CROSS-REFERENCE TO RELATED APPLICATION

This application makes reference to and claims the benefit of priority of the Singapore Patent Application No. 10201 708739P filed on 24 October 2017, the content of which is incorporated herein by reference for all purposes, including an incorporation of any element or part of the description, claims or drawings not contained herein and referred to in Rule 20.5(a) of the PCT, pursuant to Rule 4.18 of the PCT.

FIELD OF THE INVENTION

The present invention relates generally to pharmaceutical compositions comprising thermosensitive hydrogels and agent-loaded particles dispersed therein and ocular applications using said pharmaceutical compositions.

BACKGROUND OF THE INVENTION

Treatment of ocular diseases suffers from significant challenges such as rapid clearance of the administered drugs by tear flows. In fact, only about 5% of free drugs applied on the ocular surface may successfully penetrate through the cornea to reach the aqueous humor, falling below the therapeutic limit. Therefore, repeated administration of drugs is often necessary, causing undesirable side effects. In this context, there is a considerable need for technologies that improve drug bioavailability in ocular applications by, for example, prolonging drug retention in the anterior segment of the eye.

SUMMARY OF THE INVENTION

The present invention satisfies the aforementioned need by providing novel pharmaceutical compositions for sustained ocular delivery of an agent as well as methods of using the same.

In a first aspect, the invention relates to a pharmaceutical composition for sustained ocular delivery of an agent, comprising :

(a) a thermosensitive hydrogel; and

(b) particles comprising the agent, stably dispersed in the thermosensitive hydrogel.

In various embodiments, the thermosensitive hydrogel comprises, consists essentially of, or consists of at least one biodegradable and biocompatible polymer and water. In various embodiments, the at least one polymer is selected from the group consisting of poloxamers, polyacrylic acid (polyacrylate), poly-(N-isopropylacrylamide), poly-(vinyl alcohol), PEG-PLGA-PEG copolymers, PEG-PLA-PEG copolymers, PEG-PCL-PEG copolymers, chitosan and chitosan-based derivatives and copolymers (chitosan/gelatin, chitosan/glycerophosphate, chitosan/pectin and chitosan/PEG), poly(N-isopropylacrylamide)-based (PNIPAAM) copolymers, poly(ethylene oxide)/poly(propylene oxide) (PEO/PPO)-based copolymers, poly(ether urethane) (NHP407)-based copolymers, PCGA-PEG-PCGA triblock copolymers, PEG/pHPMAIac/HAMA triblock copolymers, hydroxyethyl methacrylate-co-oligo(trimethylene carbonate), and methacrylate poly(ethylene oxide) methoxy ester, and combinations thereof. In various embodiments, the at least one polymer comprises a poloxamer, preferably poloxamer 407 and/or poloxamer 188.

In various embodiments, the at least one polymer is poloxamer 407. In various embodiments, poloxamer 407 is present in the thermosensitive hydrogel at a concentration of 16-30% by weight.

In various embodiments, poloxamer 407 is present in the thermosensitive hydrogel at a concentration of 20-25%, preferably about 24% by weight.

In various embodiments, the thermosensitive hydrogel has a gelation temperature of 21 -37°C.

In various embodiments, the particles are liposomes or polymersomes, preferably liposomes. In various embodiments, the particles are less than 200 nm in diameter.

In various embodiments, the particles are liposomes comprising a shell-forming lipid material selected from the group consisting of fatty acids, lysolipids, dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine, phosphatidic acid, sphingomyelin, cholesterol, cholesterol hemisuccinate, tocopherol hemisuccinate, phosphatidylethanolamine, phosphatidylinositol, lysolipids, sphingomyelin, glycosphingolipids, glucolipids, glycolipids, sulphatides, lipids with ether and ester-linked fatty acids, polymerized lipids, and combinations thereof.

In various embodiments, the particles are liposomes comprising DPPC.

In various embodiments, the agent is encapsulated within the particles.

In various embodiments, the agent is a compound for the treatment or prevention of a disease, disorder, or condition of an eye, preferably a mammalian eye.

In various embodiments, the disease, disorder, or condition is selected from the group consisting of macular degeneration, diabetic retinopathy, ocular inflammation, glaucoma, chronic dry eye, keratitis, conjunctivitis, fibrosis, bacterial infection, and fungal infection. In various embodiments, the agent is selected from the group consisting of antibiotics, steroids, chemotherapeutic drugs, immunomodulators, anti-inflammatory agents, drugs for the treatment of glaucoma or ocular hypertension, therapeutic peptides or proteins or monoclonal antibodies such as anti-VEGF antibodies, siRNAs, and plasmids, or combinations thereof. In various embodiments, the agent is an inhibitor of calcium-activated potassium channel Kc _a 3.1 , preferably Senicapoc.

In various embodiments, the pharmaceutical composition is poloxamer 407 hydrogel comprising Senicapoc-loaded DPPC liposomes dispersed therein.

