the sphingolipids include Sphingomyelin; non-limiting examples of the cationic lipids are 1 ,2-dioleoyl-3- trimethylammonium-propane (DOTAP), 1 ,2-dioleoyl-3-dimethylammonium- propane (DODAP); non-limiting examples of the neutral lipids include dioleoylphosphatidylcholine (DOPC); non-limiting examples of the ionizable lipids include SM-102, DLin-MC3-DMA; and non-limiting examples of the polyethylene glycol (PEG) conjugated lipids include 1 ,2-dimyristoyl-rac-glycerol -PEG2000 (DMG-PEG2000), DSPE-PEG2000.

[0165] When a prodrug colloid hydrogel formulation is to be synthesized, a biocompatible hydrogel is formed using polymers which may be natural polymers or synthetic polymers or a mixture of both. A non-limiting example of a natural biocompatible hydrogel is a hyaluronan-based hydrogel. Alternatively, the biocompatible hydrogel may be a polyethylene glycol-based hydrogel. In the case when a mixed natural and synthetic hydrogel is preferred, a non-limiting example of such a mixture is a hyaluronan-based hydrogel and polyethylene glycol-based hydrogel.

[0166] Being embedded in the hydrogel allows for localized, controlled, and sustained release of the drug from the prodrug, since the hydrogel formulation may be administered into selected local areas of the anatomy and the release rate depends on the size, structure, molecular conformation, and composition of the promoiety. According to an embodiment, the release rate further depends on the number of carbons in the alkyl carbon chains, aryl groups, or carbon chains. The alkyl carbon chains, aryl groups, or cycloalkyl carbon chains may contain at least six carbons, for example between about six and about eighteen carbons, where a higher number of carbons generally corresponds to a longer release time and a lower number corresponds to a shorter release time. To control the release rate of the drug, the number of carbons in the hydrocarbon promoiety is selected within this range. In some embodiments, the promoiety may contain fewer than six carbons, for example from one to about six carbons or from about four to about six carbons. In another embodiment, the promoiety may contain more than eighteen carbons, for example, between about six and about twenty-four carbons, between about six to about thirty carbons, about six to about forty-four carbons, about six to hundred carbons or hundreds of carbons.

[0167] While the examples described herein employ alkyl promoieties of defined chain lengths (e.g., C4, C8, and Ci6), analogous prodrugs bearing more hydrophobic, longer, more branched, and / or sterically bulkier alkyl chains, for example Ci8-C24or higher, are also contemplated. Such prodrugs, having more hydrophobic, longer, more branched and / or sterically bulkier alkyl promoieties may form colloidal drug aggregates and may provide progressively slower drug release due to increased hydrophobicity and reduced solubility. The observed relationship between alkyl chain length and release kinetics in the exemplified prodrugs is expected to extend to these longer, more branched and / or sterically bulkier promoieties, such that increasing the number of carbons beyond the tested examples may afford additional tunability toward more prolonged release.

[0168] In addition to alkyl promoieties composed solely of carbon and hydrogen, the present disclosure also encompasses alkyl-based and non-alkyl promoieties in which the carbon backbone and / or ring system is optionally interrupted or substituted by one or more heteroatoms (for example, O, N, S, F, Cl, Br, I) and / or incorporates additional functional groups, such as ether, thioether, amide, carbamate, urea, carbonate, or ester linkages. In certain embodiments, the promoiety may further comprise aryl, heteroaryl, cycloalkyl, heterocycloalkyl, or polyethylene glycol) (PEG) segments, or combinations thereof. Such heteroatom-containing and mixed-scaffold promoieties are likewise expected to promote colloidal drug aggregate formation and to modulate drug release kinetics by adjusting overall hydrophobicity, polarity, conformational flexibility, and the steric environment around the cleavable linkage, thereby providing additional tunability of sustained-release profiles beyond that achievable with straight-chain hydrocarbon alkyl groups alone.

[0169] For therapeutic applications, the selected number of carbons in the hydrocarbons is chosen to be sufficient to maintain therapeutic levels of the drug in the targeted tissue for a period of time of at least 24 hours postadministration. The numbers of carbons may be tuned to give periods of time of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days. Alternatively, the numbers of carbons may be tuned to give periods of time greater than about 7 days and may range from about 7 days to about 14 days, or to about 1 month, or to about 3 months, or to about 6 months, or up to 1 year. In another embodiment, the degree of branching and / or steric bulkiness, together with the promoiety length, are selected to maintain therapeutic drug levels in the targeted tissue for at least 24 hours after administration. The degree of branching and / or steric bulkiness, together with the promoiety length may be tuned to give periods of time of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days. Alternatively, the numbers of carbons may be tuned to give periods of time greater than about 7 days and may range from about 7 days to about 14 days, or to about 1 month, or to about 3 months, or to about 6 months, or up to 1 year. The release rate is influenced not only by the number of carbons but also by the chemical structure of the hydrocarbon chain. For example, an 18- carbon alkyl chain would provide a different release profile compared to an 18- carbon aryl or cyclo-alkyl chain, even if they contain the same number of carbons. This is because the molecular structure and conformation of each type of hydrocarbon impact the drug’s solubility, lipophilicity, and steric hindrance, all of which affect how quickly the drug is hydrolyzed or released from the colloid. Alkyl chains are typically linear or branched, and their release rate is primarily influenced by their hydrophobicity and chain length. In contrast, aryl groups are bulkier and more rigid, which could result in slower release rates due to increased steric hindrance and reduced accessibility for enzymatic hydrolysis, hydrolysis or other degradation mechanisms. Cyclo-alkyl carbon chains, being more conformationally constrained than alkyl chains but less bulky than aryl groups, would likely offer an intermediate release profile. Accordingly, the nature of the promoiety, e.g., nature of the hydrocarbon group or heteroatom hydrocarbon is an important factor in promoting colloid formation and defining the release kinetics of long-acting formulations and may be selected and tailored to tune release behavior and achieve the desired duration of therapeutic effect. The prodrug colloids are produced by reacting a drug with one or more alkyl carbon chains, aryl groups, or cycloalkyl carbon chains or heteroatom alkyl, aryl or cycloalkyl carbon chains that typically contain at least six carbons, for example from about six to about eighteen carbons, or alternatively from about six to about twenty-four carbons. The term “reacting” refers to the process of covalent modification of the drug through well- established organic chemistry techniques. Depending on the functional groups present on the drug and the hydrocarbon, the reaction could involve esterification, amidation, or other types of chemical bonding, such as hydrazone formation or oxime bond formation. For example, if an ester bond is desired, the drug with a hydroxyl or carboxyl functional group would be reacted with a hydrocarbon possessing the complementary functional group (e.g., a carboxylic acid or alcohol) under conditions that promote ester bond formation. This reaction is typically carried out in the presence of an activating agent, such as dicyclohexylcarbodiimide (DCC), and a catalyst like 4-dimethylaminopyridine (DMAP), in an appropriate organic solvent. Other reaction types, such as amide bond formation, may use similar coupling agents or direct condensation under appropriate conditions, such as using carbodiimides for carboxyl to amine coupling.

[0170] Once the prodrug is synthesized, the prodrug is mixed with lipid excipients in one of the above-mentioned organic solvents to form a mixture, upon which the mixture is suspended in one of the above-mentioned aqueous buffer solutions to induce formation of prodrug colloidal nanoparticles, due to the amphiphilic nature of the lipid excipients and the hydrophobic nature of the prodrug, specifically due to the amphiphilic interactions between the lipids and the prodrug. Finally, the nanoparticles typically undergo buffer exchange via centrifugal filtration to remove residual organic solvent, prodrug, or lipid excipients, and to achieve a stable colloidal suspension.

[0171] To form a prodrug colloid hydrogel, the prodrug colloidal nanoparticles are concentrated to form a suspension of concentrated colloidal nanoparticles. The concentrated colloidal nanoparticles are then mixed with a biocompatible hydrogel precursor, whereupon gelation of the hydrogel precursors is induced to encapsulate the concentrated colloidal nanoparticles in a biocompatible hydrogel formed by crosslinking of the biocompatible hydrogel precursor to form the prodrug colloid hydrogel.

[0172] The step of concentrating the colloidal nanoparticles to form a suspension of concentrated colloidal nanoparticles is performed using any one of centrifugal filters, dialysis bags, or tangential flow filtration to remove excess solvent and unincorporated materials, thereby concentrating the nanoparticle suspension while maintaining the stability and integrity of the colloids.

[0173] For the case of producing a HA-oxime gel, gelation is induced by mixing the components of the HA-oxime gel in an aqueous environment under mild conditions. Specifically, a solution containing 1.00% (w / v) hyaluronan-ketone (HAK) is mixed with 0.25% (w / v) hyaluronan-aldehyde (HAA) in the presence of 50 mol% of a crosslinking agent, such as 4-arm polyethylene glycol-tetra- oxyamine (PEGOA4). The crosslinking agent reacts with the aldehyde and ketone groups present on the HAK and HAA, respectively, forming stable oxime bonds through a condensation reaction. The gelation process occurs in situ where the HAA reacts more quickly with the crosslinker compared to HAK, allowing for controlled modulation of the gelation rate. The reaction progresses over a time frame that may be tuned by adjusting the concentrations of HAK, HAA, and crosslinker, as well as the temperature and pH of the solution. Full gelation typically occurs within minutes to hours, depending on the desired application, leading to the formation of a biocompatible, bioresorbable hydrogel matrix.

[0174] In certain embodiments, the prodrug colloid formulations described above are applied to beta-blocker drugs to form beta-blocker prodrug colloid formulations with analogous sustained-release properties.

[0175] According to an embodiment of the present disclosure, the beta-blocker prodrug colloid formulations disclosed herein comprise colloidal nanoparticles which comprise a beta-blocker prodrug formed by a beta-blocker drug chemically conjugated to promoiety of hydrocarbon. The hydrocarbons may be alkyl carbon chains, aryl groups, cyclo-alkyl carbon chains, or any combination of these, wherein the hydrocarbons contain a minimum of six carbons. The beta-blocker drug is conjugated to the alkyl carbon chains, aryl groups, or cyclo-alkyl carbon chains. Conjugation is achieved through chemical bonds which may include ester bonds, amide bonds, carbamate bonds, hydrazone bonds, disulfide bonds, boronic ester bonds, borate ester bonds, phosphonate bonds, glycosidic bonds, anhydride bonds, thioester bonds, phosphate bonds, urea bonds, thiourea bonds, imine bonds, oxime bonds, acetal bonds, ketal bonds, peptidic bonds, succinimide bonds, ether bonds, azo bonds, lactone bonds, lactam bonds, carbonate bonds, sulfonic ester bonds, sulfonamide bonds, siloxane bonds, pyrophosphate bonds, phosphoramide bonds, phosphoramidate bonds, thioether bonds, orthoester bonds, oxoester bonds, vinyl ether bonds, thionoester bonds, and sulfenamide bonds. Those skilled in the art of organic and medicinal chemistry will readily understand how to form these bonds, using well-established methods and reaction conditions known in the literature. For instance, esterification or amidation may be used to conjugate beta-blocker drugs to alkyl chains, while hydrazone and disulfide bonds may be formed using hydrazine derivatives or thiol-based chemistry, respectively.

