Indian Patents. 279458:A METHOD FOR PREPARING PARTICLES OF CYCLOSPORINE A

Title of Invention	A METHOD FOR PREPARING PARTICLES OF CYCLOSPORINE A
Abstract	The present invention provides ophthalmic formulations containing cyclosporine, methods for preparing the formulation, and methods for using the formulation.

Full Text	OPHTHALMIC PREPARATIONS CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to US Patent Application No. 12/007,902 filed January 16, 2008, the disclosure of which is incorporated herein by reference in its entirety. FIELD OF THE INVENTION [0002] The invention is directed to ophthalmic formulations, methods for preparing the ophthalmic formulation, and methods of using the ophthalmic formulation. BACKGROUND [0003] Most ocular medications may be administered topically to treat surface as well as intraocular disorders. This route is often preferred for the management of various pathological diseases that affect the anterior chamber of the eye, for two main reasons: (1) it is more conveniently administered and (2) provides a higher ratio of ocular to systemic drug levels. To be administered topically and to achieve the necessary patient compliance, the medication must present a good local tolerance. [0004] Cyclosporine is a known immunosuppressant, which acts by reducing inflammatory cells such as activated lymphocytes in the conjunctiva (Kunert et al., Arch Ophthalmol., 118:1489-96, 2000) or by increasing the number of mucin secreting goblet cells (Kunert et al., Arch Ophthalmol., 120:330-7, 2002). Over the years, Cyclosporine A (CsA) has been evaluated for numerous potential applications in ophthalmology. [0005] New developments in the topical delivery of CsA can be divided in two general areas of research: new delivery systems, such as solutions, ointments, colloidal carriers and drug impregnated contact lenses, and chemical modifications of drugs or prodrugs. [0006] At the present time, only one ophthalmic formulation of CsA is commercially available, which is currently marketed as Restasis®. The extensive literature on the delivery of CsA to the eye (Lallemand et al., European Journal of Pharmaceutics and Biopharmaceutics 56: 307-318, 2003; Nussenblatt et al., Am. J. Ophthalmol. 112: 138- 146, 1991; Masuda et al, Lancet 1: 1093-1096,1989; Georganas etai, Clin. Rheumatol. 15: 189-192, 1996; Prummel etai, N. Engl. J. Med. 321: 1353-1359, 1989; Reinhard et al, Ophthalmologe 94: 496-500, 1997) reflects the great medical interest and the pharmaco-economical aspects of this challenge. Despite a poor intraocular penetration, topical CsA has been successfully used in a variety of immune-mediated ocular surface phenomena such as vernal conjunctivitis, dry eye syndrome and the prevention of corneal allograft rejection. For example, Cross et al. reported that topical cyclosporine eye drop therapy not only improved the signs and symptoms of dry eye disease, but also resulted in high patient satisfaction, fewer patients with chronic dry eye visiting the ophthalmologist, and less ancillary drug use (Manag. Care Interface 2002;15:44-49). [0007] One obstacle to preparing an ophthalmic CsA formulation is that CsA cannot be prepared in formulations based on the commonly used aqueous ophthalmic vehicles because of both its hydrophobicity (log P = 3.0) and its extremely low aqueous solubility at 6.6 mg/ml. Therefore, in most studies, CsA was dissolved and administered in vegetable oils. However, these media are poorly tolerated, resulting in relatively low ocular availability. Also, formulations prepared in these media have short shelf lives. [0008] Oil-in-water emulsions are particularly useful in the delivery of lipophilic drugs. In vivo data from early studies confirmed that emulsions could be effective topical ophthalmic drug delivery systems, with a potential for sustained drug release. [0009] The product currently on the market, Restasis®, is packaged in single unit doses to avoid microbial contamination because it does not contain any preservatives. It would be highly desired to have a preparation to be dispensed in a multi-dose container. [0010] Therefore, there is a need for an ideal topical ocular formulation of CsA, which fulfills several requirements: (1) the formulation must be well tolerated, or non-irritating to the eye, (2) the formulation must be easy to administer, (3) the formulation ideally has an increased CsA residence time in the eye, (4) systemic absorption of the formulation should be avoided because the toxic CsA concentration in blood is above 300 ng/ml, (5) the formulation should have a long shelf life, and (6) the formulation should be easily manufactured. [0011] The present invention satisfies the needs in the field by providing stable, non- irritating emulsion formulations of CsA suitable for ophthalmic application, and methods for preparing and using the formulations. SUMMARY [0012] One aspect of the invention relates to an ophthalmic formulation comprising: (a) cyclosporine or a derivative thereof, (b) at lest one solvent, (c) at least one oil, (d) at least one surfactant, (e) at least one preservative, and (f) water or buffer. In one embodiment, the cyclosporine (Cs) or a derivative thereof can be present in (a) a solid nanoparticulate state; (b) a solid microparticulate state; (c) solubilized; or (d) any combination thereof. An exemplary cyclosporine useful in the invention is cyclosporine A (CsA). [0013] In one embodiment of the invention, the ophthalmic formulation further comprises a viscosity modifier, such as a cellulose derivative, a polysaccharide or a synthetic polymer. [0014] In another embodiment of the invention, the formulation is a mixture of particles of cyclosporine or a derivative thereof suspended in emulsion droplets and stoically stabilized particulate cyclosporine or a derivative thereof in water or buffer. [0015] Another aspect of the invention is directed to an opthalmic cyclosporine composition which is cationic. The cationic nature of the formulation results in an increased residency in the eye, producing a more effective dosage form. The cationic nature of the dosage form can result, for example, from the inclusion of a cationic preservative. [0016] In yet another embodiment, the formulation comprises globules of oil comprising dissolved cyclosporine or a derivative thereof. The globules can have a diameter, for example, of less than about 10 microns, less than about 9 micros, less than about 8 microns, less than about 7 microns, less than about 6 microns, less than about 5 microns, less than about 4 microns, less than about 3 microns, less than about 2 microns, less than about 1000 nm, less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 290 nm, less than about 280 nm, less than about 270 nm, less than about 260 nm, less than about 250 nm, less than about 240 nm, less than about 230 nm, less than about 220 nm, less than about 210 nm, less than about 200 nm, less than about 190 nm, less than about 180 nm, less than about 170 nm, less than about 160 nm, less than about 150 nm, less than about 140 nm, less than about 130 nm, less than about 120 nm, less than about 110 nm, less than about 100 nm, less than about 90 nm, less than about 80 nm, less than about 70 nm, less than about 60 nm, less than about 50 nm, less than about 40 nm, less than about 30 nm, less than about 20 nm, or less than about 10 nm. [0017] In some embodiments, the oil is selected from the group consisting of, but not limited to, sweet almond oil, apricot seed oil, borage oil, canola oil, coconut oil, corn oil, cotton seed oil, fish oil, jojoba bean oil, lard oil, boiled linseed oil, Macadamia nut oil, medium chain triglycerides (Crodamol GTCC), mineral oil, olive oil, peanut oil, safflower oil, sesame oil, soybean oil, squalene, sunflower seed oil, tricaprylin (1,2,3-trioctanoyl glycerol), and wheat germ oil. [0018] In other embodiments, the solvent is selected from the group consisting of, but not limited to, isopropyl myristate, triacetin, N-methyl pyrrolidinone, aliphatic and aromatic alcohols, ethanol, dimethyl sulfoxide, dimethyl acetamide, ethoxydiglycol, polyethylene glycols, and propylene glycol. Other examples of useful solvents are long- chain alcohols. Ethanol is an example of a preferred alcohol that may be used in the present invention. [0019] In further embodiments, the surfactant is selected from the group consisting of, but not limited to, sorbitan esters (such as Span or Arlacel), glycerol esters (such as glycerin monostearate), polyethylene glycol esters (such as polyethylene glycol stearate), block polymers (such as Pluronics®), acrylic polymers (such as Pemulen ), ethoxylated fatty esters (such as Cremophor® RH-40), ethoxylated alcohols (such as Brij®), ethoxylated fatty acids (such as Tween or Tween 20), monoglycerides, silicon based surfactants and polysorbates. In a preferred embodiment, the surfactant is Polysorbate 80. [0020] In one embodiment of the invention, the ophthalmic formulations of the invention have anti-microbial properties. In some embodiments, the formulation comprises a preservative suitable for opthalmic administration. Examples of such a preservative include, but are not limited to, quaternary ammonium compounds, such as cetyltrimethylammonium bromide, cetylpyridinium chloride, benzethonium chloride, and benzalkonium chloride. Preferably, the preservative is benzalkonium chloride. [0021] In a preferred embodiment, the ophthalmic formulation comprises: (a) cyclosporine A, (b) ethanol, (c) medium chain triglycerides, (d) Polysorbate 80, (e) benzalkonium chloride, and (f) phosphate buffer. [0022] Another aspect of the invention is directed to a method for preparing particles of Cs or a derivative thereof. The method comprises: (a) forming an emulsion base by suspending cyclosporine or a derivative thereof in a mixture of oil, solvent, surfactant, preservative, and water or buffer, and (b) homogenizing or vigorously stirring the emulsion base, wherein the resultant composition is a mixture of particles of cyclosporine or a derivative thereof suspended in emulsion droplets and sterically stabilized microcrystalline or nanoparticulate cyclosporine or a derivative thereof in the media. Optionally, the emulsion base of step (a) further comprises a viscosity modifier, such as a cellulose derivative, a polysaccharide or a synthetic polymer. In one embodiment, the particles of cyclosporine or a derivative thereof have a diameter of less than about 10 microns, less than about 9 microns, less than about 8 microns, less than about 7 microns, less than about 6 microns, less than about 5 microns, less than about 4 microns, less than about 3 microns, less than about 2 microns, less than about 1 micron, or even smaller in size. In another embodiment, the homogenizing step is performed via a high-pressure system at 1,000 to 40,000 psi. [0023] Another aspect of the invention is directed to a method for preparing particles of Cs or a derivative thereof, comprising: (a) dissolving cyclosporine or a derivative thereof in a mixture of oil, solvent, and surfactant to form an emulsion pre-mix, (b) adding preservative, and water or buffer to the emulsion pre-mix, and (c) homogenizing or vigorously stirring the mixture, whereby cyclosporine or a derivative thereof is precipitated into particles. Optionally, the mixture of step (a) or that of step (b) further comprises a viscosity modifier, such as a cellulose derivative, a polysaccharide or a synthetic polymer. In one embodiment, the diameter of the particles of cyclosporine or a derivative thereof is less than about 10 microns, less than about 9 microns, less than about 8 microns, less than about 7 microns, less than about 6 microns, less than about 5 microns, less than about 4 microns, less than about 3 microns, less than about 2 microns, or less than about 1 micron. In another embodiment, the homogenizing step is performed via a high-pressure system at 1,000 to 40,000 psi. [0024] In one embodiment, the particles of cyclosporine or a derivative thereof, droplets comprising cyclosporine or a derivative thereof, or a combination thereof, have a mean particle size of less than about 10 microns, less than about 9 microns, less than about 8 microns, less than about 7 microns, less than about 6 microns, less than about 5 microns, less than about 4 microns, less than about 3 microns, less than about 2900 nm, less than about 2800 nm, less than about 2700 nm, less than about 2600 nm, less than about 2500 nm, less than about 2400 nm, less than about 2300 nm, less than about 2200 nm, less than about 2100 nm, less than about 2000 nm, less than about 1900 nm, less than about 1800 nm, less than about 1700 nm, less than about 1600 nm, less than about 1500 nm, less than about 1400 nm, less than about 1300 nm, less than about 1200 nm, less than about 1100 nm, less than about 1000 nm, less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, or less than about 100 nm, less than about 90 nm, less than about 80 nm, less than about 70 nm, less than about 60 nm, less than about 50 nm, less than about 40 nm, less than about 30 nm, less than about 20 nm, or less than about 10 nm. Preferably, the particles of cyclosporine or a derivative thereof, droplets comprising cyclosporine or a derivative thereof, or a combination thereof have a mean particle size of less than about 3 microns in diameter. [0025] In a related aspect, the invention provides a method of treating a subject suffers from a condition, such as an aqueous deficient dry-eye state, phacoanaphylaxis endophthalmitis, uveitis, keratoconjunctivitis, allergic eye disorders, or immune- modulated eye diseases. The method comprises administering a therapeutically effective amount of the CsA formulations of the invention to the eye of a subject in need. [0026] Both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the invention as claimed. Other objects, advantages, and novel features will be readily apparent to those skilled in the art from the following detailed description of the invention. BRIEF DESCRIPTION OF THE DRAWINGS [0027] Figures 1A, 1B, and 1C show mean particle size and mean zeta potential for Restasis®, for the cationic micellar nanoparticle (cMNP) formulation of cyclosporine A prepared according to Example 1, and for the neutral micellar nanoparticle (nMNP) formulation prepared according to Example 2, respectively. [0028] Figure 2 shows the mean corneal concentration of cyclosporine in a Franz cell- human cornea model. High level of cyclosporine was detected in the cornea for the cMNP formulation, but not for the nMNP formulation or Restasis®. [0029] Figure 3 depicts the stability data for the cMNP formulation stored at 25°C, 40°C and 60°C, respectively, in terms of cyclosporine potency and mean particle size. [0030] Figure 4 depicts the stability data for the nMNP formulation stored at 25°C, 40°C and 60°C, respectively, in terms of cyclosporine potency and mean particle size. DETAILED DESCRIPTION A. Overview of the Invention [0031] The invention relates to ophthalmic formulations comprising cyclosporine or a derivative thereof and methods of making and using the same. One aspect of the present invention is directed to an ophthalmic formulation comprising: (a) cyclosporine or a derivative thereof, (b) at lest one solvent, (c) at least one oil, (d) at least one surfactant, (e) at least one preservative, and (f) water or buffer. In one embodiment, the cyclosporine (Cs) or a derivative thereof can be present in (a) a solid nanoparticulate state; (b) a solid microparticulate state; (c) solubilized; or (d) any combination thereof. In another embodiment, the ophthalmic formulation further comprises