In various embodiments, the agent is a selective small molecule Kv1 .3 blocker, preferably 5-(4- phenoxybutoxy)psoralen (PAP-1 ).

In various embodiments, the pharmaceutical composition is poloxamer 407 hydrogel comprising PAP- 1 loaded DPPC liposomes dispersed therein.

In various embodiments, the pharmaceutical composition is a liquid at 4 °C or 20 °C that can be administered to a subject in the form of an eye drop or an eye injection. In various embodiments, the pharmaceutical composition disclosed herein is for use in the delivery of the agent to an eye.

In various embodiments, the pharmaceutical composition disclosed herein is for use in the treatment or prevention of an ocular disease, disorder, or condition.

In a second aspect, the invention relates to a method for sustained delivery of an agent to an eye in a subject, comprising topically administering to the ocular surface or subconjunctival^ injecting a pharmaceutical composition disclosed herein comprising the agent in the particles of the pharmaceutical composition.

In various embodiments, the pharmaceutical composition is administered in the form of an eye drop or an eye injection.

In a third aspect, the invention relates to a method for treating or preventing an ocular disease, disorder, or condition, comprising topically administering to the ocular surface or subconjunctival^ injecting an effective amount of a pharmaceutical composition disclosed herein comprising an agent for treating or preventing said ocular disease, disorder, or condition in the particles of the pharmaceutical composition. In various embodiments, the pharmaceutical composition is administered in the form of an eye drop or an eye injection.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings.

Figure 1 : Size (A) and Drug Loading (B) Stability of Senicapoc-loaded Liposomal Formulation at 4 °C and 37 °C Storage Conditions. Figure 2: Cumulative percentage release (A) and Daily mass release (B) of Senicapoc from drug- loaded Liposomal Formulation.

Figure 3: Cumulative percentage release (A) and Daily mass release (B) of Senicapoc from drug- loaded liposomes dispersed in 18% hydrogel formulation.

Figure 4: Residence of free fluorescein-tagged liposomes and hydrogel formulations in eyes of anesthetized Sprague Dawley rats.

Figure 5: Fluorotron Master scan of fluorescein-tagged liposomes in Viscous Formulation and Gel Formulation.

Figure 6: Senicapoc concentration in flushed tears after topical administration of hydrogel formulations in anesthetized Sprague Dawley rats. Figure 7: Residual Senicapoc concentration in sub-conjunctiva tissues.

Figure 8: In vitro Gelation of Pluronic F-127.

Figure 9: PAP-1 nanoliposome formulation stability at 4 ^S C and 37 ^S C.

Figure 10: Cumulative percentage release (A) and Daily mass release (B) of PAP-1 from drug-loaded Liposomal Formulation.

DETAILED DESCRIPTION OF THE INVENTION

The following detailed description refers to, by way of illustration, specific details and embodiments in which the invention may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention. Other embodiments may be utilized and structural, and logical changes may be made without departing from the scope of the invention. The various embodiments are not necessarily mutually exclusive, as some embodiments can be combined with one or more other embodiments to form new embodiments.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The singular terms "a", "an", and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "comprises" means "includes." In case of conflict, the present specification, including explanations of terms, will control.

Disclosed herein are pharmaceutical compositions comprising thermosensitive hydrogels and particles dispersed therein. The inventors have surprisingly found that drugs comprised in the particles of said pharmaceutical compositions topically administered to the ocular surface or subconjunctival^ injected benefit from enhanced retention in vivo and hence improved bioavailability and treatment efficacy. Therefore, such pharmaceutical compositions can be used as a novel system for sustained delivery of agents comprised in the particles to an eye and in the treatment or prevention of an ocular disease, disorder, or condition.

In a first aspect, the invention relates to a pharmaceutical composition for sustained ocular delivery of an agent, comprising :

(a) a thermosensitive hydrogel; and

(b) particles comprising the agent, stably dispersed in the thermosensitive hydrogel.

The term "pharmaceutical composition" refers to a composition that is, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The term "hydrogel" as used herein refers to water insoluble, crosslinked, three-dimensional networks of polymer chains plus water that fills the voids between polymer chains. Crosslinking facilitates insolubility in water and provides required mechanical strength and physical integrity. Hydrogels may be mostly water, which means that the mass fraction of water may be much greater than that of polymer. The ability of a hydrogel to hold significant amount of water implies that the polymer chains must have at least moderate hydrophilic character.

"Stably dispersed", as used herein, means that the particles are dispersed as solids in the continuous fluid phase of the composition, i.e. the liquid hydrogel. The term "stably" preferably relates to the particles not sedimenting to the bottom of the composition or floating to the surface of the composition over typical time periods of storage. The hydrogel of the invention may be composed of synthetic polymers, natural polymers, or a combination thereof. It is thermosensitive in that it has phase change behavior in response to a change in temperature, i.e. it undergoes a liquid-to-gel transition, or gelation, in response to an increase in temperature above a certain threshold, described herein as the gelation temperature or transition temperature. At a temperature lower than the gelation temperature, the hydrogel is in a liquid form. At a temperature higher than or equal to the gelation temperature, however, the hydrogel transforms into a gel or gel-like structure.