[0176] Similarly, oxime bonds may be achieved through the reaction of aldehydes or ketones with hydroxylamines under mild conditions. The conjugation approach may be tailored depending on the desired bond type, which is informed by considerations such as the prodrug’s hydrolysis rate, stability, and pharmacokinetics.

[0177] In terms of utility, the colloidal nanoparticles formed from these betablocker prodrugs may be used in a variety of medical applications. These nanoparticles provide enhanced stability and solubility of the prodrug, leading to prolonged drug release and improved bioavailability. Additionally, they may enhance targeted delivery to ocular tissues, improving the therapeutic outcomes for conditions like glaucoma. The colloidal formulation is also beneficial for minimizing systemic absorption and side effects, particularly those related to the beta-blocker class of drugs, such as bradycardia. Furthermore, these nanoparticles may be administered via various routes, including subconjunctival injection, topical application, or even intravenous administration, depending on the clinical indication.

[0178] The beta-blocker prodrug is mixed with lipid excipients in an organic solvent to form a mixture which is suspended in an aqueous buffer solution to induce formation of prodrug colloidal nanoparticles. The lipid excipients enhance the stability and delivery of the beta-blocker prodrug, and a mass ratio of the beta-blocker prodrug to lipid excipients is in a range from 1 to 99% w / w to 99 to 1 % w / w.

[0179] Non-limiting examples of organic solvents include, but are not limited to, any one or combination of ethanol, methanol, isopropanol, butanol, DCM, chloroform, DMSO, acetone, acetonitrile, THF, DMF, and toluene.

[0180] Non-limiting examples of aqueous buffer solutions include, but are not limited to, any one or combination of PBS, TBS, HEPES buffer, citrate buffer, acetate buffer, borate buffer, sodium bicarbonate buffer, MES buffer, and malate buffer.

[0181] Non-limiting examples of lipid excipients include any one or combination of phosphatidylcholines, phosphatidylethanolamines, PS, phosphatidylglycerols, PI, PA, sphingolipids, cholesterol and cholesterol derivatives, triglycerides and diglycerides, cationic lipids, neutral lipids, ionizable lipids, PEG conjugated lipids, ceramides and derivatives, fatty acids, and fatty acid esters.

[0182] Non-limiting examples of the phosphatidylcholines include DPPC, DSPC, SPC; non-limiting examples of the phosphatidylethanolamines include DSPE, DMPE; non-limiting examples of the phosphatidylglycerols include DOPG; PI; non-limiting examples of the sphingolipids include Sphingomyelin; non-limiting examples of the cationic lipids are DOTAP, DODAP; non-limiting examples of the neutral lipids include DOPC; non-limiting examples of the ionizable lipids include SM-102, DLin-MC3-DMA; and non-limiting examples of the PEG conjugated lipids include DMG-PEG2000, DSPE-PEG2000.

[0183] When a beta-blocker prodrug colloid hydrogel formulation is to be synthesized, a biocompatible hydrogel is formed using polymers which may be natural polymers or synthetic polymers or a mixture of both. A non-limiting example of a natural biocompatible hydrogel is a hyaluronan-based hydrogel. Alternatively, the biocompatible hydrogel may be a polyethylene glycol-based hydrogel. In the case when a mixed natural and synthetic hydrogel is preferred, a non-limiting example of such a mixture is a hyaluronan-based hydrogel and polyethylene glycol-based hydrogel.

[0184] Being embedded in the hydrogel allows for localized, controlled, and sustained release of the beta-blocker drug from the beta-blocker prodrug, since the hydrogel formulation may be administered into selected local areas of the anatomy and the release rate depends on the size, structure, molecular conformation, and composition of the promoiety. According to an embodiment, the release rate further depends on the number of carbons in the alkyl carbon chains, aryl groups, or cycloalkyl carbon chains. The alkyl carbon chains, aryl groups, or cycloalkyl carbon chains may contain at least six carbons, for example between about six and about eighteen carbons, where a higher number of carbons generally corresponds to a longer release time and a lower number corresponds to a shorter release time. To control the release rate of the drug, the number of carbons in the hydrocarbon promoiety is selected within this range. In some embodiments, the promoiety may contain fewer than six carbons, for example from one to about six carbons or from about four to about six carbons. In another embodiment, the promoiety may contain more than eighteen carbons, for example, between about six and about twenty-four carbons, between about six to about thirty carbons, about six to about forty-four carbons, about six to hundred carbons or hundreds of carbons.

[0185] While the examples described herein employ alkyl promoieties of defined chain lengths (e.g., C4, C8, and Ci6), analogous beta-blocker prodrugs bearing more hydrophobic, longer, more branched, and / or sterically bulkier alkyl chains, for example Ci8-C24or higher, are also contemplated. Such prodrugs, having more hydrophobic, longer, more branched and / or sterically bulkier alkyl promoieties may form colloidal drug aggregates and may provide progressively slower drug release due to increased hydrophobicity and reduced solubility. The observed relationship between alkyl chain length and release kinetics in the exemplified prodrugs is expected to extend to these longer, more branched and / or sterically bulkier promoieties, such that increasing the number of carbons beyond the tested examples may afford additional tunability toward more prolonged release.

[0186] In addition to alkyl promoieties composed solely of carbon and hydrogen, the present disclosure also encompasses alkyl-based and non-alkyl promoieties in which the carbon backbone and / or ring system is optionally interrupted or substituted by one or more heteroatoms (for example, O, N, S, F, Cl, Br, I) and / or incorporates additional functional groups, such as ether, thioether, amide, carbamate, urea, carbonate, or ester linkages. In certain embodiments, the promoiety may further comprise aryl, heteroaryl, cycloalkyl, heterocycloalkyl, or polyethylene glycol) (PEG) segments, or combinations thereof. Such heteroatom-containing and mixed-scaffold promoieties are likewise expected to promote colloidal drug aggregate formation and to modulate drug release kinetics by adjusting overall hydrophobicity, polarity, conformational flexibility, and the steric environment around the cleavable linkage, thereby providing additional tunability of sustained-release profiles beyond that achievable with hydrocarbon alkyl groups alone.

[0187] Non-limiting examples of beta-blocker drugs that may be incorporated into the hydrogel include, but are not limited to, any one or combination of acebutolol, atenolol, betaxolol, bisoprolol, carvedilol, labetalol, metoprolol, nebivolol, nadolol, pindolol, propranolol, sotalol, and timolol, such that the betablocker prodrug colloid hydrogel formulation is formulated for ophthalmic administration for lowering IOP.

[0188] According to an embodiment, the beta-blocker is timolol and the betablocker prodrug comprises at least one compound selected from the group consisting of timolol octanoate, timolol palmitate, timolol decylbenzoate., and timolol-abietate. According to another embodiment, the beta-blocker is timolol and the beta-blocker prodrug timolol palmitate.

[0189] Beta-blocker prodrugs in colloidal formulations may be used for various therapeutic indications, including cardiovascular conditions (e.g., hypertension, arrhythmias, and heart failure), anxiety disorders, migraine prophylaxis, and other conditions in which the use of beta blockers is therapeutically beneficial. Beta-blockers like propranolol and sotalol, which are used for systemic conditions, may be formulated as colloidal nanoparticles for purposes beyond IOP lowering.

[0190] The beta-blocker prodrug colloid hydrogel formulations may have incorporated therein non-beta-blocker lOP-lowering drugs to give a combination of beta-blocker and non-beta-blocker drugs in the hydrogel. The addition of these additional drugs may be based on previously known combinations in use so that they are used to provide combinations of these drugs that are useful and have been used before. The selection of non-beta-blocker drugs to pair with beta-blockers is guided by their complementary mechanisms for reducing IOP and their established clinical use in combination therapies. Common non- beta-blocker drugs used in glaucoma management include prostaglandin analogs (e.g., latanoprost, bimatoprost), carbonic anhydrase inhibitors (e.g., dorzolamide, brinzolamide), alpha agonists (e.g., brimonidine), and rho kinase inhibitors (e.g., netarsudil). These drugs are chosen because they act through different pathways than beta-blockers, thus offering an additive or synergistic effect when used together. For instance, prostaglandin analogs work by increasing aqueous humor outflow through the uveoscleral pathway, while betablockers like timolol decrease aqueous humor production by inhibiting adrenergic receptors in the ciliary body. Combining these two classes results in both reduced production and enhanced outflow of aqueous humor, providing greater lOP-lowering effects than either drug alone. Similarly, carbonic anhydrase inhibitors reduce the production of aqueous humor by inhibiting carbonic anhydrase enzymes, offering an alternative mechanism that complements beta-blockers. These combinations are typically selected to optimize IOP reduction, especially in patients with more advanced glaucoma who may not respond sufficiently to monotherapy. Furthermore, combination therapy may reduce the frequency of drug administration, which enhances patient compliance by simplifying the treatment regimen. Additionally, the inclusion of non-beta-blocker drugs in the colloidal formulation may help to achieve prolonged release and sustained therapeutic effects, minimizing the need for frequent re-administration and reducing the risk of systemic side effects.

[0191] For ophthalmic administration applications, non-limiting examples of non- beta-blocker intraocular pressure-lowering drugs include any one or combination of carbonic anhydrase inhibitors (CAIs), Rho kinase (ROCK) inhibitors, alpha-agonists, and prostaglandin analogs.

