US 2015O190359A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2015/0190359 A1 Venkatraman et al. (43) Pub. Date: Jul. 9, 2015

(54) STABLE LIPOSOMAL FORMULATIONS FOR Publication Classification OCULAR DRUG DELVERY (51) Int. Cl. (71) Applicants:Nanyang Technological University, A63L/216 (2006.01) Singapore (SG); Singapore Health A 6LX 9/27 (2006.01) Services Pte Ltd., Bowyer Block (SG) (52) U.S. Cl. CPC ...... A61 K3I/216 (2013.01); A61K 9/127 (72) Inventors: Subramanian Venkatraman, Singapore (2013.01) (SG); Jayaganesh V. Natarajan, Singapore (SG); Tina Howden, (57) ABSTRACT Singapore (SG); Freddy Boey, Singapore (SG) A stable liposomal formulation for ocular delivery. The for mulation contains a liposome that includes at least one lipid (21) Appl. No.: 14/149,159 bilayer containing a phosphatidylcholine, and a prostaglan din F, encapsulated in the liposome. Also provided is a (22) Filed: Jan. 7, 2014 method for treating an ocular disorder with the formulation. Patent Application Publication Jul. 9, 2015 Sheet 1 of 3 US 2015/O190359 A1

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STABLE LIPOSOMAL FORMULATIONS FOR be apparent from the description and from the claims. All OCULAR DRUG DELVERY references cited herein are hereby incorporated by reference in their entirety. BACKGROUND BRIEF DESCRIPTION OF THE DRAWINGS 0001 Intraocular pressure (IOP) in the eye is maintained by a continuous flow of aqueous humor produced by the 0010. The description below refers to the accompanying ciliary body. Excess fluid flows out of the eye through the drawings, of which: trabecular meshwork. If the outflow is blocked, aqueous 0011 FIG. 1 is a plot of cumulative release of latanoprost humor builds up inside the eye leading to increased IOP and from a palmitoyl oleoyl phosphatidylcholine (POPC) lipo ocular hypertension. The ocular hypertension can damage the Some versus time; optic nerve, resulting in an optic neuropathy and irreversibly 0012 FIG. 2a is a plot of the percentage of the starting impaired vision. This condition, known as glaucoma, affects amount of latanoprost remaining in liposomes versus time for more than 60 million people worldwide and is the second egg phosphatidyl choline (PC)-containing liposomes and leading cause of blindness. Increased IOP is the key modifi POPC-containing liposomes; able risk factor for glaucoma. 0013 FIG. 2b is a plot of liposome size versus time of 0002 Conventional treatments for ocular hypertensive storage at 4°C.; and and glaucoma patients include ocular Surgery and topical eye 0014 FIG. 3 is a plot of intraocular pressure versus days drop instillation. These treatment modalities have drawbacks. after a single Subconjunctival injection of latanoprost encap For example, not all patients are candidates for ocular Sur sulated in a POPC-containing liposome gery. Additionally, although topical eye drops are generally considered to be effective, patients long-term compliance DETAILED DESCRIPTION with instillation schedules is a major issue. Ocular hyperten 0015. As mentioned above, a stable liposomal formulation sion and glaucoma cannot be well controlled if patients do not for ocular delivery is provided. The formulation includes a adhere to the proper topical eye drop instillation schedule. liposome containing at least one lipid bilayer that includes a 0003 Topical eye drops contain drugs for controlling IOP phosphatidylcholine. In an embodiment, the liposome is a which typically act by reducing fluid production by the eye, unilamellar vesicle. In a preferred embodiment, the phos increasing fluid outflow, or by both mechanisms. Prostaglan phatidyl choline is palmitoyl oleoyl phosphatidyl choline din analogues, e.g., latanoprost, are potent drugs which can (POPC). reduce IOP by increasing aqueous outflow through the uveo 0016. The formulation also contains a prostaglandin F, Scleral pathway. encapsulated in the liposome. The prostaglandin F, can be 0004 Typically, only 5% of free drug applied to the cor latanoprost, bimatoprost, travoprost, or carboprost. In a pre neal epithelium via eye drops successfully penetrates through ferred embodiment, the prostaglandin F is latanoprost. the cornea. As a result, the amount of drug reaching the 0017. The mole ratio of prostaglandin F, to POPC can be aqueous humor often falls below the therapeutically effective 0.01:1 to 0.5:1. In an embodiment, the mole ratio is 0.01:1 to concentration. This necessitates repeated administration. 