WO2018169950A1

WO2018169950A1 - Injectable multidrug delivery hydrogel and uses thereof

Info

Publication number: WO2018169950A1
Application number: PCT/US2018/022172
Authority: WO
Inventors: Maziar MOHAMMADI; Kisha K. PATEL; Seyedeh P. ALAIE; Nisha HOLLINGSWORTH; Jordan J. Green; Cagri Besirli; Ronald G. LARSON
Original assignee: The Johns Hopkins University; The Regents Of The University Of Michigan
Priority date: 2017-03-13
Filing date: 2018-03-13
Publication date: 2018-09-20

Abstract

The present invention provides a novel sustained drug release system comprising microparticles incorporated into a bulk hydrogel that is engineered to be in liquid form at room temperature for simple delivery into the eye or other tissue site, and form a hydrogel at physiological body temperatures (approximately 37 °C) to act as a depot release platform. Upon injection or implantation, this novel drug delivery system immediately gels in situ to form a depot that releases a number of different drug molecules at predetermined rates and times to provide optimal intraocular or intra-tissue concentrations for effective treatment.

Description

INJECTABLE MULTIDRUG DELIVERY HYDROGEL AND USES THEREOF

REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application Nos. 62/470,506, filed on March 13, 2017, and 62/561,796 filed on September 22, 2017, both of which are hereby incorporated by reference for all purposes as if fully set forth herein.

STATEMENT OF GOVERNMENTAL INTEREST

[0002] This invention was made with government support under grant no. 1403335, awarded from the National Science Foundation, and grant no. EY026148, awarded from the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] Careful management with topical medications is crucial for preventing postoperative complications including vision loss after ocular surgery.¹ Successful surgical outcomes require frequent applications of one or more ophthalmic drops over several days to several weeks and depends heavily on patient compliance. Similar to most other surgical procedures, incisional ophthalmic procedures carry a small risk of infection^{2 3} and a more common physiologic reaction of inflammation in the immediate post-operative period.^{4 5} In the later stages of surgical recovery, secondary complications including rebound

inflammation and elevated intraocular pressure (IOP) may cause morbidity, suboptimal vision, and on rare occasions may lead to vision loss and further surgery. To prevent postsurgical complications, topical antibiotic and corticosteroid formulations are administered routinely after ophthalmic procedures.^6"8 Post-surgical inflammation as well as

corticosteroids induce IOP increase in many patients,^{1 9"11} requiring additional topical treatment with ocular hypotensives, adding to the overall burden of treatment and increasing patient non-adherence.

[0004] The vast majority of ophthalmic drugs are marketed as topical formulations. The delivery of topical drug formulations is greatly hindered by ocular surface barrier and availability is limited by rapid clearance from the ocular surface by the tear flow.^{12 13} Despite these inherent shortcomings, the most critical barrier for ophthalmic drug delivery remains to be poor patient adherence, limiting effective dosing and delaying visual recovery. Patient adherence is affected by numerous factors, including ocular surface irritation and allergic reaction with pain and discomfort caused by topical ophthalmic agents.¹⁴

[0005] Alternative ocular drug delivery mechanisms have been developed to replace topical drop administration, improving drug dosing and minimizing side effects. These mechanisms employ distinct approaches to the drug delivery problem and include contact lenses, implants and hydrogels.^{12 14"18} Although most are still in the pre-clinical development phase, a handful of products have received FDA approval and are available for patient use.¹⁴

[0006] Ocular drug implants can be categorized into different groups based on several characteristics including release duration, location of administration, and type of molecule delivered. One of the pioneering technologies in ophthalmic therapeutics was Vitrasert (Bausch & Lomb, Rochester, NY), a non-biodegradable intravitreal implant delivering ganciclovir to treat cytomegalovirus retinitis.¹² Vitrasert uses two polymeric layers composed of poly vinyl alcohol (PVA) and ethylene vinyl alcohol (EVA) to deliver ganciclovir at a constant rate for five to eight months.¹² This system had several

disadvantages due to non-biodegradable design, including suboptimal biocompatibility and immunogenicity compared with a biodegradable system, and the need for vitreoretina surgery for implantation and removal.^{19 20} Retisert (Bausch & Lomb, Rochester, NY) is another FDA-approved, non-biodegradable intravitreal implant that is indicated for the treatment of chronic non-infectious uveitis. This implant is composed of a PVA and silicon container and delivers fluocinolone acetonide up to 2.5 years.¹² Similar to Vitrasert, Retisert is inserted into the vitreous cavity via transscleral route during vitreoretinal surgery. Some of the implant and surgical procedure-related complications of Retisert include elevated IOP, cataract formation, and retinal detachment.¹²'^{21 22}

[0007] Iluvien (Alimera Sciences, Alpharetta, GA) is the next generation nonbiodegradable intravitreal corticosteroid implant which was recently approved by FDA. Unlike Retisert, Iluvien does not require surgical implantation and is delivered into the vitreous cavity via transscleral injection. The implant releases fluocinolone acetonide for the treatment of diabetic macular edema up to 3 years.¹² Similar to other corticosteroids, Iluvien causes cataract formation in 82% of patients and leads to elevated IOP in 34% of cases.^{23 24} While the Iluvien implant is not biodegradable, surgical removal is typically not required once the drug is depleted.

[0008] Ozurdex (Allergan, Irvine, CA) is an intravitreal implant for delivering dexamethasone to treat macular edema secondary to diabetes and retinal vein occlusion, as well as non-infectious posterior uveitis. Ozurdex is the first biodegradable implant approved by FDA for ocular indications.¹² Dexamethasone is encapsulated in a poly(D,L-lactide-co- glycolide) (PLGA) matrix, and the implant is administered via intravitreal injection. Steroid is released from the implant for up to six months.¹² The adverse effects of Ozurdex is similar to other implants releasing corticosteroid agents and include cataract formation and elevated

JOp 25,26

[0009] Other biodegradable implants are currently under development, including Verisome (Icon Bioscience, Sunnyvale, CA). This drug delivery technology encapsulates various drug molecules and can maintain drug release for up to a year after intravitreal injection.¹² Dexycu implant based on Verisome technology releases dexamethasone and is currently being tested in a phase III clinical trial to treat post-operative inflammation after cataract surgery.²⁷

[0010] Hirani et al. designed triamcinolone acentonide loaded PEG-PLGA nanoparticles and loaded the nanoparticles in a PLGA-PEG-PLGA hydrogel to treat age related macular degeneration (AMD).²⁸ The authors reported drug release from PLGA-PEG-PLGA hydrogel for up to 10 days, though most drug release occurred in the first 10 hours. This initial burst release of drug molecule followed by rapid reduction in drug concentration is not favorable for the treatment of ocular diseases. Using nanoparticles for a sustained release strategy is ineffective. Drug release is governed by diffusion of molecules, thus nanoparticles are too small for the controlled release of the drug. In addition, to further sustain the drug release, using more hydrophobic polymers that degrade slower including poly(lactic acid) and poly(caprolactone) are essential.

[0011] None of the current drug delivery technologies discussed above are designed to release multiple drug molecules while regulating release durations. A multidrug delivery platform that temporally regulates drug release would be ideal for the treatment of many ophthalmic conditions, including post-operative management requiring multiple topical agents for inflammation, infection, and elevated IOP. For other ocular diseases including glaucoma, previous studies have shown that combination therapy is more effective than single drug therapy in many patients.^{12 29} SUMMARY OF THE INVENTION

[0012] To address the shortcomings of existing technologies and fulfill an unmet need in ocular drug delivery, the present invention provides a novel drug delivery platform designed to release multiple drug molecules with precise temporal regulation. The inventive drug delivery system is administered via intraocular or periocular injection, and is composed of biocompatible and biodegradable materials for optimal ocular safety.

[0013] In accordance with some embodiments, the present invention provides a sustained drug release system comprising microparticles incorporated into a bulk hydrogel that is engineered to be in liquid form at room temperature for simple delivery into the eye or other tissue site, and form a hydrogel at physiological body temperatures (approximately 37 °C) to act as a depot release platform. Upon injection or implantation, this novel drug delivery system immediately gels in situ to form a depot that releases a number of different drug molecules at predetermined rates and times. In some embodiments, the hydrogel

compositions can be formed to release from 2 to 5 different biologically active agents or drugs for between 1 to 365 days.

[0014] In some embodiments the drug release system of the present invention can be used to provide optimal intraocular concentrations for effective post-operative treatment.

[0015] In an embodiment, the inventive injectable, multi drug delivery system addresses the unmet need of a simple, effective, and patient-independent post-operative treatment regimen in which the daily intraocular dosage and the duration of release of each ocular agent can be adjusted based on the patient needs. After experimenting with the polymer encapsulating the drug molecules, the amount of microparticles embedded within the hydrogel, and the type of triblock copolymer used, the inventors arrived at an ideal drug delivery system that effectively prevents immediate and late complications after ophthalmic surgery, requiring minimal patient compliance and improve post-operative success rates for ophthalmic surgeons.

[0016] In some embodiments, the thermos ensitive hydrogel compositions of the present invention are configured to release at least one broad-spectrum antibiotic, at least one potent corticosteroid, and at least one ocular antihypertensive, three ophthalmic therapeutic agents essential for post-operative management. In some embodiments, the thermosensitive hydrogel compositions of the present invention are designed to release one or more antibiotics for up to a week, at least one corticosteroid for up to two months or more and at least one anti-hypertensive agent for at least one month.

[0017] In accordance with an embodiment, the present invention provides a

thermosensitive hydrogel composition comprising one or more biologically compatible triblock copolymers and one or more biologically active agents.

[0018] In accordance with an embodiment, the present invention provides a

thermosensitive hydrogel composition comprising at least two or more biologically compatible triblock copolymers and one or more biologically active agents; and wherein the composition having a gelation temperature dependent on the weight % ratio of the at least two or more triblock copolymers in said mixture.

[0019] In accordance with an embodiment, the present invention provides a

thermosensitive hydrogel composition comprising: a) one or more biologically compatible triblock copolymers selected from the group consisting of: poly(D,L-lactide-co-glycolide) (PLGA)-poly(ethylene glycol) (PEG)-(PLGA); poly(lactic acid) (PLA)-(PEG)-(PLA); poly(lactide-co-caprolactone) (PLCL)-(PEG)-(PLCL); wherein the at least one or more triblock copolymers are combined a mixture; b) at least one or more biologically active agents; and wherein the composition having a gelation temperature dependent on the weight % ratio of the triblock copolymers in said mixture.

[0020] In accordance with an embodiment, the present invention provides a

thermosensitive hydrogel composition comprising: a) two or more biologically compatible triblock copolymers selected from the group consisting of: poly(D,L-lactide-co-glycolide) (PLGA)-poly(ethylene glycol) (PEG)-(PLGA); poly(lactic acid) (PLA)-(PEG)-(PLA); poly(lactide-co-caprolactone) (PLCL)-(PEG)-(PLCL); wherein the at least two or more triblock copolymers are combined a mixture; b) at least one or more biologically active agents; and wherein the composition having a gelation temperature dependent on the weight % ratio of the at least two or more triblock copolymers in said mixture.

[0021] In accordance with another embodiment, the present invention provides a thermosensitive hydrogel composition comprising: a) three biologically compatible triblock copolymers consisting of: poly(D,L-lactide-co-glycolide) (PLGA)-poly(ethylene glycol) (PEG)-(PLGA); poly(lactic acid) (PLA)-(PEG)-(PLA); poly(lactide-co-caprolactone) (PLCL)-(PEG)-(PLCL); wherein the three triblock copolymers are combined a mixture; b) three biologically active agents; and wherein the composition having a gelation temperature dependent on the weight % ratio of the three triblock copolymers in said mixture. [0022] In accordance with a further embodiment, the present invention provides a thermosensitive hydrogel composition comprising: a) at least one, two, or more biologically compatible triblock copolymers selected from the group consisting of: poly(D,L-lactide-co- glycolide) (PLGA)-poly(ethylene glycol) (PEG)-(PLGA); poly(lactic acid) (PLA)-(PEG)- (PLA); poly(lactide-co-caprolactone) (PLCL)-(PEG)-(PLCL); wherein at least one, two, or more triblock copolymers are combined a mixture; b) at least two or more biologically active agents; and wherein the composition having a gelation temperature dependent on the weight % ratio of the at least two or more triblock copolymers in said mixture.

[0023] In accordance with another embodiment, the present invention provides a thermosensitive hydrogel composition comprising: a) three biologically compatible triblock copolymers consisting of: poly(D,L-lactide-co-glycolide) (PLGA)-poly(ethylene glycol) (PEG)-(PLGA); poly(lactic acid) (PLA)-(PEG)-(PLA); poly(lactide-co-caprolactone) (PLCL)-(PEG)-(PLCL); wherein the three triblock copolymers are combined a mixture; b) two or three or four biologically active agents; and wherein the composition having a gelation temperature dependent on the weight % ratio of the three triblock copolymers in said mixture.

[0024] In accordance with still another embodiment, the present invention provides a method for treatment of post-operative conditions of surgery of the eye comprising administering to a subject in need thereof, an effective amount of the thermosensitive hydrogel compositions of the present invention.

[0025] In accordance with a further embodiment, the present invention provides a method for treatment of infection, and/or inflammation and/or intra-ocular pressure in an eye of a subject in need thereof comprising administering to a subject in need thereof, an effective amount of the thermosensitive hydrogel compositions of the present invention.

[0026] In accordance with still another embodiment, the present invention provides a method for treatment of retinal edema secondary to surgery of the eye comprising

administering to a subject in need thereof, an effective amount of the thermosensitive hydrogel compositions of the present invention.

[0027] In accordance with an embodiment, the present invention provides a method for treatment of retinal edema secondary to vitreoretinal disease of the eye, including but not limited to diabetic macular edema and retinal vein occlusion, comprising administering to a subject in need thereof, an effective amount of the thermosensitive hydrogel compositions of the present invention. [0028] In accordance with another embodiment, the present invention provides a method for treatment of abnormal retinal and/or choroidal vascularization, including but not limited to diabetic retinopathy, retinal vein occlusion, macular degeneration, choroidal neovascular membrane, comprising administering to a subject in need thereof, an effective amount of the thermosensitive hydrogel compositions of the present invention.

[0029] In accordance with a further embodiment, the present invention provides a method for treatment of increased intraocular pressure secondary to glaucoma comprising administering to a subject in need thereof, an effective amount of the thermosensitive hydrogel compositions of the present invention.

[0030] In accordance with yet another embodiment, the present invention provides for the prevention, amelioration, or treatment of a disease or condition associated with oxidative stress in an eye of a subject in need thereof, comprising administration of a therapeutically effective amount of an antioxidant agent to the eye of the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 depicts a phase diagram for the blended PLGA-PEG-PLGA triblock copolymer.