a viscosity modifier, such as a cellulose derivative, a polysaccharide or a synthetic polymer. [0032] The cyclosporine can be, for example, cyclosporine A, and the cyclosporine or a derivative thereof can be in an amorphous form, semi- amorphous form, crystalline form, semi-crystalline form, or any combination thereof. [0033] In one embodiment of the invention, the opthalmic cyclosporine composition is cationic. The cationic nature of the formulation can result in an increased residency in the eye, producing a more effective dosage form. Moreover, the increased residency in the eye can result in a need for fewer applications, a decreased drug dosage for effectiveness, and increased patient compliance (as fewer applications are desirable to patients). The cationic nature of the dosage form can result, for example, from the inclusion of a cationic preservative. An exemplary cationic preservative is benzalkonium chloride. [0034] In another embodiment of the invention, the formulation is a mixture of particles of cyclosporine or a derivative thereof suspended in emulsion droplets and sterically stabilized particulate cyclosporine or a derivative thereof in water or buffer. [0035] Another aspect of the invention encompasses a method of making a tri-phasic composition comprising a lipophilic phase, water or a buffer, and particulate cyclosporine or a derivative thereof. The invention also encompasses compositions comprising an oil phase that has at least one oil, at least one solvent, and a surfactant for cyclosporine or a derivative thereof. Two specific methods of making the composition of the invention are described. In the first method ("Route I"), cyclosporine or a derivative thereof is milled in an emulsion base. This method requires that cyclosporine or a derivative thereof is poorly soluble or insoluble in all phases of the oil phase/lipophilic phase and the water or buffer. In die second method ("Route II"), simultaneous milling and precipitation of cyclosporine or a derivative thereof in an emulsion base is observed. The second method requires mat cyclosporine or a derivative thereof is soluble or partially soluble in one or more phases of the emulsion base. [0036] One benefit of the invention is to provide a method applicable to cyclosporine or a derivative thereof, which is poorly water-soluble, since the conventional method, such as wet milling, is not effective. Another benefit of the invention is that it does not require grinding media or specialized grinding process or equipment. The use of such grinding media can add cost and complexity to a particle size reduction process for cyclosporine. [0037] For Route I, cyclosporine or a derivative thereof is first suspended in a mixture of a non-miscible liquid, such as an oil, solvent, preservative and water or buffer, to form an emulsion base, followed by homogenization or vigorous stirring of the emulsion base. Optionally, the emulsion base further comprises a viscosity modifier, such as a cellulose derivative, a polysaccharide or a synthetic polymer. Nanoparticles can be produced with reciprocating syringe instrumentation, continuous flow instrumentation, or high speed mixing equipment. High velocity homogenization or vigorous stirring, producing forces of high shear and cavitation, are preferred. High shear processes are preferred as low shear processes can result in larger particle sizes of cyclosporine or a derivative thereof. The resultant composition is a composite mixture of cyclosporine or a derivative thereof suspended in the emulsion droplet (nanoemulsion fraction) and sterically stabilized micro- /nano-crystalline cyclosporine or a derivative thereof in the media. This tri-phasic system comprises particulate cyclosporine or a derivative thereof, oil, preservative, and water or buffer. Preferably, the resultant micro/nano-particulate cyclosporine or a derivative thereof has a mean particle size of less than about 3 microns. Smaller particulate cyclosporine or a derivative thereof can also be obtained, as described below. [0038] Cyclosporine or a derivative thereof can be precipitated out from the oil droplets by adding more of the non-miscible liquid. The precipitated cyclosporine or a derivative thereof typically has a mean particle size of less than about 3 microns. If desired, the particles of cyclosporine or a derivative thereof can be prevented from aggregating or clumping together by incorporating a surfactant or emulsifier, e.g., a "surface stabilizer." [0039] Route II is utilized for cyclosporine or a derivative thereof because it is soluble in at least one part of the emulsion base, such as the solvent, in particular, ethanol. For Route II, cyclosporine or a derivative thereof is dissolved in a mixture of oil, solvent, and surfactant to form an emulsion pre-mix. Cyclosporine or a derivative thereof remains in soluble form if preservative and water or buffer are not added to the mixture. Upon the addition of preservative, a viscosity modifier, and water or buffer and the application of shear forces, cyclosporine or a derivative thereof is precipitated into micro/nano-particles having a mean particle size of less than about 3 microns. Nanoparticles can be produced with reciprocating syringe instrumentation, continuous flow instrumentation, or high speed mixing equipment. High energy input, through high velocity homogenization or vigorous stirring, is a preferred process. The high energy processes reduce the size of the emulsion droplets, thereby exposing a large surface area to the surrounding aqueous environment. High shear processes are preferred, as low shear processes can result in larger particle sizes. This is followed by precipitation of nanoparticulate cyclosporine or a derivative thereof previously embedded in the emulsion base. The end product comprises cyclosporine or a derivative thereof in solution and particulate suspension, both distributed between the solvent, oil, preservative, and water or buffer. Nanoparticulate cyclosporine or a derivative thereof has at least one surface stabilizer associated with the surface thereof. [0040] If desired, the water miscible oil droplets and nanoparticles of cyclosporine or a derivative thereof prepared using Route I or Route II may be filtered through either a 0.2 or 0.45 micron filter. Larger oil droplets and/or particles of cyclosporine or a derivative thereof can be created by simply increasing the water content, decreasing the oil-stabilizer- solvent content, or reducing the shear in forming the oil droplets. [0041] For the 50X concentrated emulsion base used in Route I or Route II, the content of cyclosporine is about 0.1%-10%, the content of solvent is about 0.1%-20%, the content of oil is about 5%-50%, the content of surfactant is about 0.1%-20%, the content of preservative is about 0.1 %-5%, and the content of the aqueous medium is about 20%-80%, all in w/w percentage. Optionally, the viscosity modifier is present in the emulsion base in the amount of about 0.1% to about 10% (w/w). The content of each ingredient in the final product is the amount above divided by 50, with the aqueous medium being the major component, at about 98% or more. B. Definitions [0042] The present invention is described herein using several definitions, as set forth below and throughout the application. [0043] As used herein, "about" will be understood by persons of ordinary skill in the art and will vary to some extent depending upon the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, "about" will mean up to plus or minus 10% of the particular term. [0044] The phrase "poorly water-soluble" or "water insoluble" as used herein refers to a solubility in water of less than about 30 mg/mL, less than about 20 mg/mL, less than about 10 mg/mL, less than about 1 mg/mL, less than about 0.1 mg/mL, less than about 0.01 mg/mL or less than about 0.001 mg/mL at ambient temperature and pressure and at about pH7. [0045] The phrase "soluble" as used herein refers to a solubility in water or another medium selected from the group consisting of greater than about 10 mg/mL, greater than about 20 mg/mL, and greater than about 30 mg/mL. [0046] As used herein, the term "subject" is used to mean an animal, preferably a mammal, including a human or non-human. The terms "patient" and "subject" may be used interchangeably. [0047] As used herein, the phrase "therapeutically effective amount" shall mean that drug dosage that provides the specific pharmacological response for which the drug is administered in a significant number of subjects in need of such treatment. It is emphasized that a therapeutically effective amount of a drug that is administered to a particular subject in a particular instance will not always be effective in treating the conditions/diseases described herein, even though such dosage is deemed to be a therapeutically effective amount by those of skill in the art. [0048] As used herein, the phrase "cyclosporines and derivatives thereof include a group of nonpolar cyclic oligopeptides with known immunosuppressant activity, as disclosed in U.S. Patent No. 5,474,979, which is incorporated by reference in its entirety. In addition to the most common form of cyclosporine A, there are several other minor metabolites, cyclosporine B through I, identified. The present invention intends to include any individual member of the cyclosporine family, as well as a mixture of two or more members of the family, because the commercial cyclosporines may contain a mixture of several forms of cyclosporines, which all share a cyclic peptide structure consisting of eleven amino acids with a total molecular weight of about 1200. The cyclosporines and derivatives thereof also include the natural or synthetic product, as well as any substituents or different configurations of the amino acids, as long as they maintain the immunosuppressive activity and capability of enhancing or restoring the lacrimal gland tearing. The present invention further encompasses the cyclosporine A derivatives, e.g., methylthio-substitued cyclosporine A and other alkylthio-substitued cyslosporine A derivatives, as disclosed in U.S. Patent No. 6,254,860 and 6,350,442, each of which is incorporated by reference in its entirety. C. Compositions of the Invention 1. Exemplary Compositions [0049] The methods of the invention can produce several different types of compositions. A first composition comprises: (1) microparticulate and/or nanoparticulate cyclosporine or a derivative thereof having a mean particle size of less than about 10 microns and, optionally for nanoparticulate cyclosporine or a derivative thereof, having associated with the surface thereof at least one surface stabilizer; (2) at least one preservative; (3) water or a buffer; and (4) an emulsion pre-mix or oil phase or lipophilic phase comprising at least one oil and optionally at least one solvent, and/or a viscosity modifier. The composition may additionally comprise microcrystalline cyclosporine or a derivative thereof. The particulate cyclosporine or a derivative thereof can be present in the water or buffer, oil, solvent, preservative, or a combination thereof. Such a composition is made utilizing Route I. [0050] A second composition comprises: (1) microparticulate and/or nanoparticulate cyclosporine or a derivative thereof having a mean particle size of less than about 10 microns and, optionally for nanoparticulate cyclosporine or a derivative thereof having associated with the surface thereof at least one surface stabilizer; (2) a preservative; (3) water or a buffer; and (4) an emulsion pre-mix or oil phase or lipophilic phase comprising at least one oil, optionally at least one solvent, solubilized cyclosporine or a derivative thereof, and/or a viscosity modifier. The composition may additionally comprise microcrystalline cyclosporine or a derivative thereof. The solubilized cyclosporine or a derivative thereof may be present in oil, solvent, or a combination thereof. In addition, nanoparticulate cyclosporine or a derivative thereof can be present in the water or buffer, oil, solvent, or a combination thereof. Such a composition is made utilizing Route II. [0051] In a further embodiment of the invention, the solubilized cyclosporine or a derivative thereof can be precipitated out from the emulsion droplets. The precipitated microparticulate cyclosporine or a derivative thereof has a mean particle size of less than about 10 microns, less than about 9 microns, less than about 8 microns, less than about 7 microns, less than about 6 microns, less than about 5 microns, less than about 4 microns, less than about 3 microns, less than about 2 microns, or about 1 micron or greater. In other embodiments of the invention, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% of the particles of cyclosporine or a derivative thereof can have a diameter less than the size listed above, e.g., less than about 10 microns, less than about 9 microns, etc. [0052] In yet another embodiments of the invention, the nanoparticles of cyclosporine or a derivative thereof have a diameter of less than about 1000 nm, less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 290 nm, less than about 280 nm, less than about 270 nm, less than about 260 nm, less than about 250 nm, less than about 240 nm, less than about 230 nm, less than about 220 nm, less than about 210 nm, less than about 200 nm, less than about 190 nm, less than about 180 nm, less than about 170 nm, less than about 160 nm, less than about 150 nm, less than about 140 nm, less than about 130 nm, less than about 120 nm, less than about 110 nm, less than about 100 nm, less than about 90 nm, less than about 80 nm, less than about 70 nm, less than about 60 nm, less than about 50 nm, less than about 40 nm, less than about 30 nm, less than about 20 nm, or less than about 10 nm. In other embodiments of the invention, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% of the particles of cyclosporine or a derivative thereof can have a diameter less than the size listed above, e.g., less than about 1000 nm, less than about 900 nm, etc. [0053] The tri-phasic compositions of the invention are beneficial for several reasons. First, formulations resulting from the Route II method comprise both solid and solubilized forms of cyclosporine or a derivative thereof. This enables a resultant pharmaceutical formulation to provide both immediate release and controlled release of me component cyclosporine or a derivative thereof, providing for fast onset of activity combined with prolonged activity of cyclosporine or a derivative thereof. [0054] The different components of the two types of compositions described above can be separated and used independently. 