0.35:1. In a preferred embodiment, the stable liposomal for Additionally, a Substantial portion of the drug can enter the mulation includes latanoprost and POPC at a mole ratio of circulation via the conjunctival sac, causing undesirable sys 0.01:1 to 0.175:1. Additionally, the liposome can be formu temic side effects. lated to include between 2 and 25 wt % of the prostaglandin F2c. 0005. As an alternative to topical eye drops, subconjunc 0018. The liposome can have a diameter of less than 2 Lum. tival injection of a Sustained release IOP-reducing drug can be In an embodiment, the diameter of the liposome is 100 nm to used to deliver drugs directly to the site of action. Such a 300 nm, e.g., 100 nm, 150 nm, 200 nm, 250 nm, and 300 nm. delivery modality solves the problems of patient non-compli In a preferred embodiment, the diameter is 100 nm. These ance with instillation schedules and inefficient drug transport nano-sized drug-loaded liposomes can release the prostaglan across the cornea. din F, slowly over time. 0006. The need exists for a stable drug formulation for 0019. In an embodiment, the liposomal formulation Subconjunctival injection requiring a minimal frequency of described above can be a pre-constituted, ready-to-inject administration for long-term stable control of IOP aqueous formulation for Sub-conjunctival injection. 0020. Also within the scope of the invention is a method SUMMARY for treating an ocular disorder that relies on administering the liposomal formulation described above. The ocular disorder 0007 To address the need for an improved treatment for can be ocular hypertension or glaucoma. Preferably, the dis intraocular pressure (IOP), a stable liposomal formulation for order is glaucoma. In an embodiment, the liposomal formu ocular delivery is provided. The formulation includes a lipo lation is administered by Subconjunctival injection. Some containing at least one lipid bilayer that includes a 0021 Administering the liposomal formulation described phosphatidylcholine. The formulation also contains a pros above can significantly reduce IOPat one hour post injection. taglandin Fencapsulated in the liposome. Additionally, the More specifically, administering the liposomal formulation liposome has a diameter of less than 2 Jum. results in a reduction in IOP of at least 30% (e.g., 30%, 40%, 0008 Also provided is a method for treating an ocular and 50%) compared to the pretreatment IOP. Advanta disorder by administering the formulation described above. geously, the IOP remains reduced for as long as 4-6 months 0009. The details of one or more embodiments of the following a single injection. invention are set forth in the drawings and description below. 0022. As mentioned above, the injected liposomal formu Other features, objects, and advantages of the invention will lation can slowly release the prostaglandin F, e.g., latano US 2015/O 190359 A1 Jul. 9, 2015

prost, over several months. For example, 50% of the initial latanoprost:POPC mole ratio of 0.175:1 was used to prepare amount of drug in the liposomes can be released within 10 the drug-loaded LUVs. Latanoprost-loaded POPC LUVs days after injection of the formulation into the eye. Prefer were sterile filtered at room temperature using a 0.2 um ably, 65% of the drug is released within 28 days of injection. syringe filter and stored at 4° C. until analyzed. A similar Chronic topical application of anti-glaucoma drugs often method was used to prepare latanoprost-loaded egg PC lipo induces ocular surface disease. The slow-release feature of SOS. the liposomal formulation obviates the need for daily appli cations of these drugs, thereby reducing or eliminating the Example 3 concomitant side effects. 0023 Typically, drugs which are not stable are lyophilized Characterization of Drug Loaded POPC Liposomes or frozen at a temperature of -20°C. or lower to prevent loss 0028 Drug Release studies were performed by dialysing of activity over time. Notably, lyophilizing or freezing sus latanoprost-loaded POPCliposomes prepared as described in tained release formulations of drug-loaded liposomes can EXAMPLE 1 above against PBSata pH of 7.4 and measuring adversely affect the drug release profile, resulting in an unpre by HPLC the amount of latanoprost released. The results are dictable variability in the drug effectiveness. shown in FIG.1. Over a 28 day period, approximately 65% of 0024 Advantageously, the liposomal formulation is stable the latanoprost was released from the latanoprost-loaded for extended periods of time. For example, the formulation POPC liposomes in a time-dependent manner. can be stored at 4°C. for up to 6 months (e.g., 1, 2, 3, 4, 5, and 0029. The physical characteristics of the latanoprost 6 months) without aggregation of the liposomes or loss of loaded POPC liposomes were determined essentially as drug from them. Thus, the liposomal formulation can be described in Venkatraman et al., International Application stored in a vial at 4°C. for extended periods and then can be Publication No. 2012/021107, the content of which is incor directly injected straight from the vial. porated herein by reference in its entirety. 