In certain embodiments, the gelation temperature of a hydrogel is higher than the body temperature such that the pharmaceutical composition remains as a viscous solution after in vivo administration. However, in preferred embodiments, the gelation temperature is between room temperature and the body temperature such that the pharmaceutical composition is in a liquid form at room temperature but gels at body temperature when administered in vivo. In preferred embodiments, the thermosensitive hydrogel may have a gelation temperature of 21 -37 °C, for example, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, or 37 °C. The gels may break down over several minutes, hours, or days in vivo. Varying the components and concentrations in the composition can allow fine-tuning of the properties, such as gelation temperature or degradation rate, of the hydrogels.

In various embodiments, the thermosensitive hydrogel comprises, consists essentially of, or consists of at least one biodegradable and biocompatible polymer and water.

The term "biodegradable" as used herein refers to the property of a material that it can be completely, or substantially completely, degraded, dissolved, and/or eroded over time when exposed to physiological conditions (pH, temperature, and fluid or other environment), and can be gradually resorbed, absorbed and/or eliminated by the body, or that it can be degraded into fragments that can be eliminated from the body by the kidneys. The term "biocompatible" as used herein refers to the property of being relatively inert with respect to provoking a response from a host immune system.

In various embodiments, the at least one polymer is selected from the group consisting of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PPO) triblock copolymers (i.e. poloxamers), polyacrylic acid (polyacrylate), poly-(N-isopropylacrylamide), poly-(vinyl alcohol), PEG-PLGA-PEG copolymers (with "PEG" for "polyethyleneglycol" and "PLGA" for "poly(co-glycolic acid)"), PEG-PLA-PEG copolymers (with "PLA" for "poly(lactic acid)"), PEG-PCL-PEG copolymers (with "PCL" for "polycaprolactone"), chitosan and chitosan-based derivatives and copolymers (chitosan/gelatin, chitosan/glycerophosphate, chitosan/pectin and chitosan/PEG), poly(N- isopropylacrylamide)-based (PNIPAAM) copolymers, poly(ethylene oxide)/poly(propylene oxide) (PEO/PPO)-based copolymers, poly(ether urethane) (NHP407)-based copolymers, PCGA-PEG- PCGA triblock copolymers, PEG/pHPMAIac/H AM A triblock copolymers, hydroxyethyl methacrylate-co- oligo(trimethylene carbonate), and methacrylate poly(ethylene oxide) methoxy ester, and combinations thereof.

The thermosensitive hydrogel may also be formed by natural polymers. Many natural polymers exhibit thermos-sensitivity, biocompatibility and biodegradation. However, some natural polymers lack intrinsic thermosensitive properties or have thermosensitivity outside the physiological temperature. In these cases, modifications are necessary to improve the thermoresponsive gelation behavior of the polymer. The natural polymers that may be used for such modifications include, but are not limited to, chitosan and related derivatives, methylcellulose, alginate, hyaluronic acid, dextran, and xyloglucan. In various embodiments, the at least one polymer comprises a poloxamer. Poloxamers are FDA- approved thermosensitive synthetic polymers. Poloxamers are nonionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxy ethylene (poly(ethylene oxide)). Biocompatible poloxamers have been widely used for drug delivery and tissue engineering. Poloxamer-based hydrogels allow reversible gelation under certain physiological temperature and pH by adjusting the composition of PEO and PPO, and the overall molecular weight and concentration. The poloxamers that have been used for drug delivery include, but are not limited to, poloxamer 188, poloxamer 237, poloxamer 238, pluronic F-98, poloxamer 124, poloxamer 184, poloxamer 338, poloxamer 401 , and poloxamer 407. The physicochemical characteristics and gel-forming properties of some selected poloxamers are known in the art.

The at least one polymer may be present in the thermosensitive hydrogel in an amount of between about 5% to about 50%, about 1 0% to about 40%, or about 20% to about 30% by weight or by volume of the hydrogel. In some embodiments, the at least one polymer is present in the hydrogel in an amount of about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 1 1 %, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21 %, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% by weight or by volume of the hydrogel. In preferred embodiments, the at least one polymer is poloxamer 407 and/or poloxamer 188. In more preferred embodiments, the at least one polymer is poloxamer 407.

In various embodiments, poloxamer 407 is present in the thermosensitive hydrogel at a concentration of 16-30% by weight. In preferred embodiments, poloxamer 407 is present in the thermosensitive hydrogel at a concentration of 20-25%, preferably about 24% by weight. In some embodiments, poloxamer 407 is present in the thermosensitive hydrogel at a concentration of about 16%, about 17%, about 18%, about 19%, about 20%, about 21 %, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%,or about 30% by weight. The term "particles" as used herein encompasses all particles for pharmaceutical agent delivery irrespective of their three-dimensional shape or conformation. The particles of the invention may be such that they fully or partially encapsulate the pharmaceutical agent or entrap the pharmaceutical agent within the polymer matrix of the particle. Alternatively, the agent to be delivered may be coupled to the exterior surface of the particles.