[0192] When the formulations are formulated for ophthalmic administration for lowering intraocular pressure, the hydrogel formulation may be administered in several ways including by subconjunctival, intravitreal, topical, transscleral, intracameral, suprachoroidal, subtenon, or subretinal injection for the treatment of ocular hypertension or glaucoma. In order to provide control over the release rates of the beta-blocker drug, the number of carbons in the alkyl carbon chains, aryl groups, or carbon chains. The alkyl carbon chains, aryl groups, or cycloalkyl carbon chains may contain at least six carbons, for example between about six and about eighteen carbons, where a higher number of carbons generally corresponds to a longer release time and a lower number corresponds to a shorter release time. To control the release rate of the beta-blocker drug, the number of carbons in the hydrocarbon promoiety is selected within this range. In some embodiments, the promoiety may contain fewer than six carbons, for example from one to about six carbons or from about four to about six carbons. In another embodiment, the promoiety may contain more than eighteen carbons, for example, between about six and about twenty-four carbons, between about six to about thirty carbons, about six to about forty-four carbons, about six to hundred carbons or hundreds of carbons.

[0193] For therapeutic applications, the selected number of carbons in the hydrocarbons is chosen to be sufficient to maintain therapeutic levels of the drug in the eye for a period of time of at least 24 hours post-administration. The numbers of carbons may be tuned to give periods of time of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days. Alternatively, the numbers of carbons may be tuned to give periods of time greater than about 7 days and may range from about 7 days to about 14 days, or to about 1 month, or to about 3 months, or to about 6 months, or up to 1 year. In another embodiment, the degree of branching and / or steric bulkiness, together with the promoiety length, are selected to maintain therapeutic drug levels in the eye for at least 24 hours after administration. The degree of branching and / or steric bulkiness, together with the promoiety length may be tuned to give periods of time of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days. Alternatively, the numbers of carbons may be tuned to give periods of time greater than about 7 days and may range from about 7 days to about 14 days, or to about 1 month, or to about 3 months, or to about 6 months, or up to 1 year. The release rate is influenced not only by the number of carbons but also by the chemical structure of the hydrocarbon chain. For example, an 18-carbon alkyl chain would provide a different release profile compared to an 18-carbon aryl or cyclo-alkyl chain, even if they contain the same number of carbons. This is because the molecular structure and conformation of each type of hydrocarbon impact the drug’s solubility, lipophilicity, and steric hindrance, all of which affect how quickly the drug is hydrolyzed or released from the colloid. Alkyl chains are typically linear or branched, and their release rate is primarily influenced by their hydrophobicity and chain length. In contrast, aryl groups are bulkier and more rigid, which could result in slower release rates due to increased steric hindrance and reduced accessibility for enzymatic hydrolysis or other degradation mechanisms. Cyclo-alkyl carbon chains, being more conformationally constrained than alkyl chains but less bulky than aryl groups, would likely offer an intermediate release profile. Accordingly, the nature of the promoiety, e.g., nature of the hydrocarbon group or heteroatom hydrocarbon is an important factor in promoting colloid formation and defining the release kinetics of long- acting formulations and may be selected and tailored to tune release behavior and achieve the desired duration of therapeutic effect.

[0194] The beta-blocker prodrug colloids are produced by reacting a betablocker with one or more alkyl carbon chains, aryl groups, or cycloalkyl carbon chains or heteroatom alkyl, aryl or cycloalkyl carbon chains that typically contain at least six carbons, for example from about six to about eighteen carbons, or alternatively from about six to about twenty-four carbons. The term “reacting” refers to the process of covalent modification of the beta-blocker drug through well-established organic chemistry techniques. Depending on the functional groups present on the beta-blocker drug and the hydrocarbon, the reaction could involve esterification, amidation, or other types of chemical bonding, such as hydrazone formation or oxime bond formation. For example, if an ester bond is desired, the beta-blocker drug with a hydroxyl or carboxyl functional group would be reacted with a hydrocarbon possessing the complementary functional group (e.g., a carboxylic acid or alcohol) under conditions that promote ester bond formation. This reaction is typically carried out in the presence of an activating agent, such as DCC, and a catalyst like DMAP, in an appropriate organic solvent. Other reaction types, such as amide bond formation, may use similar coupling agents or direct condensation under appropriate conditions, such as using carbodiimides for carboxyl to amine coupling.

[0195] Once the beta-blocker prodrug is synthesized, the prodrug is mixed with lipid excipients in one of the above-mentioned organic solvents to form a mixture, upon which the mixture is suspended in one of the above-mentioned aqueous buffer solutions to induce formation of prodrug colloidal nanoparticles, due to the amphiphilic nature of the lipid excipients and the hydrophobic nature of the prodrug, specifically due to the amphiphilic interactions between the lipids and the prodrug. Finally, the nanoparticles typically undergo buffer exchange via centrifugal filtration to remove residual organic solvent, prodrug, or lipid excipients, and to achieve a stable colloidal suspension.

[0196] To form a beta-blocker prodrug colloid hydrogel, the prodrug colloidal nanoparticles are concentrated to form a suspension of concentrated colloidal nanoparticles. The concentrated colloidal nanoparticles are then mixed with a biocompatible hydrogel precursor, whereupon gelation of the hydrogel precursors is induced to encapsulate the concentrated colloidal nanoparticles in a biocompatible hydrogel formed by crosslinking of the biocompatible hydrogel precursor to form the beta-blocker prodrug colloid hydrogel.

[0197] The step of concentrating the colloidal nanoparticles to form a suspension of concentrated colloidal nanoparticles is performed using any one of centrifugal filters, dialysis bags, or tangential flow filtration to remove excess solvent and unincorporated materials, thereby concentrating the nanoparticle suspension while maintaining the stability and integrity of the colloids.

[0198] For the case of producing a HA-oxime gel, gelation is induced by mixing the components of the HA-oxime gel in an aqueous environment under mild conditions. Specifically, a solution containing 1.00% (w / v) HAK is mixed with 0.25% (w / v) HAA in the presence of 50 mol% of a crosslinking agent, such as PEGOA4. The crosslinking agent reacts with the aldehyde and ketone groups present on the HAK and HAA, respectively, forming stable oxime bonds through a condensation reaction. The gelation process occurs in situ where the HAA reacts more quickly with the crosslinker compared to HAK, allowing for controlled modulation of the gelation rate. The reaction progresses over a time frame that may be tuned by adjusting the concentrations of HAK, HAA, and crosslinker, as well as the temperature and pH of the solution. Full gelation typically occurs within minutes to hours, depending on the desired application, leading to the formation of a biocompatible, bioresorbable hydrogel matrix.

[0199] The beta-blocker prodrug colloid hydrogel may be formed with non-beta- blocker intraocular pressure-lowering drugs mentioned above which are mixed in with the concentrated colloidal nanoparticles prior to gelation of the hydrogel precursors.

[0200] The beta-blocker prodrug colloid hydrogel will now be illustrated with the following non-limiting example.

[0201] Timolol maleate, the commercial form, did not spontaneously form CDAs (Figure 1), prompting us to develop timolol prodrugs with hydrophobic tails to encourage colloidal aggregation (Figure 2).

[0202] Timolol was modified using various hydrophobic moieties to facilitate the spontaneous formation of CDAs in an aqueous environment.

[0203] Timolol ester prodrugs were prepared from (S)-timolol maleate and the corresponding acyl chloride (butyryl, octanoyl, palmitoyl, or 4-decylbenzoyl chloride) using a common procedure.

[0204] The chemical modification of timolol (Figure 3A) starts with removal of the maleate salt (1) using 2 M NaOH to enhance the basicity of the hydroxyl group (1 H NMR and mass spectrum in Figure 4). Timolol (2) was then chemically reacted with one of: butyryl chloride, octanoyl chloride, palmitoyl chloride, or 4-decylbenzoyl chloride, resulting in the formation of timolol butanoate (3a), octanoate (3b), palmitate (3c), and decyl-benzoate (3d), respectively. These derivatives were successfully synthesized as confirmed with 1 H NMR and mass spectrometry analyses (Figures 5, 6, 7 and 8, respectively). A distinct 1 H-NMR shift was observed in the neighboring protons of the hydroxyl group of timolol with ester formation. In another embodiment, timolol maleate (1) was reacted directly with acyl chloride, such as palmitoyl chloride, resulting in the formation of timolol ester, such as timolol palmitate (3c). This derivative was successfully synthesized as confirmed with 1 H NMR and mass spectrometry analyses.

[0205] The formation of CDAs was assessed using dynamic light scattering (DLS). Timolol prodrug stocks were dissolved in ethanol and then diluted 100- fold in PBS. Critical aggregate concentrations (CACs) were determined as the inflection point in a plot of scattering intensity vs. drug concentration, as described previously (Donders et al. 2019a). Timolol butanoate prodrug 3a did not form a colloid (Figure 3B), which was attributed to either its hydrophilicity and / or rapid ester hydrolysis. Conversely, timolol prodrugs 3b, 3c, and 3d all formed colloids with CACs of 12.10 ± 6.31 pM, 2.72 ± 0.94 pM, and 0.66 ± 0.39 pM, respectively. Notably, an inverse correlation was observed between CAC and hydrophobicity of the timolol derivatives. CDA hydrodynamic diameter and polydispersity index (PDI) were measured via dynamic light scattering (DLS) at 37 °C, over time, as an indication of stability. Timolol octanoate, 3b colloids displayed dense, macroscopic aggregates with decreased scattering intensity: the hydrodynamic diameter of 3b colloids increased within the first 48 h (Figure 9A), a behavior typically observed during flocculation (Ganesh et al. 2018). By the end of 7 d, the scattering intensity of 3b colloids decreased significantly, resulting in undetectable colloids due to precipitation (Figure 9B). Colloids of timolol palmitate 3c and timolol decyl-benzoate 3d exhibited a continuous, slow increase in hydrodynamic diameter over 28 d.

[0206] To overcome the inherent instability of timolol prodrug colloids, The use of lipid-based excipients, which have been previously employed in other drug delivery vehicles including lipid nanoparticles (LNPs) was investigated. Specifically, timolol prodrug CDAs were stabilized with 1 DSPC and DMG- PEG2000, which are commonly used phospholipids to stabilize nanoparticles (Donders et al. 2019b). With lipid excipients incorporated into the timolol formulation, consistent hydrodynamic diameters and scattering intensities were observed over 28 d for 3b, 3c and 3d colloids following incubation at 37 °C (Figure 9C and 9D). In the lipid excipient formulation, a timolol prodrug concentration of 100 pM exhibited high loading of 69% w / w. Stabilized timolol prodrug CDAs of 3b, 3c and 3d formed nanoparticles with diameters of 191 ± 43.0 nm, 184 ± 13.2 nm and 154 ± 9.01 nm, respectively, with relatively low PDIs (<0.25, Figure 3C), consistent with FDA guidelines (Danaei et al. 2018) and the literature values for translational nanoparticles (Hoseini et al. 2023). Cryogenic transmission electron microscopy (cryo-TEM) was used to characterize CDA morphology, which were amorphous and spherical (Figure 3D). Together, these results show that timolol esters 3b, 3c and 3d form lipid- stabilized CDAs.