[0032] Figure 2 is a graph showing the rheological properties of a PLGA-PEG-PLGA triblock copolymer embodiment used in Experimental Study 1.

[0033] Figure 3 is a graph depicting the cumulative moxifloxacin release from a hydrogel embodiment used in Experimental Study 1.

[0034] Figure 4 shows the release profile of dexamethasone and brinzolamide from a hydrogel embodiment developed in Experimental Study 1.

[0035] Figures 5A-5B are SEM micrographs on A) dexamethasone and B) brinzolamide loaded microparticles made in this study. Scale bars show a distance of 2 μιτι.

[0036] Figure 6 is a graph depicting cumulative release of moxifloxacin from a hydrogel of the present invention.

[0037] Figure 7 is a graph showing dexamethasone and brinzolamide release from the Experimental Study 2 hydrogel system.

[0038] Figures 8A-8B are SEM micrographs of A) dexamethasone and B) levobunolol hydrochloride loaded microparticles made in Experimental Study 3. Scale bar shows a distance of 2 μιτι. [0039] Figure 9 depicts a phase diagram for a PLCL-PEG-PLCL hydrogel embodiment.

[0040] Figure 10 is a graph showing the variation of rheological properties of some PLCL-PEG-PLCL hydrogels of the present invention with respect to temperature.

[0041] Figure 11 is a graph depicting the cumulative release of moxifloxacin in the Experimental Study 3 embodiment of the present invention.

[0042] Figure 12 is a graph depicting the cumulative release of dexamethasone from a hydrogel and from microparticles alone in Experimental Study 3.

[0043] Figure 13 is a graph depicting the cumulative release of levobunolol

hydrochloride from a hydrogel and microparticles alone in Experimental Study 3.

[0044] Figures 14A-14E (fig 1 of data) SEM micrographs on a) levobunolol HCl-loaded

MPs with 75/25 (lactic/gly colic acid ratio) PLGA, deprotonated levobunolol-loaded MPs with b) 60/40, c) 75/25 and d) 85/15 PLGA and e) dexamethasone-loaded MPs with 50/50

PLGA. All of the images were taken at a magnification of 1000, and scale bar shows a distance of 10 μιτι. Figure 14 confirms the presence of spherical particles after loading different drug molecules and using different polymer types.

[0045] Figures 15A-15C. Phase diagram for different triblock co-polymers used in this research, A) PLGA-PEG-PLGA, B) PLA-PEG-PLA, C) PLCL-PEG-PLCL.

[0046] Figures 16A-16B. Rheological results for different triblock copolymers; a) Storage modulus, b) Loss modulus. Results were obtained at IHz oscillation frequency. The PLGA-PEG-PLGA triblock copolymer was a 3/1 blend of PLGA-PEG-PLGA

Mw 1,500: 1,500: 1,500 Da, 6: 1 LA:GA (86%/14% LA/GA) (w:w) and PLGA-PEG-PLGA LG 50:50 (w:w) (M_n -1,000: 1,000: 1,000 Da). The PLA-PEG-PLA was P(DL)LA-PEG- P(DL)LA 1700-1500-1700DA only. The PLCL-PEG-PLCL was a 6/1 blend of PLCL-PEG- PLCL MW -1600-1500-1600 DA, 75:25 CL:LA and PLCL-PEG-PLCL (-1700-1500-1700 DA, 60:40 CL:LA).

[0047] Figures 17A-17B. Variations of A) storage and B) loss moduli with oscillation frequency at different temperatures for PLCL-PEG-PLCL polymer solutions.

[0048] Figures 18A-18B. (A) Comparison of release of levobunolol with levobunolol HC1 from MPs. (B) Release of dexamethasone from samples containing different levobunolol type. For these samples no hydrogel was present and drug release from MPs alone was compared. In this and all of the subsequent figures, error bars represent standard deviations between duplicate measurements and data points represent average values of duplicate measurements. [0049] Figures 19A-19B. Comparison of drug released from MP-loaded in PLCL-PEG-

PLCL hydrogels with that from MPs alone, (A) levobunolol, (B) dexamethasone.

[0050] Figures 20A-20C. Drug release kinetics for different types of hydrogels, A)

Moxifloxacin, B) Levobunolol and C) Dexamethasone. Data points are the average between duplicate measurements and error bars represent standard deviation.

[0051] Figure 21. Effect of polymer type on regulation of levobunolol release from the drug delivery system.

[0052] Figures 22A-22C. Comparison of A) moxifloxacin, B) levobunolol and C) dexamethasone release profile when the loaded drug content is decreased by a factor of 2.

[0053] Figure 23. A depiction of a multi-drug delivery hydrogel embodiment capable of releasing three different drug molecules at different release rates.

DETAILED DESCRIPTION OF THE INVENTION

[0054] As indicated above, in one or more embodiments, the present invention provides a novel multidrug delivery platform that temporally regulates drug release via selective thermosensitive hydrogel compositions comprising biologically active agents that can either be mixed within the hydrogel composition directly, or mixed in the composition after being made in microparticle form with biologically compatible and degradable polymers. The thermosensitive hydrogel compositions are liquid at room temperature, and then become a viscous hydrogel when exposed to tissues at body temperatures. Such a platform is ideal for the treatment of many ophthalmic and other physiological conditions and diseases.

[0055] In accordance with an embodiment, the present invention provides a

thermosensitive hydrogel composition comprising at least one, two, or more biologically compatible triblock copolymers and one or more or more biologically active agents, and wherein the composition having a gelation temperature dependent on the weight % ratio of the at least two or more triblock copolymers in said mixture.

[0056] In some embodiments the weight % ratios of the mixture of two or more triblock copolymers can be in the range from 1/1, 2/1, 3/1, 3/2, 4/1, 4/3, 5/1, 5/2, 6/1, 7/1, 7/3, 8/1, 9/1 up to 10/1 depending on the gelation temperature desired. For example, a 3/1 ratio of PLGA- PEG-PLGA Mw 1,500: 1,500: 1,500 Da, 6: 1 LA:GA (86%/14% LA/GA) (w:w) PLGA-PEG- PLGA to PLGA-PEG-PLGA LG 50:50 (W:W) (MN -1,000: 1,000: 1,000 DA), when mixed together, provides a gelation temperature of 37 °C. Another example is blending PLCL- PEG-PLCL copolymers PLCL-PEG-PLCL MW -1600-1500-1600 DA, 75:25 CL:LA with PLCL-PEG-PLCL (-1700-1500-1700 DA, 60:40 CL:LA) at a ratio of 6/1 gave a gelation temperature in the desired range. It will be understood by those of skill in the art that in some embodiments, one triblock copolymer may have the right gelation temperature. For example, it was found that the PLA-PEG-PLA copolymer P(DL)LA-PEG-P(DL)LA 1700-1500-1700 DA had the desired range without mixing with another copolymer.

[0057] As used herein, the term "triblock copolymers" means a biodegradable polymer of the formula A-block-B-block-A. Examples of such polymers useful in the composition of the present invention include, but are not limited to, poly(D,L-lactide-co-glycolide) (PLGA)- poly(ethylene glycol) (PEG)-(PLGA); (PEG)- (PLGA)-(PEG); poly(lactic acid) (PLA)- (PEG)-(PLA); (PEG)-(PLA)-(PEG); poly(lactide-co-caprolactone) (PLCL)-(PEG)-(PLCL); (PEG)-(PLCL)-(PEG); poly(caprolactone) (PCL)-(PEG)-(PCL); (PEG)-(PCL)-(PEG);

poly(caprolactone)-b-poly(tetrahydrofuran)-b-poly(caprolactone) (PCL)-(PTHF)-(PCL); and poly(glycolide)-b-poly(ethylene glycol)-b-poly(glycolide) (PGA)-(PEG)-(PGA); (PEG)- (PGA)-(PEG).

[0058] A biologically compatible polymer refers to a polymer which is functionalized to serve as a composition for creating an injectable drug. The polymer is one that is a naturally occurring polymer or one that is not toxic to the host. The polymer may be a homopolymer where all monomers are the same or a hetereopolymer containing two or more kinds of monomers, such as triblock copolymers. The terms "biocompatible polymer,"

"biocompatible cross-linked polymer matrix" and "biocompatibility" when used in relation to the instant polymers are art-recognized and are considered equivalent to one another, including to biologically compatible polymer. For example, biocompatible polymers include polymers that are neither toxic to the host (e.g., an animal or human), nor degrade (if the polymer degrades) at a rate that produces monomeric or oligomeric subunits or other byproducts at toxic concentrations in the host.

[0059] Polymer is used to refer to molecules composed of repeating monomer units, including homopolymers, block copolymers, heteropolymers, random copolymers, graft copolymers and so on. "Polymers" also include linear polymers as well as branched polymers, with branched polymers including highly branched, dendritic, and star polymers. [0060] A monomer is the basic repeating unit in a polymer. A monomer may itself be a monomer or may be dimer or oligomer of at least two different monomers, and each dimer or oligomer is repeated in a polymer.

[0061] Suitable polymers for the triblock copolymers useful in the thermosensitive hydrogels could include, for example, biocompatible monomers with recurring units found in poly(phosphoesters), poly(lactides), poly(glycolides), poly(caprolactones), poly(anhydrides), poly(amides), poly(urethanes), poly(esteramides), poly(orthoesters), poly(dioxanones), poly(acetals), poly(ketals), poly(carbonates), poly(orthocarbonates), poly(phosphazenes), poly(hydroxybutyrates), poly(hydroxyl valerates), poly(alkylene oxalates), poly(alkylene succinates), poly(malic acids), poly(amino acids), polyvinylalcohol, poly(vinylpyrrolidone), poly(ethylene glycol), poly(hydroxy cellulose), copolymers, terpolymers or combinations or mixtures of the above materials.

[0062] As used herein, the terms "stability" and "stable" in the context of a liquid formulation comprising a biopolymer of interest that is resistant to thermal and chemical aggregation, degradation or fragmentation under given manufacture, preparation, transportation and storage conditions, such as, for one month, for two months, for three months, for four months, for five months, for six months or more. The "stable" formulations of the invention retain biological activity equal to or more than 80%, 85%, 90%, 95%, 98%, 99% or 99.5% under given manufacture, preparation, transportation and storage conditions. The stability of said preparation can be assessed by degrees of aggregation, degradation or fragmentation by methods known to those skilled in the art.

[0063] Biocompatible polymer and biocompatibility are art-recognized. For example, biocompatible polymers include polymers that are neither themselves toxic to the host (e.g., and animal or human), nor degrade (if the polymer degrades) at a rate that produces monomeric or oligomeric subunits or other byproducts at toxic concentrations in the host. In certain embodiments of the present invention, biodegradation generally involves degradation of the polymer in an organism, e.g., into its monomeric subunits, which may be known to be effectively non-toxic. Intermediate oligomeric products resulting from such degradation may have different toxicological properties, however, or biodegradation may involve oxidation or other biochemical reactions that generate molecules other than monomeric subunits of the polymer. Consequently, in certain embodiments, toxicology of a biodegradable polymer intended for in vivo use, such as implantation or injection into a patient, may be determined after one or more toxicity analyses. It is not necessary that any subject composition have a purity of 100% to be deemed biocompatible; indeed, it is only necessary that the subject compositions be biocompatible as set forth above. Hence, a subject composition may comprise polymers comprising 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75% or even less of biocompatible polymers, e.g., including polymers and other materials and excipients described herein, and still be biocompatible.

[0064] To determine whether a polymer or other material is biocompatible, it may be necessary to conduct a toxicity analysis. Such assays are well known in the art, using, for example, chemical means or enzymatic means. An aliquot of the treated sample products are placed in culture plates previously seeded with the cells. The sample products are incubated with the cells. The results of the assay may be plotted as % relative growth vs. concentration of degraded sample.

[0065] In addition, monomers, polymers, polymer matrices, and formulations of the present invention may also be evaluated by well-known in vivo tests, such as subcutaneous implantations or intraocular/subconjunctival/subtenon/suprachoroidal injections to the eye of rodents, rabbits, mini pigs, or non-human primates, etc. to confirm that they do not cause significant levels of irritation or inflammation at the implantation/injection sites.

[0066] "Biodegradable" is art-recognized, and includes monomers, polymers, polymer matrices, gels, compositions and formulations, such as those described herein, that are intended to degrade during use, such as in vivo. Biodegradable polymers and matrices typically differ from non-biodegradable polymers in that the former may be degraded during use. In certain embodiments, such use involves in vivo use, such as in vivo therapy, and in other certain embodiments, such use involves in vitro use. In general, degradation attributable to biodegradability involves the degradation of a biodegradable polymer into its component subunits, or digestion, e.g., by a biochemical process, of the polymer into smaller, non-polymeric subunits. In certain embodiments, two different types of biodegradation may generally be identified. For example, one type of biodegradation may involve cleavage of bonds (whether covalent or otherwise) in the polymer backbone. In such biodegradation, monomers and oligomers typically result, and even more typically, such biodegradation occurs by cleavage of a bond connecting one or more of subunits of a polymer. In contrast, another type of biodegradation may involve cleavage of a bond (whether covalent or otherwise) internal to a side chain or that connects a side chain, functional group and so on to the polymer backbone. For example, a therapeutic agent, biologically active agent, or other chemical moiety attached as a side chain to the polymer backbone may be released by biodegradation. In certain embodiments, one or the other or both general types of biodegradation may occur during use of a polymer. As used herein, the term

"biodegradation" encompasses both general types of biodegradation.

[0067] The degradation rate of a biodegradable polymer often depends in part on a variety of factors, including the chemical identity of the linkage responsible for any degradation, the molecular weight, crystallinity, biostability, and degree of cross-linking of such polymer, the physical characteristics of the implant, shape and size, and the mode and location of administration. For example, the greater the molecular weight, the higher the degree of crystallinity, and/or the greater the biostability, the biodegradation of any biodegradable polymer is usually slower. The term "biodegradable" is intended to cover materials and processes also termed "bioerodible."

[0068] In certain embodiments, the biodegradation rate of such polymer may be characterized by the presence of enzymes, for example, a chondroitinase. In such circumstances, the biodegradation rate may depend on not only the chemical identity and physical characteristics of the polymer matrix, but also on the identity of any such enzyme.

[0069] In accordance with one or more embodiments, the present invention provides compositions comprising one or more biologically active agents incorporated into a biologically compatible microparticles, and wherein the microparticles are then incorporated into the thermosensitive hydrogel compositions comprising at least one, two, or more biologically compatible triblock copolymers and which can optionally have one or more or more biologically active agents dissolved within the hydrogel composition, and wherein the composition having a gelation temperature dependent on the weight % ratio of the at least two or more triblock copolymers in said mixture.