The particles of the invention may include such entities commonly referred to as, for example, liposomes, polymersomes, polymer particles, transferosomes, niosomes, micelles, bubbles, microbubbles, microspheres, microballoons, microcapsules, aerogels, clathrate bound particles, hexagonal HII phase structures, and the like. Particles previously classified as either micro- or nanoparticles are included within this definition, so that average particle diameter may vary from between about 10 nm and 900 μηι. However, it should be noted that the particles used should be suitable for ocular applications. For example, they should have reduced light scattering and adequate transparency such that they will not obscure the vision when applied in vivo. In various embodiments, the particles are less than 200 nm in diameter. The particle size may be measured using any means available in the art, for the example, by gel filtration, dynamic light scattering, or electron microscopy.

The particles, including the comprised agents or not, may be present in the pharmaceutical composition in any amount that is suitable for the intended use. For example, the particles may be present in the pharmaceutical composition of about 0.1 % to about 50%, about 5% to about 40%, or about 10% to about 30% by weight or by volume of the composition. In some embodiments, the particles are present in the pharmaceutical composition in an amount of about 0.1 %, about 0.5%, about 1 %, about 5%, about 8%, about 10%, about 1 1 %, about 12%, about 13%, about 14%, about 1 5%, about 16%, about 17%, about 18%, about 19%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% by weight or by volume of the composition.

In various embodiments, the particles are liposomes or polymersomes, preferably liposomes. The term "liposome" as used herein refers to a stable, spherical or spheroidal cluster or aggregate of lipid-based amphiphilic compounds stacked in bilayer arrays. Phospholipids having ionic polar head groups are the most common building blocks for liposomes. Liposomes of this type are typically negatively charged (anionic liposomes) due to presence of the phosphate groups. Liposomes having net positive charges (cationic liposomes) are also well known in the art.

The term "polymersome" as used herein refers to particles defined by a membrane formed from amphiphilic synthetic block copolymers. In various embodiments, the particles are liposomes comprising a shell-forming lipid material selected from the group consisting of fatty acids, lysolipids, dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine, phosphatidic acid, sphingomyelin, cholesterol, cholesterol hemisuccinate, tocopherol hemisuccinate, phosphatidylethanolamine, phosphatidylinositol, lysolipids, sphingomyelin, glycosphingolipids, glucolipids, glycolipids, sulphatides, lipids with ether and ester-linked fatty acids, polymerized lipids, and combinations thereof.

In various embodiments, the particles are liposomes comprising DPPC.

Upon incorporation of pharmaceutical agents, the hydrogel systems disclosed herein can act as sustained drug release depot in situ. In accordance with the present invention, such pharmaceutical agents may be comprised in particles stably dispered in the hydrogels.

In various embodiments, the agent is encapsulated within the particles. The term "agent" as used herein refers to a chemical compound, a biological macromolecule (such as a nucleic acid, an antibody, an antibody fragment, a protein, or a peptide), or a combination thereof. The activity of such agents may render them suitable as a therapeutic agent which is a biologically, physiologically, or pharmacologically active substance that acts locally or systemically in a subject. A pharmaceutical composition disclosed herein may comprise one or more such agents.

In various embodiments, the agent is a compound for the treatment or prevention of a disease, disorder, or condition of an eye, preferably a mammalian eye.

In various embodiments, the disease, disorder, or condition is selected from the group consisting of macular degeneration, diabetic retinopathy, ocular inflammation, glaucoma, chronic dry eye, keratitis, conjunctivitis, fibrosis, bacterial infection, and fungal infection.

In various embodiments, the agent is selected from the group consisting of antibiotics, steroids, chemotherapeutic drugs, immunomodulators, anti-inflammatory agents, drugs for the treatment of glaucoma or ocular hypertension, therapeutic peptides or proteins or monoclonal antibodies such as anti-VEGF antibodies, siRNAs, and plasmids, or combinations thereof.

In certain embodiments, the agent is an inhibitor of calcium -activated potassium channel Kc _a 3.1 , preferably Senicapoc. In certain embodiments, the pharmaceutical composition is poloxamer 407 hydrogel comprising Senicapoc-loaded DPPC liposomes dispersed therein. In certain embodiments, the agent is a selective small molecule Kv1 .3 blocker, preferably 5-(4-phenoxybutoxy)psoralen (PAP- 1 ). In certain embodiments, the pharmaceutical composition is poloxamer 407 hydrogel comprising PAP-1 loaded DPPC liposomes dispersed therein. The pharmaceutical compositions disclosed herein may additionally comprise a pharmaceutically- acceptable excipient, which, as used herein, includes, but are not limited to, any and all solvents, buffering agents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Various excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21 st Edition, A. R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference). The use of a conventional excipient medium may be contemplated within the scope of the present invention, except insofar as any conventional excipient medium may be incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition.