[0207] The hyaluronan-oxime (HA-oxime) hydrogel is comprised of HAK, HAA, and PEGOA4, each of which were characterized by 1 H-NMR (Figure 10 and 11 , respectively). CDAs (and used timolol-P as a proxy for all timolol prodrugs) were dispersed within the HA-oxime hydrogel and investigated their inclusion on the mechanical properties of the gel (Figure 12A). HAK:HAA was formulated at 1.00:0.25 w / w%, with (or without) the addition of timolol-P (3c) CDAs (1 .75 mg / mL timolol-P, equivalent to 1 .00 mg / mL timolol), and then added 0.85 w / w% PEGOA4 to initiate gelation, which was monitored by rheometry. The hydrogel gelled rapidly upon mixing with PEGOA4, displaying similar time sweep G’ and G” profiles over a 50-min period with or without embedded CDAs (Figure 12B). The injectability, stiffness, and swelling of the timolol-P 3c CDA-loaded hydrogels were evaluated: the formulation was easily injected through a 30-gauge needle commonly employed in subconjunctival injection, for up to 15 min after mixing, requiring less than the published, maximum force of 30 N (Baker et al. 2021) for hand injection (Figure 12C). Neither the stiffness nor the minimal swelling of the HA-oxime gel over 28 d were affected by inclusion of CDAs (Figure 12D and 12E), suggesting it would be appropriate for subconjunctival injection.

[0208] The capacity of the timolol prodrug CDA-loaded HA-oxime hydrogel to release timolol in vitro via ester hydrolysis was evaluated (Figure 13A). A series of formulations were compared: timolol maleate alone (the currently available eye drop drug) vs. timolol prodrug CDAs 3b-, 3c-, or 3d-loaded in the HA-oxime hydrogel, which were then incubated in PBS (pH 7.4) at 37 °C. Timolol maleate loaded-hydrogels exhibited an initial burst release within the first 24 h, with complete release at 48 h (Figure 13B to 13D). In contrast, the hydrogel loaded with timolol prodrug CDAs displayed controlled and sustained release of timolol over 28 d. Notably, the release rate correlated with the length of the hydrophobic chain in prodrugs 3b and 3c, with slower release resulting from increased hydrophobicity. The prolonged release of timolol from timolol decyl-benzoate (3d) was attributed to the steric hindrance associated with the benzyl group (Takahashi et al. 2021 ), further decreasing the rate of ester hydrolysis. All hydrogels were enzymatically degraded via hyaluronidase (100 U / mL) after the 28 d release period and the remaining timolol was quantified by ultra-high performance liquid chromatography with a UV detector (UV-UPLC) to calculate the mass balance. The amount of timolol left in the hydrogels inversely correlated with the amount released, as expected. Specifically, no detectable timolol remained in the hydrogels with timolol maleate and timolol 3b whereas up to 37% and 82% of the drug was detected in the timolol 3c and 3d CDA-loaded hydrogels, respectively, reflecting their capacity for long, sustained release (Figure 14). To further validate the mechanism of release, an amide-based timolol analog, 3c', which does not undergo hydrolysis was synsthesized. In vitro release kinetics demonstrated minimal release of timolol from the timolol palmitamide 3c’ CDA-loaded hydrogel, supporting the ester hydrolysis-mediated release mechanism from timolol prodrug CDA-loaded hydrogels (Figure 13E and 13F). While both 3c and 3d timolol prodrug CDAs provide prolonged release from HA-oxime hydrogels, timolol-palmitate 3c hydrolyzes to timolol and palmitic acid, which is generally recognized as safe (GRAS) by the FDA (Mortensen et al. 2017), making 3c the safer choice for further development. Thus, to maximize in vivo drug bioavailability while maintaining long-acting behavior, timolol-P 3c CDAs in HA-oxime for subsequent animal studies were investigated.

[0209] Next, 6-week-old, healthy Long Evans rats were utilized to investigate the sustained lOP-lowering effect of our timolol-CDA-loaded hydrogels injected in the subconjunctiva space. Initial IOP measurements were taken prior to injection (at t = -2, -1 , 0 d) to obtain baseline readings and allow the rats to acclimatize to the measurement procedure using a tonometer (Figure 15). Subsequently, rats received a single subconjunctival injection of either saline, hydrogel alone, timolol maleate (0.137% w / v, equivalent to 0.1 % w / v timolol), or timolol-P 3c CDA-loaded hydrogel (0.175% w / v timolol-P, equivalent to 0.1 % w / v timolol). Each rat was administered the same treatment in both eyes to mitigate any contralateral IOP effects. Over a span of 56 d post-administration, IOP was monitored and blood samples (-100 pL) were collected from the rat tail vein for pharmacokinetic (PK) analysis. At the end of the study, rats were euthanized, perfused with 4% paraformaldehyde, and eyes were enucleated, embedded, cryosectioned and stained to assess in vivo biocompatibility (Figure 16A). The cornea (Figure 16B), retina (Figure 17A), and trabecular meshwork (Figure 17F) of all the rat eyes showed no differences across all treatment groups, as determined with hematoxylin and eosin (H&E) staining, indicating that timolol-CDA-loaded HA-oxime was well tolerated in the subconjunctival space. Importantly, both the corneal and corneal epithelial thicknesses were unchanged across all experimental groups (Figure 16C and 16D), underscoring the advantage of the subconjunctival space for injection. Additionally, thicknesses of outer nuclear layer (ONL), inner nuclear layer (INL) and ganglion cell layer (GCL) in the retina were similar across all groups (Figure 17B to 17E). Timolol-P 3c CDA-loaded gel significantly reduced timolol systemic exposure compared to bolus injection of timolol maleate at 6 h post-injection, as measured by UPLC-MS / MS (Figure 16E). In addition, the systemic exposure to timolol released from CDA-loaded hydrogel continued to decrease progressively after the initial 6 h post-injection, ultimately reaching non- detectable levels by 21 d (Figure 18), yet IOP remained lower much longer. These findings suggest that utilizing the hydrogel composite system may mitigate the risk of systemic toxicity associated with twice-daily administration of timolol maleate eye drops at 0.5% w / v in clinical settings (Abbas et al. 2020). Impressively, the timolol-P 3c CDA-loaded gel exhibited a prolonged IOP- lowering effect compared to timolol maleate alone (Figure 16F and 16G). While the administration of timolol maleate alone initially lowered IOP as expected, the effect was transient, with a significant reduction observed at 6 h but not 24 h. In contrast, the timolol-P 3c CDA-loaded gel consistently reduced IOP, maintaining its effect up to 49 d (Figure 16G). This sustained efficacy is attributed to the prolonged release kinetics facilitated by the composite, colloid- hydrogel delivery system, which has the potential to mitigate the increase in IOP associated with disease progression and vision deterioration in glaucoma.

[0210] In certain embodiments, the prodrug colloid formulations described above are applied to Rho kinase (ROCK) inhibitor drugs to form ROCK-inhibitor prodrug colloid formulations with analogous sustained-release properties.

[0211] In certain embodiments, the preparation of prodrug CDAs, prodrug colloidal nanoparticles, and colloidal nanoparticle hydrogel formulations described herein are applied to Rho kinase (ROCK) inhibitor drugs, following the same principles previously detailed for modifying parent drugs into prodrugs that form CDAs and for modifying beta-blocker drugs into beta-blocker prodrugs that form CDAs and their colloidal nanoparticle hydrogel formulations. In such embodiments, the therapeutic agent is a ROCK inhibitor, and the prodrug is a ROCK inhibitor prodrug that is capable of forming CDAs under the preparation conditions described herein and is embedded within a biocompatible hydrogel for sustained local delivery to tissues, for example ocular tissues.

[0212] According to an embodiment of the present disclosure, the ROCK inhibitor prodrug nanoparticles disclosed herein comprise colloidal nanoparticles which comprise a ROCK inhibitor prodrug formed by a ROCK inhibitor drug conjugated to promoiety of hydrocarbon. The hydrocarbons may be alkyl carbon chains, aryl groups, cyclo-alkyl carbon chains, or any combination of these, wherein the hydrocarbons contain a minimum of six carbons. The ROCK inhibitor drug is conjugated to the alkyl carbon chains, aryl groups, or cyclo-alkyl carbon chains. Conjugation is achieved through chemical bonds which may include ester bonds, amide bonds, carbamate bonds, hydrazone bonds, disulfide bonds, boronic ester bonds, borate ester bonds, phosphonate bonds, glycosidic bonds, anhydride bonds, thioester bonds, phosphate bonds, urea bonds, thiourea bonds, imine bonds, oxime bonds, acetal bonds, ketal bonds, peptidic bonds, succinimide bonds, ether bonds, azo bonds, lactone bonds, lactam bonds, carbonate bonds, sulfonic ester bonds, sulfonamide bonds, siloxane bonds, pyrophosphate bonds, phosphoramide bonds, phosphoramidate bonds, thioether bonds, orthoester bonds, oxoester bonds, vinyl ether bonds, thionoester bonds, and sulfenamide bonds. Those skilled in the art of organic and medicinal chemistry will readily understand how to form these bonds, using well-established methods and reaction conditions known in the literature. For instance, esterification or amidation may be used to conjugate ROCK inhibitor drugs to alkyl chains, while hydrazone and disulfide bonds may be formed using hydrazine derivatives or thiol-based chemistry, respectively. Similarly, oxime bonds may be achieved through the reaction of aldehydes or ketones with hydroxylamines under mild conditions. The conjugation approach may be tailored depending on the desired bond type, which is informed by considerations such as the prodrug’s hydrolysis rate, stability, and pharmacokinetics.

[0213] In terms of utility, the colloidal nanoparticles formed from these ROCK inhibitor prodrugs may be used in a variety of medical applications. These nanoparticles provide enhanced stability and solubility of the prodrug, leading to prolonged drug release and improved bioavailability. Additionally, they may enhance targeted delivery to ocular tissues, improving the therapeutic outcomes for conditions like glaucoma and ocular hypertension. The colloidal formulation is also beneficial for minimizing systemic absorption and side effects, particularly those related to the ROCK inhibitor class of drugs. Furthermore, these nanoparticles may be administered via various routes, including subconjunctival injection, topical application, or even intravenous administration, depending on the clinical indication.