[0070] As used herein, the term "biologically compatible microparticles" means biologically compatible and degradable polymers in micro or nano particle form which have been made using oil/water emulsion and solvent evaporation methods, water/oil/water emulsion and solvent evaporation methods, nanoprecipitation methods, coacervation and phase separation methods, multiorifice centrifugal methods, pan coating methods, and spray drying or spray congealing methods known in the art. For example, PLGA microparticles used in the compositions of the present invention, can be made by dissolving PLGA in an organic solvent, such as dichloromethane. The biologically active agents of interest, such as, for example, dexamethasone, is dissolved in DMSO, and then the two solutions are mixed together. The mixture is then added to an aqueous solution of polyvinyl alcohol and agitated continuously with a high speed propeller type blade to create a high shear in the solution which results in an emulsion with small particle sizes. This mixture is then allowed to evaporate the dichloromethane or placed under vacuum. The microspheres are collected by ultrafiltration, centrifugation and then lyophilization.

[0071] In addition, the drug elution profiles of the microparticle formulations of the present invention can vary depending of the weight ratio of the components of the copolymer used in the microparticle. For example, the weight ratio of lactic acid to gly colic acid can be varied in the PLGA copolymer particles. The weight ratios can vary from as much as 0%/100% lactic acid to gly colic acid, to 100%/0% lactic acid to gly colic acid, including for example, ratios of 85%/15%, 75%/25% and 50%/50%. In some embodiments, when the proportion of lactic to gly colic acid is similar, the faster the drug eluted from the

microparticle and vice versa. In addition, other types of polymers (e.g. PCL, PLCL) could potentially be used to encapsulate the drug molecules.

[0072] In certain embodiments, hydrogel polymeric formulations of the present invention biodegrade within a period that is acceptable in the desired application. In certain embodiments, such as in vivo therapy, such degradation occurs in a period usually less than about five years, one year, six months, three months, one month, fifteen days, five days, three days, or even one day on exposure to a physiological solution with a pH between 6 and 8 having a temperature of between about 25 and 40 °C. In other embodiments, the polymer degrades in a period of between about one hour and several weeks, depending on the desired application. In some embodiments, the polymer or polymer matrix may include a detectable agent that is released on degradation.

[0073] It will be understood by those of skill in the art, that the degradation or drug eluting properties of the hydrogels can be modified by adjusting the ratios of the two or more block copolymers used in the composition. For example, by blending the ratio (PLGA)- (PEG)-(PLGA), with a triblock copolymer of same type but different properties (e.g.

molecular weight, lactic to gly colic acid ratio) or with (PLA)-(PEG)-(PLA) and/or (PLCL)- (PEG)-(PLCL), one can get a desired degradation and drug elution profile.

[0074] In some embodiments, it is understood that the degradation of the block copolymers produces acidic byproducts which lower the pH of the surrounding tissue microenvironment. In embodiments wherein microparticles of biocompatible polymers with drugs or other biologically active agents are incorporated in the hydrogels, the acidic microenvironment of the degrading hydrogel can accelerate the degradation of the microparticle polymer formulation.

[0075] It will also be understood by those of skill in the art, that the amount of biologically active agent added during the manufacturing of the hydrogel can be varied as well. Typically, the amount of drug added can be in the range of 0.1 mg to about 100 mg, up to about 1,000 mg.

[0076] Moreover, the drug elution profile can also be manipulated by the hydrophilicity or hydrophobicity of the biologically active agent added, its molecular weight, and/or chemical structure. In some embodiments, by choosing an agent which is more hydrophobic, the drug will elute from the composition more slowly. In some embodiments, a larger molecular weight agent may also slow the release of the agent. When the molecule has some interaction with hydrogel network, its release can favorably be slowed down.

[0077] In a similar manner, it will be understood by those of skill in the art, that the hydrogels can be modified by adjusting the ratios of the two or more block copolymers used in the composition to adjust the gelation temperature profile.

[0078] In some embodiments, a desired degradation and drug elution profile can be further altered by first encapsulating the drug or compound of interest in a microparticle comprised of a biodegradable and biocompatible polymer. The resulting microparticles containing the drug of interest can then be incorporated into the triblock copolymer composition and mixed and then inj ected into the site of interest, such as in the eye.

[0079] Methods for the synthesis of the polymers described above are known to those skilled in the art, see, e.g., Concise Encyclopedia of Polymer Science and Polymeric Amines and Ammonium Salts, E. Goethals, editor (Pergamen Press, Elmsford, N.Y. 1980). Many polymers, such as PLGA, are commercially available. Naturally occurring polymers can be isolated from biological sources as known in the art or are commercially available. Naturally occurring and synthetic polymers may be modified using chemical reactions available in the art and described, for example, in March, "Advanced Organic Chemistry," 4th Edition, 1992, Wiley-Interscience Publication, New York.

[0080] "Gel" refers to a state of matter between liquid and solid, and is generally defined as a cross-linked polymer network swollen in a liquid medium. Typically, a gel is a two- phase colloidal dispersion containing both solid and liquid, wherein the amount of solid is greater than that in the two-phase colloidal dispersion referred to as a "sol." As such, a "gel" has some of the properties of a liquid (i.e., the shape is resilient and deformable) and some of the properties of a solid (i.e., the shape is discrete enough to maintain three dimensions on a two-dimensional surface).

[0081] Hydrogels consist of hydrophilic polymers cross-linked to from a water-swollen, insoluble polymer network. Cross-linking can be initiated by many physical or chemical mechanisms. The hydrogels of interest also are configured to have a viscosity that will enable the gelled hydrogel to remain affixed on or in the cell, tissue or organ, or surface. Viscosity can be controlled by the monomers and polymers used, by the level of water trapped in the hydrogel, and by incorporated thickeners, such as biopolymers, such as proteins, lipids, saccharides and the like.

[0082] The terms "incorporated," "encapsulated,", "entrapped" and "impregnated" are art-recognized when used in reference to a therapeutic agent, dye, or other material and a polymeric composition, such as a composition of the present invention. In certain embodiments, these terms include incorporating, formulating or otherwise including such agent into a composition that allows for sustained release of such agent in the desired application. The terms may contemplate any manner by which a therapeutic agent or other material is incorporated into a polymer matrix, including, for example, attached to a monomer of such polymer (by covalent or other binding interaction) and having such monomer be part of the polymerization to give a polymeric formulation, distributed throughout the polymeric matrix, appended to the surface of the polymeric matrix (by covalent or other binding interactions), encapsulated inside the polymeric matrix, etc. The term "co-incorporation" or "co-encapsulation" refers to the incorporation of a therapeutic agent or other material and at least one other therapeutic agent or other material in a subject composition.

[0083] More specifically, the physical form in which any therapeutic agent or other material is encapsulated in polymers may vary with the particular embodiment. For example, a therapeutic agent or other material may be first encapsulated in a microsphere and then combined with the polymer in such a way that at least a portion of the microsphere structure is maintained. Alternatively, a therapeutic agent or other material may be sufficiently immiscible in the polymer of the invention that it is dispersed as small droplets, rather than being dissolved in the polymer. Any form of encapsulation or incorporation is contemplated by the present invention, in so much as the sustained release of any encapsulated therapeutic agent or other material determines whether the form of encapsulation is sufficiently acceptable for any particular use. [0084] The term, "carrier," refers to a diluent, adjuvant, excipient or vehicle with which the therapeutic is administered. Such physiological carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Saline solutions and aqueous dextrose and glycerol solutions also can be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0085] An active agent and a biologically active agent are used interchangeably herein to refer to a chemical or biological compound that induces a desired pharmacological and/or physiological effect, wherein the effect may be prophylactic or therapeutic. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of those active agents specifically mentioned herein, including, but not limited to, salts, esters, amides, prodrugs, active metabolites, analogs and the like. When the terms "active agent,"

"pharmacologically active agent" and "drug" are used, then, it is to be understood that the invention includes the active agent per se as well as pharmaceutically acceptable,

pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs etc. The active agent can be a biological entity, such as a virus or cell, whether naturally occurring or manipulated, such as transformed.

[0086] Pharmaceutically acceptable salts are art-recognized, and include relatively nontoxic, inorganic and organic acid addition salts of compositions of the present invention, including without limitation, therapeutic agents, excipients, other materials and the like. Examples of pharmaceutically acceptable salts include those derived from mineral acids, such as hydrochloric acid and sulfuric acid, and those derived from organic acids, such as ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like. Examples of suitable inorganic bases for the formation of salts include the hydroxides, carbonates, and bicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium, aluminum, zinc and the like. Salts may also be formed with suitable organic bases, including those that are non-toxic and strong enough to form such salts. For purposes of illustration, the class of such organic bases may include mono-, di-, and trialkylamines, such as methylamine,

dimethylamine, and triethylamine; mono-, di-, or trihydroxyalkylamines such as mono-, di-, and triethanolamine; amino acids, such as arginine and lysine; guanidine; N- methylglucosamine; N-methylglucamine; L-glutamine; N-methylpiperazine; morpholine; ethylenediamine; N-benzylphenthylamine; (trihydroxymethyl) aminoethane; and the like, see, for example, J. Pharm. Sci., 66: 1-19 (1977).

[0087] In accordance with an embodiment, the present invention provides a

thermosensitive hydrogel composition comprising: a) at least one, two, or more biologically compatible triblock copolymers selected from the group consisting of: poly(D,L-lactide-co- glycolide) (PLGA)-poly(ethylene glycol) (PEG)-(PLGA); poly(lactic acid) (PLA)-(PEG)- (PLA); poly(lactide-co-caprolactone) (PLCL)-(PEG)-(PLCL); wherein the at least one, two, or more triblock copolymers are combined a mixture; b) at least two or more biologically active agents; and wherein the composition having a gelation temperature dependent on the weight % ratio of the at least one, two, or more triblock copolymers in said mixture.

[0088] In accordance with another embodiment, the present invention provides a thermosensitive hydrogel composition comprising: a) three biologically compatible triblock copolymers consisting of: poly(D,L-lactide-co-glycolide) (PLGA)-poly(ethylene glycol) (PEG)-(PLGA); poly(lactic acid) (PLA)-(PEG)-(PLA); poly(lactide-co-caprolactone) (PLCL)-(PEG)-(PLCL) and the like; wherein the three triblock copolymers are combined a mixture; b) three biologically active agents; and wherein the composition having a gelation temperature dependent on the weight % ratio of the three triblock copolymers in said mixture.

[0089] It will be understood by those of skill in the art, that the biologically active agents can be incorporated into the same or different triblock copolymers of the composition, and can also be incorporated into a microparticle prior to being incorporated into the triblock copolymers of the inventive thermosensitive hydrogel.

[0090] It will be also understood by those of skill in the art, that an increase in the molecular weights of the polymers used in the triblock copolymer will increase the gelation temperature of the composition.

[0091] The biologically active agent may vary widely with the intended purpose for the composition. The term active is art-recognized and refers to any moiety that is a biologically, physiologically, or pharmacologically active substance that acts locally or systemically in a subject. Examples of biologically active agents, that may be referred to as "drugs", are described in well-known literature references such as the Merck Index, the Physicians' Desk Reference, and The Pharmacological Basis of Therapeutics, and they include, without limitation, medicaments; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure or mitigation of a disease or illness; substances which affect the structure or function of the body; or pro-drugs, which become biologically active or more active after they have been placed in a physiological environment. Various forms of a biologically active agent may be used which are capable of being released the subject composition, for example, into adjacent tissues or fluids upon administration to a subject.

[0092] In other embodiments, a biologically active agent may be used in the

thermosensitive hydrogels of this invention, to treat, ameliorate, inhibit, or prevent a disease or symptom, in conjunction with, for example, promoting ocular healing, reducing inflammation, fighting infection, and reducing intra-ocular pressure.

[0093] Further examples of biologically active agents include, without limitation, enzymes, receptor antagonists or agonists, hormones, growth factors, peptides, antibiotics, antimicrobial agents, antibodies, proteins and antibody-drug conjugates.

[0094] In certain embodiments, the subject compositions comprise about 1% to about 75% or more by weight of the total composition, alternatively about 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60% or 70%, of a biologically active agent.

[0095] Non-limiting examples of biologically active agents include following: adrenergic blocking agents, anabolic agents, androgenic steroids, anti-allergenic materials, anticholinergics and sympathomimetics anti-hypertensive agents, anti-infective agents, antiinflammatory agents such as steroids, non-steroidal anti-inflammatory agents, anti-pyretic and analgesic agents, antihistamines, benzophenanthridine alkaloids, biologicals, decongestants, diagnostic agents, estrogens, mucolytic agents, growth factors, peripheral vasodilators, progestational agents, prostaglandins, vitamins and prodrugs.

[0096] Examples of NS AIDS used in the methods of the present invention include mefenamic acid, aspirin, Diflunisal, Salsalate, Ibuprofen, Naproxen, Fenoprofen, Ketoprofen, Deacketoprofen, Flurbiprofen, Oxaprozin, Loxoprofen, Indomethacin, Sulindac, Etodolac, Ketorolac, Diclofenac, Nabumetone, Piroxicam, Meloxicam, Tenoxicam, Droxicam, Lornoxicam, Isoxicam, Meclofenamic acid, Flufenamic acid, Tolfenamic acid, elecoxib, Rofecoxib, Valdecoxib, Parecoxib, Lumiracoxib, Etoricoxib, Firocoxib, Sulphonanilides, Nimesulide, Niflumic acid, and Licofelone.

[0097] Examples of steroids used in the compositions described herein include dexamethasone, betamethasone, fluocinolone, fluocinolone acetonide, difluprednate, fluorometholone, loteprednol, prednisolone, medrysone, triamcinolone, triamcinolone acetonide, and rimexolone. [0098] Examples of ophthalmic antibiotics used in the compositions described herein include levofloxacin, natamycin, tobramycin, polymyxin b/trimethoprim, ciprofloxacin, trifiuridine, moxifloxacin, gatifloxacin, besifloxacin, ganciclovir, azithromycin,

chloramphenicol, erythromycin, gentamicin, vancomycin, ceftazidime, amikacin, and ofloxacin.

[0099] Examples of ophthalmic hypotensive agents used in the compositions described herein include timolol, brimonidine, apraclonidine, zioptan, pilocarpine, brinzolamide, bimatoprost, dorzolamide, levobunolol, betimol, betaxolol, lopidine, betagan, metipranol, carteolol, travoprost, latanoprost and tafluprost.

[0100] Any form of the biologically active agents may be used. These include, without limitation, such forms as uncharged or charged molecules, molecular complexes, salts, ethers, esters, amides, prodrug forms and the like, which are biologically activated when implanted, injected or otherwise placed into a subject.

[0101] Buffers, acids and bases may be incorporated in the compositions to adjust pH. Agents to increase the diffusion distance of agents released from the composition may also be included.

[0102] The charge, lipophilicity or hydrophilicity of a composition may be modified by employing an additive. For example, surfactants may be used to enhance miscibility of poorly miscible liquids. Examples of suitable surfactants include dextran, polysorbates and sodium lauryl sulfate. In general, surfactants are used in low concentrations, generally less than about 5%.