Exemplary preservatives may include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and/or other preservatives. Exemplary antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabi sulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabi sulfite, and/or sodium sulfite. Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and/or trisodium edetate. Exemplary antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and/or thimerosal. Exemplary antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and/or sorbic acid. Exemplary alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and/or phenylethyl alcohol. Exemplary acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and/or phytic acid. Other preservatives known in the art may also be used.

Exemplary buffering agents include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, d-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, etc., and/or combinations thereof.

Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods may include the step of encapsulating an agent within particles, coupling it to the exterior or interior surface of the particles, or integrating it into the circumferential membrane of the particles, followed by dispersing the agent-loaded particles in the thermosensitive hydrogel.

In various embodiments, the pharmaceutical composition is a liquid at 4 °C or 20 °C that can be administered to a subject in the form of an eye drop or an eye injection. In various embodiments, the pharmaceutical composition disclosed herein is for use in the delivery of the agent to an eye.

The term "delivery" as used herein refers to providing a therapeutically effective amount of a pharmaceutically active agent to a specific location within a host causing a therapeutically effective concentration of the pharmaceutically active agent at a particular location.

In various embodiments, the pharmaceutical composition disclosed herein is for use in the treatment or prevention of an ocular disease, disorder, or condition. The term "treating" as used herein refers to partially or completely alleviating, ameliorating, improving, relieving, delaying onset of, inhibiting progression of, reducing severity of, and/or reducing incidence of one or more symptoms or features of a particular disease, disorder, or condition. The pharmaceutical composition may also be administered to a subject who does not exhibit signs of a disease, disorder, or condition for prevention thereof and/or to a subject who exhibits only early signs of a disease, disorder, or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, or condition.

In a second aspect, the invention relates to a method for sustained delivery of an agent to an eye in a subject, comprising topically administering to the ocular surface or subconjunctival^ injecting a pharmaceutical composition disclosed herein comprising the agent in the particles of the pharmaceutical composition.

In various embodiments, the pharmaceutical composition is administered in the form of an eye drop or an eye injection. In a third aspect, the invention relates to a method for treating or preventing an ocular disease, disorder, or condition, comprising topically administering to the ocular surface or subconjunctivally injecting an effective amount of a pharmaceutical composition disclosed herein comprising an agent for treating or preventing said ocular disease, disorder, or condition in the particles of the pharmaceutical composition.

In various embodiments, the pharmaceutical composition is administered in the form of an eye drop or an eye injection. As will be readily understood by those skilled in the art, the exact amount of the pharmaceutical composition required may vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like. The present invention is further illustrated by the following examples. However, it should be understood, that the invention is not limited to the exemplified embodiments.

EXAMPLES

Materials

Senicapoc and TRAM-34 were gifts from Prof. Heike Wulff, University of California, Davis. Additional TRAM-34 was purchased from Sigma-Aldrich. Dipalmitoylphosphatidylcholine (DPPC) and 1 ,2- Dioleoyl-sn-Glycero-3-Phosphoethanolamine-N-(Carboxyfluoresc ein) were purchased from Avanti Polar Lipids. Whatmann drain discs and polycarbonate membrane filters were purchased from GE Healthcare Life Sciences. Chloroform (HPLC Grade) was purchased from Tedia Chemicals. Acetonitrile and Methanol (LCMS Grades) were purchased from Fisher Scientific. Cellulose ester dialysis tubings (100 kDa MWCO) and tubing closures were obtained from Spectrum Laboratories. Salts used to make Phosphate Buffered Saline (PBS) includes sodium chloride (NaCI), potassium chloride (KCI), Di-sodium hydrogen phosphate (Na2HP04) and Potassium dihydrogen phosphate (KH2PO4) are all purchased from Sigma-Aldrich. Zinc Sulphate Monohydrate salt for analytical processing was also purchased from Sigma-Aldrich. Soft tissue homogenizing kits (CK14, 0.5 ml) used for tissue analysis were purchased from Bertin-lnstruments.

Animals

Female Sprague Dawley rats between 6-8 weeks old (InVivos, Singapore) were used in this experiment. Animals were handled according to institutional guidelines and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The study protocol was approved by the Institutional Animal Care and Use Committee of SingHealth.