2. Emulsion Globules Comprising Cyclosporine Nanoparticles and/or Solubilized Cyclosporine [0055] The emulsion globules comprising solubilized cyclosporine or a derivative thereof, nanoparticles of cyclosporine or a derivative thereof, or a combination thereof can also be isolated from the surrounding aqueous or buffer phase and used in therapeutic dosage forms. The emulsion globules can be made using food grade, USP or NF grade materials suitable for human use applications. Nanoparticulate oil globules comprising solubilized active pharmaceutical ingredient (API) and methods of making the same are described in U.S. Patent No. 5,629,021 ("the '021 patent"), which is incorporated herein by reference. The emulsion globules of the invention typically comprise (1) solubilized cyclosporine or a derivative thereof, particulate cyclosporine or a derivative thereof, or a combination thereof; (2) at least one oil; (3) at least one solvent; (4) at least one preservative; and (5) at least one surface stabilizer or surfactant. Optionally, the emulsion globules further comprises a viscosity modifier, such as a cellulose derivative, a polysaccharide or a synthetic polymer. Emulsion globules comprising solubilized cyclosporine or a derivative thereof, particulate cyclosporine or a derivative thereof, or a combination thereof can be isolated by, for example, filtration. [0056] In general, the emulsion globules comprising solubilized cyclosporine or a derivative thereof, nanoparticles of cyclosporine or a derivative thereof, or a combination thereof comprise a significant quantity of cyclosporine or a derivative thereof and have diameters of about 10 to about 1000 nm, with a mean a diameter of less than about 1 micron preferred, and with the smallest globules filterable through a 0.2 micron filter, such as is typically used for microbiological purification. The range of concentration of cyclosporine or a derivative thereof in the globules in the diluted final product can be from about 0.01% to about 5%. The emulsion globules can be stored at between about -20°C and about 40°C. In one embodiment of the invention, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the globules in the preparation have diameters of less than about 1 micron, less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, or less than about 100 nm. [0057] By varying different parameters of Route I and Route II, the size and integrity of such globules can be modified. Hence, the stability of globules comprising dissolved cyclosporine or a derivative thereof can be altered to enable the release of cyclosporine or a derivative thereof, either as a solution or precipitate. This is a microreservoir- dissolution-controlled system, where the drug solids act as depot and, as the solubilized fraction is depleted, more drug is drawn into solution form the particulate depot. Thus, the emulsion globules comprising solubilized cyclosporine or a derivative thereof enable controlled cyclosporine or a derivative thereof release over time. [0058] In addition, the emulsion globules comprising solubilized cyclosporine or a derivative thereof, nanoparticles of cyclosporine or a derivative thereof, or a combination thereof can be diluted with aqueous solutions without stability loss. This enables the use of high concentration of cyclosporine or a derivative thereof, e.g., up to about 10%, in products which can be diluted to obtain the final product. [0059] In a preferred embodiment, 50X concentrated cyclosporine formulation containing 2.5% w/w cyclosporine is manufactured aseptically. The particles of cyclosporine in the concentrated product have a mean particle size of less than 200 mn. Subsequently, the concentrated product is diluted with sterile PBS/saline to achieve 0.05% w/w cyclosporine in the final product, which has similar appearance to Restasis®. The final product is packaged and sealed in 0.4 ml per dose. D. Methods of Making the Inventive Compositions Three methods for making the compositions of the invention are described herein. One benefit of the invention is to provide methods applicable to cyclosporine or a derivative thereof, which is poorly water-soluble. Another benefit of the methods of the invention is that they do not require grinding media or specialized grinding process or equipments. The use of such grinding media can add cost and complexity to a particle size reduction process for an API. Additionally, the methods of the invention can accommodate amorphous or semi-amorphous forms of cyclosporine or a derivative thereof. In summary, the three methods are as follows: Route I: cyclosporine or a derivative thereof is iasoluble or slightly soluble in any of the components of the formulation; Route II: cyclosporine or a derivative thereof is soluble or partially soluble in at least one of the components of the formulation; and Route III: cyclosporine or a derivative thereof is completely soluble in all of the components of the formulation. 1. Route I [0060] The method of Route I essentially comprises milling cyclosporine or a derivative thereof in an emulsion base. This method requires that cyclosporine or a derivative thereof is poorly soluble or insoluble in all phases of the oil phase/lipophilic phase and the water or buffer. Hence, cyclosporine or a derivative thereof is first suspended in a mixture of a non-miscible liquid, which can comprise at least one oil, at least one solvent, at least one preservative, at least one viscosity modifier, and at least one buffer or water to form an emulsion base, followed by homogenization or vigorous stirring of the emulsion base. Nanoparticles of cyclosporine or a derivative thereof can be produced with reciprocating syringe instrumentation, continuous flow instrumentation, or high speed mixing equipment. High velocity homogenization or vigorous stirring, producing forces of high shear and cavitation, are preferred. High shear processes are preferred as low shear processes can result in larger particle sizes. [0061] The resultant composition is a composite mixture of cyclosporine or a derivative thereof suspended in the emulsion droplet (nanoemulsion fraction of cyclosporine or a derivative thereof) and sterically stabilized microcrystalline or microparticulate cyclosporine or a derivative thereof in the media. This tri-phasic system comprises particulate cyclosporine, oil, preservative and water or buffer. [0062] In one embodiment of the invention, the resultant microparticulate cyclosporine or a derivative thereof has a diameter of less than about 10 microns, less than about 9 microns, less than about 8 microns, less than about 7 microns, less than about 6 microns, less than about 5 microns, less than about 4 microns, less than about 3 microns, less than about 2 microns, or greater than about 1 micron. [0063] In another embodiment of the invention, the nanoparticulate cyclosporine or a derivative thereof can have a diameter of less than about 1000 nm, less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 290 nm, less than about 280 nm, less than about 270 nm, less than about 260 nm, less than about 250 nm, less than about 240 nm, less than about 230 nm, less than about 220 nm, less than about 210 nm, less than about 200 nm, less than about 190 nm, less than about 180 nm, less than about 170 nm, less than about 160 nm, less than about 150 nm, less than about 140 nm, less than about 130 nm, less than about 120 nm, less than about 110 nm, less than about 100 nm, less than about 90 nm, less than about 80 ran, less than about 70 nm, less than about 60 nm, less than about 50 nm, less than about 40 nm, less than about 30 nm, less than about 20 nm, or less than about 10 nm. [0064] In other embodiments of the invention, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% of the microcrystalline or microparticulate cyclosporine or a derivative thereof in a composition can have a diameter of less than about 10 microns, less than about 9 microns, less than about 8 microns, less than about 7 microns, less than about 6 microns, less than about 5 microns, less than about 4 microns, less than about 3 microns, less than about 2 microns, about 1 micron, or greater than about 1 micron and less than about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 microns. [0065] In yet other embodiments of the invention, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% of the nanoparticulate cyclosporine or a derivative thereof can have a diameter of less than about 1000 nm, less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 290 nm, less than about 280 nm, less than about 270 nm, less than about 260 nm, less than about 250 nm, less than about 240 nm, less than about 230 nm, less than about 220 nm, less than about 210 nm, less than about 200 nm, less than about 190 nm, less than about 180 nm, less than about 170 nm, less than about 160 nm, less than about 150 nm, less than about 140 nm, less than about 130 nm, less than about 120 nm, less than about 110 nm, less than about 100 nm, less than about 90 nm, less than about 80 nm, less than about 70 nm, less than about 60 nm, less than about 50 nm, less than about 40 nm, less than about 30 nm, less than about 20 nm, or less than about 10 nm in size. [0066] Cyclosporine or a derivative thereof can be precipitated out from the oil droplets by adding more of the non-miscible liquid. The precipitated particles of cyclosporine or a derivative thereof typically have a diameter of less than about 10 microns, less than about 9 microns, less than about 8 microns, less than about 7 microns, less than about 6 microns, less than about 5 microns, less than about 4 microns, less than about 3 microns, less than about 2 microns, or less than about 1 micron. If desired, the particles cyclosporine or a derivative thereof can be prevented from aggregating or clumping together by incorporating a surfactant or emulsifier, e.g., a "surface stabilizer." 2. Route II and Route III [0067] Routes II and III require that cyclosporine or a derivative thereof is soluble or partially soluble in at least one (Route II) or all of the phases (Route III) of the emulsion base; e.g., that cyclosporine or a derivative thereof is soluble in at least one oil, at least one solvent, at least one preservative, or water or buffer. In some embodiments, Route II or III can comprise the simultaneous milling and precipitation of cyclosporine or a derivative thereof in an emulsion base. [0068] Route II is utilized when cyclosporine or a derivative thereof is soluble in at least one part of the emulsion base, such as the solvent, and Route III is utilized when cyclosporine or a derivative thereof is soluble in all of the components of the emulsion base, such as oil and a solvent. For Routes II and III, cyclosporine or a derivative thereof is dissolved in a mixture of oil, solvent, preservative, viscosity modifier and stabilizer to form an emulsion pre-mix. Cyclosporine or a derivative thereof remains in soluble form if water or buffer is not added to the mixture. Upon the addition of water or buffer and the application of shear forces, cyclosporine or a derivative thereof is precipitated into microparticles having a diameter of less than about 10 microns, and nanoparticles having a diameter of less than about 1 micron (as described above in Route I; the same particle sizes are applicable to Routes II and HI). Nanoparticles can be produced with reciprocating syringe instrumentation, continuous flow instrumentation, or high speed mixing equipment. High energy input, through high velocity homogenization or vigorous stirring, is a preferred process. The high energy processes reduce the size of the emulsion droplets, thereby exposing a large surface area to the surrounding aqueous environment. High shear processes are preferred, as low shear processes can result in larger particle sizes. [0069] This can be followed by precipitation of nanoparticulate cyclosporine or a derivative thereof previously embedded in the emulsion base. The end product comprises cyclosporine or a derivative thereof in solution and particulate suspension, both distributed between the solvent, oil, and water or buffer. In one embodiment, nanoparticulate cyclosporine or a derivative thereof has at least one surface stabilizer associated with the surface thereof. [0070] If desired, the water miscible oil droplets and nanoparticles of cyclosporine or a derivative thereof prepared using Route I, Route II, or Route III may be filtered through either a 0.2 or 0.45 micron filter. Larger oil droplets and/or particles of cyclosporine or a derivative thereof can be created by simply increasing the water content, decreasing the oil-stabilizer-solvent content, or reducing the shear in forming the oil droplets. [0071] For the 50X concentrated emulsion base used in Route I, Route II, or Route III, the content of cyclosporine is about 0.1%-10%, the content of solvent is about 0.1%-20%, the content of oil is about 5%-50%, the content of surfactant is about 0.1%-20%, the content of preservative is about 0.1%-5%, and the content of the aqueous medium is about 20%-80%, all in w/w percentage. Optionally, the viscosity modifier is present in the emulsion base in the amount of about 0.1 % to about 10% (w/w). The content of each ingredient in the final product is the amount above divided by 50, with the aqueous medium being the major component, at about 98% or more. E. Components of the Methods and Compositions of the Invention 1. Cyclosporine a. Suitable Cyclosporine and Derivatives Thereof The phrase "cyclosporines and derivatives thereof is defined supra, as disclosed in U.S. Patent Nos. 5,474,979, 6,254,860, and 6,350,442. The cyclosporines useful in the present invention include naturally occurring cyclosporines, preferably cyclosporines A, B,C, D and G, as well as synthetic and semi-synthetic cyclosporines and cyclosporine derivatives disclosed in U.S. Patent Nos. 4,108,985, 4,210,581,4,220,641, 4,384,996, 4,764,503, 4,703,033 and U.K. Patent Application No. 2,206, 119A, which are hereby incorporated by reference in their entirety. Preferably, the cyclosporine is cyclosporine A. b. Particle Size of Cyclosporine [0072] As used herein, the particle size is determined on the basis of the weight average particle size as measured by conventional techniques well known to those skilled in the art, such as sedimentation field flow fractionation, laser diffraction, photon correlation spectroscopy (also known as dynamic light scattering), electroacoustic spectroscopy, or disk centrifugation. [0073] As used herein, "nanoparticulate cyclosporine or a derivative thereof refers to cyclosporine or a derivative thereof having a diameter of less than about 1 micron. An exemplary cyclosporine useful in the invention is cyclosporine A. "Macrocrystalline cyclosporine or a derivative thereof refers to cyclosporine or a derivative thereof having a diameter of greater than about 1 micron. In other embodiments of the invention, microparticulate cyclosporine or a derivative thereof have a diameter of less than about 10 microns, less than about 9 microns, less than about 8 microns, less than about 7 microns, less than about 6 microns, less than about 5 microns, less than about 4 microns, less than about 3 microns, less than about 2 microns, or about 1 micron or greater. In other embodiments of the invention, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% of the microparticles of cyclosporine or a derivative thereof can have a diameter less than the size listed above, e.g., less than about 10 microns, less than about 9 microns, etc. [0074] In yet other embodiments of the invention, nanoparticulate cyclosporine or a derivative thereof has a diameter of less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 290 nm, less than about 280 nm, less than about 270 nm, less than about 260 nm, less than about 250 nm, less than about 240 nm, less than about 230 nm, less than about 220 nm, less than about 210 nm, less than about 200 nm, less than about 190 nm, less than about 180 nm, less than about 170 nm, less than about 160 nm, less than about 150 nm, less than about 140 nm, less than about 130 nm, less than about 120 nm, less than about 110 nm, less than about 100 nm, less than about 90 nm, less than about 80 nm, less than about 70 rim, less than about 60 nm, less than about 50 nm, less than about 40 nm, less than about 30 nm, less than about 20 nm, or less than about 10 nm. In other embodiments of the invention, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% of the nanoparticles of cyclosporine or a derivative thereof can have a diameter less than the size listed above, e.g., less than about 900 nm, less than about 800 nm, etc. [0075] In other embodiments of the invention, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% of the particles of cyclosporine or a derivative thereof, or droplets comprising solubilized cyclosporine or a derivative thereof, have a size less than the mean particle size, less than about 3 microns, less than about 2900 nm, less than about 2800 nm, etc. 2. Oils [0076] For both the methods of Route I and Route II and the compositions of the invention, any suitable oil can be used. Exemplary oils that can be used include, for example, vegetable oils, nut oils, fish oils, lard oil, mineral oils, squalane, tricaprylin, and mixtures thereof. Specific examples of oils that may be used include, but are not limited to, almond oil (sweet), apricot seed oil, borage oil, canola oil, coconut oil, com oil, cotton seed oil, fish oil, jojoba bean oil, lard oil, linseed oil (boiled), Macadamia nut oil, medium chain triglycerides, mineral oil, olive oil, peanut oil, safflower oil, sesame oil, soybean oil, squalene, sunflower seed oil, tricaprylin (1,2,3-trioctanoyl glycerol), wheat germ oil, and mixtures thereof. 