[0103] Buffering agents help to maintain the pH in the range which approximates physiological conditions. Buffers are preferably present at a concentration ranging from about 2 mM to about 50 mM. Suitable buffering agents for use with the instant invention include both organic and inorganic acids, and salts thereof, such as citrate buffers (e.g., monosodium citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-monosodium citrate mixture etc.), succinate buffers (e.g., succinic acid monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid-disodium succinate mixture etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaric acid- potassium tartrate mixture, tartaric acid-sodium hydroxide mixture etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture, fumaric acid-disodium fumarate mixture, monosodium fumarate-disodium fumarate mixture etc.), gluconate buffers (e.g., gluconic acid-sodium glyconate mixture, gluconic acid-sodium hydroxide mixture, gluconic acid- potassium gluconate mixture etc.), oxalate buffers (e.g., oxalic acid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate mixture etc.), lactate buffers (e.g., lactic acid-sodium lactate mixture, lactic acid-sodium hydroxide mixture, lactic acid-potassium lactate mixture etc.) and acetate buffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide mixture etc.). Phosphate buffers, carbonate buffers, histidine buffers, trimethylamine salts, such as Tris, HEPES and other such known buffers can be used.

[0104] Preservatives may be added to retard microbial growth, and may be added in amounts ranging from 0.2%-l % (w/v). Suitable preservatives for use with the present invention include phenol, benzyl alcohol, m-cresol, octadecyldimethylbenzyl ammonium chloride, benzyaconium halides (e.g., chloride, bromide and iodide), hexamethonium chloride, alkyl parabens, such as, methyl or propyl paraben, catechol, resorcinol,

cyclohexanol and 3-pentanol.

[0105] Isotonicifiers are present to ensure physiological isotonicity of liquid

compositions of the instant invention and include polhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol. Polyhydric alcohols can be present in an amount of between about 0.1 % to about 25%, by weight, preferably 1 % to 5% taking into account the relative amounts of the other ingredients.

[0106] Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which solubilizes the therapeutic agent or helps to prevent denaturation or adherence to the container wall. Typical stabilizers can be polyhydric sugar alcohols; amino acids, such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine etc. ; organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, arabitol, erythritol, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol and the like, including cyclitols such as inositol; polyethylene glycol; amino acid polymers; sulfur containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, a-monothioglycerol and sodium thiosulfate; low molecular weight polypeptides (i.e., <10 residues); proteins, such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone, saccharides, monosaccharides, such as xylose, mannose, fructose or glucose; disaccharides, such as lactose, maltose and sucrose; trisaccharides, such as raffinose; polysaccharides, such as, dextran and so on. Stabilizers can be present in the range from 0.1 to 10,000 w/w per part of biopolymer.

[0107] Additional miscellaneous excipients include bulking agents, (e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbic acid, methionine or vitamin E) and cosolvents.

[0108] Non-ionic surfactants or detergents (also known as "wetting agents") may be added to help solubilize the therapeutic agent, as well as to protect the therapeutic protein against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stresses without causing denaturation of the protein. Suitable non-ionic surfactants include polysorbates (20, 80 etc.), polyoxamers (184, 188 etc.), Pluronic® polyols and polyoxyethylene sorbitan monoethers (TWEEN-20®, TWEEN-80® etc.). Non-ionic surfactants may be present in a range of about 0.05 mg/ml to about 1.0 mg/ml, preferably about 0.07 mg/ml to about 0.2 mg/ml.

[0109] The instant invention encompasses formulations, such as, liquid formulations having stability at temperatures found in a commercial refrigerator and freezer found in the office of a physician or laboratory, such as from about 20 °C to about 2 °C, said stability assessed, for example, by microscopic analysis, for storage purposes, such as for about 60 days, for about 120 days, for about 180 days, for about a year, for about 2 years or more. The liquid formulations of the present invention also exhibit stability, as assessed, for example, by particle analysis, at room temperatures, for at least a few hours, such as one hour, two hours or about three hours or more prior to use.

[0110] The formulations to be used for in vivo administration must be sterile. That can be accomplished, for example, by filtration through sterile filtration membranes. For example, the formulations of the present invention may be sterilized by filtration.

[0111] The thermosensitive hydrogel compositions will be formulated, dosed and administered in a manner consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The "therapeutically effective amount" of the biopolymer to be administered will be governed by such considerations, and can be the minimum amount necessary to prevent, ameliorate or treat a disorder of interest. As used herein, the term "effective amount" is an equivalent phrase refers to the amount of a therapy (e.g., a prophylactic or therapeutic agent), which is sufficient to reduce the severity and/or duration of a disease, ameliorate one or more symptoms thereof, prevent the advancement of a disease or cause regression of a disease, or which is sufficient to result in the prevention of the development, recurrence, onset, or progression of a disease or one or more symptoms thereof, or enhance or improve the prophylactic and/or therapeutic effect(s) of another therapy (e.g., another therapeutic agent) useful for treating a disease.

[0112] In another embodiment, an effective amount of a therapeutic or a prophylactic agent of interest reduces the symptoms of a disease, such as a post-operative inflammation, infection, or increased intraocular pressure, by at least 5%, preferably at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%. Also used herein as an equivalent is the term, "therapeutically effective amount."

[0113] As used herein, the term "treat," as well as words stemming there from, includes preventative as well as disorder remitative treatment. The terms "reduce," "suppress," "prevent," and "inhibit," as well as words stemming there from, have their commonly understood meaning of lessening or decreasing. These words do not necessarily imply 100% or complete treatment, reduction, suppression, or inhibition.

[0114] Embodiments of the invention also include a process for preparing pharmaceutical products comprising the inventive thermosensitive hydrogel compositions. The term

"pharmaceutical product" means a composition suitable for pharmaceutical use

(pharmaceutical composition), as defined herein. Pharmaceutical compositions formulated for particular applications comprising the compounds of the present invention are also part of this invention, and are to be considered an embodiment thereof. Examples of such products can include hydrogel compositions incorporating drugs or other biologically active agents, and also the hydrogel compositions comprising microparticles having drugs or other biologically active agents encapsulated within the microparticles in the hydrogel

composition.

[0115] It will be understood by those of skill in the art that the hydrogel compositions of the present invention can be used to deliver a multitude of FDA approved agents for ocular and intraocular indications. Examples of drugs or biologically active agents that can be formulated with the hydrogel compositions of the present invention include, but are not limited to, naphazoline; amikacin; amphotericin B; besifloxacin; ofloxacin; vancomycin; ketorolac tromthamine; pemirolast potassium; cidofovir; azithromycin; gangcyclovir;

valgancyclovir; travaprost; bimatoprost; cyclosporine; pegaptanib; ranbizumab;

difluprednate; dexamethasone; triamcinolone acetonide; foscarnet; methotrexate; bepotastine besilate; ceftazidime; voriconazole; aflibercept; tafluprost; ocriplasmin; cysteamine HC1; phenylephrine; adalimumab; lifitegrast; carbachol; and epinephrine.

[0116] An article of manufacture containing thermosensitive hydrogel compositions useful for the treatment of the disorders described above is provided. The article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes and test tubes. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is effective for preventing or treating, for example, a wound or a joint disease and may have a sterile access port (for example, the container may be a vial having a stopper pierceable by a hypodermic injection needle). The label on or associated with the container indicates that the composition is used for treating the condition of choice. The article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as phosphate- buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes and package inserts with instructions for use.

[0117] In accordance with another embodiment, the present invention provides a method for treatment of post-operative conditions of surgery of the eye comprising administering to a subject in need thereof, an effective amount of the thermosensitive hydrogel compositions of the present invention.

[0118] In accordance with another embodiment, the present invention provides a method for treatment of infection, and/or inflammation and/or elevation of intra-ocular pressure in an eye of a subject in need thereof comprising administering to a subject in need thereof, an effective amount of the thermosensitive hydrogel compositions of the present invention.

[0119] In an embodiment, the term "administering" means that the compounds of the present invention are introduced into a subject, preferably a subject receiving treatment for a inflammatory related disease of the eye, infection related disease of the eye, hypertension related disease of the eye, etc. and the compounds are allowed to come in contact with the one or more disease related cells or population of cells in vivo, using one or more known routes of administration. In some embodiments, the preferred route of administration is injection. In accordance with still another embodiment, the present invention provides a method for treatment of post-operative conditions of surgery of the eye comprising administering to a subject in need thereof, an effective amount of the thermosensitive hydrogel compositions of the present invention.

[0120] In accordance with an embodiment, the present invention provides a method for treatment of retinal edema secondary to vitreoretinal disease of the eye, including but not limited to diabetic macular edema and retinal vein occlusion, comprising administering to a subject in need thereof, an effective amount of the thermosensitive hydrogel compositions of the present invention.

[0121] In accordance with another embodiment, the present invention provides a method for treatment of increased intraocular pressure secondary to glaucoma comprising administering to a subject in need thereof, an effective amount of the thermosensitive hydrogel compositions of the present invention.

[0122] In accordance with an embodiment, the present invention provides a method for treatment of abnormal retinal and/or choroidal vascularization, including but not limited to diabetic retinopathy, retinal vein occlusion, macular degeneration, choroidal neovascular membrane, comprising administering to a subject in need thereof, an effective amount of the thermosensitive hydrogel compositions of the present invention.

[0123] In accordance with yet another embodiment, the present invention provides for the prevention, amelioration, or treatment of a disease or condition associated with oxidative stress in an eye of a subject in need thereof, comprising administration of a therapeutically effective amount of an antioxidant agent to the eye of the subject.

[0124] As used herein, the term "a disease or condition associated with oxidative stress in an eye" include, but are not limited to, retinitis pigmentosa, macular degeneration including age related macular degeneration (AMD) both wet and dry, diabetic retinopathy, Lebers optic neuropathy, and optic neuritis.

[0125] Retinitis pigmentosa ("RP") comprises a large group of inherited vision disorders that cause progressive loss of photoreceptor cells of the retina, leading to severe vision impairment and often incurable blindness. The most common form of RP is a rod-cone dystrophy, in which the first symptom is night blindness, followed by progressive loss in the peripheral visual field in daylight, and eventually leading to blindness after several decades. As a common pathology, rod photoreceptors die early, whereas light-insensitive, morphologically altered cone photoreceptors persist longer. [0126] Currently, there is no approved therapy that stops the evolution of the disease or restores vision. The therapeutic approach is restricted to slowing down the degenerative process by sunlight protection, vitamin A supplementation, treating complications (cataract and macular edema), and helping patients to cope with the social and psychological impact of blindness.

[0127] Glutathione (GSH) is a tripeptide, c-L-glutamyl-L-cysteinyl-glycine, found in all mammalian tissues. It has several important functions including detoxification of

electrophiles, scavenging ROS, maintaining the thiol status of proteins, and regeneration of the reduced forms of vitamins C and E. GSH is the dominant non-protein thiol in mammalian cells; as such it is essential in maintaining the intracellular redox balance and the essential thiol status of proteins. Also, it is necessary for the function of some antioxidant enzymes such as the glutathione peroxidases.

[0128] Intracellular GSH levels are determined by the balance between production and loss. Production results from de novo synthesis and regeneration of GSH from GSSG by GSSG reductase. Generally there is sufficient capacity in the GSSG reductase system to maintain all intracellular GSH in the reduced state, so little can be gained by ramping up that pathway. The major source of loss of intracellular GSH is transport out of cells. Intracellular GSH levels range from 1-8 mM while extracellular levels are only a few μΜ; this large concentration gradient essentially precludes transport of GSH into cells and once it is transported out of cells, it is rapidly degraded by γ-glutamyltranspeptidase. Inhibition of GSH transporters could theoretically increase intracellular GSH levels, but is potentially problematic because the transporters are not specific for GSH and their suppression could lead imbalance of other amino acids and peptides. Thus, intracellular GSH levels are modulated primarily by changes in synthesis.

[0129] As used herein "amelioration" or "treatment" is understood as meaning to lessen or decrease at least one sign, symptom, indication, or effect of a specific disease or condition. For example, amelioration or treatment of retinitis pigmentosa (RP) can be to reduce, delay, or eliminate one or more signs or symptoms of RP including, but not limited to, a reduction in night vision, a reduction in overall visual acuity, a reduction in visual field, a reduction in the cone density in one or more quadrants of the retina, thinning of retina, particularly the outer nuclear layer, reduction in a- or b-wave amplitudes on scotopic or photopic

electroretinograms (ERGs); or any other clinically acceptable indicators of disease state or progression. Amelioration and treatment can require the administration of more than one dose of an agent, either alone or in conjunction with other therapeutic agents and interventions. Amelioration or treatment does not require that the disease or condition be cured.

[0130] "Antioxidant" as used herein is understood as a molecule capable of slowing or preventing the oxidation of other molecules. Oxidation is a chemical reaction that transfers electrons from a substance to an oxidizing agent. Such reactions can be promoted by or produce superoxide anions or peroxides. Oxidation reactions can produce free radicals, which start chain reactions that damage cells. Antioxidants terminate these chain reactions by removing free radical intermediates, and inhibit other oxidation reactions by being oxidized themselves. As a result, antioxidants are often reducing agents such as thiols, ascorbic acid or polyphenols. Antioxidants include, but are not limited to, a-tocopherol, ascorbic acid, Mn(III)tetrakis (4-benzoic acid) porphyrin, a-lipoic acid, n-acetylcysteine, and n- acetylcysteineamide.

[0131] "Co-administration" as used herein is understood as administration of one or more agents to a subject such that the agents are present and active in the subject at the same time. Co-adminsitration does not require a preparation of an admixture of the agents or

simultaneous administration of the agents.

[0132] Examples of pharmaceutically acceptable antioxidants include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxy toluene (BHT), lecithin, propyl gallate, a-tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0133] As used herein, the term "subject" refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. It is preferred that the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). It is more preferred that the mammals are from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). It is most preferred that the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). An especially preferred mammal is the human.

[0134] It will be understood by those of ordinary skill that a dosing regimen used in the inventive methods can be any length of time sufficient to provide a reduction in the inflammatory, infection and/or hypertension disease in the eyes of the subject. The term "chronic" as used herein, means that the length of time of the dosage regimen can be hours, days, weeks, months, or possibly years.

[0135] The multidrug delivery platform of the present invention which temporally regulates drug release is ideal for the treatment of many ophthalmic conditions, including post-operative management requiring multiple topical agents for inflammation, infection, and elevated IOP. For other ocular diseases including glaucoma, previous studies have shown that combination therapy is more effective than single drug therapy in many patients.^{12 29} [0136] In an embodiment, the inventive drug delivery system is administered via intraocular injection, and is composed of biocompatible and biodegradable materials for optimal ocular safety.