Preparation of Fluorescein-tagged DPPC Liposomal Suspension The 20 mM fluorescein-tagged DPPC liposomes were prepared by dissolving 1 1 .4 mg (2 mM) of 1 ,2- Dioleoyl-sn-Glycero-3-Phosphoethanolamine-N-(Carboxyfluoresc ein) and 66.1 mg (18 mM) of DPPC lipids and in chloroform : methanol with a 2:1 ratio in a round-bottom flask (RBF). The RBF was secured to a rotary evaporator and held for 60 minutes within a 40 °C water bath for the rapid removal of the organic solvents. A thin layered film of the lipid mixture was eventually formed on the inner surface of the round-bottomed flask. The spontaneous formation of multilaminar particles (MLVs) was achieved upon rehydration with 5 ml of phosphate buffered saline (PBS, pH 7.4). The MLVs were then subjected to extrusion using a 10 ml LIPEX extruder heated at 60 °C with a circulating water bath and extruded with 5 cycles of 200 nm membrane filters followed by 10 cycles of 80 nm membrane filter to form small unilaminar particles (SUVs). The loss of buffer through the extrusion process was measured and reconstituted with PBS to 5 ml of final stock liposomal formulation. The stock liposomal formulation was subsequently stored at 4 °C for future use.

Preparation of Senicapoc-loaded DPPC Liposomal Suspension

The 10 mol% Senicapoc-loaded DPPC liposomes were prepared by dissolving 3.233 mg of Senicapoc and 73.4 mg (20 mM) of DPPC lipids in chloroform : methanol with a 2:1 ratio in a round-bottomed flask (RBF). The subsequent steps for the preparation were identical to the fabrication methodology as described above.

Preparaton of Pluronic F-127 Hydrogel Formulations

Senicapoc-loaded liposomes were subsequently loaded into Pluronic F-127 (a trade name of poloxamer 407) hydrogels by hydrating 160 mg and 240 mg of Pluronic F-127 polymer with 1 ml of the final liposomal suspension and dissolved overnight at 4 °C to form a 16% viscous solution and a 24% formulation that is capable of gelation in situ, respectively. Drug Loading of Senicapoc-loaded Liposomal Suspension

Loss of Senicapoc during the extrusion process was quantified by measuring the remaining volume of the liposomal suspension. Characterization of drug loading were performed by lysing 5ul of liposomes suspension with PBS and acetonitrile respectively to a final ratio of 2:8. Samples were filtered through a 0.22 μιτι RC filter and subsequently analyzed using Liquid Chromatography Mass Spectroscopy (LCMS, Waters Corporation) with multiple reaction monitoring, through a BEH 1 .7 μιτι C-18 column, 2.1 mm x 50 mm using an in situ mixed Isocratic mobile phase of 80% Acetonitrile + 0.1 % Formic Acid and 20% Dl Water + 0.1 % Formic Acid, at a flowrate of 0.25 ml/min.

In vitro Stability of Senicapoc-loaded Liposomal Suspension

Senicapoc-loaded liposomes were monitored for stability based on size and drug loading over a period of 28 days. Samples from the stock liposomal formulation stored at 4 °C and aliquot stock formulations stored at 37 °C, were periodically considered for analysis. Characterization of liposome sizes by intensity were performed with dynamic light scattering (DLS) measurements using Malvern Nano Zetasizer. Characterization for drug loading at periodic time points were performed identically using the method as described above.

In vitro Release Studies of Senicapoc-loaded Liposomes & Hydrogel Formulations

Sample volumes by mass, containing 1 12.5 μg of Senicapoc, were dispensed into individual dialysis tubing before being secured and placed submerged within wide-mouth bottles containing 30 ml of PBS each. The bottles were subsequently placed in a 37 °C incubator with a shaking speed of 50 rpm to mimic physiological eye conditions. The release assays were also performed for 28 days in triplicates to ensure consistency. Samples were aliquoted before being replaced on a daily basis with fresh PBS to maintain sink conditions. 200 μΙ of sample was mixed thoroughly with 800 μΙ acetonitrile and filtered before characterizing the quantity released using the LCMS method as mentioned above.

In vivo Residence of Pluronic F-127 Hydrogel

Three rats were anesthetized with ketamine (75 mg/kg) and xylazine (10 mg/kg). Ten μΐε of fluorescein-tagged free liposome, viscous formulation (16%) and gel formulation (24%) were applied into the conjunctival sac of rat eye. To determine the retention time of the fluorescein-tagged liposome on ocular surface, rat eye was examined by fluorescein imaging and fluorophotometry Fluorescein imaging on the rat ocular surface was performed with Micron IV (Phoenix Research labs, Pleasanton, USA) platform with a cobalt blue filter. The rat eye was imaged every 5 min, and to prevent dessication of rat eyes, lids were passively blinked every 30 sees between two examinations.

Fluorophotometry of the rat eye was carried out with Fluorotron Master™ (OcuMetrics, Mountain View, CA) with the rat lens. This equipment recorded the fluorescence signal along the visual axis in the posterior to anterior direction of the eye. Before applying the fluorescent formulation, a baseline measurement of the rat eye was obtained. After application of the formulation, the rat was held on an adjustable stand and the cornea of rat eye was adjusted to face the lens. Eye was imaged every 3 min, without any blinking between scans.