[0214] The ROCK inhibitor prodrug is mixed with lipid excipients in an organic solvent to form a mixture which is suspended in an aqueous buffer solution to induce formation of prodrug colloidal nanoparticles. The lipid excipients enhance the stability and delivery of the ROCK inhibitor prodrug, and a mass ratio of the ROCK inhibitor prodrug to lipid excipients is in a range from 1 to 99% w / w to 99 to 1 % w / w.

[0215] Non-limiting examples of organic solvents include, but are not limited to, any one or combination of ethanol, methanol, isopropanol, butanol, DCM, chloroform, DMSO, acetone, acetonitrile, THF, DMF, and toluene.

[0216] Non-limiting examples of aqueous buffer solutions include, but are not limited to, any one or combination of PBS, TBS, HEPES buffer, citrate buffer, acetate buffer, borate buffer, sodium bicarbonate buffer, MES buffer, and malate buffer.

[0217] Non-limiting examples of lipid excipients include any one or combination of phosphatidylcholines, phosphatidylethanolamines, PS, phosphatidylglycerols, PI, PA, sphingolipids, cholesterol and cholesterol derivatives, triglycerides and diglycerides, cationic lipids, neutral lipids, ionizable lipids, PEG conjugated lipids, ceramides and derivatives, fatty acids, and fatty acid esters.

[0218] Non-limiting examples of the phosphatidylcholines include DPPC, DSPC, SPC; non-limiting examples of the phosphatidylethanolamines include DSPE, DMPE; non-limiting examples of the phosphatidylglycerols include DOPG; PI; non-limiting examples of the sphingolipids include Sphingomyelin; non-limiting examples of the cationic lipids are DOTAP, DODAP; non-limiting examples of the neutral lipids include DOPC; non-limiting examples of the ionizable lipids include SM-102, DLin-MC3-DMA; and non-limiting examples of the PEG conjugated lipids include DMG-PEG2000, DSPE-PEG2000.

[0219] When a ROCK inhibitor prodrug colloid hydrogel formulation is to be synthesized, a biocompatible hydrogel is formed using polymers which may be natural polymers or synthetic polymers or a mixture of both. A non-limiting example of a natural biocompatible hydrogel is a hyaluronan-based hydrogel. Alternatively, the biocompatible hydrogel may be a polyethylene glycol-based hydrogel. In the case when a mixed natural and synthetic hydrogel is preferred, a non-limiting example of such a mixture is a hyaluronan-based hydrogel and polyethylene glycol-based hydrogel.

[0220] Being embedded in the hydrogel allows for localized, controlled, and sustained release of the ROCK inhibitor drug from the ROCK inhibitor prodrug, since the hydrogel formulation may be administered into selected local areas of the anatomy and the release rate depends on enzymatic cleavage of the benzyl ester, in addition to the size, structure, molecular conformation, and composition of the promoiety. According to an embodiment, the release rate further depends on the number of carbons in the alkyl carbon chains, aryl groups, or cycloalkyl carbon chains. The alkyl carbon chains, aryl groups, or cycloalkyl carbon chains may contain at least six carbons, for example between about six and about eighteen carbons, where a higher number of carbons generally corresponds to a longer release time and a lower number corresponds to a shorter release time. To control the release rate of the drug, the number of carbons in the hydrocarbon promoiety is selected within this range. In some embodiments, the promoiety may contain fewer than six carbons, for example from one to about six carbons or from about four to about six carbons. In another embodiment, the promoiety may contain more than eighteen carbons, for example, between about six and about twenty-four carbons, between about six to about thirty carbons, about six to about forty-four carbons, about six to hundred carbons or hundreds of carbons.

[0221] While the examples described herein employ alkyl promoieties of defined chain lengths (e.g., C4, C8, and Ci6), analogous ROCK inhibitor prodrugs bearing more hydrophobic, longer, more branched, and / or sterically bulkier alkyl chains, for example Ci8-C24or higher, are also contemplated. Such prodrugs, having more hydrophobic, longer, more branched and / or sterically bulkier alkyl promoieties may form colloidal drug aggregates and may provide progressively slower drug release due to increased hydrophobicity and reduced solubility. The observed relationship between alkyl chain length and release kinetics in the exemplified prodrugs is expected to extend to these longer, more branched and / or sterically bulkier promoieties, such that increasing the number of carbons beyond the tested examples may afford additional tunability toward more prolonged release.

[0222] In addition to alkyl promoieties composed solely of carbon and hydrogen, the present disclosure also encompasses alkyl-based and non-alkyl promoieties in which the carbon backbone and / or ring system is optionally interrupted or substituted by one or more heteroatoms (for example, O, N, S, F, Cl, Br, I) and / or incorporates additional functional groups, such as ether, thioether, amide, carbamate, urea, carbonate, or ester linkages. In certain embodiments, the promoiety may further comprise aryl, heteroaryl, cycloalkyl, heterocycloalkyl, or polyethylene glycol) (PEG) segments, or combinations thereof. Such heteroatom-containing and mixed-scaffold promoieties are likewise expected to promote colloidal drug aggregate formation and to modulate drug release kinetics by adjusting overall hydrophobicity, polarity, conformational flexibility, and the steric environment around the cleavable linkage, thereby providing additional tunability of sustained-release profiles beyond that achievable with straight-chain hydrocarbon alkyl groups alone.

[0223] Non-limiting examples of ROCK inhibitor drugs that may be incorporated into the hydrogel include, but are not limited to, any one or combination of netarsudil, netarsudil-M1 , ripasudil, fasudil, and pharmaceutically acceptable salts, prodrugs, or derivatives thereof, such that the ROCK inhibitor prodrug colloid hydrogel formulation is formulated for ophthalmic administration for lowering intraocular pressure.

[0224] In certain embodiments, the ROCK inhibitor parent drug is netarsudil-M1 , and the prodrug is netarsudil, which is an ester prodrug of netarsudil-M1 . Netarsudil comprises netarsudil-M1 covalently conjugated to a 2,4- dimethylbenzoate promoiety via an ester linkage. The 2,4-dimethylbenzoate group is an aryl promoiety comprising a benzene ring substituted at the 2- and 4-positions with methyl groups and bearing a carboxylic acid-derived ester functional group and thus constitutes an aryl hydrocarbon promoiety having at least six carbons within the scope of the promoieties described herein. In some embodiments, the 2,4-dimethylbenzoate (2,4-dimethylbenzoyl) promoiety increases the hydrophobicity of the netarsudil prodrug, thereby facilitating the formation and stabilization of CDAs under the preparation conditions described herein and enhancing its incorporation into lipid-stabilized colloidal nanoparticles and hydrogel matrices. Upon administration, hydrolytic cleavage of the ester bond between netarsudil-M1 and the 2,4-dimethylbenzoate promoiety, i.e. , the enzymatic cleavage of the benzyl ester bond, regenerates the active parent ROCK inhibitor netarsudil-M1 and releases 2,4- dimethylbenzoic acid. In certain embodiments, the rate of ester hydrolysis and, consequently, the release kinetics of netarsudil-M1 from the colloidal nanoparticles and hydrogel matrix are controlled by the structure and hydrophobicity of the 2,4-dimethylbenzoate promoiety, in combination with the other formulation parameters described herein. In such embodiments, netarsudil CDAs embedded within the hydrogel provide sustained local delivery of netarsudil-M1 to ocular tissues for reduction of intraocular pressure. ROCK inhibitor prodrugs in colloidal formulations may be used for various therapeutic indications, including ocular hypertension and glaucoma, cerebral vasospasm and other ischemic or neurovascular disorders (such as subarachnoid hemorrhage-associated vasospasm), vascular and cardiovascular disorders (including systemic or pulmonary arterial hypertension and other vasospastic conditions), chronic graft-versus-host disease (cGvHD), fibrotic diseases (including pulmonary, hepatic, renal, or cardiac fibrosis), diabetic microvascular complications, and other conditions in which modulation of Rho kinase signaling is therapeutically beneficial.

[0225] The ROCK inhibitor prodrug colloid hydrogel formulations may have incorporated therein one or more additional intraocular pressure-lowering drugs to give a combination of ROCK inhibitor and non-ROCK-inhibitor drugs in the hydrogel. The addition of these additional drugs may be based on previously known combinations in use so that they are used to provide combinations of these drugs that are useful and have been used before. The selection of non- ROCK-inhibitor drugs to pair with ROCK inhibitors is guided by their complementary mechanisms for reducing IOP and their established clinical use in combination therapies. Common non-ROCK-inhibitor drugs used in glaucoma management include beta-blockers (e.g., timolol), prostaglandin analogs (e.g., latanoprost, bimatoprost), carbonic anhydrase inhibitors (e.g., dorzolamide, brinzolamide), and alpha agonists (e.g., brimonidine). These drugs are chosen because they act through different pathways than ROCK inhibitors, thus offering an additive or synergistic effect when used together. For instance, ROCK inhibitors primarily enhance aqueous humor outflow through the trabecular meshwork and distal outflow pathways, while beta-blockers decrease aqueous humor production by inhibiting adrenergic receptors in the ciliary body. Combining these two classes results in both reduced production and enhanced outflow of aqueous humor, providing greater lOP-lowering effects than either drug alone. Similarly, carbonic anhydrase inhibitors reduce the production of aqueous humor by inhibiting carbonic anhydrase enzymes, offering an alternative mechanism that complements ROCK inhibitors. These combinations are typically selected to optimize IOP reduction, especially in patients with more advanced glaucoma who may not respond sufficiently to monotherapy. Furthermore, combination therapy may reduce the frequency of drug administration, which enhances patient compliance by simplifying the treatment regimen. Additionally, the inclusion of non-ROCK-inhibitor drugs in the colloidal formulation may help to achieve prolonged release and sustained therapeutic effects, minimizing the need for frequent re-administration and reducing the risk of systemic side effects.

[0226] For ophthalmic administration applications, non-limiting examples of additional lOP-lowering drugs include any one or combination of beta-blockers, CAIs, alpha-agonists, and prostaglandin analogs.

[0227] When the formulations are formulated for ophthalmic administration for lowering intraocular pressure, the hydrogel formulation may be administered in several ways including by subconjunctival, intravitreal, topical, transscleral, intracameral, suprachoroidal, subtenon, or subretinal injection for the treatment of ocular hypertension or glaucoma.