3. Stabilizers or Surfactants [0077] The stabilizer used in the methods and compositions of the invention associates with, or adsorbs, to the surface of the nanoparticulate cyclosporine or a derivative thereof, but does not covalently bind to cyclosporine or a derivative thereof. In addition, the individual stabilizer molecules are preferably free of cross-linkages. The stabilizer is preferably soluble in water. One or more stabilizers may be used in the compositions and methods of the invention. As used herein, the terms "stabilizer", "surface stabilizer", and "surfactant" are used interchangeably. [0078] Any suitable nonionic or ionic surfactant may be utilized in the compositions of the invention, including anionic, cationic, and zwitterionic surfactants. Exemplary stabilizers or surfactants that may be used in both Routes I and II include, but are not limited to, non-phospholipid surfactants, such as the Tween (polyoxyethylene derivatives of sorbitan fatty acid esters) family of surfactants {e.g., Tween 20, Tween 60, and Tween 80), nonphenol polyethylene glycol ethers, sorbitan esters (such as Span and Arlacel), glycerol esters (such as glycerin monostearate), polyethylene glycol esters (such as polyethylene glycol stearate), block polymers (such as Pluronics®), acrylic polymers (such as Pemulen®), ethoxylated fatty esters (such as Cremophor® RH-40), ethoxylated alcohols (such as Brij®), ethoxylated fatty acids, monoglycerides, silicon based surfactants, polysorbates, Tergitol NP-40 (Poly(oxy-l,2-ethanediyl), a -(4-nonylphenol)-.omega.- hydroxy, branched [molecular weight average 1980]), and Tergitol NP-70 (a mixed surfactant-AQ=70%). 4. Solvents [0079] Any suitable solvent can be used in the methods and compositions of the invention. Exemplary solvents include, but are not limited, to isopropyl myristate, triacetin, N-methyl pyrrolidinone, aliphatic or aromatic alcohols, polyethylene glycols, propylene glycol. An example of an alcohol useful in the present invention includes, but is not limited to ethanol. Other short chain alcohols and/or amides may be used. Other solvents include dimethyl sulfoxide, dimethyl acetamide, and ethoxydiglycol. Mixtures of solvents can also be used in the compositions and methods of the invention. 5. Water or Buffer [0080] If the methods and/or compositions of the invention use or comprise water or a buffer, the aqueous solution is preferably a physiologically compatible solution such as water or phosphate buffered saline. 6. Preservatives [0081] The formulations of the invention have anti-microbial properties. The anti- microbial properties can be associated with the formulation. As an example, the formulations described herein, in the absence of cyclosporine, can exhibit antimicrobial activity. [0082] Antimicrobial agents or preservatives are added to nonsterile dosage forms to protect them from microbiological growth or from microorganisms that are introduced inadvertently during or subsequent to the manufacturing process. In the case of sterile articles used in multi-dose containers, antimicrobial preservatives are added to inhibit the growth of microorganisms that may be introduced by repeatedly withdrawing individual doses. [0083] U.S. Food and Drug Administration guidelines require that antimicrobial effectiveness, whether inherent in the product (e.g., for an antibiotic agent) or whether produced because of the addition of an antimicrobial agent, must be demonstrated for all injections packaged in multiple-dose containers or for other products containing antimicrobial preservatives. Antimicrobial effectiveness must be demonstrated for multiple-dose topical and oral dosage forms, and for other dosage forms such as ophthalmic, otic, nasal, irrigation, and dialysis fluids. See USP 25, Section 51, "Antimicrobial Effectiveness Testing." [0084] The formulations of the invention meet the Antimicrobial Effectiveness Test as described in the United States Pharmacopeia (USP - General Chapter #51). The standard USP testing requires evaluation in five microorganisms: Aspergillus niger (ATCC 16404), Candida albicans (ATCC 10231), Escherichia coli (ATCC 8739), Pseudomonas aeruginosa (ATCC 9027) and Staphylococcus aureus (ATCC 6538). [0085] The formulations of the invention comprise preservatives, e.g., water soluble quaternary ammonium compounds, such as cetyltrimethylammonium bromide, cetylpyridinium chloride, benzethonium chloride, and benzalkonium chloride. Preferably, the preservative is benzalkonium chloride. [0086] BKC has been used as a preservative in ophthalmic formulations for many years. The formulation of the present invention contains BKC at a concentration that is recognized to be sufficient to qualify the USP Preservative Efficacy testing. There is sufficient literature evidence available to prove preservative efficacy for BKC used in ophthalmic formulations (Handbook of Pharmaceutical Excipients, 3ri edition (2000), edited by Arthur H. Kibbe, published by American Pharmaceutical Association and Pharmaceutical Press, pp. 33-35). Hence, the formulation of the invention is suitable for multi-dose packaging. [0087] In the present invention, BKC unexpectedly functions as a cationic agent, which contributes to the prolonged drug residence time. This property of the compositions of the invention enable fewer applications of the dosage form to the eye, which is generally preferred by patient populations. Moreover, the dosage applied is more effective, as the cationic property of the dosage form results in the drug "adhering" to the eye, rather than having much of the dosage form run out of the eyes following administration. [0088] It is believed that the beneficial aspects of the novel opthalmic cyclosporine compositions of the invention result from a synergistic combination of: (1) particle size of cyclosporine; (2) potential presence of cyclosporine in both a solubilized and particulate form in the dosage; (3) the cationic property of the dosage form, and (4) the stable, non- irritating final product suitable for multi-dose packaging. [0089] In general, tissues in the body are considered to be negatively charged. As a result, a positively-charged (cationic) product is considered to have better chance of improved interaction of the product with the tissue. This improved interaction between the cationic product and body tissues is not always observed, however. [0090] Prior art describes that for a biphasic system, oil-soluble cationic lipids, e.g.. stearylamine, has been used to impart a net positive charge onto oil droplet, whereby the droplet being electrostatically attracted to the anionic surface of the eye. See, for example, U.S. Patent No. 6,656,460. [0091] When water-soluble, quaternary ammonium compounds are used, they will not impart positive charge on the emulsion. Instead, they are likely to be adsorbed or loosely bound onto the surface and may result in a charge. Typically, when quaternary ammonium compounds are used, the acidic pH needs to be maintained for protonation of the groups to impart positive charge. In the present invention, it is unexpectedly found that BKC induces positive charge, under about neutral pH, in a multi-phasic composition having a solid particulate phase and an oil phase dispersed in aqueous milieu. Surprisingly, the cationic emulsion prepared using BKC shows significantly prolonged residence time in the eye using human cornea mounted on a Franz cell. [0092] Due to the presence of the preservative, the formulations of the present invention can be packaged into multi-dose containers. Moreover, as demonstrated in the following working examples, the presence of cyclosporine was only detected locally and administration of the formulations of the invention does not cause any irritation to the eye. Hence, the formulations of the invention are suitable for daily application. 7. Viscosity Modifier [0093] The ophthalmic formulation may further comprise a viscosity modifier, such as a cellulose derivative, a polysaccharide or a synthetic polymer. The viscosity of the formulations of the present invention can be increased without compromising other properties. [0094] The following examples are given to illustrate the present invention. It should be understood, however, that the invention is not to be limited to the specific conditions or details described in these examples. Throughout the specification, any and all references to a publicly available document, including U.S. patents, are specifically incorporated by reference. Example 1 [0095] The purpose of this example was to prepare formulations according to the invention comprising cyclosporine and a cationic preservative, benzalkonium chloride. [0096] The commercial Restasis® standard, which contains cyclosporine A (0.05% w/w) and inactive ingredients like glycerin, castor oil, polysorbate 80, carbomer 1342, purified water and sodium hydroxide, was used as a control for the purpose of comparing the properties of the formulation. [0097] Cyclosporine is commercially available under the trade names SANDIMMUNE81 and NEORAL®. It is a cyclic polypeptide immunosuppressant agent consisting of 11 amino acids. It is produced as a metabolite by the fungus species Beauveria nlyea. Chemically, cyclosporine is designated as [R-[R,R-(E)]]-cyclic(L-alanyl-D- alanyl-N- methyl-L-leucyl-N-methyl-L-leucyl-N-methyl-L-valyl-3-hydroxy-N, 4-dimethyl-L-2- amino-6-octenoyl-L-a-amino-butyryl-N-methylglycyl-N-methyl-L-leucyl-L-valyl-N- methyl-L-leucyl). The chemical structure of cyclosporine (also known as cyclosporin A) is: [0098] Cyclosporine was dissolved in ethanol at room temperature. Polysorbate 80 and crodamol (Table 1) were then added to the cyclosporine solution and mixed well with a paddle stirrer. Then benzalkonium chloride solution and water were added and mixing was continued for about 5 minutes. The coarse emulsion was then fed into a high pressure homogenizer (APV Invensys, model APV-1000) and homogenized at 10,000 psi for three passes. The resultant 50X concentrated composition, described below in Table 1, comprised cyclosporine dissolved in the solvent ethanol and nanoparticulate cyclosporine particles associated with the surface stabilizer polysorbate 80 present in the water portion of the emulsion. The concentrated emulsion was collected and diluted with phosphate buffered saline (PBS), pH 7.2, at the ratio of 1 part of concentrated emulsion with 49 parts of PBS to obtain the final product, cationic micellar nanoparticle formulation (cMNP) of cyclosporine. The cMNP and Restasis® have similar appearance as milky white emulsion. The viscosity of the formulation of the invention can be increased without compromising otiier properties. [0099] The resultant particle size and zeta potential of cMNP of cyclosporine were measured and compared with those of the commercial Restasis® standard, as shown in Figure 1A and 1B, as well as in Table 2. Example 2 [01001 The purpose of this example was to prepare formulations comprising cyclosporine and a permeation enhancer, vitamin E TPGS. The formulations of this example are used as a comparison to the formulations of the invention prepared according to Example 1. [0101] Cyclosporine and Restasis® standard are the same as those in Example 1. Vitamin E TPGS (d-alpha-tocopheryl polyethylene glycol-1000 succinate, obtained from Eastman Chemical Company) is a water-soluble form of natural-source vitamin E. [0102] Cyclosporine was dissolved in ethanol at room temperature. Polysorbate 80 and crodamol (Table 3) were then added to the cyclosporine solution and mixed well with a paddle stirrer. Then vitamin E TPGS solution and water were added and mixing was continued for about 5 minutes. The coarse emulsion was then fed into a high pressure homogenizer (APV Invensys, model APV-1000) and homogenized at 10,000 psi for three passes. The resultant 50X concentrated composition, described below in Table 3, comprised cyclosporine dissolved in the solvent ethanol and nanoparticulate cyclosporine particles associated with the surface stabilizer polysorbate 80 present in the water portion of the emulsion. The concentrated emulsion was collected and diluted with phosphate buffered saline (PBS), pH 7.2, at the ratio of 1 part of concentrated emulsion with 49 parts of PBS to obtain the final product, neutral micellar nanoparticle formulation (nMNP) of cyclosporine. The nMNP and Restasis® have similar appearance as milky while emulsion. The viscosity of the formulation of the invention can be increased without compromising other properties. [0103] The resultant particle size and zeta potential of nMNP of cyclosporine were measured and compared with those of the commercial Restasis® standard, as shown in Figure 1A and 1C, as well as in Table 4. Example 3 [0104] The purpose of this example was to test the formulations prepared in Example 1 and Example 2 for drug residence on cornea, for possibility of once-daily application, and for systemic absorption, using an in vitro Franz cell-human cornea assembly. [0105J The protocol is as follows: isolated human cornea was obtained from an eye bank. The corneas were collected from donors and refrigerated in a suitable preservation medium. The preserved cornea was allowed to reach room temperature for about 20 minutes, and then rinsed with PBS to remove the preservation medium. The corneas were mounted horizontally between the donor and receptor halves of the Franz diffusion cells. The surface area of the cornea exposed to the formulation in the donor chamber is 0.64 cm2, and the receptor cell volume was 5.0 ml. The receptor compartment was filled with 0.01M PBS, pH 7.4, and ethanol at a ratio of 9:1. A double water circulation jacket at 37°C surrounds the receptor cell in order to have the cornea temperature maintained at physiologic level. The cMNP and nMNP formulations prepared according to Example 1 and Example 2, as well as the Restasis® standard, were applied over the surface of the cornea gravimetrically using a syringe. Periodic samples of 1.0 ml each were taken from the receptor cell to measure the amount of drug transporting across the cornea at time points of 0, 2,4, 8, and 24 hours, respectively. At each time point, the donor sample was discarded and fresh donor sample was replenished. At the end of 24 hours, the cornea were collected, washed and homogenized in PBS:ethanol (9:1) and assayed for drug residence. [0106] No drug presence in the receptor compartment was detected at any time point. Hence, both formulations are suitable for once-daily application and the systemic availability of cyclosporine for both formulations is expected to be at comparable level to that of Restasis®. Moreover, Figure 2 illustrates that significantly high level of cyclosporine was retained in the cornea for the cMNP formulation prepared according to Example 1. Therefore, the cMNP formulation is expected to have a longer retention within the cul-de-sac via electrostatic attraction with ocular tissue. Example 4 [0107] The purpose of this example was to test the formulations prepared in Example 1 and Example 2 for biocompatibility. [0108] To determine the in vivo irritant and/or corrosive effects, the cMNP and nMNP formulations prepared according to Example 1 and Example 2 were instilled into the rabbit eye upon once-a-day application for 7 consecutive days. [0109] Six healthy New Zealand White rabbits were assigned to two groups of three animals per group. One group was dosed with the cMNP formulation, while the other was dosed with the nMNP formulation. All animals were dosed daily for seven consecutive days with 0.1 ml of the formulation into the conjunctival sac of the left eye of each rabbit. The right eye served as the untreated control. The treated eyes were examined for irritation using the Draize technique, pretest, prior to each subsequent dose and at the time points of 24, 48 and 72 hours, respectively, following the final dose. Observations for mortality, toxicity and pharmacologic effects were made prior to each ocular observation period. Body weights were recorded pretest. The animals were humanely sacrificed using CO2, following the final observation. [0110] The results show that there was no evidence of ocular irritation noted in any eye dosed with either the cMNP or the nMNP formulation and that there were no abnormal physical signs noted during the study. Hence, both the cMNP and the nMNP formulations are well-tolerated. Example 5 [0111] The purpose of this example was to test the formulations prepared in Example 1 and Example 2 for stability. [0112] Both formulations were subjected to an in-house or informal non-ICH stability study. The storage temperatures and time points taken for the stability test are as follows: 1. 