0025. Without further elaboration, it is believed that one 0030. The stability of latanoprost-loaded POPC lipo skilled in the art can, based on the description above, utilize somes prepared as described in EXAMPLE 2 above was the present invention to its fullest extent. The specific measured and compared to similarlatanoprost-loaded egg PC examples below are to be construed as merely illustrative, and liposomes. Stability of the liposomes stored at 4° C. was not limitative of the remainder of the disclosure in any way assessed for a period of 6–9 months. Stability was assessed by whatsoever. measuring the average size of the liposomes as well as mea Suring by HPLC the concentration of latanoprost in the lipo Example 1 somes. The results are depicted in FIG. 2A and FIG. 2B. As shown in FIG. 2A, a significant amount of latanoprost was Preparation of Large Unilamellar Vesicles (LUVs) lost from egg PC liposomes over time, demonstrating a 45% for Drug Release Studies reduction of the starting amount of latanoprost after 3 months 0026. A thin film hydration technique was used to formu of storage at 4°C. Unexpectedly, no measureable amount of late latanoprost-loaded POPC liposomes for drug release latanoprost was lost from POPC liposomes even after 6 studies. Briefly, POPC was weighed and dissolved in a chlo months of storage at 4°C. Turning to particle size, the results roform:methanol (2:1 v/v) solvent mixture. Latanoprost (2 depicted in FIG. 2B reveal that the average size of latano mg/ml stock solution in acetonitrile) was added to the lipid prost-loaded egg PC liposomes got progressively smaller solvent mixture at a drug:lipid mole ratio of 0.175:1 and over a 9 month period of storage at 4°C. Again unexpectedly, maintained at 40° C. The solvent mixture was added to a latanoprost-loaded POPC liposomes did not change measur round bottomed flask in a rotary evaporator connected to a ably even after 6 months at 4°C. Notably, a change in size of water bath maintained at 40°C. The flask was rotated at 100 a drug-loaded liposome will significantly alter the drug rpm under low pressure for 1 h to remove the solvent, thereby release kinetics. forming a thin drug-loaded lipid film. Isotonic phosphate buffered saline (PBS: 150 mM, pH 5.5) was added to this film Example 4 to form multilamellar vesicles (MLVs). The MLVs were extruded ten times through a polycarbonate filter (0.2 um-0. In Vivo Activity of Latanoprost Liposomal 08 um). The extrusions resulted in the formation of LUVs Formulation having a size distribution of 0.09-0.12 um in diameter. 0031. The efficacy of the latanoprost liposomal formula tion described above was tested in an animal model of glau Example 2 coma, namely, long tailed macaques (Macaca fascicularis) having ocular hypertension, defined as an IOP-18 mm Hg. Preparation of LUVs for Drug Stability Studies Animal studies were performed in accordance with the state 0027 Drug-loaded LUVs were also prepared for stability ment for the use of animals in ophthalmic and vision research studies. POPC was dissolved in PBS at a pH of 6.7 with approved by the Association for Research in Vision and Oph constant stifling at room temperature for 2 h to form MLVs. thalmology. Additionally, the guidelines of the Animal Ethics The MLVs were extruded 3-5 times using three 80 nm sized Committee of the Singhealth Singapore Association for polycarbonate membranes stacked together on a bench top Assessment and Accreditation of Laboratory Animal Care extruder to form POPCLUVs. Latanoprost was dissolved in were also followed. ethanol and the resulting solution, maintained in a round 0032. The macaques were anesthetized by intramuscular bottomed flask at 50° C. in a water bath, was dried under a injection of a mixture containing ketamine (5-10 mg/kg body stream of nitrogen gas to form a thin drug film. The drug film weight) and acepromazine maleate (0.25 mg/kg body was hydrated with the POPC LUVs at room temperature for weight), together with a subcutaneous injection of atropine 2-3 huntil no oil droplets were observed on the flask walls. A Sulfate (0.125 mg/kg body weight). Their airway, respiration, US 2015/O 190359 A1 Jul. 9, 2015