[0228] In order to provide control over the release rates of the ROCK inhibitor drug, the number of carbons in the alkyl carbon chains, aryl groups, or cycloalkyl carbon chains is controlled. The alkyl carbon chains, aryl groups, or cycloalkyl carbon chains may contain at least six carbons, for example between about six and about eighteen carbons, where a higher number of carbons generally corresponds to a longer release time and a lower number corresponds to a shorter release time. To control the release rate of the ROCK inhibitor drug, the number of carbons in the hydrocarbon promoiety is selected within this range. In some embodiments, the promoiety may contain fewer than six carbons, for example from one to about six carbons or from about four to about six carbons. In another embodiment, the promoiety may contain more than eighteen carbons, for example, between about six and about twenty-four carbons, between about six to about thirty carbons, about six to about forty-four carbons, about six to hundred carbons or hundreds of carbons.

[0229] For therapeutic applications, the number of carbons in the hydrocarbons is chosen to be sufficient to maintain therapeutic levels of the drug in the eye for a period of time of at least 24 hours post-administration. The numbers of carbons may be tuned to give periods of time of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days. Alternatively, the numbers of carbons may be tuned to give periods of time greater than about 7 days and may range from about 7 days to about 14 days, or to about 1 month, or to about 3 months, or to about 6 months, or up to 1 year. In another embodiment, the degree of branching and / or steric characteristics, together with the promoiety length, are selected to maintain therapeutic drug levels in the eye for at least 24 hours after administration. The degree of branching and / or steric bulkiness, together with the promoiety length may be tuned to give periods of time of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days. Alternatively, the numbers of carbons may be tuned to give periods of time greater than about 7 days and may range from about 7 days to about 14 days, or to about 1 month, or to about 3 months, or to about 6 months, or up to 1 year. The release rate is influenced not only by the number of carbons but also by the chemical structure of the hydrocarbon chain. For example, an 18-carbon alkyl chain would provide a different release profile compared to an 18-carbon aryl or cyclo-alkyl chain, even if they contain the same number of carbons. This is because the molecular structure and conformation of each type of hydrocarbon impact the drug’s solubility, lipophilicity, and steric hindrance, all of which affect how quickly the drug is hydrolyzed or released from the colloid. Alkyl chains are typically linear or branched, and their release rate is primarily influenced by their hydrophobicity and chain length. In contrast, aryl groups are bulkier and more rigid, which could result in slower release rates due to increased steric hindrance and reduced accessibility for enzymatic hydrolysis or other degradation mechanisms. Cyclo-alkyl carbon chains, being more conformationally constrained than alkyl chains but less bulky than aryl groups, would likely offer an intermediate release profile. Accordingly, the nature of the promoiety, e.g., nature of the hydrocarbon group or heteroatom hydrocarbon is an important factor in promoting colloid formation and defining the release kinetics of long- acting formulations and may be selected and tailored to tune release behavior and achieve the desired duration of therapeutic effect.

[0230] The ROCK inhibitor prodrug colloids are produced by reacting a ROCK inhibitor drug with one or more alkyl carbon chains, aryl groups, or cycloalkyl carbon chains or heteroatom alkyl, aryl or cycloalkyl carbon chains that typically contain at least six carbons, for example from about six to about eighteen carbons, or alternatively from about six to about twenty-four carbons. The term “reacting” refers to the process of covalent modification of the ROCK inhibitor drug through well-established organic chemistry techniques. Depending on the functional groups present on the ROCK inhibitor drug and the hydrocarbon, the reaction could involve esterification, amidation, or other types of chemical bonding, such as hydrazone formation or oxime bond formation. For example, if an ester bond is desired, the ROCK inhibitor drug with a hydroxyl or carboxyl functional group would be reacted with a hydrocarbon possessing the complementary functional group (e.g., a carboxylic acid or alcohol) under conditions that promote ester bond formation. This reaction is typically carried out in the presence of an activating agent, such as DCC, and a catalyst like DMAP, in an appropriate organic solvent. Other reaction types, such as amide bond formation, may use similar coupling agents or direct condensation under appropriate conditions, such as using carbodiimides for carboxyl to amine coupling.

[0231] Once the ROCK inhibitor prodrug is synthesized, the prodrug is mixed with lipid excipients in one of the above-mentioned organic solvents to form a mixture, upon which the mixture is suspended in one of the above-mentioned aqueous buffer solutions to induce formation of prodrug colloidal nanoparticles, due to the amphiphilic nature of the lipid excipients and the hydrophobic nature of the prodrug, specifically due to the amphiphilic interactions between the lipids and the prodrug. Finally, the nanoparticles typically undergo buffer exchange via centrifugal filtration to remove residual organic solvent, prodrug, or lipid excipients, and to achieve a stable colloidal suspension.

[0232] To form a ROCK inhibitor prodrug colloid hydrogel, the prodrug colloidal nanoparticles are concentrated to form a suspension of concentrated colloidal nanoparticles. The concentrated colloidal nanoparticles are then mixed with a biocompatible hydrogel precursor, whereupon gelation of the hydrogel precursors is induced to encapsulate the concentrated colloidal nanoparticles in a biocompatible hydrogel formed by crosslinking of the biocompatible hydrogel precursor to form the ROCK inhibitor prodrug colloid hydrogel. The step of concentrating the colloidal nanoparticles to form a suspension of concentrated colloidal nanoparticles is performed using any one of centrifugal filters, dialysis bags, or tangential flow filtration to remove excess solvent and unincorporated materials, thereby concentrating the nanoparticle suspension while maintaining the stability and integrity of the colloids.

[0233] For the case of producing a HA-oxime gel, gelation is induced by mixing the components of the HA-oxime gel in an aqueous environment under mild conditions. Specifically, a solution containing 1.00% (w / v) HAK is mixed with 0.25% (w / v) HAA in the presence of 70 mol% of a crosslinking agent, such as PEGOA4. The crosslinking agent reacts with the aldehyde and ketone groups present on the HAK and HAA, respectively, forming stable oxime bonds through a condensation reaction. The gelation process occurs in situ where the HAA reacts more quickly with the crosslinker compared to HAK, allowing for controlled modulation of the gelation rate. The reaction progresses over a time frame that may be tuned by adjusting the concentrations of HAK, HAA, and crosslinker, as well as the temperature and pH of the solution. Full gelation typically occurs within minutes to hours, depending on the desired application, leading to the formation of a biocompatible, bioresorbable hydrogel matrix.

[0234] The ROCK inhibitor prodrug colloid hydrogel may be formed with additional intraocular pressure-lowering drugs mentioned above which are mixed in with the concentrated colloidal nanoparticles prior to gelation of the hydrogel precursors.

[0235] The ROCK inhibitor prodrug colloid hydrogel will now be illustrated with the following non-limiting example.

[0236] Netarsudil was evaluated for its ability to form CDAs and for its compatibility with a hyaluronan-based hydrogel platform enabling sustained drug release (Figure 19). Colloids were generated by diluting netarsudil dissolved in DMSO into aqueous buffer. Serial dilutions demonstrated a clear scattering-intensity threshold, confirming formation of netarsudil CDAs. Curve fitting identified a CAC of approximately 12-13 pM. Bare netarsudil colloids were unstable and rapidly flocculated, leading to increased particle size, loss of detectable colloidal features, and visible macroscopic aggregation. To overcome this instability, lipids (DSPC and DMG- PEG2000) were incorporated during colloid formation (Figure 20A-20B). This produced smaller, stable colloids (-200-250 nm) that retained consistent scattering intensity, size, and PDI for at least 21 days at 37 °C. The lipid- stabilized colloids also showed high drug loading (nearly 50%), significantly higher than typical nanoparticle formulations.

[0237] A hyaluronan-oxime hydrogel was employed to embed these stable colloids for sustained release (Figure 21A-21C). Colloids were mixed uniformly into the polymer solution prior to gelation, ensuring consistent drug loading across replicates. After gelation, buffer was layered onto the hydrogel and sampled over time to quantify release (Figure 21 A). In buffer alone, only -25% of the loaded netarsudil was released over the first week, after which the release plateaued. Analysis confirmed that this initial release corresponded to free (non-colloidal) drug present in the gel; the encapsulated colloids remained largely retained within the hydrogel.

[0238] To simulate ocular metabolism and promote further release, the hydrogel-colloid system was subsequently incubated with esterase-containing buffer (using butyrylcholinesterase) (Figure 21 B-21C). Netarsudil, being an ester prodrug, underwent enzymatic hydrolysis to its more hydrophilic active metabolite, netarsudil-M1. Introduction of esterase triggered dissolution of colloids within the hydrogel, increased the release rate, and ultimately resulted in near-complete release (-100%) of the originally loaded drug mass by day 28.

[0239] Accordingly, in certain embodiments the formulations comprise CDAs of any drug or prodrug capable of forming colloidal drug aggregates under the preparation conditions described herein, embedded within a hydrogel for sustained local delivery, without limitation to p-blockers, ROCK inhibitors or any other specific therapeutic class. References

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[0241] Baker, Alexander E.G., Hong Cui, Brian G. Ballios, Sonja Ing, Peng Yan, Joe Wolfer, Thomas Wright, et al. 2021. “Stable Oxime-Crosslinked Hyaluronan- Based Hydrogel as a Biomimetic Vitreous Substitute.” Biomaterials 271 (March): 120750. https: / / doi.Org / 10.1016 / j. biomaterials.2021 .120750.

[0242] Danaei, M, M Dehghankhold, S Ataei, F Hasanzadeh Davarani, R Javanmard, A Dokhani, S Khorasani, and M R Mozafari. 2018. “Impact of Particle Size and Polydispersity Index on the Clinical Applications of Lipidic Nanocarrier Systems.” Pharmaceutics 10 (2). https: / / d0i.0rg / l 0.3390 / pharmaceutics10020057.

[0243] Donders, Eric N., Ahil N. Ganesh, Hayarpi Torosyan, Parnian Lak, Brian K. Shoichet, and Molly S. Shoichet. 2019a. “Triggered Release Enhances the Cytotoxicity of Stable Colloidal Drug Aggregates.” ACS Chemical Biology 14 (7): 1507-14. https: / / doi.org / 10.1021 / acschembio.9b00247.

[0244] Donders, Eric N, Ahil N Ganesh, Hayarpi Torosyan, Parnian Lak, Brian K Shoichet, and Molly S Shoichet. 2019b. “Triggered Release Enhances the Cytotoxicity of Stable Colloidal Drug Aggregates.” ACS Chemical Biology 14 (7): 1507-14. https: / / doi.org / 10.1021 / acschembio.9b00247.