[0137] As a proof of concept, the present inventors designed the inventive drug delivery system to address the challenges of multi-drug treatment during postoperative management of cataract surgery. In routine care after cataract surgery, topical antibiotics are administered for 7 days to reduce risk of infection, whereas topical corticosteroids are applied up to a month with decreasing frequency to reduce inflammation.^6"8 Often an ocular hypotensive is added to the post-operative treatment regimen to reduce IOP increase secondary to inflammation and/or corticosteroid use.^{1 9"11} The inventors designed the present novel drug delivery platform to replace this topical treatment paradigm.

[0138] In an embodiment, a broad-spectrum antibiotic, the 4^th generation

fluoroquinolone, moxifloxacin, is delivered over a period of 7 days to prevent post-operative infection. In addition, a potent corticosteroid, dexamethasone, is released from the drug delivery system over a two-month period to suppress inflammatory reaction. Because increased IOP is seen commonly with intraocular steroid implants, an ocular hypotensive agent, such as the carbonic anhydrase inhibitor brinzolamide,³⁰' ¹ or beta-blocker levobunolol hydrochloride,^{32 3} is delivered intraocularly to control ocular pressure.

[0139] In an embodiment, the anti-inflammatory and hypotensive drugs are encapsulated in PLGA microparticles, which act as barriers through which drug release can be controlled. It will be understood by those of skill in the art that other biocompatible and degradable polymers known in the art can be used to manufacture the microparticles. The particles are then embedded into the inventive thermosensitive hydrogel compositions of the present invention to further regulate the control of drug release. With the innovative design, drug release rate and time for each agent can be individually controlled to for specific ocular indications or other diseases by changing the amount and loading of microparticles, composition of the polymer used to synthesize the particles as well as the hydrogels. Since the drug delivery platform is in liquid phase at room temperature, implantation can be accomplished through direct injection into the anterior chamber or vitreous cavity after surgical procedure or in office.

EXAMPLE 1

[0140] Experimental Study 1 : Materials and Methods.

[0141] Synthesis of one microparticle embodiment.

[0142] 55 mg PLGA (Resomer 503H, molecular weight: 24-38 kDa, lactic acid (LA) to gly colic acid (GA) ratio of 50/50) was mixed with 5 mg PEGylated PLGA (Polyscitech, PEG and PLGA molecular weights: 5 and 20 kDa respectively.) and they were dissolved in dichloromethane (DCM) at a concentration of 10 mg/ml. About 6 mg dexamethasone was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mg/ml. The polymer-drug solutions were mixed and sonicated at an amplitude of 60% for 45 seconds and quickly transferred to a 50 ml of 1% PVA solution that was homogenized at a speed of 15,000 rpm for 1 minute. The resulting microparticles (MPs) were transferred to a larger bath of PVA (volume: 100 ml) at a concentration of 0.5% that was being stirred at a speed of 500 rpm for 3.5 hrs. The MPs were pelleted by centrifugation at a speed of 4000 rpm for 5 mins, and washed thrice with miliQ water, and lyophilized for later use. Brinozolamide loaded MPs were made using the same protocol except 3 mg of Brinzolamide was used at a concentration of 10 mg/ml in DMSO.

[0143] Preparation of the hydrogel composition.

[0144] The following triblock copolymers were used in some exemplary embodiments: PLGA-PEG-PLGA (with MWs of 1000: 1000: 1000 Da), ratio of lactic acid (LA) to glycolic acid (GA): 1/1; and PLGA-PEG-PLGA (with MWs of 1500: 1500: 1500 Da), LA/GA: 6/1.

[0145] The triblock copolymers were dissolved in milliQ water at a concentration of 28.57 wt% by shaking for 3 days. The drug release experiments in the Experimental Study 1 were done with a hydrogel made up of the following compositions.

[0146] A blend of 94.5 μΐ of PLGA-PEG-PLGA Mw 1,500: 1,500: 1,500 Da, 6: 1 LA:GA (86% 14% LA/GA) (w:w) (at a concentration of 28.57 wt%) and 31.5 μΐ of PLGA-PEG- PLGA LG 50:50 (W:W) (MN -1,000: 1,000: 1,000 DA) (at a concentration of 28.57 wt%). PLGA-PEG-PLGA Mw 1,500: 1,500: 1,500 Da, 6: 1 LA:GA (86% 14% LA/GA) (w:w) has a gelation temperature of 40 ^°C and PLGA-PEG-PLGA LG 50:50 (W:W) (MN -1,000: 1,000: 1,000 DA) has a gelation temperature of 20 ^°C. The resulting blend gels at 37 ^°C. By blending different amounts of triblock copolymers, gelation temperature can be finely tuned.

[0147] About 18 μΐ of phosphate buffered saline (PBS) at a concentration of 10X.

Maintaining the hydrogel in IX PBS concentration, keeps the osmotic pressure of hydrogel the same as body cells (isotonic concentration).

[0148] About 36 μΐ of water. Addition of water decreases the polymer concentration. Strength of the hydrogel can be adjusted in this step. If higher mechanical strength is desired, polymer concentration should be higher, and vice versa.

[0149] About 144 μg of antibiotic (moxifloxacin). Expected duration of release of moxifloxacin is approximately one week. In some embodiments, the antibiotics are directly added to the hydrogel to maintain the planned shorter drug release duration.

[0150] About 8.1 mg of dexamethasone loaded MPs and 4.5 mg of brinzolamide loaded MPs. A longer duration of sustained release is desired for dexamethasone and brinzolamide. To achieve this goal, these small molecule drugs are encapsulated by MPs first, and then MPs are mixed into the hydrogel network. This method creates two barriers against burst release and premature escape of drug molecules.

[0151] The final inventive hydrogel composition had a total volume of 180 μΐ and was kept in a 1.5 ml centrifuge tube. 1.2 ml of PBS was poured on it as the release media. 1 ml of PBS was replaced with fresh PBS at certain intervals.

[0152] Drug release characterization.

[0153] The release samples taken from hydrogels or microparticles were lyophilized and drug molecules were dissolved in the DMSO-methanol (1-27 or 1-10 ratios). The samples were analyzed with the high performance liquid chromatography (HPLC) to quantify the amount of drug release. The mobile phase for HPLC was composed of mixture of acetonitrile (24%) and water (76%) for the first 11 minutes. The proportion of acetonitrile was raised to 36% during the last 9 minutes to elute dexamethasone, which was a more hydrophobic compound compared to other drugs studied. The flow rate of solvent system was set to 1 ml/min, and injection volume of the drug solution was 25 μΐ.

[0154] Results.

[0155] Figure 1 illustrates the phase diagram for the blended PLGA-PEG-PLGA triblock copolymers. To determine the phase for each polymer solution, copolymer was incubated at each set temperature point for 15 minutes. Subsequently, the vial was placed upside-down for 30 seconds. If the polymer solution was not able to flow during this time, it was considered as a gel. Otherwise, it was a liquid or precipitate.

[0156] Two different triblock copolymers were blended to make sure the resulting polymer solution is a liquid at room temperature and gels at body temperature. Figure 1 confirms this and shows that at lower temperatures the polymer solution is a low viscosity liquid when manufactured and it will gel at body temperature. Figure 1 also indicates that at very high temperatures, the polymers precipitate out of solution and can no longer form a gel network. During all drug release experiments the polymer concentration for all the hydrogels was 20% wt/vol.

[0157] Figure 2 depicts the variation of storage and loss moduli for the PLGA-PEG- PLGA triblock blended copolymers. The polymer solution was formulated at 20% wt/vol in IX PBS. Figure 2 shows that the resulting blend of triblock copolymers had a low value for both the storage and loss moduli at room temperature, indicating liquid like behavior.

However, with increasing the temperature, mechanical properties of gel shift as gelation occurs and the moduli are enhanced and it gels at 37 °C. As temperature is further increased, the polymer precipitates and the desirable mechanical properties decrease. Quantitative rheological observations support the qualitative observations depicted in phase diagram.

[0158] Figure 3 depicts moxifloxacin release from the drug delivery platform developed in Experimental Study 1. The duration of moxifloxacin release shown in Fig. 3 is as expected.

[0159] Release profiles of dexamethasone (anti-inflammatory agent) and brinzolamide (hypotensive drug) from this hydrogel are shown in Fig. 4. Brinzolamide is a relatively hydrophilic drug molecule and quickly escaped the particles and hydrogels within a week. However, for dexamethasone, drug release was detectable up to 70 days. Nevertheless, the daily release of both drugs was below our preset target doses to provide maximum ocular effect. Therefore, the loading for both drugs in the MPs was increased and more MPs were loaded in the hydrogel in the subsequent trials.

EXAMPLE 2

[0160] Experimental Study 2: Materials and Methods.

[0161] Synthesis of microparticles. From the previous study, it was determined that the loading of drug molecules in the hydrogel should be increased. For this phase of development of drug delivery system, MPs were made with similar synthesis protocol as before, except the following measures were taken to enhance the drug loading. First, the PEG-PLGA to PLGA mass proportion was reduced from 8.3% to 1%. Having PEG helps reducing non-specific interactions, however it could lower the drug loading. That is the reason for reduction in the PEG-PLGA portion. Second, the concentration of drugs

(dexamethasone and brinzolamide) in DMSO was enhanced to 20 mg/ml. Third, target drug loading for brinzolamide was increased to 10%. Fourth, the polymer concentration in DCM was raised to 40 mg/ml. The effect of different synthesis parameters was screened and it was noticed that this concentration is very critical in the MP loading and size. The greater the concentration, the larger the drug loading would be. However, very high polymer concentrations could lead to poor particle size. Figure 5 illustrates the SEM images taken from the microparticles made in Experimental Study 2.

[0162] As shown in Fig. 5, particles have a monodispersed size distribution and the particle size is between 1-2 μιτι.

[0163] Synthesis of a hydrogel embodiment.

[0164] The protocol to make hydrogels was similar to Experimental Study 1. The following improvements were made to enhance drug loading in the hydrogel: 1) about 450 μg of antibiotic (moxifioxacin) was added to the triblock copolymer solution. This higher dosage is more relevant to actual clinical applications. It should be noted that this loading could be further enhanced, if needed. Up to 5 mg free drug in this hydrogel formulation has been loaded with success. 2) With the modified synthesis protocol, the loading of dexamethasone and brinzolamide in MPs has enhanced considerably. Also, the amount of dexamethasone and brinzolamide loaded microparticles in the hydrogel was increased to 13.5 and 6.8 mg, respectively.

[0165] Hydrogel was kept in a 1.5 ml centrifuge tube. 1.1 ml of PBS (at IX

concentration) was poured on it as the release media. About 1 ml of PBS was replaced with fresh PBS at certain intervals. The taken PBS samples were analyzed with high performance liquid chromatography (HPLC) to determine daily release of drug molecules from the hydrogels.

[0166] Results.

[0167] As the relative ratio of triblock copolymers was the same as Experimental Study 1, the phase diagram and rheological properties of the triblock copolymer solution were the same. Moxifloxacin release from Experimental Study 2 hydrogel is shown in Fig. 6. The amount of daily release of moxifloxacin from the hydrogel in this Experimental Study and its release duration are in accordance with clinical requirements for this drug. The release of dexamethasone and brinzolamide from this hydrogel are shown in Fig. 7. Comparing with Experimental Study 1 release data, for both dexamethasone and brinzolamide, the daily drug release has been considerably enhanced and has become closer to clinical requirements. For brinzolamide, even though the drug loading in the hydrogel has increased, the drug was still able to escape the microparticles and hydrogel too fast. In addition, the release of dexamethasone in the first two weeks is in accordance with clinical requirements, however, the amount of drug released is not favorable after this period. This triggered the initiation of Experimental Study 3.

EXAMPLE 3

[0168] Experimental Study 3: Materials and Methods.

[0169] Synthesis of a second microparticle embodiment.

[0170] From the previous studies, it was found that the loading of drug molecules in the hydrogel should be increased. Additionally, measures were taken to avoid premature drug leakage. For this Experimental Study, MPs were made with similar synthesis protocol as before, except the following measures were taken to enhance the drug loading and avoid drug escape. Around 50 different microparticles batches were made and their size and loading was characterized to find the optimized parameters to get high loading and favorable particle size.

[0171] In this experiment, the PEG-PLGA proportion of the polymer was reduced to 0. This increased the drug loading in the microparticles. Instead of brinzolamide, levobunolol hydrochloride was chosen. Levobunolol hydrochloride is more hydrophobic than brinzolamide, and it was found that the increased hydrophobicity prevents its escape from MPs and hydrogel. Furthermore, levobunolol hydrochloride is cheaper than brinzolamide, easing the transition of the technology into clinical settings. The concentration of drugs (dexamethasone and levobunolol hydrochloride) in DMSO was raised to around 250 mg/ml. Target drug loading for dexamethasone and levobunolol hydrochloride was increased to 25 and 15% wt% respectively. The polymer concentration in DCM was raised to about 68 mg/ml. The PVA volume and concentration were decreased to 40 ml and 0.75% wt% for homogenization step. [0172] Drug loading was found to depend on polymer mass used to make MPs. Two homogenization steps were performed to make MPs each for 50 mg of polymer mass. To avoid escape of levobunolol hydrochloride, a more hydrophobic version of PLGA was used with the LA/GA ratio of 85/15. In a separate synthesis, polycaprolactone as a slower degrading polymer was used to make MPs too. However, due to the very low loading of levobunolol hydrochloride in the synthesized MPs, the resultant loading of levobunolol was insignificant.

[0173] Figure 8 illustrates the SEM images taken from the microparticles made in this Experimental Study. As shown in Fig. 8, size of dexamethasone loaded microparticles ranges from 1 to 3 microns. However, levobunolol loaded microparticles are bigger in size (between 2-8 microns). It will be shown how larger particle size for levobunolol hydrochloride will enable a more sustained drug release compared to brinzolamide hydrogel system developed in Experimental Study 2.

[0174] Synthesis of another hydrogel embodiment.

[0175] In order to avoid premature drug escape, a more hydrophobic and slower degrading triblock copolymer was used to make the hydrogel. It was a blend of

poly(caprolactone) and poly(lactic acid):

Poly(lactide-co-caprolactone)-b-Poly(ethylene glycol)-b-Poly(lactide-co-caprolactone) (PLCL-PEG-PLCL with MWs of 1700 and 1500 Da for PLCL and PEG, respectively). The ratio of caprolactone to lactic acid was 60/40.

[0176] A list of ingredients of the hydrogel made in this trial is provided below:

140 μΐ of PLCL-PEG-PLCL (-1700-1500-1700 DA, 60:40 CL:LA) (at a concentration of

28.57 wt%);

20 μΐ of phosphate buffered saline (PBS) at a concentration of 10X;

40 μΐ of water;

500 μg of antibiotic (moxifloxacin); and

12.5 mg of dexamethasone loaded MPs and 12.5 mg of levobunolol hydrochloride loaded MPs.