In vivo PK Analysis of Flushed Tears

Ten μΐε of viscous formulation (16%) and gel formulation (24%) were applied to the conjunctival sac of the right and left eye of a rat respectively. The rat eyes were passively blinked every 30 sees to prevent desiccation of the ocular surface. Eye-flush tears were collected at 4 specific time points after 10 min, 30 min, 60 min and 90 min. At each time point, ten μΐε of PBS was instilled on rat cornea, the rat eye was passively blinked 3 times, and then flush tears collected by a 10-μΙ_ capillary tube (Sigma- Aldrich, St. Louis, Missouri, USA) and stored under -80°C. Baseline eye-flush tears collected before formulation instillation was used as control. Senicapoc in tear samples was quantified by mixing 1 μΙ of the tear sample with specific volumes of PBS and acetonitrile to achieve a final ratio of 1 :4 and subsequently analyzed using the above-described LCMS method developed by the inventors.

In vivo PK Analysis of Sub-conjunctival Injection Eighteen rats were anesthetized prior to the injection of Senicapoc nanoliposome (5 μΙ_) into the subconjunctiva of right eye. Left eye were injected with the same volume of PBS as control. Subconjunctiva injection was peformed by gently pulling conjunctiva from the sclera with a pair of forceps, and nanoliposome or PBS was injected into the superior subconjunctival region using a 50 μΙ_ Hamilton syringe with a 30G needle. Injected rats were randomly divided into 3 groups according to the study plan as shown in Table 1 .

Conjunctival tissues covering the eye globe from limbus to fornix were harvested from both eyes at 1 hr, 24 hrs and 3 weeks after injection. Harvested tissues were stored at -80°C until use. Conjunctival tissues from another 3 control rats were used for LC-MS baseline and calibration. Senicapoc in conjunctival tissues were extracted after tissue homogenization with PBS and acetonitrile in a ratio of 1 :4 at 10,000 rpm on 30s On' followed by 30s Off Interval for 2 cycles, before quantification by the LC- MS. TRAM-34 was added as an internal standard for tissue analysis.

Statistical Analysis

A descriptive statistical tool was used to evaluate the comparisons between formulations. Experimental data and calculations are reported as mean ± standard deviation. Two-tailed t-test involving ANOVA distribution followed by homoscedastic post hoc analysis were used to compare means between groups. A P-value of <0.05 was considered statistically significant. Example 1 : In vitro Size and Drug Loading Stability of Senicapoc-loaded Liposomes

The inventors monitored the liposome size and drug loading stability at two storage conditions of 4°C and 37°C for a period of 28 days (Fig. 1 ). It was observed that 20 mM Senicapoc-loaded DPPC liposomes had no significant changes in its size when stored at 4 °C and 37 °C. The liposomal suspension maintained drug loading capacity at 89.9% ± 4.7% at 4°C and 82.1 % ± 7.5% at 37°C over a 28 day storage period, respectively.

Example 2: In vitro Release of Senicapoc-loaded Liposomes

The cumulative drug release of Senicapoc from DPPC liposomes (Fig. 2) was observed over a period of 28 days before achieving a cumulative released amount of 86.3%. Therapeutic dosages of 1 μΜ of Senicapoc, established for the suppression of 90% naive and central memory T cells had been sustainably achieved within the initially 5 days, and possibly up to 7 days before an additional instillation is required.

Example 3: In vitro Release of Senicapoc-loaded Liposomal Hydroqel Formulation

The release of Senicapoc from the Senicapoc-loaded liposomal dispersion within an 18% hydrogel (Fig. 3) exhibited a sustained release profile over a period of 28 days before achieving a cumulative release of 81 .2%. For the first day, Senicapoc release was significantly lower (p = 0.02) from the liposomal hydrogel dispersion formulation compared to the free liposomal formulation (Fig. 2). Subsequent release from days 2 to 5 were also lower for the liposomal hydrogel dispersion formulation though not significantly different as compared to the formulation without the hydrogel matrix. The differences in the rates of release of Senicapoc between the formulations (Fig 2 & 3) could be related to increase in viscosity induced by the Pluronic F-127 polymer, hindering diffusion of released Senicapoc across the hydrogel matrix within the dialysis bag into the release medium. However, the daily release of Senicapoc from the hydrogel formulation (Fig. 3) also achieved dosages within therapeutic range for the initial 3 days.

Example 4: In vivo Residence of Hydrogel on Ocular Surface of Spraque Dawlev rats

The fluorescein-tagged free liposomes, viscous formulation and gel formulation were applied onto separate anesthetized rat eyes to determine the retention time on the ocular surface. Fluorescein signals from these formulations were recorded by the Micron IV imaging system under cobalt blue filter at different time points.

As seen in Figure 4, the free liposomes could not be observed on rat ocular surface 5 min after application, whereas the viscous formulation could be observed for up to 30 min, and the observed retention of gel formulation was the longest at 60min. Clearance of added substances from ocular surface is mainly through eye blinking and the tear drainage system. During eye blinking, free liposome and viscous formulations spread more rapidly on the ocular surface than gel formulation, thus were cleared faster. The blinking of the rat's eyes has a tendency to expel the excessive formulation, which explained the observed fluorescence on the eyelids and the eyelashes for all formulations.