[0245] Ganesh, Ahil N., Eric N. Donders, Brian K. Shoichet, and Molly S. Shoichet. 2018. “Colloidal Aggregation: From Screening Nuisance to Formulation Nuance.” Nano Today 19:188-200. https: / / doi.Org / 10.1016 / j.nantod.2018.02.011.

[0246] Hoseini, Benyamin, Mahmoud Reza Jaafari, Amin Golabpour, Amir Abbas Momtazi-Borojeni, Maryam Karimi, and Saeid Eslami. 2023. “Application of Ensemble Machine Learning Approach to Assess the Factors Affecting Size and Polydispersity Index of Liposomal Nanoparticles.” Scientific Reports 13 (1): 18012. https: / / doi.Org / 10.1038 / S41598-023-43689-4.

[0247] Mortensen, Alicja, Fernando Aguilar, Riccardo Crebelli, Alessandro Di Domenico, Birgit Dusemund, Maria Jose Frutos, Pierre Galtier, et al. 2017. “Re- Evaluation of Fatty Acids (E 570) as a Food Additive.” EFSA Journal. European Food Safety Authority 15 (5): e04785. https: / / doi.Org / 10.2903 / j.efsa.2017.4785. Takahashi, Masato, Ibuki Hirota, Tomoyuki Nakano, Tomoyuki Kotani, Daisuke Takani, Kana Shiratori, Yura Choi, Masami Haba, and Masakiyo Hosokawa. 2021 . “Effects of Steric Hindrance and Electron Density of Ester Prodrugs on

[0248] Controlling the Metabolic Activation by Human Carboxylesterase.” Drug Metabolism and Pharmacokinetics 38: 100391 . https: / / doi.Org / 10.1016 / j.dmpk.2021.100391.

Claims

THEREFORE WHAT IS CLAIMED IS:1 . A colloidal nanoparticle comprising: a therapeutic agent capable of forming colloidal drug aggregates (CDAs) under conditions effective to form CDAs, the therapeutic agent being a prodrug of a parent drug that is chemically conjugated to a promoiety comprising(i) one or more hydrocarbon groups selected from alkyl, aryl, and cycloalkyl carbon chains each having at least six carbons, and / or(ii) an alkyl-based or non-alkyl segment having a carbon backbone and / or ring system interrupted or substituted by one or more heteroatoms selected from oxygen, nitrogen, and sulfur, or by one or more halogens selected from fluorine, chlorine, bromine, and iodine, the parent drug being conjugated to the promoiety via a bond selected from ester, amide, carbamate, hydrazone, disulfide, boronic ester, borate ester, phosphonate, glycosidic, anhydride, thioester, phosphate, urea, thiourea, imine, oxime, acetal, ketal, peptidic, succinimide, ether, azo, lactone, lactam, carbonate, sulfonic ester, sulfonamide, siloxane, pyrophosphate, phosphoramide, phosphoramidate, thioether, orthoester, oxoester, vinyl ether, thionoester, and sulfenamide bonds; and lipid excipients present in an amount effective to enhance the stability and delivery of the therapeutic agent, wherein a mass ratio of the therapeutic agent to the lipid excipients is from about 1 :99 to about 99:1 (w / w).

2. The colloidal nanoparticle of claim 1 , wherein the promoiety comprises one or more functional groups selected from ether, thioether, amide, carbamate, urea, carbonate, and ester linkages.

3. The colloidal nanoparticle of claim 1 or 2, wherein the promoiety further comprises one or more segments selected from aryl, heteroaryl, cycloalkyl, heterocycloalkyl, and polyethylene glycol) (PEG) segments, or combinations thereof.

4. The colloidal nanoparticle according to claim 1 , 2, or 3, wherein the parent drug is a beta-blocker drug, and the prodrug is beta-blocker prodrug or wherein the parent drug is a ROCK inhibitor drug and the pro-drug is a ROCK inhibitor prodrug.

5. The colloidal nanoparticle according to claim 4, wherein the beta-blocker is timolol and the beta-blocker prodrug comprises at least one compound selected from the group consisting of timolol octanoate, timolol palmitate, timolol decylbenzoate, and timolol-abietate.

6. The colloidal nanoparticle according to claim 5, wherein the beta-blocker is timolol and the beta-blocker prodrug timolol palmitate.

7. The colloidal nanoparticle according to claim 4, wherein the ROCK inhibitor drug is netarsudil-M1 and the ROCK-inhibitor is netarsudil.

8. The colloidal nanoparticle according to any one of claims 1 to 7, wherein the lipid excipients comprise one or more lipids selected from the group consisting of phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines (PS), phosphatidylglycerols, phosphatidylinositols (PI), phosphatidic acids (PA), sphingolipids, cholesterol and cholesterol derivatives, triglycerides, diglycerides, cationic lipids, neutral lipids, ionizable lipids, polyethylene glycol (PEG) conjugated lipids, ceramides and derivatives, fatty acids, and fatty acid esters.

9. A colloidal nanoparticle hydrogel formulation comprising: the colloidal nanoparticles according to any one of claims 1 to 8; and a biocompatible hydrogel matrix,wherein the biocompatible hydrogel matrix is formed by crosslinking of a biocompatible hydrogel precursor in solution and the colloidal nanoparticles are embedded within the biocompatible hydrogel matrix forming a colloid nanoparticle hydrogel formulation for controlled release of the parent drug and wherein the release of the parent drug from the colloidal nanoparticle is mediated by hydrolysis of the bonds releasing the parent drug from the promoiety.

10. The colloidal nanoparticle hydrogel formulation according to claim 9, wherein the release rate of the parent drug is modulated by a factor selected from the number of carbons of the promoiety, the length of the promoiety, the degree of branching of the promoiety, the steric bulkiness of the promoiety, and any combination thereof.11 . The colloidal nanoparticle hydrogel formulation according to claim 9 or 10, wherein the release rate of the parent drug is modulated by the number of carbons in the promoiety, with shorter alkyl chains and smaller cyclo-alkyl or aryl groups resulting in a faster release and longer alkyl chains and larger cyclo-alkyl or aryl groups resulting in a slower release.

12. The colloidal nanoparticle hydrogel formulation according to claims 9, 10 or 11 , wherein the biocompatible hydrogel is formed from one or more polymers selected from the group consisting of natural polymers and synthetic polymers.

13. The colloidal nanoparticle hydrogel formulation according to claim 12, wherein the biocompatible hydrogel is a hyaluronan-based hydrogel.

14. The colloidal nanoparticle hydrogel formulation according to claim 12, wherein the biocompatible hydrogel is a polyethylene glycol-based hydrogel.

15. The colloidal nanoparticle hydrogel formulation according to claim 12, wherein the biocompatible hydrogel is a mixed hydrogel comprising a hyaluronan-based hydrogel and a polyethylene glycol-based hydrogel.

16. The colloidal nanoparticle hydrogel formulation according to any one of claims 9 to 115, further comprising at least one additional drug.

17. The colloidal nanoparticle hydrogel formulation according to claim 16, wherein the parent drug is a beta-blocker and the at least one additional drug comprises at least one non-beta-blocker drug.

18. The colloidal nanoparticle hydrogel formulation according to claim 17, wherein the beta-blocker comprises at least one compound selected from the group consisting of acebutolol, atenolol, betaxolol, bisoprolol, carvedilol, labetalol, metoprolol, nebivolol, nadolol, pindolol, propranolol, sotalol, timolol, and combinations thereof, and wherein the colloidal nanoparticle hydrogel formulation is formulated for ophthalmic administration for lowering intraocular pressure.

19. The colloidal nanoparticle hydrogel formulation according to claim 18, wherein the beta-blocker is timolol and the beta-blocker prodrug comprises at least one compound selected from the group consisting of timolol octanoate, timolol palmitate, timolol decylbenzoate, and timolol-abietate.

20. The colloidal nanoparticle hydrogel formulation according to claim 19, wherein the beta-blocker is timolol and the beta-blocker prodrug timolol palmitate.21 . The colloidal nanoparticle hydrogel formulation according to any one of claims 16 to 20, wherein the at least one additional drug comprises at least one non-beta-blocker intraocular pressure-lowering drug.

22. The colloidal nanoparticle hydrogel formulation according to claim 21 , wherein the at least one non-beta-blocker intraocular pressure-lowering drug comprises one or more agents selected from the group consisting of carbonic anhydrase inhibitors (CAIs), Rho kinase (ROCK) inhibitors, alpha-agonists and prostaglandin analogs.

23. The colloidal nanoparticle hydrogel formulation according to claim 9 to 16, wherein the parent drug is a ROCK Inhibitor and the at least one additional drug comprises at least one non-ROCK-inhibitor drug.

24. The colloidal nanoparticle hydrogel formulation according to claim 23, wherein the ROCK inhibitor is netarsudil-M1 , and wherein the colloidal nanoparticle hydrogel formulation is formulated for ophthalmic administration for lowering intraocular pressure.

25. The colloidal nanoparticle hydrogel formulation according to claim 23 or 24, wherein the at least one additional drug comprises at least one non-ROCK- inhibitor intraocular pressure-lowering drug.

26. The colloidal nanoparticle hydrogel formulation according to claim 25, wherein the at least one non-ROCK inhibitor intraocular pressure-lowering drug comprises one or more agents selected from the group consisting of beta blockers carbonic anhydrase inhibitors (CAIs), alpha-agonists and prostaglandin analogs.

27. The colloidal nanoparticle hydrogel formulation according to any one of 9 to 26, wherein the colloidal nanoparticle hydrogel formulation is administered by a route selected from the group consisting of subconjunctival injection, intravitreal injection, topical application, transscleral injection, intracameral injection, suprachoroidal injection, subtenon injection, subretinal injection, intravenous administration.

28. The colloidal nanoparticle hydrogel formulation according to claim 27 wherein the route of administration is selected for the treatment of ocular hypertension or glaucoma.

29. The colloidal nanoparticle hydrogel formulation according to claim 28, wherein the selected factor modulating the release rate of the parent drug is sufficient to maintain therapeutic levels of the drug in the eye for a period of at least 24 hours post-administration.

30. The colloidal nanoparticle hydrogel formulation according to claim 29, wherein the selected factor modulating the release rate of the parent drug is the number of carbons in the promoiety.31 . The colloidal nanoparticle hydrogel formulation according to claim 29 or30, wherein the period of time is greater than about 7 days and up to about 1 year.

32. The colloidal nanoparticle hydrogel formulation according to claim 31 , wherein the period of time is from about 7 days to about 6 months.