[0177] The hydrogel was kept in a 1.5 ml centrifuge tube. About 1.1 ml of PBS (at IX concentration) was poured on it as the release media. About 1 ml of PBS was replaced with fresh PBS at certain intervals. The PBS samples were lyophilized, reconstituted in DMSO- methanol and analyzed with high performance liquid chromatography (HPLC) to determine daily release of drug molecules from the hydrogels. The release of dexamethasone and levobunolol hydrochloride from the hydrogel was compared with their release from MPs alone. To do so, 7 mg of dexamethasone loaded MPs and 7 mg of levobunolol loaded MPs were re-suspended in 1.1 ml PBS, and then were constantly rotated in an inverter to simulate their Brownian motion upon injection to the eye. About 1 ml of PBS was taken every day, to analyze with HPLC and determine the amount of drug release.

[0178] Results.

[0179] Figure 9 depicts the phase diagram for PLCL-PEG-PLCL hydrogels. For each polymer concentration, Fig. 9 indicates the temperatures where the polymer solution is a liquid, gel and precipitate. This figure also indicates that the polymer solution is a hydrogel at body temperatures. Figure 10 illustrates the rheological properties of PLCL-PEG-PLCL hydrogels. Qualitative observations for phase diagram are backed by quantitative rheological measurements shown in Fig. 10. This figure also indicates that the current hydrogel formulation has high mechanical properties at body temperature.

[0180] The release of moxifioxacin from the Experimental Study 3 hydrogel is shown in Fig. 11. The release of moxifloxacin is as expected and most of it is released in about a week. Figure 12 and 13 compare the release of dexamethasone and levobunolol

hydrochloride from Experimental Study 3 hydrogel with microparticles alone. According to Fig. 12, MPs alone have a huge burst release of dexamethasone on day 1. Additionally, for MPs, daily dexamethasone release is decreased over time, and most of the drug is released in 10 days. However, for this embodiment of the inventive composition, burst drug release is eliminated and daily release of dexamethasone is constant up to 3 weeks. The hydrogel has been able to release the dexamethasone for more than 37 days. The hydrogel is still releasing the dexamethasone on physiologically relevant concentrations.

[0181] An outstanding feature of the hydrogel system developed in this invention is enhancement in the release of levobunolol hydrochloride after 3 weeks. Because the anti- glaucoma drug is embedded in the hydrogel to prevent the side effect of releasing steroids and elevating ocular pressure, increase in daily release of anti-glaucoma drugs after 3 weeks is extremely desirable.

EXAMPLE 4

[0182] The inventors were interested in further enhancing the loading and release of levobunolol from the hydrogel. In addition, this and subsequent examples feature the ability of the multidrug delivery hydrogel in finely tuning the release of different drug molecules. [0183] Preparation of microparticles comprising levobunolol and deprotonated levobunolol alone and with dexamethasone.

[0184] To make the hydrogels of the optimum formulation (liquid at room temperature and a hydrogel at body temperature), the following triblock co-polymers were used:

PLGA-PEG-PLGA (with MWs of 1000: 1000: 1000 Da, ratio of lactic to glycolic acid (LA/GA): 1/1);

PLGA-PEG-PLGA (with MWs of 1500: 1500: 1500 Da, LA/GA: 6/1);

PLA-PEG-PLA (with MWs of 1700: 1500: 1700 Da);

PLCL-PEG-PLCL (with MWs of 1600: 1500: 1600 Da, caprolactone (CL)/LA: 3/1); and PLCL-PEG-PLCL (with MWs of 1700: 1500: 1700 Da, CL/LA: 3/2).

[0185] Different types of PLGA were used to make drug loaded MPs. For levobunolol loaded MPs, PLGA (with LA/GA of 60/40 (Product number: AP43), 75/25 (Product number: AP18), or 85/15 (Product number: AP 87), all with number averaged molecular weight of 45- 55kDa) was purchased from PolySciTech. For dexamethasone-loaded MPs, PLGA (Resomer 503H with LA/GA ratio of 50/50 and weight averaged molecular weight of 24-38 kDa) was purchased from Evonik Corporation (Essen, Germany). Dexamethasone (Product number: 46165) and moxifloxacin (Product number: PHR1542) were obtained from Sigma Aldrich (St. Louis, MO), while levobunolol hydrochloride (HC1) (Product number: 1359801) was purchased from United States Pharmacopeia (Rockville, MD). Poly(vinyl alcohol) (PVA, with molecular weight of 13-23 kDa, product number: 363170) was obtained from Sigma Aldrich. All other materials were obtained from Sigma Aldrich. Unless noted otherwise, the materials were used as received without further purification.

[0186] Synthesis of microparticle embodiments.

[0187] Deprotonation of levobunolol hydrochloride.

[0188] To remove the HC1 salt from levobunolol HC1, 30 mg of the drug was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 97 mg/ml. A one-to-one mole ratio of triethylamine (TEA) was added to that and the solution was constantly inverted at a speed of 11 rpm for 3 hrs to obtain deprotonated levobunolol. The resulting drug solution was kept at room temperature protected from light and was used to make the MPs the next day.

[0189] Levobunolol loaded microparticles.

[0190] About 100 mg PLGA (60/40, 75/25 or 85/15) was dissolved in dichloromethane (DCM) at a concentration of 61 mg/ml. This solution was added to 10.7 mg levobunolol previously dissolved in DMSO and deprotonated in the presence of TEA (total DMSO plus TEA volume: 115 μΐ). The polymer-drug solutions were then mixed and sonicated with a bath sonicator for 45 seconds and split in half. Each half was transferred to a 40 ml aqueous solution of 0.75% PVA during homogenization at a speed of 15,000 rpm for 1 minute. The resulting MPs were transferred to a larger bath of PVA (80 ml at a concentration of 0.5%) while stirring at a speed of 990 rpm for 3.5 hours. The MPs were pelleted by centrifugation at a speed of 3300 RCF for 5 mins, and washed thrice with miliQ water. The MPs were then lyophilized and stored at -20 °C. To synthesize blank MPs, the same DMSO/TEA volume (115 μΐ) with no drug was mixed with PLGA solution in DCM.

[0191] Levobunolol hydrochloride-loaded microparticles.

[0192] To make MPs loaded with levobunolol HC1, all the steps mentioned in the previous section were followed except that 10.7 mg of levobunolol HC1 dissolved in DMSO at a concentration of 97 mg/ml was used as the drug solution, in place of simple levobunolol.

[0193] Dexamethasone-loaded microparticles.

[0194] Dexamethasone-loaded MPs were made following the same protocol as described for levobunolol, except that PLGA (50/50) was dissolved in DCM at a higher concentration of 68 mg/ml. In addition, 20 mg of dexamethasone was dissolved in DMSO at a

concentration of 250 mg/ml and used as the drug solution.

[0195] Preparation of another hydrogel embodiment.

[0196] To make the thermogels, the polymer solution is dissolved at a higher

concentration than the intended concentration, so that liquid used to dilute the polymer solution to the right concentration, can also be used to add other components (e.g., addition of a drug molecule to polymer network or tonicity adjustment). Thus, even though the intended triblock co-polymer concentration was 20 % wt/vol, the triblock co-polymers were dissolved in milliQ water at a concentration of 28.6 % wt/vol by shaking while cold (2-8°C) for 3 days. The triblock co-polymer solutions were then diluted to reach the intended polymer concentration by addition of excess water and 10X PBS. The volume of 10X PBS addition was chosen so that the final formulations were at IX PBS concentration to minimize any osmotic pressure difference with the biological environment (isotonic concentration).

[0197] Three different hydrogel classes were developed, namely, PLGA-PEG-PLGA, PLA-PEG-PLA and PLCL-PEG-PLCL hydrogels by blending them with different triblock co-polymers. The ideal triblock co-polymer solution is one that is a liquid at room

temperature to ensure injectability but forms a hydrogel network at body temperature. To achieve this, different polymer solutions need to be blended so that the gelation temperature is at around 37°C. The PLGA-PEG-PLGA was a 3/1 blend of PLGA-PEG-PLGA Mw 1,500: 1,500: 1,500 Da, 6: 1 LA:GA (86%/14% LA/GA) (w:w) and PLGA-PEG-PLGA LG 50:50 (w:w) (M_n -1,000: 1,000: 1,000 Da) triblock co-polymer solutions. The PLA-PEG-PLA hydrogel was made with P(DL)LA-PEG-P(DL)LA 1700-1500-1700 DA triblock co-polymer solution only, as its gelation temperature was in the right range. Furthermore, PLCL-PEG- PLCL hydrogels were a 6/1 blend of PLCL-PEG-PLCL MW -1600-1500-1600 DA, 75:25 CL:LA/PLCL-PEG-PLCL (-1700-1500-1700 DA, 60:40 CL:LA) triblock co-polymer solutions. By blending different amounts of triblock co-polymers, the gelation temperature can be finely tuned.

EXAMPLE 5

[0198] Hydrogel characterization.

[0199] To characterize the hydrogels, two different schemes were implemented, namely a qualitative vial inversion test to determine the gelation temperature for each triblock copolymer solution and quantitative rheological measurements to determine the mechanical strength of the hydrogel at different temperatures.

[0200] To determine the phase diagram, co-polymer solution at different concentrations of 10, 15, 20, and 25 % wt/vol was incubated at each set temperature point for 15 minutes. Subsequently, the vial was placed upside-down for 30 seconds and the triblock co-polymer solution was visually inspected. If the polymer solution was not able to flow during this time, it was considered a gel. Otherwise, it was a liquid or precipitate.

[0201] Rheological experiments were performed using an ARES-G2 rheometer (TA Instruments) with a stainless steel rheometer plate with a diameter of 40 mm. This rheometer plate with a large diameter was chosen to maximize the torque signal generated by the triblock co-polymer solution and enhance the accuracy of the data. Rheological experiments were performed with a temperature step of 3 °C. At each specific temperature, a strain sweep experiment was done at a frequency of 0.1 Hz to determine the linear viscoelastic region of the material. Afterwards, a frequency sweep was performed in the linear viscoelastic region to determine the values of moduli at different frequencies. The elastic modulus (G') is a measure of elasticity of the material (solid like behavior), while the viscose modulus (G") is a measure of viscosity of the material (viscose liquid like behavior). EXAMPLE 6

[0202] Drug release studies.

[0203] For the moxifloxacin, fast release of the drug in a week was desired, and as a result 500 μg of moxifloxacin was directly added to the hydrogel network. To do so, moxifloxacin solution at 12.5 mg/ml was prepared and 40 μΐ was added to the triblock copolymer solution. At the concentration of 12.5 mg/ml, dissolution of moxifloxacin in water did not increase the overall solution volume. A longer duration of release is desired for dexamethasone and levobunolol. In addition, for each of these two drug molecules a different release profile is required. For dexamethasone, the amount of drug release should be high initially and gradually decrease over time. However, levobunolol release should be low initially and increase later to suppress any ocular pressure increase in the postoperative treatment period. To achieve this goal, these small drug molecules are encapsulated in MPs first and then these MPs are loaded in the hydrogel network. This provides two barriers against burst release and premature escape of the drug molecules. Unless noted otherwise, 7 mg of dexamethasone-loaded MPs and 17 mg of levobunolol-loaded MPs were loaded into the hydrogel network for drug release studies.

[0204] Hydrogels were made with 200 μΐ triblock co-polymer solution and were kept in a 1.5 ml centrifuge tube. After incubation of hydrogel at 37 °C for half an hour, 1.2 ml of "pre- warmed" PBS was poured on it as the release media and the drug release was initiated. 1 ml of PBS was replaced with fresh PBS at certain intervals to make sure hydrogel was exposed to an effectively infinite-volume bath to release the drugs and to determine the released amount over time.

[0205] To assess the effectiveness of hydrogels in sustaining the release of drugs, the drug release from the MP-loaded hydrogels was compared with that of MPs alone. To do so, drug release from MPs was studied by incubating the same amount of MPs (7 mg of dexamethasone-loaded MPs and 17 mg of levobunolol-loaded MPs) in 1.2 ml IX PBS. At certain time points, the tubes were centrifuged at 3000 RCF and 1 ml of PBS was replaced with fresh PBS. For the microparticle release samples, release media could evaporate over time (less than 7% in the worst case), even though the tubes were sealed with parafilm. The effect of evaporation of media on the release results was taken into account. All drug- releasing hydrogels and MPs were shaken in a 37 °C incubator. [0206] In addition to drug-releasing hydrogels and MPs, three hydrogel samples representing PLGA-PEG-PLGA, PLA-PEG-PLA, and PLCL-PEG-PLCL hydrogels loaded with blank MPs as well as a blank MP sample without hydrogel were made and incubated to serve as the negative controls and any signal from them (which was very small in most cases) was subtracted from that of drug-releasing samples.

EXAMPLE 7

[0207] Drug release characterization.

[0208] The release media taken from hydrogels or MP-containing samples were lyophilized and drug molecules were reconstituted in DMSO/methanol (at 1/10 volumetric ratio) and were analyzed with high performance liquid chromatography (HPLC). The mobile phase for HPLC was composed of mixture of acetonitrile (20%) and water (80%) for the first 9.5 minutes to elute levobunolol (peak position: between 3.8 to 4.6 minutes) and

moxifioxacin (peak position: between 5.6 to 7.5 minutes). Then a linear compositional ramp was induced and the proportion of acetonitrile was raised to 34% in 1 minute. The acetonitrile was then kept at 34% for 6.5 minutes to elute dexamethasone (peak position: between 13.6 and 14.1 minutes), which was a more hydrophobic compound than the other drugs studied. Levobunolol, moxifioxacin and dexamethasone signals were read at 221, 295 and 240 nm, respectively. Peak position for each drug could shift slightly from one day to another, but the drug content would always be checked with its UV absorption spectra. The flow rate of solvent was set to 1 ml/min, and injection volume of the drug solution was 25 μΐ.

[0209] To determine the standard curve for the analysis of release samples, moxifioxacin and dexamethasone were dissolved in PBS at different concentrations, incubated at 37 °C for 24 hrs (to simulate exposure of released drugs to 37 °C environment during drug release experiments), lyophilized and reconstituted in DMSO/methanol (1/10 volumetric ratio). However, since levobunolol was in DMSO after deprotonation, the drug in DMSO at very high concentration was diluted with excess PBS. The rest of steps for determining the standard curve were the same as for moxifioxacin and dexamethasone.

EXAMPLE 8

[0210] Scanning electron microscopy. [0211] To determine the relative size of MPs and their surface morphology, a LEO/Zeiss scanning electron microscope (SEM) was utilized. In this regard, MPs were deposited on a SEM mount, and were coated with a thin (<20 nm) layer of gold. Operating voltage was set to IkV to minimize sample damage during microscopy. At least four SEM micrographs from different parts of the samples at a magnification of 1000X were taken and the results shown are chosen to be representatives.