and pulse were monitored during all procedures. One to two What is claimed is: drops of 1% Xylocaine was used as a topical anesthesia to 1. A stable liposomal formulation for ocular delivery, the reduce possible discomfort to the animals during Subconjunc formulation comprising a liposome including at least one tival injection. The animals' pupils were dilated with 2.5% lipid bilayer that contains a phosphatidylcholine, and a pros phenylephrine hydrochloride and 1% tropicamide drops (Al taglandin F, encapsulated in the liposome, wherein the lipo Some has a diameter of less than 2 Lum. con Laboratories, French’s Forest, NSW, Australia). 2. The stable liposomal formulation of claim 1, wherein the 0033. Latanoprost-loaded POPC liposomes were pre phosphatidylcholine is palmitoyl oleoyl phosphatidylcholine pared by the technique described in EXAMPLE 2 above. A (POPC). latanoprost liposomal formulation (100 ul) having an initial 3. The stable liposomal formulation of claim 2, wherein the drug/lipid mole ratio of 0.175:1 was introduced by subcon prostaglandin F, is latanoprost, bimatoprost, travoprost, or junctival injection into both eyes of three animals. IOP was carboprost. measured prior to injection and at daily and weekly intervals 4. The stable liposomal formulation of claim3, wherein the post-injection. prostaglandin F, is latanoprost. 5. The stable liposomal formulation of claim 4, wherein the 0034. The monkeys were slightly anesthetized with ket formulation has a moleratio of latanoprost to POPC of 0.01:1 amine (5 mg/kg body weight) before measurement of IOP. to 0.5:1. Again, 1-2 drops of 1% Xylocaine was used as topical anes 6. The stable liposomal formulation of claim 5, wherein the thesia to reduce possible discomfort to the animals involved mole ratio of latanoprost to POPC is 0.01:1 to 0.175:1. during the measurement procedure. IOP was measured using 7. The stable liposomal formulation of claim 5, wherein the a calibrated tonometer (Tono-PenR XL, Reichert Technolo liposome has a diameter of 100 nm to 300 nm. gies, Depew, N.Y.). IOP was monitored for three days pre 8. The stable liposomal formulation of claim 6, wherein the liposome has a diameter of 100 nm to 300 nm. injection and for 120 days post-injection. The results are 9. A method for treating an ocular disorder, comprising shown in FIG. 3. A single subconjunctival injection led to an administering to an eye of a Subject in need thereof the for initial rapid drop in intraocular pressure during the first day mulation of claim 1. following the injection. The reduced IOP remained stable for 10. The method of claim 9, wherein the ocular disorder is at least 120 days post-injection. ocular hypertension or glaucoma. 11. The method of claim 10, wherein the formulation is Other Embodiments administered via Subconjunctival injection. 12. A method for treating an ocular disorder, comprising 0035 All of the features disclosed in this specification administering to an eye of a Subject in need thereof the for may be combined in any combination. Each feature disclosed mulation of claim 7. in this specification may be replaced by an alternative feature 13. The method of claim 12, wherein the ocular disorder is serving the same, equivalent, or similar purpose. Thus, unless ocular hypertension or glaucoma. 14. The method of claim 13, wherein the formulation is expressly stated otherwise, each feature disclosed is only an administered via Subconjunctival injection. example of a generic series of equivalent or similar features. 15. A method for treating an ocular disorder, comprising 0036. From the above description, one skilled in the art administering to an eye of a Subject in need thereof the for can easily ascertain the essential characteristics of the present mulation of claim 8. invention, and without departing from the spirit and scope 16. The method of claim 15, wherein the ocular disorder is thereof, can make various changes and modifications of the ocular hypertension or glaucoma. invention to adapt it to various usages and conditions. Thus, 17. The method of claim 16, wherein the formulation is other embodiments are also within the scope of the following administered via Subconjunctival injection. claims. k k k k k