25°C: 0, 1, 2, 4, 12, and 40 weeks; 2. 40°C: 1, 2, 4 and 12 weeks; 3. 60°C: 1week [0113] The stability data, in terms of the potency of cyclosporine and the mean particle size, is depicted in Figure 3 and Figure 4, for the cMNP formulation and the nMNP formulation, respectively. For the cMNP formulation, the results show that there was no change in physical appearance of the emulsion for any storage condition at any time point tested. pH was stable at 7.3±0.3 across all storage conditions and time pointes tested. The cyclosporine content dropped by approximately 10% for 60°C storage condition at 1-week time point. However, the 25°C and 40°C storage conditions did not cause any significant decrease in potency during the test period. The mean particle size did not change during the test period for any storage condition tested. Hence, the cMNP formulation of cyclosporine will likely be stable for two years at room temperature. Figure 4 shows that the nMNP formulation is stable for up to 12 weeks at 25°C or at 40°C. Example 6 [0114] The purpose of this example was to manufacture the cyclosporine formulation. [0115] 50X concentrated cyclosporine formulation containing 2.5% w/w cyclosporine is manufactured aseptically according to the description in Example 1 or Example 2. The particles of cyclosporine in the concentrated product have a mean particle size of less than 200 nm. Subsequently, the concentrated composition is diluted with sterile PBS/saline to achieve 0.05% w/w cyclosporine in the final product, which has similar appearance to Restasis®. The final product is packaged and sealed in 0.4 ml per dose. [0116] It will be apparent to those skilled in the art that various modifications and variations can be made in the methods and compositions of the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents. WHAT IS CLAIMED IS: 1. An ophthalmic formulation comprising: (a) cyclosporine or a derivative thereof, (b) at least one solvent, (c) at least one oil, (d) at least one surfactant, (e) at least one preservative, and (f) water or buffer. 2. The opthalmic formulation of claim 1, wherein the cyclosporine is cyclosporine A. 3. The ophthalmic formulation of claim 1, wherein cyclosporine or a derivative thereof is in a solid particulate state and in a soluble state. 4. The ophthalmic formulation of claim 3, wherein the formulation is a mixture of particles of cyclosporine or a derivative thereof suspended in emulsion droplets and sterically stabilized particulate cyclosporine or a derivative thereof in the water or buffer. 5. The ophthalmic formulation of claim 1, wherein the formulation comprises globules of oil comprising dissolved cyclosporine or a derivative thereof, wherein the globules have a diameter of less than about 10 microns. 6. The ophthalmic formulation of claim 5, wherein the globules have a diameter selected from the group consisting of less than about 9 microns, less than about 8 microns, less than about 7 microns, less than about 6 microns, less than about 5 microns, less than about 4 microns, less than about 3 microns, less than about 2 microns, less than about 1000 nm, less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 290 nm, less than about 280 nm, less than about 270 nm, less than about 260 nm, less than about 250 nm, less than about 240 nm, less than about 230 nm, less than about 220 nm, less than about 210 nm, less than about 200 nm, less than about 190 nm, less than about 180 nm, less than about 170 nm, less than about 160 nm, less than about 150 nm, less than about 140 nm, less than about 130 nm, less than about 120 nm, less than about 110 nm, less than about 100 nm, less than about 90 nm, less than about 80 nm, less than about 70 nm, less than about 60 nm, less than about 50 nm, less than about 40 nm, less than about 30 nm, less than about 20 nm, or less than about 10 nm. 7. The formulation of claim 1, wherein the oil is selected from the group consisting of sweet almond oil, apricot seed oil, borage oil, canola oil, coconut oil, corn oil, cotton seed oil, fish oil, jojoba bean oil, lard oil, boiled linseed oil, Macadamia nut oil, medium chain triglycerides (Crodamol GTCC), mineral oil, olive oil, peanut oil, safflower oil, sesame oil, soybean oil, squalene, sunflower seed oil, tricaprylin (1,2,3-trioctanoyl glycerol), and wheat germ oil. 8. The formulation of claim 1, wherein the solvent is selected from the group consisting of isopropyl myristate, triacetin, N-methyl pyrrolidinone, aliphatic and aromatic alcohols, ethanol, dimethyl sulfoxide, dimethyl acetamide, ethoxydiglycol, polyethylene glycols, and propylene glycol. 9. The formulation of claim 1, wherein the surfactant is selected from the group consisting of sorbitan esters, glycerol esters, polyethylene glycol esters, block polymers, acrylic polymers, ethoxylated fatty esters, ethoxylated alcohols, ethoxylated fatty acids, monoglycerides, silicon based surfactants, and polysorbates. 10. The formulation of claim 9, wherein the surfactant is Polysorbate 80. 11. The formulation of claim 9, wherein the sorbitan ester surfactant is Span or Arlacel, wherein the glycerol ester is glycerin monostearate, wherein the polyethylene glycol ester is polyethylene glycol stearate, wherein the block polymer is a Pluronic, wherein the acrylic polymer is Pemulen, wherein the ethoxylated fatty ester is Cremophor RH-40, wherein the ethoxylated alcohol is Brij, and wherein the ethoxylated fatty acid is Tween 20. 12. The formulation of claim 1, wherein formulation is cationic. 13. The formulation of claim 1, wherein the preservative is cationic. 14. The formulation of claim 1, wherein the preservative is a quaternary ammonium compound. 15. The formulation of claim 1, wherein the preservative is selected from the group consisting of cetyltrimethylammonium bromide, cetylpyridinium chloride, benzethonium chloride, and benzalkonium chloride. 16. The formulation of claim 1, wherein the preservative is benzalkonium chloride. 17. The formulation of claim 1, further comprising a viscosity modifier. 18. The formulation of claim 17, wherein the viscosity modifier is selected from the group consisting of a cellulose derivative, a polysaccharide and a synthetic polymer. 19. An ophthalmic formulation comprising: (a) cyclosporine A, (b) ethanol, (c) medium chain triglycerides, (d) Polysorbate 80, (e) benzalkonium chloride, and (f) phosphate buffer. 20. A method of treating a subject in need comprising administering the formulation of claim 1 or claim 19 to the eye of a subject. 21. The method according to claim 20, wherein the subject suffers from a condition selected from the group consisting of an aqueous deficient dry-eye state, phacoanaphylaxis endophthalmitis, and uveitis. 22. A method for preparing particles of cyclosporine or a derivative thereof, comprising: (a) forming an emulsion base by suspending cyclosporine or a derivative thereof in a mixture of oil, solvent, surfactant, preservative, and water or buffer to form an emulsion base, and (b) homogenizing the emulsion base to form particles of cyclosporine or a derivative thereof, droplets comprising solubilized cyclosporine or a derivative thereof, or a combination thereof. 23. The method of claim 22, wherein the homogenizing step is performed via a high-pressure system at 1,000 to 40,000 psi. 24. The method of claim 22, wherein the cyclosporine or a derivative thereof, droplets have an average or mean particle size selected from the group consisting of less than about 10 microns, less than about 9 microns, less than about 8 microns, less than about 7 microns, less than about 6 microns, less than about 5 microns, less than about 4 microns, less than about 3 microns, less than about 2 microns, and about 1 micron or greater. 25. The method of claim 22, wherein the cyclosporine or derivative thereof, droplets have a mean particle size selected from the group consisting of less than about 1 micron, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 290 nm, less than about 280 nm, less than about 270 nm, less than about 260 nm, less than about 250 nm, less than about 240 nm, less than about 230 nm, less than about 220 nm, less than about 210 nm, less than about 200 nm, less than about 190 nm, less than about 180 nm, less than about 170 nm, less than about 160 nm, less than about 150 nm, less than about 140 nm, less than about 130 nm, less than about 120 nm, less than about 110 nm, less than about 100 nm, less than about 90 nm, less than about 80 nm, less than about 70 nm, less than about 60 nm, less than about 50 nm, less than about 40 nm, less than about 30 nm, less than about 20 nm, or less than about 10 nm. 26. The method of claim 22, wherein the emulsion base of step (a) further comprises a viscosity modifier. 27. The method of claim 26, wherein the viscosity modifier is selected from the group consisting of a cellulose derivative, a polysaccharide and a synthetic polymer. 28. A method for preparing particles of cyclosporine or a derivative thereof, comprising: (a) dissolving cyclosporine or a derivative thereof in a mixture of (i) at least one oil, (ii) at least one solvent, and (iii) at least one surfactant to form an emulsion pre- mix, (b) adding at least one preservative, and water or buffer to the emulsion pre- mix, and (c) homogenizing or vigorously stirring the mixture of step (b), whereby cyclosporine A or a derivative thereof is precipitated into particles. 29. The method of claim 28, wherein the homogenizing step is performed via a high-pressure system at 1,000 to 40,000 psi. 30. The method of claim 28, wherein the cyclosporine or a derivative thereof, droplets have an average or mean particle size selected from the group consisting of less than about 10 microns, less than about 9 microns, less than about 8 microns, less than about 7 microns, less than about 6 microns, less than about 5 microns, less than about 4 microns, less than about 3 microns, less than about 2 microns, and about 1 micron or greater. 31. The method of claim 28, wherein the cyclosporine A or derivative thereof, droplets have a mean particle size selected from the group consisting of less than about 1 micron, less than about 800 run, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 290 nm, less than about 280 nm, less than about 270 nm, less than about 260 nm, less than about 250 nm, less than about 240 nm, less than about 230 nm, less than about 220 nm, less than about 210 nm, less than about 200 nm, less than about 190 nm, less than about 180 nm, less than about 170 nm, less than about 160 nm, less than about 150 nm, less than about 140 nm, less than about 130 nm, less than about 120 nm, less than about 110 nm, less than about 100 nm, less than about 90 nm, less than about 80 nm, less than about 70 nm, less than about 60 nm, less than about 50 nm, less than about 40 nm, less than about 30 nm, less than about 20 nm, or less than about 10 nm. 32. The method of claim 28, wherein the mixture of step (a) or the mixture of step (b) further comprises a viscosity modifier. 33. The method of claim 32, wherein the viscosity modifier is selected from the group consisting of a cellulose derivative, a polysaccharide and a synthetic polymer. 34. A method for preparing particles of cyclosporine or a derivative thereof, comprising: (a) dissolving cyclosporine or a derivative thereof in ethanol to form a solution, (b) adding Polysorbate 80 and medium chain triglyceride to the solution of step (a), (c) adding benzalkonium chloride dissolved in water to the solution of (b), and (d) subjecting the solution of (c) to high-pressure homogenization to produce particles of cyclosporine or a derivative thereof. The present invention provides ophthalmic formulations containing cyclosporine, methods for preparing the formulation, and methods for using the formulation.

Full Text

OPHTHALMIC PREPARATIONS
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to US Patent Application No. 12/007,902 filed
January 16, 2008, the disclosure of which is incorporated herein by reference in its
entirety.
FIELD OF THE INVENTION
[0002] The invention is directed to ophthalmic formulations, methods for preparing the
ophthalmic formulation, and methods of using the ophthalmic formulation.
BACKGROUND
[0003] Most ocular medications may be administered topically to treat surface as well as
intraocular disorders. This route is often preferred for the management of various
pathological diseases that affect the anterior chamber of the eye, for two main reasons: (1)
it is more conveniently administered and (2) provides a higher ratio of ocular to systemic
drug levels. To be administered topically and to achieve the necessary patient compliance,
the medication must present a good local tolerance.
[0004] Cyclosporine is a known immunosuppressant, which acts by reducing
inflammatory cells such as activated lymphocytes in the conjunctiva (Kunert et al., Arch
Ophthalmol., 118:1489-96, 2000) or by increasing the number of mucin secreting goblet
cells (Kunert et al., Arch Ophthalmol., 120:330-7, 2002). Over the years, Cyclosporine A
(CsA) has been evaluated for numerous potential applications in ophthalmology.
[0005] New developments in the topical delivery of CsA can be divided in two general
areas of research: new delivery systems, such as solutions, ointments, colloidal carriers
and drug impregnated contact lenses, and chemical modifications of drugs or prodrugs.