[0212] Error analysis.

[0213] The experiments were performed in duplicate. Error bars represent the standard deviations between data points. The error values were typically small, and average error for each plot was below 10% for most of the cases.

EXAMPLE 9

[0214] Particle characterization.

[0215] Figures 14A-14E illustrate the SEM micrographs taken from the MPs loaded with levobunolol or dexamethasone in this invention. Figure 14 confirms the presence of spherical particles after loading different drug molecules and using different polymer types.

EXAMPLE 10

[0216] Hydrogel characterization.

[0217] Figures 15A-15C illustrate the phase diagram for different triblock co-polymers used in this study. All the formulations tested were at IX PBS concentration. For PLGA- PEG-PLGA and PLCL-PEG-PLCL, two different triblock co-polymers with different molecular weight and ratio of lactic-to-caprolactone (as discussed above) were blended to make sure that the resulting polymer solution was a liquid at room temperature and a gel at body temperature. PLA-PEG-PLA polymer solutions already possessed this property.

Figure 15 confirms this and shows that at lower temperatures all of the polymer solutions are liquid. However, they will change into the gel form at body temperature. Figure 15 also indicates that at very high temperatures, the polymers precipitate out of solution and can no longer form a gel network. According to Fig. 15, for each triblock co-polymer concentration, there is a gelation temperature window. When the polymer concentration is increased, this gelation window becomes wider. At very high polymer concentrations, there are enough micelles associated with each other that the triblock co-polymer solution could form a hydrogel network even at room temperature. On the other hand, at lower polymer concentrations, the gelation window is too narrow to work with. As a result, in this research, the polymer concentration for all the hydrogels was set to 20% wt/vol. Interestingly, PLCL- PEG-PLCL polymer solutions have a much wider gelation window at each concentration, compared to PLGA-PEG-PLGA and PLA-PEG-PLA polymer solutions.

[0218] Figures 16A-16B depict the variation of storage and loss moduli for the different triblock co-polymers blended in this study. The polymer solution was formulated at 20% wt/vol in IX PBS. Figure 16 shows that the resulting blend of triblock co-polymers had a low value for both the storage and loss moduli at room temperature, indicating liquid-like behavior. However, with increasing temperatures, the mechanical properties of the gels shift as gelation occurs and the moduli are maximized near body temperature (37 °C). As temperature is further increased, the polymer precipitates and the desirable mechanical properties decrease. Quantitative rheological observations support the qualitative

observations depicted in phase diagram.

[0219] Figures 17A-17B depict the variations of the moduli with frequency at different temperatures for the PLCL-PEG-PLCL triblock co-polymer solution. According to Fig. 17, regardless of oscillation frequency, the moduli are maximum at body temperature.

EXAMPLE 11

[0220] Effect of deprotonation on levobunolol release.

[0221] For ocular hypotensives, daily release on the order of 1 μg is required to ensure effectiveness of the molecule in reducing the ocular pressure.^{35, 6} Thus, a high loading of levobunolol in MPs was required to ensure the proper effect. To enhance the loading, levobunolol was deprotonated in the presence of TEA. A strong base like TEA attracts H+ ions, thus removing the HC1 salt from the levobunolol and making it more hydrophobic due to removal of charged species. To determine the effect of deprotonation on the drug release profile, two samples were made: one with 17 mg levobunolol-loaded MPs and the other with 17 mg of levobunolol HCl-loaded MPs. Both samples had 7 mg of dexamethasone-loaded MPs added to them to check whether the presence of levobunolol would impact the release of dexamethasone. Release results from these samples are compared in Fig. 18. As can be seen, the daily release of deprotonated levobunolol is more than 2-fold higher than the sample with levobunolol HC1. Thus, deprotonation enabled enhancing the daily release of levobunolol from the MPs. Figure 18 also shows that daily release of dexamethasone from the two samples is the same, indicating that the presence of levobunolol MPs doesn't impact the dexamethasone release.

EXAMPLE 12

[0222] Effect of hydrogel on sustaining drug release.

[0223] Next, the effect of the hydrogel network on sustaining the drug release from MPs was determined by comparing results with MPs in hydrogel with those for MPs alone. To do so, two samples were made: one containing 17 mg of deprotonated levobunolol MPs with 75/25 PLGA and 7 mg of dexamethasone loaded MPs with 50/50 PLGA, and the other a PLCL-PEG-PLCL hydrogel containing the same amount and type of microparticles plus 500 μg of moxifloxacin added directly to the hydrogel network. Figures 19A-19B compare the amount of dexamethasone and levobunolol released from each sample.

[0224] As can be seen in Fig. 19A, levobunolol release from MPs loaded in the hydrogel is nearly the same as that from MPs alone. Thus, the hydrogel does not seem to have much influence on the release profile of levobunolol. On the other hand, release of dexamethasone from MPs in the hydrogel is considerably slowed down compared to release from MPs alone. Evident in Fig. 19B is the huge burst release of dexamethasone from MPs on day 1, which might be toxic for ocular cells. Also, for MPs, daily dexamethasone release is decreased abruptly over time and most of the drug is released within the first 5 days. However, for the MPs loaded in the hydrogel, burst drug release is eliminated and daily release of

dexamethasone is constant for at least a week. Beyond the first week, daily drug release decreases over time (which is greatly favorable for the clinical application). Dexamethasone release from MPs was detectable for up to 36 days, while for MPs in the hydrogel, the drug release was detectable for up to 51 days. As will be described in the subsequent section, if required, the drug release profile could be manipulated by varying the hydrophobicity of the polymer encapsulating the drug molecules. However, throughout the first month, daily release of dexamethasone was enough to have a biologically significant effect. Notably, the amount of dexamethasone released to the vitreous cavity by implants should be between 0.2- 1.2 μg daily in the first week, and the amount of drug should decrease gradually over time for postoperative management following cataract surgeries. [0225] Without being held to any particular theory, it is thought that the mechanism behind sustaining the dexamethasone release by the hydrogel mainly stems from their chemical interaction. Dexamethasone has a fluorine and several hydroxyl and double bonded oxygen groups that could form hydrogen bonds with excess water content in the hydrogel or with its PEG block. However, levobunolol does not have as many groups capable of forming hydrogen bonds. In addition, dexamethasone is very hydrophobic compared to levobunolol, and the tendency of dexamethasone to diffuse out of the hydrogel network and go into release media is therefore low. A-B-A triblock co-polymers form a hydrogel network with large pore sizes (-50-100 μηι), ⁷ and therefore can't physically avoid or slow down the release of small drug molecules studied in this research. As will be discussed in the subsequent section, the mechanical properties of the hydrogel network do not seem to effect drug release kinetics, since drug release kinetics were found to be similar in hydrogels with significantly different moduli (Fig. 20A).

[0226] Even though the PLCL-PEG-PLCL hydrogel did not have any effect on sustaining the release of levobunolol, still having the MPs in the hydrogel is more advantageous than administering MPs alone in the eye, since the hydrogel could hold the MPs in place, while the free MPs could potentially diffuse throughout the eye especially to the lens, interfere with vision and induce inflammation.

EXAMPLE 13 [0227] Effect of hydrogel type on drug release profile.

[0228] Figures 20A-20C depict the effect of different hydrogel types on the release kinetics of moxifioxacin, dexamethasone and levobunolol, respectively.

[0229] Moxifloxacin is hydrophilic (water solubility: 24 mg/ml), and was added directly to the polymer solution, and upon hydrogel formation, it will be held "loosely" within the hydrogel network. Its release profile as shown in Fig. 20A contains a high burst release followed by gradual decrease in daily drug release amount with the majority of the drug being released within a week. To explain the release requiring a week despite the absence of microparticles, we suggest that there might be some chemical interactions between the drug and hydrogel network such as hydrogen bonding between fluorine, hydroxyl and double- bonded oxygen groups in Moxifloxacin with excess water content in hydrogel or its PEG part that has slowed down the release of this drug. This release profile was favorable for meeting clinical requirements to avoid infection. Figure 20A also shows that there is not much difference in release kinetics of moxifloxacin between different hydrogel types. This is expected, as moxifloxacin was added directly to the hydrogel network and there was not a strong barrier against its release.

[0230] On the other hand, for levobunolol and dexamethasone, drug release from PLGA- PEG-PLGA hydrogels is faster than from PLA-PEG-PLA for the first three weeks. Also, the drug release from PLA-PEG-PLA seems to be faster than from PLCL-PEG-PLCL hydrogels over this period of time. This trend is more noticeable for levobunolol. Compared with release from microparticles alone, it seems that the PLGA-PEG-PLGA hydrogels have accelerated the release of levobunolol (Fig. 20B). This seems counter-intuitive, since the hydrogels are a barrier and if anything they should slow down the drug release rate.

However, this trend makes sense when one considers the fact that by hydrolysis and cleavage of ester bonds of PLGA in PLGA-PEG-PLGA hydrogels, the degradation products make the environment acidic, leading to faster degradation of microparticles that are embedded in the hydrogel network. The same phenomenon should happen by degradation of PLA-PEG-PLA and PLCL-PEG-PLCL hydrogels, but due to their higher hydrophobicity and slower degradation rate, it takes those hydrogels a longer time to make the environment acidic and thus their impact on acceleration of drug release is less pronounced.

EXAMPLE 14

[0231] Use of polymer type to fine-tune the drug release profile.

[0232] Depending on the specific disease model and its progression, ophthalmologists might be interested in having different drug dosages delivered to the eye over the course of treatment. To do so, the drug delivery platform should have the ability to finely tune the drug release profile. One way to achieve this goal is to vary the type of polymer encapsulating the drug molecule. To demonstrate this, deprotonated levobunolol loaded MPs were made out of PLGA with differing hydrophobicity and degradation rate with LA/GA ratios of 60/40, 75/25, and 85/15. A higher ratio of lactic to gly colic acid leads to greater hydrophobicity and slower degradation rate. Three hydrogel release samples were prepared each containing 17 mg levobunolol-loaded MPs with different PLGA polymer types mentioned above, plus 7 mg dexamethasone-loaded MPs and 500 μg moxifloxacin in all samples. PLCL-PEG-PLCL hydrogels were used for this experiment. Figure 21 depicts the levobunolol release profile from these hydrogels.

[0233] Evident in Fig. 21 is the ability of the drug delivery platform to finely tune the drug release profile by variation of polymer used to encapsulate the drug molecule. PLGA with a LA/GA ratio of 60/40 degrades faster and thus enables rapid and linear drug release kinetics. For this polymer, the majority of levobunolol release happens within 25 days. On the other hand, 85/15 PLGA is the most hydrophobic polymer and releases the drug molecule more slowly and sustain the drug release for up to 60 days. Interestingly, for this polymer an increase in the drug release content was observable on the fourth week of drug release. Without being held to any particular theory, it is thought that because after 4-weeks of incubation at 37 °C, most of the polymers that create a solid shell on the exterior of the MPs made with 85/15 PLGA are degraded and the encapsulated drug molecules will be able to escape the particles. Drug release from 75/25 PLGA is somewhere in between 60/40 and 85/15 PLGA, with an increase in the drug release content in the second week. During the postoperative treatment period, elevation of ocular pressure as a side effect of steroids happens during the second week for most of the patients. Therefore, it is extremely desirable to have a boost in release of levobunolol in this window for the proper postoperative management of ocular surgeries. By combining different mass ratios of levobunolol loaded MPs (50% 60/40 PLGA+50% 85/15 PLGA) in the hydrogel, one can obtain intermediate release profiles as well.

EXAMPLE 15

[0234] Varying the microparticle or moxifloxacin mass to tune the daily drug release.

[0235] In the previous section, the ability to finely tune the drug release profile by varying the type of polymer encapsulating levobunolol was highlighted. Another flexibility of the presented drug delivery system is the freedom to vary the mass of dexamethasone or levobunolol loaded MPs or moxifloxacin encapsulated in the same amount of hydrogel to change the daily drug release dosage while keeping the overall release profile relatively constant. In this regard, two PLCL-PEG-PLCL hydrogels were synthesized. In the first hydrogel group, 17 mg deprotonated levobunolol loaded MPs made with 75/25 PLGA, 7 mg dexamethasone loaded MPs made with 50/50 PLGA and 500 μg moxifloxacin were loaded. This group will be regarded as "full drug dosage." While, in the second hydrogel category, half of the MPs and moxifloxacin mass were loaded in the hydrogel network and this hydrogel will be referred to as "half drug dosage." Figures 22A-22C compares the drug release from these two hydrogels.

[0236] As can be seen in Fig. 22, by decreasing the amount of moxifloxacin dissolved in the triblock copolymer solution or by changing the mass of levobunolol or dexamethasone loaded MPs embedded in the hydrogel, one can vary the daily drug release content of each drug proportionally. However, the drug release profile remains relatively constant.

EXAMPLE 16

[0237] Multi-drug delivery hydrogel embodiments.

[0238] Figure 23 depicts the variation in percent drug release over time for three different drug molecules loaded in the present multi-drug delivery platform. To determine the percent of drug release, drug release at each time point is divided by the maximum detected drug release from the hydrogel.

[0239] Conspicuous in Fig. 23 is the ability of the present drug delivery system to release different drug molecules regardless of their hydrophobicity over different durations chosen according to the application. Among the drug molecules chosen for this study, moxifloxacin was the most hydrophilic (water solubility: 24 mg/ml) and dexamethasone was the most hydrophobic drug (water solubility: <100 μg/ml). ⁸ Drug release duration could be adjusted on demand depending on the direct addition of drug to the polymer network (rapid drug release) or encapsulating the drug molecules in MPs and embedding the MPs in the hydrogel network (slow drug release). Even with the drug molecules loaded in the particles, the drug release profile could be modified depending on the polymer used to encapsulate the drug molecules. Using very hydrophobic polymer (85/15), one could achieve a slow drug release in the beginning followed by a rapid enhancement in drug release amount later on. However, with less hydrophobic polymers, more drugs could be released early on during the course of treatment.

[0240] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. [0241] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and

"containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0242] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0243] References:

1 Tyson, S. L., Bailey, R., Roman, J. S., Zhan, T., Hark, L. A., & Haller, J. A. . Clinical outcomes after injection of a compounded pharmaceutical for prophylaxis after cataract surgery: a large-scale review. Current Opinion in Ophthalmology 28, 73-80 (2017).

2 Garg, P., Roy, A., & Sharma, S. . Endophthalmitis after cataract surgery: epidemiology, risk factors, and evidence on protection. Current Opinion in Ophthalmology 28, 67-72 (2017). Lundstrom, M., Friling, E., & Montan, P. . Risk factors for endophthalmitis after cataract surgery: Predictors for causative organisms and visual outcomes. Journal of Cataract & Refractive Surgery 41, 2410-2416 (2015).