33. The colloidal nanoparticle hydrogel formulation according to claim 32, wherein the period of time is from about 7 days to about 3 months.

34. The colloidal nanoparticle hydrogel formulation according to claim 33, wherein the period of time is from about 7 days to about 1 month.

35. The colloidal nanoparticle hydrogel formulation according to claim 34, wherein the period of time is from about 7 days to about 14 days.

36. The colloidal nanoparticle hydrogel formulation according to any one of claims 9 to 35, wherein the promoiety comprises from about six to about hundred carbons, wherein higher numbers of carbons are associated with longer release times and lower numbers of carbons are associated with shorter release times.

37. The colloidal nanoparticle hydrogel formulation according to claim 36, wherein the promoiety has from about six to about twenty-four carbons.

38. A method of producing a colloidal nanoparticle hydrogel formulation for controlled and sustained release of a parent drug, the formulation comprising colloidal nanoparticles that comprise a prodrug of the parent drug, the prodrug being capable of forming colloidal drug aggregates, the method comprising: mixing the prodrug with lipid excipients in an organic solvent to form a mixture; suspending the mixture in an aqueous buffer solution to induce formation of prodrug colloidal nanoparticles; concentrating the prodrug colloidal nanoparticles to form a suspension of concentrated colloidal nanoparticles; mixing the suspension of concentrated colloidal nanoparticles with a biocompatible hydrogel precursor; and inducing gelation of the hydrogel precursor to embed the concentrated colloidal nanoparticles within a biocompatible hydrogel formed by crosslinking of the biocompatible hydrogel precursor, thereby forming , thereby forming the colloidal nanoparticle hydrogel formulation, wherein controlled and sustained release of the parent drug is mediated by hydrolysis of the bonds releasing the parent drug from the alkyl carbon chains, aryl groups, or cyclo-alkyl carbon chains of the promoiety.

39. The method according to claim 38, further comprising the step of synthesizing the prodrug by reacting the parent drug with promoiety comprising hydrocarbons selected from the group consisting of alkyl carbon chains, aryl groups, and cyclo-alkyl carbon chains, the alkyl carbon chains, aryl groups, or cyclo-alkyl carbon chains having at least six carbons, the parent drug being conjugated to the alkyl carbon chains, aryl groups, or cyclo-alkyl carbon chains through bonds selected from the group consisting of ester bonds, amide bonds, carbamate bonds, hydrazone bonds, disulfide bonds, boronic ester bonds, borate ester bonds, phosphonate bonds, glycosidic bonds, anhydride bonds, thioester bonds, phosphate bonds, urea bonds, thiourea bonds, imine bonds, oxime bonds, acetal bonds, ketal bonds, peptidic bonds, succinimide bonds,ether bonds, azo bonds, lactone bonds, lactam bonds, carbonate bonds, sulfonic ester bonds, sulfonamide bonds, siloxane bonds, pyrophosphate bonds, phosphoramide bonds, phosphoramidate bonds, thioether bonds, orthoester bonds, oxoester bonds, vinyl ether bonds, thionoester bonds, and sulfenamide bonds.

40. The method according to claim 38 or 39, wherein the promoiety has from about six to about hundred carbons.41 . The method according to claims 38, 39 or 40, wherein the release rate of the parent drug is modulated by a factor selected from the number of carbons of the promoiety, the length of the promoiety, the degree of branching of the promoiety, the steric bulkiness of the promoiety, and any combination thereof.

42. The method according to claim 41 , wherein the release rate of the parent drug is modulated by the number of carbons of the promoiety, with shorter alkyl chains and smaller cyclo-alkyl or aryl groups resulting in a faster release and longer alkyl chains and larger cyclo-alkyl or aryl groups resulting in a slower release.

43. The method according to any one of claims 38 to 42, wherein the biocompatible hydrogel is formed from one or more polymers selected from the group consisting of natural polymers and synthetic polymers.

44. The method according to claim 43, wherein the biocompatible hydrogel is a hyaluronan-based hydrogel.

45. The method according to claim 43, wherein the biocompatible hydrogel is a polyethylene glycol-based hydrogel.

46. The method according to claim 43, wherein the biocompatible hydrogel is a mixed hydrogel comprising a hyaluronan-based hydrogel and a polyethylene glycol-based hydrogel.

47. The method according to any one of claims 38 to 46, wherein the lipid excipients comprise one or more lipids selected from the group consisting of phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines (PS), phosphatidylglycerols, phosphatidylinositols (PI), phosphatidic acids (PA), sphingolipids, cholesterol and cholesterol derivatives, triglycerides, diglycerides, cationic lipids, neutral lipids, ionizable lipids, polyethylene glycol (PEG) conjugated lipids, ceramides and derivatives, fatty acids, and fatty acid esters.

48. The method according to claim 47, wherein the lipid excipients comprise one or more lipids selected from the group consisting of phosphatidylcholines selected from Dipalmitoylphosphatidylcholine (DPPC), Distearoylphosphatidylcholine (DSPC), and Soybean Phosphatidylcholine (SPC); phosphatidylethanolamines selected from Distearoylphosphatidylethanolamine (DSPE) and Dimyristoylphosphatidylethanolamine (DMPE); phosphatidylglycerols selected from Dioleoylphosphatidylglycerol (DOPG); phosphatidylinositols (PI); sphingolipids selected from Sphingomyelin; cationic lipids selected from 1 ,2- dioleoyl-3-trimethylammonium-propane (DOTAP) and 1 ,2-dioleoyl-3- dimethylammonium-propane (DODAP); neutral lipids selected from Dioleoylphosphatidylcholine (DOPC); ionizable lipids selected from SM-102 and DLin-MC3-DMA; and polyethylene glycol (PEG) conjugated lipids selected from 1 ,2-dimyristoyl-rac-glycerol-PEG2000 (DMG-PEG2000) and DSPE-PEG2000.

49. The method according to any one of claims 38 to 48, wherein the organic solvent comprises one or more solvents selected from the group consisting of ethanol, methanol, isopropanol, butanol, dichloromethane (DCM), chloroform, dimethyl sulfoxide (DMSO), acetone, acetonitrile, tetrahydrofuran (THF), dimethylformamide (DMF), and toluene.

50. The method according to any one of claims 38 to 49, wherein the aqueous buffer solution comprises one or more buffers selected from the group consisting of phosphate-buffered saline (PBS), Tris-buffered saline (TBS), HEPES buffer, citrate buffer, acetate buffer, borate buffer, sodium bicarbonate buffer, MES buffer, and malate buffer.51 . The method according to any one of claims 38 to 50, wherein concentrating the colloidal nanoparticles to form a suspension of concentrated colloidal nanoparticles comprises using one or more of centrifugal filters, dialysis bags, or tangential flow filtration to remove excess solvent and unincorporated materials, thereby concentrating the nanoparticle suspension while maintaining the stability and integrity of the colloids.

52. The method according to any one of claims 38 to 51 , wherein the parent drug is a beta-blocker drug, and the prodrug is beta-blocker prodrug.

53. The method according to claim 52, wherein the beta-blocker comprises at least one compound selected from the group consisting of acebutolol, atenolol, betaxolol, bisoprolol, carvedilol, labetalol, metoprolol, nebivolol, nadolol, pindolol, propranolol, sotalol, timolol, and combinations thereof.

54. The method according to claim 53, wherein the beta-blocker is timolol and the beta-blocker prodrug comprises at least one compound selected fromthe group consisting of timolol octanoate, timolol palmitate, timolol decylbenzoate, and timolol-abietate.

55. The method according to claim 54, wherein the beta-blocker is timolol and the beta-blocker prodrug timolol palmitate.

56. The method according to any one of claims 52 to 55, further comprising the step of adding at least one non-beta-blocker intraocular pressure-lowering drug.

57. The method according to claim 56, wherein the at least one non-beta- blocker intraocular pressure-lowering drug is added during the step of mixing the suspension of concentrated colloidal nanoparticles with the biocompatible hydrogel precursor.

58. The method according to claim 56 or 57, wherein the at least one non- beta-blocker intraocular pressure-lowering drug comprises one or more agents selected from the group consisting of carbonic anhydrase inhibitors (CAIs), Rho kinase (ROCK) inhibitors, alpha-agonists and prostaglandin analogs.

59. The method according to any one of claims 38 to 51 , wherein the parent drug is a ROCK inhibitor drug, and the pro-drug is a ROCK inhibitor prodrug.

60. The method according to claim 59, wherein the ROCK inhibitor is netarsudil-M1.61 . The method according to claim 59 or 60, further comprising the step of adding at least one non-ROCK-inhibitor intraocular pressure-lowering drug.

62. The method according to claim 61 , wherein the at least one non-ROCK- inhibitor intraocular pressure-lowering drug is added during the step of mixing the suspension of concentrated colloidal nanoparticles with the biocompatible hydrogel precursor.

63. The method according to claim 61 or 62, wherein the at least one non- ROCK inhibitor intraocular pressure-lowering drug comprises one or more agents selected from the group consisting of beta blockers carbonic anhydrase inhibitors (CAIs), alpha-agonists and prostaglandin analogs.

64. A colloidal nanoparticle hydrogel formulation being produced according to any one of claims 38 to 63.

65. The colloidal nanoparticle hydrogel formulation according to claim 64, wherein the parent drug is a beta-blocker drug, and the prodrug is beta-blocker prodrug or wherein the parent drug is a ROCK inhibitor, and the prodrug is ROCK-inhibitor prodrug.

66. A method of treating ocular hypertension in a patient, comprising administering to an eye of the patient the colloidal nanoparticle hydrogel formulation according to any one of claims 9 to 37 or 64 or 65.

67. The method according to claim 66, wherein the colloidal nanoparticle hydrogel formulation is administered by subconjunctival, intravitreal, topical, transscleral, intracameral, suprachoroidal, subtenon, or subretinal injection for the treatment of ocular hypertension or glaucoma.68 The method according to claim 66 or 67, wherein the number of carbons in the hydrocarbons of the promoiety is sufficient to maintain therapeutic levels of the drug in the eye for a period of at least 24 hours post-administration.

69. The method according to claim 68, wherein the period of time is greater than about 7 days and up to about 1 year.

70. The method according to claim 69, wherein the period of time is from about 7 days to about 6 months.71 . The method according to claim 70, wherein the period of time is from about 7 days to about 3 months.

72. The method according to claim 71 , wherein the period of time is from about 7 days to about 1 month.

73. The method according to claim 72, wherein the period of time is from about 7 days to about 14 days.

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