The retention time of viscous formulation and gel formulation on ocular surface of anesthetized rat was also examined by fluorophotometry. As the scanning software of Fluorotron Master™ was designed according to human eye, the actual magnitude of the measurements for the rat eye distances in the horizontal axis is not applicable for this purpose, but this does not affect the interpretation of fluorescence signals in the vertical axis.

In Fig 5, the fluorescence signal of the viscous formulation could be observed at the surface of the eye even after 60min, in the absence of blinking. It is also interesting to note that the fluorescence peak for gel formulation shifted posteriorly towards the back of the eye within the initial 3 mins after instillation and eventually residing within the eye for up to 30 mins into the study.

Example 5: In vivo Pharmacokinetic Analysis of Eye-flush Tears

The topical instillation of 10 μΙ Senicapoc-loaded liposomal hydrogel formulations into the eyes of Sprague Dawley rats were well-tolerated with no significant sign of irritation or redness being observed (data not shown). Eye-flush tears were collected and analyzed as described in the materials and method section. Fig. 6 shows the higher concentrations of Senicapoc were present in the eye at all- time points consistent with increased residence time provided by the 24% gel formulation (shown previously in Figure 4). This has implications for improvement in therapeutic bioavailability of the drug compared to drug associated with free liposomal eye drops.

Example 6: In vivo Pharmacokinetic Analysis of Sub-coniunctival Injection

The delivery of nanoliposomes without gels via sub-conjunctiva injection was investigated as an alternative measure to increase bioavailability to the ocular surface. It was found that Senicapoc from the Senicapoc-loaded DPPC liposomes in the sub-conjunctiva tissues were detectable and quantifiable up to 24 hrs (Figure 7), indicating a rapid but sustained delivery of Senicapoc, associated with a rapid clearance rate. Several distributional pathways of the nanoparticles have been identified within the eye, including nasolacrimal and lymphatic clearance routes via lymph nodes (Feng, et al., Ocular delivery of pRNA nanoparticles: distribution and clearance after subconjunctival injection, Pharm Res, 31 (2014) 1 046- 1058). Feng et al found that the internalization and distribution of their pRNA nanoparticles had different terminal locations (Conjunctiva, Cornea, Sclera, and Retina), related to the shape and size of the nanoparticles, and encouragingly, nanoparticles remained detectable by fluorescence up to 20 hrs. Another study that used sub-conjunctiva injection of PLGA nanoparticles containing brinzolamine for glaucoma treatment showed positive efficacy data in the reduction of intraocular pressure of up to 7 days after a single injection, which was attributed to the longer mean retention time of the PLGA nanoparticles compared to eye drops (Salama, et al., PLGA Nanoparticles as Subconjunctival Injection for Management of Glaucoma, AAPS PharmSciTech, (201 7)). Collectively, these data highly suggest that minimally invasive sub-conjunctiva injections could reduce the distributional clearance of Senicapoc-loaded liposomes within the eye, and could serve as depots for sustained release in treatment of ocular diseases. In summary, hydrogel formulations were shown to significantly increase bioavailability as a result of increased viscosity, compared to a less viscous formulation of Pluronic F-127 for up to 30 mins. Therapeutic dosages were also achievable within the eye for close to 60 mins for hydrogel formulations, or 6 times longer than viscous formulations (Fig. 6). This is much longer than what is attainable with currently available free drug topical solutions. The liposomal hydrogel formulation (Fig. 3) also showed a gentler profile of drug release compared to the free liposomal formulation (Fig. 2), reducing the spiking effect and hence contributing to a more controlled daily drug release profile.

The sub-conjunctival injection provided Senicapoc-loaded liposomes with a natural depot within connective tissues, prolonging liposomal residence by weak hydrophobic epithelial-stromal interactions (Akbarzadeh, et al., Liposome classification, preparation, and applications, Nanoscale Research Letters, 8 (2013)). From Figure 5, it is also possible that migration of nanoliposome occurs within the eye, leading to low detectability of Senicapoc after 24h (Fig 7). The low but still measurable tissue concentrations of Senicapoc at 24h suggests that sub-conjunctival injection could be a viable method to prolong drug residence and maintain bioavailability of drugs compared to conventional topical eye drops.

The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. Other embodiments are within the following claims.

One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. Further, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The compositions, methods, procedures, treatments, molecules and specific compounds described herein are presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention are defined by the scope of the claims. The listing or discussion of a previously published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including," containing", etc. shall be read expansively and without limitation. The word "comprise" or variations such as "comprises" or "comprising" will accordingly be understood to imply the inclusion of a stated integer or groups of integers but not the exclusion of any other integer or group of integers. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by exemplary embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention. The content of all documents and patent documents cited herein is incorporated by reference in their entirety.