Kessel, L., Tendal, B., Jorgensen, K. J., Emgaard, D., Flesner, P., Andresen, J. L., & Hjortdal, J. Post-cataract prevention of inflammation and macular edema by steroid and nonsteroidal anti-inflammatory eye drops: a systematic review. Ophthalmology 121, 1915-1924 (2014).

Taravati, P., Lam, D. L., Leveque, T., & Van Gelder, R. N. Postcataract surgical inflammation. Current opinion in ophthalmology 23, 12-18 (2012).

Shoss, B. L., & Tsai, L. M. . Postoperative care in cataract surgery. Current opinion in ophthalmology 24, 66-73 (2013 ).

Gower, E. W., Lindsley, K., Nanji, A. A., Leyngold, I., & McDonnell, P. J. Perioperative antibiotics for prevention of acute endophthalmitis after cataract surgery. The Cochrane Library (2013).

DeCroos, F. C, & Afshari, N. A. Perioperative antibiotics and anti-inflammatory agents in cataract surgery. Current opinion in ophthalmology 19, 22-26 (2008).

Pleyer, U., Ursell, P. G., & Rama, P. Intraocular pressure effects of common topical steroids for post-cataract inflammation: are they all the same? Ophthalmology and Therapy !, 55-72 (2013).

Chang, D. F., Tan, J. J., & Tripodis, Y. Risk factors for steroid response among cataract patients. Journal of Cataract & Refractive Surgery 37, 675-681 (2011).

Fan, F., Luo, Y., Lu, Y., & Liu, X. Reasons for early ocular hypertension after uneventful cataract surgery. European journal of ophthalmology 24, 712-717 (2013). Yasin, M. N., Svirskis, D., Seyfoddin, A., & Rupenthal, I. D. . Implants for drug delivery to the posterior segment of the eye: A focus on stimuli-responsive and tunable release systems. Journal of Controlled Release 196, 208-221 (2014).

Rawas-Qalaji, M., & Williams, C. A. . Advances in ocular drug delivery Current eye research 37, 345-356 (2012 ).

Kim, Y. C, Chiang, B., Wu, X., & Prausnitz, M. R. . Ocular delivery of macromolecules. Journal of Controlled Release 190 172-181 (2014 ).

Maity, P., Moin, A., Gowda, D.V., Osmani, R A M. . Ophthalmic drug delivery by contact lenses. Journal of Chemical and Pharmaceutical Research 8, 644-651 (2016). Hui, A., Willcox, M. . In vivo studies evaluating the use of contact lenses for drug delivery. Optometry and Vision Science 93, 367-376 (2016).

Maulvi, F. A., Soni, T.G., Shah, D.O. A review on therapeutic contact lenses for ocular drug delivery. Drug Delivery 23, 3017-3026 (2016).

Kirchhof, S., Goepferich, A.M., Brandl, F.P. . Hydrogels in ophthalmic applications European Journal of Pharmaceutics and Biopharmaceutics 95, 227-238 (2015). Chang, M., & Dunn, J. P. Ganciclovir implant in the treatment of cytomegalovirus retinitis. Expert review of medical devices 2, 421-427 (2005).

Kappel, P. J., Charonis, A. C, Holland, G. N., Narayanan, R., Kulkarni, A. D., Yu, F., Boyer, D.S., Engstrom, R.E., Kuppermann, B.D., Southern California HIV/Eye Consortium. Outcomes associated with ganciclovir implants in patients with AIDS- related cytomegalovirus retinitis. Ophthalmology 113, 673-683 (2006).

Holbrook, J. T., Sugar, E. A., Burke, A. E., Vitale, A. T., Thorne, J. E., Davis, J. L., Jabs, D.A., and Multicenter Uveitis Steroid Treatment (MUST) Trial Research Group. . Dissociations of the Fluocinolone Acetonide Implant: The Multicenter Uveitis Steroid Treatment (MUST) Trial and Follow-up Study. American journal of ophthalmology 164, 29-36 (2016).

Jaffe, G. I, Martin, D., Callanan, D., Pearson, P. A., Levy, B., Comstock, T., & Fluocinolone Acetonide Uveitis Study Group. Fluocinolone acetonide implant (Retisert) for noninfectious posterior uveitis: thirty -four- week results of a multicenter randomized clinical study. Ophthalmology 113, 1020-1027 (2006).

Campochiaro, P. A., Brown, D. M., Pearson, A., Ciulla, T., Boyer, D., Holz, F. G, Tolentino, M., Gupta, A., Duarte, L., Madreperla, S., Gonder, J., Kapik, B., Billman, K., Kane, F., and FAME Study Group. Long-term benefit of sustained-delivery fluocinolone acetonide vitreous inserts for diabetic macular edema. Ophthalmology 118, 626-635 (2011).

Cunha-Vaz, J., Ashton, P., Iezzi, R, Campochiaro, P., Dugel, P. U., Holz, F. G, Weber, M., Danis, R.P., Kuppermann, B.D., Bailey, C, Billman, K, Kapik, B., Kane, F., Green, K, for the FAME Study Group. Sustained delivery fluocinolone acetonide vitreous implants: long-term benefit in patients with chronic diabetic macular edema. Ophthalmology 121, 1892-1903 (2014).

Tservakis, I., Koutsandrea, C, Papaconstantinou, D., Paraskevopoulos, T., & Georgalas, I. . Safety and efficacy of dexamethasone intravitreal implant (Ozurdex) for the treatment of persistent macular edema secondary to retinal vein occlusion in eyes previously treated with anti-vascular endothelial growth factors. Current drug safety 10, 145-151 (2015).

Sella, R, Oray, M., Filling, R, Umar, L., Tugal-Tutkun, I., & Kramer, M. . Dexamethasone intravitreal implant (Ozurdex®) for pediatric uveitis. Graefe's Archiveor Clinical and Experimental Ophthalmology 253, 1777-1782 (2015).

Study to Evaluate the Safety for the Treatment of Inflammation Associated With Cataract Surgery. ClinicalTrials.gov, Identifier:NCT02547623 (2015).

Hirani, A., Grover, A., Lee, Y. W., Pathak, Y., & Sutariya, V. . Triamcinolone acetonide nanoparticles incorporated in thermoreversible gels for age-related macular degeneration. Pharmaceutical development and technology 21 61-67 (2016).

Englander, M., & Kaiser, P. K. . Combination therapy for the treatment of neovascular age-related macular degeneration. Current opinion in ophthalmology 24, 233-238 (2013).

Georgakopoulos, C. D., Makri, O. E., Plotas, P., & Pharmakakis, N. Brinzolamide- timolol fixed combination for the prevention of intraocular pressure elevation after phacoemulsification. Clinical & experimental ophthalmology 41, 662-667 (2013). Cetinkaya, A., Akman, A., & Akova, Y. A. Effect of topical brinzolamide 1% and brimonidine 0.2% on intraocular pressure after phacoemulsification. Journal of Cataract & Refractive Surgery 30, 1736-1741 (2004).

Silverstone, D. E., Novack, G. D., Kelley, E. P., & Chen, K. S. Prophylactic treatment of intraocular pressure elevations after neodymium: YAG laser posterior capsulotomies and extracapsular cataract extractions with levobunolol. Ophthalmology 95, 713-718 (1988).

West, D. R., Lischwe, T. D., Thompson, V. M., & Ide, C. H. Comparative efficacy of the β-blockers for the prevention of increased intraocular pressure after cataract extraction. American journal of ophthalmology 106, 168-173 (1988).

French, D. D., Margo, C. E., Behrens, J. J., & Greenberg, P. B. . Rates of Routine Cataract Surgery Among Medicare Beneficiaries. JAMA ophthalmology 135, 163-165 (2017).

DDS. TangDLiu, S. L., J. Neff, R. Sandri. Disposition of levobunolol after an ophthalmic dose to rabbits. Journal of pharmaceutical sciences 76, 780-783 (1987). Andrew A. Acheampong, A. B., Martha Shackleton, Wendy Luo, Steve Lam, and Diane D.S. Tang-Liu. Comparison of concentration-time profiles of levobunolol and timolol in anterior and posterior ocular tissues of albino rabbits. Journal of ocular pharmacology and therapeutics 11, 489-502 (1995).

Binbin Xie, L. J., Zichao Luo, Jing Yu, Shuai Shi, Zhaoliang Zhang, Meixiao Shen, Hao Chen, Xingyi Lia, Zongming Song. An injectable thermosensitive polymeric hydrogel for sustained release of Avastin® to treat posterior segment disease.

International journal of pharmaceutics 490, 375-383 (2015).

www.drugbank.ca/drugs/DB01234.

Claims

Claims:

1. A thermosensitive hydrogel composition comprising:

a) at least one, two, or more biologically compatible triblock copolymers selected from the group consisting of: poly(D,L-lactide-co-glycolide) (PLGA)-poly(ethylene glycol) (PEG)-(PLGA); poly(lactic acid) (PLA)-(PEG)-(PLA); poly(lactide-co-caprolactone) (PLCL)-(PEG)- (PLCL); wherein at least one, two, or more triblock copolymers are combined a mixture;

b) at least two or more biologically active agents; and wherein the composition having a gelation temperature dependent on the weight % ratio of the at least two or more triblock copolymers in said mixture.

2. A thermosensitive hydrogel composition comprising:

a) three biologically compatible triblock copolymers consisting of: poly(D,L- lactide-co-glycolide) (PLGA)-poly(ethylene glycol) (PEG)-(PLGA); poly(lactic acid) (PLA)-(PEG)-(PLA); poly(lactide-co-caprolactone) (PLCL)-(PEG)-(PLCL); wherein the three triblock copolymers are combined a mixture;

b) two or three or four biologically active agents; and wherein the composition having a gelation temperature dependent on the weight % ratio of the three triblock copolymers in said mixture.

3. The thermosensitive hydrogel composition of either claims 1 or 2, wherein the molecular weight of the PLGA, PLA, and PLCL in the triblock copolymers is in the range of about 500-4,000 Daltons.

4. The thermosensitive hydrogel composition of either claims 1 or 2, wherein the molecular weight of the PEG in the triblock copolymers is in the range of about 500-2,500 Daltons.

5. The thermosensitive hydrogel composition of either claims 1 or 2, wherein the ratio of lactic acid to gly colic acid in the PLGA-PEG-PLGA triblock copolymer is in a range of about 1/1 to 10/1.

6. The thermosensitive hydrogel composition of either claims 1 or 2, wherein the ratio of caprolactone to lactic acid in the PLCL-PEG-PLCL triblock copolymer is in a range of about 1/1 to 3/1.

7. The thermosensitive hydrogel composition of any of claims 1 to 6, wherein the composition is a low viscosity liquid at room temperature.

8. The thermosensitive hydrogel composition of any of claims 1 to 7, wherein at least one or more of the biologically active agents is in microparticle form within the composition.

9. The thermosensitive hydrogel composition of any of claims 1 to 8, wherein the microparticles are comprised of at least one biologically compatible polymer.

10. The thermosensitive hydrogel composition of claim 9, wherein the microparticles are comprised of PLGA.

1 1. The thermosensitive hydrogel composition of claim 10, wherein the ratio of lactic acid to gly colic acid is between about 50/50 to about 95/5.

12. The thermosensitive hydrogel composition of claim 11 , wherein the ratio of lactic acid to gly colic acid is about 85/15.

13. The thermosensitive hydrogel composition of any of claims 1 to 12, wherein at least two or more of the biologically active agents is selected from the group consisting of an anti-microbial agent and an anti-inflammatory agent.

14. The thermosensitive hydrogel composition of claim 13, wherein the composition further comprises a third biologically active agent.

15. The thermosensitive hydrogel composition of claim 14, wherein the biologically active agent is an ocular anti-hypertensive agent.

16. The thermosensitive hydrogel composition of claim 14, wherein the biologically active agent is a carbonic anhydrase inhibitor or a beta-blocker.

17. The thermosensitive hydrogel composition of any of claims 1 to 12, wherein at least two or more of the biologically active agents is selected from the group consisting of an anti-ocular hypertensive agent and an anti-inflammatory agent.

18. The thermosensitive hydrogel composition of any of claims 1 to 12, wherein at least two or more of the biologically active agents is selected from the group consisting of an anti-viral agent and an anti-inflammatory agent.

19. The thermosensitive hydrogel composition of any of claims 1 to 12, wherein at least two or more of the biologically active agents is selected from the group consisting of an anti-fungal agent and an anti-inflammatory agent.

20. The thermosensitive hydrogel composition of any of claims 1 to 12, wherein at least two or more of the biologically active agents are antioxidant agents.

21. The thermosensitive hydrogel compositions of either of claims 1-2, in an effective amount, for use in the treatment of post-operative conditions of surgery of an eye in a subject in need thereof.

22. The thermosensitive hydrogel compositions of either of claims 1-2, in an effective amount, for use in the treatment of infection, and/or inflammation and/or elevated intraocular pressure in an eye of a subject in need thereof.

23. The thermosensitive hydrogel compositions of either of claims 1-2, wherein at least two or more of the biologically active agents is selected from the group consisting of an anti-viral agent and an anti-inflammatory agent, in an effective amount, for use in the treatment of retinal edema secondary to surgery of the eye in a subject in need thereof.

24. The thermosensitive hydrogel compositions of either of claims 1-2, wherein at least two or more of the biologically active agents is selected from the group consisting of an anti-fungal agent and an anti-inflammatory agent, in an effective amount, for use in the treatment of retinal edema secondary to vitreoretinal disease of the eye, including but not limited to diabetic macular edema and retinal vein occlusion, in a subject in need thereof.

25. The thermosensitive hydrogel compositions of either of claims 1-2, wherein at least two or more of the biologically active agents is selected from the group consisting of an anti-viral agent and an anti-inflammatory agent, in an effective amount for use in the treatment of increased intraocular pressure secondary to glaucoma in a subject in need thereof.

26. The thermosensitive hydrogel compositions of either of claims 1-2, wherein at least two or more of the biologically active agents is selected from the group consisting of an anti-viral agent and an anti-inflammatory agent, in an effective amount for use in the treatment of retinal degeneration administering in a subj ect in need thereof.

27. The thermosensitive hydrogel compositions of either of claims 1-2, wherein at least two or more of the biologically active agents is selected from the group consisting of an anti-viral agent, an anti-inflammatory agent, and an antioxidant agent, in an effective amount for use in the treatment of abnormal retinal and/or choroidal vascularization, including but not limited to diabetic retinopathy, retinal vein occlusion, macular degeneration, choroidal neovascular membrane, in a subject in need thereof.

28. The thermosensitive hydrogel compositions of either of claims 1-2, wherein at least two or more of the biologically active agents is selected from the group consisting of an anti-viral agent, an anti-inflammatory agent, and an antioxidant agent, in an effective amount for use in the prevention, amelioration, or treatment of a disease or condition associated with oxidative stress in an eye in a subject in need thereof.