[0006] At the present time, only one ophthalmic formulation of CsA is commercially
available, which is currently marketed as Restasis®. The extensive literature on the
delivery of CsA to the eye (Lallemand et al., European Journal of Pharmaceutics and
Biopharmaceutics 56: 307-318, 2003; Nussenblatt et al., Am. J. Ophthalmol. 112: 138-
146, 1991; Masuda et al, Lancet 1: 1093-1096,1989; Georganas etai, Clin. Rheumatol.
15: 189-192, 1996; Prummel etai, N. Engl. J. Med. 321: 1353-1359, 1989; Reinhard et
al, Ophthalmologe 94: 496-500, 1997) reflects the great medical interest and the
pharmaco-economical aspects of this challenge. Despite a poor intraocular penetration,
topical CsA has been successfully used in a variety of immune-mediated ocular surface
phenomena such as vernal conjunctivitis, dry eye syndrome and the prevention of corneal
allograft rejection. For example, Cross et al. reported that topical cyclosporine eye drop
therapy not only improved the signs and symptoms of dry eye disease, but also resulted in
high patient satisfaction, fewer patients with chronic dry eye visiting the ophthalmologist,
and less ancillary drug use (Manag. Care Interface 2002;15:44-49).
[0007] One obstacle to preparing an ophthalmic CsA formulation is that CsA cannot be
prepared in formulations based on the commonly used aqueous ophthalmic vehicles
because of both its hydrophobicity (log P = 3.0) and its extremely low aqueous solubility
at 6.6 mg/ml. Therefore, in most studies, CsA was dissolved and administered in
vegetable oils. However, these media are poorly tolerated, resulting in relatively low
ocular availability. Also, formulations prepared in these media have short shelf lives.
[0008] Oil-in-water emulsions are particularly useful in the delivery of lipophilic drugs.
In vivo data from early studies confirmed that emulsions could be effective topical
ophthalmic drug delivery systems, with a potential for sustained drug release.
[0009] The product currently on the market, Restasis®, is packaged in single unit doses
to avoid microbial contamination because it does not contain any preservatives. It would
be highly desired to have a preparation to be dispensed in a multi-dose container.
[0010] Therefore, there is a need for an ideal topical ocular formulation of CsA, which
fulfills several requirements: (1) the formulation must be well tolerated, or non-irritating
to the eye, (2) the formulation must be easy to administer, (3) the formulation ideally has
an increased CsA residence time in the eye, (4) systemic absorption of the formulation
should be avoided because the toxic CsA concentration in blood is above 300 ng/ml, (5)
the formulation should have a long shelf life, and (6) the formulation should be easily
manufactured.
[0011] The present invention satisfies the needs in the field by providing stable, non-
irritating emulsion formulations of CsA suitable for ophthalmic application, and methods
for preparing and using the formulations.
SUMMARY
[0012] One aspect of the invention relates to an ophthalmic formulation comprising: (a)
cyclosporine or a derivative thereof, (b) at lest one solvent, (c) at least one oil, (d) at least
one surfactant, (e) at least one preservative, and (f) water or buffer. In one embodiment,
the cyclosporine (Cs) or a derivative thereof can be present in (a) a solid nanoparticulate
state; (b) a solid microparticulate state; (c) solubilized; or (d) any combination thereof. An
exemplary cyclosporine useful in the invention is cyclosporine A (CsA).
[0013] In one embodiment of the invention, the ophthalmic formulation further
comprises a viscosity modifier, such as a cellulose derivative, a polysaccharide or a
synthetic polymer.
[0014] In another embodiment of the invention, the formulation is a mixture of particles
of cyclosporine or a derivative thereof suspended in emulsion droplets and stoically
stabilized particulate cyclosporine or a derivative thereof in water or buffer.
[0015] Another aspect of the invention is directed to an opthalmic cyclosporine
composition which is cationic. The cationic nature of the formulation results in an
increased residency in the eye, producing a more effective dosage form. The cationic
nature of the dosage form can result, for example, from the inclusion of a cationic
preservative.
[0016] In yet another embodiment, the formulation comprises globules of oil comprising
dissolved cyclosporine or a derivative thereof. The globules can have a diameter, for
example, of less than about 10 microns, less than about 9 micros, less than about 8
microns, less than about 7 microns, less than about 6 microns, less than about 5 microns,
less than about 4 microns, less than about 3 microns, less than about 2 microns, less than
about 1000 nm, less than about 900 nm, less than about 800 nm, less than about 700 nm,
less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about
300 nm, less than about 290 nm, less than about 280 nm, less than about 270 nm, less than
about 260 nm, less than about 250 nm, less than about 240 nm, less than about 230 nm,
less than about 220 nm, less than about 210 nm, less than about 200 nm, less than about
190 nm, less than about 180 nm, less than about 170 nm, less than about 160 nm, less than
about 150 nm, less than about 140 nm, less than about 130 nm, less than about 120 nm,
less than about 110 nm, less than about 100 nm, less than about 90 nm, less than about 80
nm, less than about 70 nm, less than about 60 nm, less than about 50 nm, less than about
40 nm, less than about 30 nm, less than about 20 nm, or less than about 10 nm.
[0017] In some embodiments, the oil is selected from the group consisting of, but not
limited to, sweet almond oil, apricot seed oil, borage oil, canola oil, coconut oil, corn oil,
cotton seed oil, fish oil, jojoba bean oil, lard oil, boiled linseed oil, Macadamia nut oil,
medium chain triglycerides (Crodamol GTCC), mineral oil, olive oil, peanut oil, safflower
oil, sesame oil, soybean oil, squalene, sunflower seed oil, tricaprylin (1,2,3-trioctanoyl
glycerol), and wheat germ oil.
[0018] In other embodiments, the solvent is selected from the group consisting of, but
not limited to, isopropyl myristate, triacetin, N-methyl pyrrolidinone, aliphatic and
aromatic alcohols, ethanol, dimethyl sulfoxide, dimethyl acetamide, ethoxydiglycol,
polyethylene glycols, and propylene glycol. Other examples of useful solvents are long-
chain alcohols. Ethanol is an example of a preferred alcohol that may be used in the
present invention.
[0019] In further embodiments, the surfactant is selected from the group consisting of,
but not limited to, sorbitan esters (such as Span or Arlacel), glycerol esters (such as
glycerin monostearate), polyethylene glycol esters (such as polyethylene glycol stearate),
block polymers (such as Pluronics®), acrylic polymers (such as Pemulen ), ethoxylated
fatty esters (such as Cremophor® RH-40), ethoxylated alcohols (such as Brij®),
ethoxylated fatty acids (such as Tween or Tween 20), monoglycerides, silicon based
surfactants and polysorbates. In a preferred embodiment, the surfactant is Polysorbate 80.
[0020] In one embodiment of the invention, the ophthalmic formulations of the
invention have anti-microbial properties. In some embodiments, the formulation
comprises a preservative suitable for opthalmic administration. Examples of such a
preservative include, but are not limited to, quaternary ammonium compounds, such as
cetyltrimethylammonium bromide, cetylpyridinium chloride, benzethonium chloride, and
benzalkonium chloride. Preferably, the preservative is benzalkonium chloride.
[0021] In a preferred embodiment, the ophthalmic formulation comprises: (a)
cyclosporine A, (b) ethanol, (c) medium chain triglycerides, (d) Polysorbate 80, (e)
benzalkonium chloride, and (f) phosphate buffer.
[0022] Another aspect of the invention is directed to a method for preparing particles of
Cs or a derivative thereof. The method comprises: (a) forming an emulsion base by
suspending cyclosporine or a derivative thereof in a mixture of oil, solvent, surfactant,
preservative, and water or buffer, and (b) homogenizing or vigorously stirring the
emulsion base, wherein the resultant composition is a mixture of particles of cyclosporine
or a derivative thereof suspended in emulsion droplets and sterically stabilized
microcrystalline or nanoparticulate cyclosporine or a derivative thereof in the media.
Optionally, the emulsion base of step (a) further comprises a viscosity modifier, such as a
cellulose derivative, a polysaccharide or a synthetic polymer. In one embodiment, the
particles of cyclosporine or a derivative thereof have a diameter of less than about 10
microns, less than about 9 microns, less than about 8 microns, less than about 7 microns,
less than about 6 microns, less than about 5 microns, less than about 4 microns, less than
about 3 microns, less than about 2 microns, less than about 1 micron, or even smaller in
size. In another embodiment, the homogenizing step is performed via a high-pressure
system at 1,000 to 40,000 psi.
[0023] Another aspect of the invention is directed to a method for preparing particles of
Cs or a derivative thereof, comprising: (a) dissolving cyclosporine or a derivative thereof
in a mixture of oil, solvent, and surfactant to form an emulsion pre-mix, (b) adding
preservative, and water or buffer to the emulsion pre-mix, and (c) homogenizing or
vigorously stirring the mixture, whereby cyclosporine or a derivative thereof is
precipitated into particles. Optionally, the mixture of step (a) or that of step (b) further
comprises a viscosity modifier, such as a cellulose derivative, a polysaccharide or a
synthetic polymer. In one embodiment, the diameter of the particles of cyclosporine or a
derivative thereof is less than about 10 microns, less than about 9 microns, less than about
8 microns, less than about 7 microns, less than about 6 microns, less than about 5 microns,
less than about 4 microns, less than about 3 microns, less than about 2 microns, or less
than about 1 micron. In another embodiment, the homogenizing step is performed via a
high-pressure system at 1,000 to 40,000 psi.
[0024] In one embodiment, the particles of cyclosporine or a derivative thereof, droplets
comprising cyclosporine or a derivative thereof, or a combination thereof, have a mean
particle size of less than about 10 microns, less than about 9 microns, less than about 8
microns, less than about 7 microns, less than about 6 microns, less than about 5 microns,
less than about 4 microns, less than about 3 microns, less than about 2900 nm, less than
about 2800 nm, less than about 2700 nm, less than about 2600 nm, less than about 2500
nm, less than about 2400 nm, less than about 2300 nm, less than about 2200 nm, less than
about 2100 nm, less than about 2000 nm, less than about 1900 nm, less than about 1800
nm, less than about 1700 nm, less than about 1600 nm, less than about 1500 nm, less than
about 1400 nm, less than about 1300 nm, less than about 1200 nm, less than about 1100
nm, less than about 1000 nm, less than about 900 nm, less than about 800 nm, less than
about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400 nm,
less than about 300 nm, less than about 200 nm, or less than about 100 nm, less than about
90 nm, less than about 80 nm, less than about 70 nm, less than about 60 nm, less than
about 50 nm, less than about 40 nm, less than about 30 nm, less than about 20 nm, or less
than about 10 nm. Preferably, the particles of cyclosporine or a derivative thereof,
droplets comprising cyclosporine or a derivative thereof, or a combination thereof have a
mean particle size of less than about 3 microns in diameter.
[0025] In a related aspect, the invention provides a method of treating a subject suffers
from a condition, such as an aqueous deficient dry-eye state, phacoanaphylaxis
endophthalmitis, uveitis, keratoconjunctivitis, allergic eye disorders, or immune-
modulated eye diseases. The method comprises administering a therapeutically effective
amount of the CsA formulations of the invention to the eye of a subject in need.
[0026] Both the foregoing general description and the following detailed description are
exemplary and explanatory and are intended to provide further explanation of the
invention as claimed. Other objects, advantages, and novel features will be readily
apparent to those skilled in the art from the following detailed description of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figures 1A, 1B, and 1C show mean particle size and mean zeta potential for
Restasis®, for the cationic micellar nanoparticle (cMNP) formulation of cyclosporine A
prepared according to Example 1, and for the neutral micellar nanoparticle (nMNP)
formulation prepared according to Example 2, respectively.
[0028] Figure 2 shows the mean corneal concentration of cyclosporine in a Franz cell-
human cornea model. High level of cyclosporine was detected in the cornea for the cMNP
formulation, but not for the nMNP formulation or Restasis®.
[0029] Figure 3 depicts the stability data for the cMNP formulation stored at 25°C, 40°C
and 60°C, respectively, in terms of cyclosporine potency and mean particle size.
[0030] Figure 4 depicts the stability data for the nMNP formulation stored at 25°C, 40°C
and 60°C, respectively, in terms of cyclosporine potency and mean particle size.
DETAILED DESCRIPTION
A. Overview of the Invention
[0031] The invention relates to ophthalmic formulations comprising cyclosporine or a
derivative thereof and methods of making and using the same. One aspect of the present
invention is directed to an ophthalmic formulation comprising: (a) cyclosporine or a
derivative thereof, (b) at lest one solvent, (c) at least one oil, (d) at least one surfactant, (e)
at least one preservative, and (f) water or buffer. In one embodiment, the cyclosporine
(Cs) or a derivative thereof can be present in (a) a solid nanoparticulate state; (b) a solid
microparticulate state; (c) solubilized; or (d) any combination thereof. In another
embodiment, the ophthalmic formulation further comprises a viscosity modifier, such as a
cellulose derivative, a polysaccharide or a synthetic polymer.
[0032] The cyclosporine can be, for example, cyclosporine A, and the cyclosporine or a
derivative thereof can be in an amorphous form, semi- amorphous form, crystalline form,
semi-crystalline form, or any combination thereof.
[0033] In one embodiment of the invention, the opthalmic cyclosporine composition is
cationic. The cationic nature of the formulation can result in an increased residency in the
eye, producing a more effective dosage form. Moreover, the increased residency in the
eye can result in a need for fewer applications, a decreased drug dosage for effectiveness,
and increased patient compliance (as fewer applications are desirable to patients). The
cationic nature of the dosage form can result, for example, from the inclusion of a cationic
preservative. An exemplary cationic preservative is benzalkonium chloride.
[0034] In another embodiment of the invention, the formulation is a mixture of particles
of cyclosporine or a derivative thereof suspended in emulsion droplets and sterically
stabilized particulate cyclosporine or a derivative thereof in water or buffer.
[0035] Another aspect of the invention encompasses a method of making a tri-phasic
composition comprising a lipophilic phase, water or a buffer, and particulate cyclosporine
or a derivative thereof. The invention also encompasses compositions comprising an oil
phase that has at least one oil, at least one solvent, and a surfactant for cyclosporine or a
derivative thereof. Two specific methods of making the composition of the invention are
described. In the first method ("Route I"), cyclosporine or a derivative thereof is milled in
an emulsion base. This method requires that cyclosporine or a derivative thereof is poorly
soluble or insoluble in all phases of the oil phase/lipophilic phase and the water or buffer.
In die second method ("Route II"), simultaneous milling and precipitation of cyclosporine
or a derivative thereof in an emulsion base is observed. The second method requires mat
cyclosporine or a derivative thereof is soluble or partially soluble in one or more phases of
the emulsion base.
[0036] One benefit of the invention is to provide a method applicable to cyclosporine or
a derivative thereof, which is poorly water-soluble, since the conventional method, such as
wet milling, is not effective. Another benefit of the invention is that it does not require
grinding media or specialized grinding process or equipment. The use of such grinding
media can add cost and complexity to a particle size reduction process for cyclosporine.
[0037] For Route I, cyclosporine or a derivative thereof is first suspended in a mixture of
a non-miscible liquid, such as an oil, solvent, preservative and water or buffer, to form an
emulsion base, followed by homogenization or vigorous stirring of the emulsion base.
Optionally, the emulsion base further comprises a viscosity modifier, such as a cellulose
derivative, a polysaccharide or a synthetic polymer. Nanoparticles can be produced with
reciprocating syringe instrumentation, continuous flow instrumentation, or high speed
mixing equipment. High velocity homogenization or vigorous stirring, producing forces
of high shear and cavitation, are preferred. High shear processes are preferred as low
shear processes can result in larger particle sizes of cyclosporine or a derivative thereof.
The resultant composition is a composite mixture of cyclosporine or a derivative thereof
suspended in the emulsion droplet (nanoemulsion fraction) and sterically stabilized micro-
/nano-crystalline cyclosporine or a derivative thereof in the media. This tri-phasic system
comprises particulate cyclosporine or a derivative thereof, oil, preservative, and water or
buffer. Preferably, the resultant micro/nano-particulate cyclosporine or a derivative
thereof has a mean particle size of less than about 3 microns. Smaller particulate
cyclosporine or a derivative thereof can also be obtained, as described below.
[0038] Cyclosporine or a derivative thereof can be precipitated out from the oil droplets
by adding more of the non-miscible liquid. The precipitated cyclosporine or a derivative
thereof typically has a mean particle size of less than about 3 microns. If desired, the
particles of cyclosporine or a derivative thereof can be prevented from aggregating or
clumping together by incorporating a surfactant or emulsifier, e.g., a "surface stabilizer."
[0039] Route II is utilized for cyclosporine or a derivative thereof because it is soluble in
at least one part of the emulsion base, such as the solvent, in particular, ethanol. For Route
II, cyclosporine or a derivative thereof is dissolved in a mixture of oil, solvent, and
surfactant to form an emulsion pre-mix. Cyclosporine or a derivative thereof remains in
soluble form if preservative and water or buffer are not added to the mixture. Upon the
addition of preservative, a viscosity modifier, and water or buffer and the application of
shear forces, cyclosporine or a derivative thereof is precipitated into micro/nano-particles
having a mean particle size of less than about 3 microns. Nanoparticles can be produced
with reciprocating syringe instrumentation, continuous flow instrumentation, or high speed
mixing equipment. High energy input, through high velocity homogenization or vigorous
stirring, is a preferred process. The high energy processes reduce the size of the emulsion
droplets, thereby exposing a large surface area to the surrounding aqueous environment.
High shear processes are preferred, as low shear processes can result in larger particle
sizes. This is followed by precipitation of nanoparticulate cyclosporine or a derivative
thereof previously embedded in the emulsion base. The end product comprises
cyclosporine or a derivative thereof in solution and particulate suspension, both distributed
between the solvent, oil, preservative, and water or buffer. Nanoparticulate cyclosporine
or a derivative thereof has at least one surface stabilizer associated with the surface
thereof.
[0040] If desired, the water miscible oil droplets and nanoparticles of cyclosporine or a
derivative thereof prepared using Route I or Route II may be filtered through either a 0.2
or 0.45 micron filter. Larger oil droplets and/or particles of cyclosporine or a derivative
thereof can be created by simply increasing the water content, decreasing the oil-stabilizer-
solvent content, or reducing the shear in forming the oil droplets.
[0041] For the 50X concentrated emulsion base used in Route I or Route II, the content
of cyclosporine is about 0.1%-10%, the content of solvent is about 0.1%-20%, the content
of oil is about 5%-50%, the content of surfactant is about 0.1%-20%, the content of
preservative is about 0.1 %-5%, and the content of the aqueous medium is about 20%-80%,
all in w/w percentage. Optionally, the viscosity modifier is present in the emulsion base in
the amount of about 0.1% to about 10% (w/w). The content of each ingredient in the final
product is the amount above divided by 50, with the aqueous medium being the major
component, at about 98% or more.
B. Definitions
[0042] The present invention is described herein using several definitions, as set forth
below and throughout the application.
[0043] As used herein, "about" will be understood by persons of ordinary skill in the art
and will vary to some extent depending upon the context in which it is used. If there are
uses of the term which are not clear to persons of ordinary skill in the art given the context
in which it is used, "about" will mean up to plus or minus 10% of the particular term.
[0044] The phrase "poorly water-soluble" or "water insoluble" as used herein refers to a
solubility in water of less than about 30 mg/mL, less than about 20 mg/mL, less than about
10 mg/mL, less than about 1 mg/mL, less than about 0.1 mg/mL, less than about 0.01
mg/mL or less than about 0.001 mg/mL at ambient temperature and pressure and at about
pH7.
[0045] The phrase "soluble" as used herein refers to a solubility in water or another
medium selected from the group consisting of greater than about 10 mg/mL, greater than
about 20 mg/mL, and greater than about 30 mg/mL.
[0046] As used herein, the term "subject" is used to mean an animal, preferably a
mammal, including a human or non-human. The terms "patient" and "subject" may be
used interchangeably.
[0047] As used herein, the phrase "therapeutically effective amount" shall mean that
drug dosage that provides the specific pharmacological response for which the drug is
administered in a significant number of subjects in need of such treatment. It is
emphasized that a therapeutically effective amount of a drug that is administered to a
particular subject in a particular instance will not always be effective in treating the
conditions/diseases described herein, even though such dosage is deemed to be a
therapeutically effective amount by those of skill in the art.
[0048] As used herein, the phrase "cyclosporines and derivatives thereof include a
group of nonpolar cyclic oligopeptides with known immunosuppressant activity, as
disclosed in U.S. Patent No. 5,474,979, which is incorporated by reference in its entirety.
In addition to the most common form of cyclosporine A, there are several other minor
metabolites, cyclosporine B through I, identified. The present invention intends to include
any individual member of the cyclosporine family, as well as a mixture of two or more
members of the family, because the commercial cyclosporines may contain a mixture of
several forms of cyclosporines, which all share a cyclic peptide structure consisting of
eleven amino acids with a total molecular weight of about 1200. The cyclosporines and
derivatives thereof also include the natural or synthetic product, as well as any substituents
or different configurations of the amino acids, as long as they maintain the
immunosuppressive activity and capability of enhancing or restoring the lacrimal gland
tearing. The present invention further encompasses the cyclosporine A derivatives, e.g.,
methylthio-substitued cyclosporine A and other alkylthio-substitued cyslosporine A
derivatives, as disclosed in U.S. Patent No. 6,254,860 and 6,350,442, each of which is
incorporated by reference in its entirety.
C. Compositions of the Invention
1. Exemplary Compositions
[0049] The methods of the invention can produce several different types of
compositions. A first composition comprises: (1) microparticulate and/or nanoparticulate
cyclosporine or a derivative thereof having a mean particle size of less than about 10
microns and, optionally for nanoparticulate cyclosporine or a derivative thereof, having
associated with the surface thereof at least one surface stabilizer; (2) at least one
preservative; (3) water or a buffer; and (4) an emulsion pre-mix or oil phase or lipophilic
phase comprising at least one oil and optionally at least one solvent, and/or a viscosity
modifier. The composition may additionally comprise microcrystalline cyclosporine or a
derivative thereof. The particulate cyclosporine or a derivative thereof can be present in
the water or buffer, oil, solvent, preservative, or a combination thereof. Such a
composition is made utilizing Route I.
[0050] A second composition comprises: (1) microparticulate and/or nanoparticulate
cyclosporine or a derivative thereof having a mean particle size of less than about 10
microns and, optionally for nanoparticulate cyclosporine or a derivative thereof having
associated with the surface thereof at least one surface stabilizer; (2) a preservative; (3)
water or a buffer; and (4) an emulsion pre-mix or oil phase or lipophilic phase comprising
at least one oil, optionally at least one solvent, solubilized cyclosporine or a derivative
thereof, and/or a viscosity modifier. The composition may additionally comprise
microcrystalline cyclosporine or a derivative thereof. The solubilized cyclosporine or a
derivative thereof may be present in oil, solvent, or a combination thereof. In addition,
nanoparticulate cyclosporine or a derivative thereof can be present in the water or buffer,
oil, solvent, or a combination thereof. Such a composition is made utilizing Route II.
[0051] In a further embodiment of the invention, the solubilized cyclosporine or a
derivative thereof can be precipitated out from the emulsion droplets. The precipitated
microparticulate cyclosporine or a derivative thereof has a mean particle size of less than
about 10 microns, less than about 9 microns, less than about 8 microns, less than about 7
microns, less than about 6 microns, less than about 5 microns, less than about 4 microns,
less than about 3 microns, less than about 2 microns, or about 1 micron or greater. In
other embodiments of the invention, at least about 50%, at least about 60%, at least about
70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% of
the particles of cyclosporine or a derivative thereof can have a diameter less than the size
listed above, e.g., less than about 10 microns, less than about 9 microns, etc.
[0052] In yet another embodiments of the invention, the nanoparticles of cyclosporine or
a derivative thereof have a diameter of less than about 1000 nm, less than about 900 nm,
less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about
500 nm, less than about 400 nm, less than about 300 nm, less than about 290 nm, less than
about 280 nm, less than about 270 nm, less than about 260 nm, less than about 250 nm,
less than about 240 nm, less than about 230 nm, less than about 220 nm, less than about
210 nm, less than about 200 nm, less than about 190 nm, less than about 180 nm, less than
about 170 nm, less than about 160 nm, less than about 150 nm, less than about 140 nm,
less than about 130 nm, less than about 120 nm, less than about 110 nm, less than about
100 nm, less than about 90 nm, less than about 80 nm, less than about 70 nm, less than
about 60 nm, less than about 50 nm, less than about 40 nm, less than about 30 nm, less
than about 20 nm, or less than about 10 nm. In other embodiments of the invention, at
least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about
90%, at least about 95%, or at least about 99% of the particles of cyclosporine or a
derivative thereof can have a diameter less than the size listed above, e.g., less than about
1000 nm, less than about 900 nm, etc.
[0053] The tri-phasic compositions of the invention are beneficial for several reasons.
First, formulations resulting from the Route II method comprise both solid and solubilized
forms of cyclosporine or a derivative thereof. This enables a resultant pharmaceutical
formulation to provide both immediate release and controlled release of me component
cyclosporine or a derivative thereof, providing for fast onset of activity combined with
prolonged activity of cyclosporine or a derivative thereof.
[0054] The different components of the two types of compositions described above can
be separated and used independently.
2. Emulsion Globules Comprising Cyclosporine
Nanoparticles and/or Solubilized Cyclosporine
[0055] The emulsion globules comprising solubilized cyclosporine or a derivative
thereof, nanoparticles of cyclosporine or a derivative thereof, or a combination thereof can
also be isolated from the surrounding aqueous or buffer phase and used in therapeutic
dosage forms. The emulsion globules can be made using food grade, USP or NF grade
materials suitable for human use applications. Nanoparticulate oil globules comprising
solubilized active pharmaceutical ingredient (API) and methods of making the same are
described in U.S. Patent No. 5,629,021 ("the '021 patent"), which is incorporated herein
by reference. The emulsion globules of the invention typically comprise (1) solubilized
cyclosporine or a derivative thereof, particulate cyclosporine or a derivative thereof, or a
combination thereof; (2) at least one oil; (3) at least one solvent; (4) at least one
preservative; and (5) at least one surface stabilizer or surfactant. Optionally, the emulsion
globules further comprises a viscosity modifier, such as a cellulose derivative, a
polysaccharide or a synthetic polymer. Emulsion globules comprising solubilized
cyclosporine or a derivative thereof, particulate cyclosporine or a derivative thereof, or a
combination thereof can be isolated by, for example, filtration.
[0056] In general, the emulsion globules comprising solubilized cyclosporine or a
derivative thereof, nanoparticles of cyclosporine or a derivative thereof, or a combination
thereof comprise a significant quantity of cyclosporine or a derivative thereof and have
diameters of about 10 to about 1000 nm, with a mean a diameter of less than about 1
micron preferred, and with the smallest globules filterable through a 0.2 micron filter, such
as is typically used for microbiological purification. The range of concentration of
cyclosporine or a derivative thereof in the globules in the diluted final product can be from
about 0.01% to about 5%. The emulsion globules can be stored at between about -20°C
and about 40°C. In one embodiment of the invention, at least about 50%, at least about
60%, at least about 70%, at least about 80%, or at least about 90% of the globules in the
preparation have diameters of less than about 1 micron, less than about 900 nm, less than
about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 nm,
less than about 400 nm, less than about 300 nm, less than about 200 nm, or less than about
100 nm.
[0057] By varying different parameters of Route I and Route II, the size and integrity of
such globules can be modified. Hence, the stability of globules comprising dissolved
cyclosporine or a derivative thereof can be altered to enable the release of cyclosporine or
a derivative thereof, either as a solution or precipitate. This is a microreservoir-
dissolution-controlled system, where the drug solids act as depot and, as the solubilized
fraction is depleted, more drug is drawn into solution form the particulate depot. Thus, the
emulsion globules comprising solubilized cyclosporine or a derivative thereof enable
controlled cyclosporine or a derivative thereof release over time.
[0058] In addition, the emulsion globules comprising solubilized cyclosporine or a
derivative thereof, nanoparticles of cyclosporine or a derivative thereof, or a combination
thereof can be diluted with aqueous solutions without stability loss. This enables the use
of high concentration of cyclosporine or a derivative thereof, e.g., up to about 10%, in
products which can be diluted to obtain the final product.
[0059] In a preferred embodiment, 50X concentrated cyclosporine formulation
containing 2.5% w/w cyclosporine is manufactured aseptically. The particles of
cyclosporine in the concentrated product have a mean particle size of less than 200 mn.
Subsequently, the concentrated product is diluted with sterile PBS/saline to achieve 0.05%
w/w cyclosporine in the final product, which has similar appearance to Restasis®. The
final product is packaged and sealed in 0.4 ml per dose.
D. Methods of Making the Inventive Compositions
Three methods for making the compositions of the invention are described herein.
One benefit of the invention is to provide methods applicable to cyclosporine or a
derivative thereof, which is poorly water-soluble. Another benefit of the methods of the
invention is that they do not require grinding media or specialized grinding process or
equipments. The use of such grinding media can add cost and complexity to a particle size
reduction process for an API. Additionally, the methods of the invention can
accommodate amorphous or semi-amorphous forms of cyclosporine or a derivative
thereof. In summary, the three methods are as follows: Route I: cyclosporine or a
derivative thereof is iasoluble or slightly soluble in any of the components of the
formulation; Route II: cyclosporine or a derivative thereof is soluble or partially soluble
in at least one of the components of the formulation; and Route III: cyclosporine or a
derivative thereof is completely soluble in all of the components of the formulation.
1. Route I
[0060] The method of Route I essentially comprises milling cyclosporine or a derivative
thereof in an emulsion base. This method requires that cyclosporine or a derivative
thereof is poorly soluble or insoluble in all phases of the oil phase/lipophilic phase and the
water or buffer. Hence, cyclosporine or a derivative thereof is first suspended in a mixture
of a non-miscible liquid, which can comprise at least one oil, at least one solvent, at least
one preservative, at least one viscosity modifier, and at least one buffer or water to form
an emulsion base, followed by homogenization or vigorous stirring of the emulsion base.
Nanoparticles of cyclosporine or a derivative thereof can be produced with reciprocating
syringe instrumentation, continuous flow instrumentation, or high speed mixing
equipment. High velocity homogenization or vigorous stirring, producing forces of high
shear and cavitation, are preferred. High shear processes are preferred as low shear
processes can result in larger particle sizes.
[0061] The resultant composition is a composite mixture of cyclosporine or a derivative
thereof suspended in the emulsion droplet (nanoemulsion fraction of cyclosporine or a
derivative thereof) and sterically stabilized microcrystalline or microparticulate
cyclosporine or a derivative thereof in the media. This tri-phasic system comprises
particulate cyclosporine, oil, preservative and water or buffer.
[0062] In one embodiment of the invention, the resultant microparticulate cyclosporine
or a derivative thereof has a diameter of less than about 10 microns, less than about 9
microns, less than about 8 microns, less than about 7 microns, less than about 6 microns,
less than about 5 microns, less than about 4 microns, less than about 3 microns, less than
about 2 microns, or greater than about 1 micron.
[0063] In another embodiment of the invention, the nanoparticulate cyclosporine or a
derivative thereof can have a diameter of less than about 1000 nm, less than about 900 nm,
less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about
500 nm, less than about 400 nm, less than about 300 nm, less than about 290 nm, less than
about 280 nm, less than about 270 nm, less than about 260 nm, less than about 250 nm,
less than about 240 nm, less than about 230 nm, less than about 220 nm, less than about
210 nm, less than about 200 nm, less than about 190 nm, less than about 180 nm, less than
about 170 nm, less than about 160 nm, less than about 150 nm, less than about 140 nm,
less than about 130 nm, less than about 120 nm, less than about 110 nm, less than about
100 nm, less than about 90 nm, less than about 80 ran, less than about 70 nm, less than
about 60 nm, less than about 50 nm, less than about 40 nm, less than about 30 nm, less
than about 20 nm, or less than about 10 nm.
[0064] In other embodiments of the invention, at least about 50%, at least about 60%, at
least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least
about 99% of the microcrystalline or microparticulate cyclosporine or a derivative thereof
in a composition can have a diameter of less than about 10 microns, less than about 9
microns, less than about 8 microns, less than about 7 microns, less than about 6 microns,
less than about 5 microns, less than about 4 microns, less than about 3 microns, less than
about 2 microns, about 1 micron, or greater than about 1 micron and less than about 2,
about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 microns.
[0065] In yet other embodiments of the invention, at least about 50%, at least about
60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at
least about 99% of the nanoparticulate cyclosporine or a derivative thereof can have a
diameter of less than about 1000 nm, less than about 900 nm, less than about 800 nm, less
than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400
nm, less than about 300 nm, less than about 290 nm, less than about 280 nm, less than
about 270 nm, less than about 260 nm, less than about 250 nm, less than about 240 nm,
less than about 230 nm, less than about 220 nm, less than about 210 nm, less than about
200 nm, less than about 190 nm, less than about 180 nm, less than about 170 nm, less than
about 160 nm, less than about 150 nm, less than about 140 nm, less than about 130 nm,
less than about 120 nm, less than about 110 nm, less than about 100 nm, less than about 90
nm, less than about 80 nm, less than about 70 nm, less than about 60 nm, less than about
50 nm, less than about 40 nm, less than about 30 nm, less than about 20 nm, or less than
about 10 nm in size.
[0066] Cyclosporine or a derivative thereof can be precipitated out from the oil droplets
by adding more of the non-miscible liquid. The precipitated particles of cyclosporine or a
derivative thereof typically have a diameter of less than about 10 microns, less than about
9 microns, less than about 8 microns, less than about 7 microns, less than about 6 microns,
less than about 5 microns, less than about 4 microns, less than about 3 microns, less than
about 2 microns, or less than about 1 micron. If desired, the particles cyclosporine or a
derivative thereof can be prevented from aggregating or clumping together by
incorporating a surfactant or emulsifier, e.g., a "surface stabilizer."
2. Route II and Route III
[0067] Routes II and III require that cyclosporine or a derivative thereof is soluble or
partially soluble in at least one (Route II) or all of the phases (Route III) of the emulsion
base; e.g., that cyclosporine or a derivative thereof is soluble in at least one oil, at least one
solvent, at least one preservative, or water or buffer. In some embodiments, Route II or III
can comprise the simultaneous milling and precipitation of cyclosporine or a derivative
thereof in an emulsion base.
[0068] Route II is utilized when cyclosporine or a derivative thereof is soluble in at least
one part of the emulsion base, such as the solvent, and Route III is utilized when
cyclosporine or a derivative thereof is soluble in all of the components of the emulsion
base, such as oil and a solvent. For Routes II and III, cyclosporine or a derivative thereof
is dissolved in a mixture of oil, solvent, preservative, viscosity modifier and stabilizer to
form an emulsion pre-mix. Cyclosporine or a derivative thereof remains in soluble form if
water or buffer is not added to the mixture. Upon the addition of water or buffer and the
application of shear forces, cyclosporine or a derivative thereof is precipitated into
microparticles having a diameter of less than about 10 microns, and nanoparticles having a
diameter of less than about 1 micron (as described above in Route I; the same particle
sizes are applicable to Routes II and HI). Nanoparticles can be produced with
reciprocating syringe instrumentation, continuous flow instrumentation, or high speed
mixing equipment. High energy input, through high velocity homogenization or vigorous
stirring, is a preferred process. The high energy processes reduce the size of the emulsion
droplets, thereby exposing a large surface area to the surrounding aqueous environment.
High shear processes are preferred, as low shear processes can result in larger particle
sizes.
[0069] This can be followed by precipitation of nanoparticulate cyclosporine or a
derivative thereof previously embedded in the emulsion base. The end product comprises
cyclosporine or a derivative thereof in solution and particulate suspension, both distributed
between the solvent, oil, and water or buffer. In one embodiment, nanoparticulate
cyclosporine or a derivative thereof has at least one surface stabilizer associated with the
surface thereof.
[0070] If desired, the water miscible oil droplets and nanoparticles of cyclosporine or a
derivative thereof prepared using Route I, Route II, or Route III may be filtered through
either a 0.2 or 0.45 micron filter. Larger oil droplets and/or particles of cyclosporine or a
derivative thereof can be created by simply increasing the water content, decreasing the
oil-stabilizer-solvent content, or reducing the shear in forming the oil droplets.
[0071] For the 50X concentrated emulsion base used in Route I, Route II, or Route III,
the content of cyclosporine is about 0.1%-10%, the content of solvent is about 0.1%-20%,
the content of oil is about 5%-50%, the content of surfactant is about 0.1%-20%, the
content of preservative is about 0.1%-5%, and the content of the aqueous medium is about
20%-80%, all in w/w percentage. Optionally, the viscosity modifier is present in the
emulsion base in the amount of about 0.1 % to about 10% (w/w). The content of each
ingredient in the final product is the amount above divided by 50, with the aqueous
medium being the major component, at about 98% or more.
E. Components of the Methods and Compositions of the Invention
1. Cyclosporine
a. Suitable Cyclosporine and Derivatives Thereof
The phrase "cyclosporines and derivatives thereof is defined supra, as disclosed
in U.S. Patent Nos. 5,474,979, 6,254,860, and 6,350,442. The cyclosporines useful in the
present invention include naturally occurring cyclosporines, preferably cyclosporines A,
B,C, D and G, as well as synthetic and semi-synthetic cyclosporines and cyclosporine
derivatives disclosed in U.S. Patent Nos. 4,108,985, 4,210,581,4,220,641, 4,384,996,
4,764,503, 4,703,033 and U.K. Patent Application No. 2,206, 119A, which are hereby
incorporated by reference in their entirety. Preferably, the cyclosporine is cyclosporine A.
b. Particle Size of Cyclosporine
[0072] As used herein, the particle size is determined on the basis of the weight average
particle size as measured by conventional techniques well known to those skilled in the
art, such as sedimentation field flow fractionation, laser diffraction, photon correlation
spectroscopy (also known as dynamic light scattering), electroacoustic spectroscopy, or
disk centrifugation.
[0073] As used herein, "nanoparticulate cyclosporine or a derivative thereof refers to
cyclosporine or a derivative thereof having a diameter of less than about 1 micron. An
exemplary cyclosporine useful in the invention is cyclosporine A. "Macrocrystalline
cyclosporine or a derivative thereof refers to cyclosporine or a derivative thereof having a
diameter of greater than about 1 micron. In other embodiments of the invention,
microparticulate cyclosporine or a derivative thereof have a diameter of less than about 10
microns, less than about 9 microns, less than about 8 microns, less than about 7 microns,
less than about 6 microns, less than about 5 microns, less than about 4 microns, less than
about 3 microns, less than about 2 microns, or about 1 micron or greater. In other
embodiments of the invention, at least about 50%, at least about 60%, at least about 70%,
at least about 80%, at least about 90%, at least about 95%, or at least about 99% of the
microparticles of cyclosporine or a derivative thereof can have a diameter less than the
size listed above, e.g., less than about 10 microns, less than about 9 microns, etc.
[0074] In yet other embodiments of the invention, nanoparticulate cyclosporine or a
derivative thereof has a diameter of less than about 900 nm, less than about 800 nm, less
than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400
nm, less than about 300 nm, less than about 290 nm, less than about 280 nm, less than
about 270 nm, less than about 260 nm, less than about 250 nm, less than about 240 nm,
less than about 230 nm, less than about 220 nm, less than about 210 nm, less than about
200 nm, less than about 190 nm, less than about 180 nm, less than about 170 nm, less than
about 160 nm, less than about 150 nm, less than about 140 nm, less than about 130 nm,
less than about 120 nm, less than about 110 nm, less than about 100 nm, less than about 90
nm, less than about 80 nm, less than about 70 rim, less than about 60 nm, less than about
50 nm, less than about 40 nm, less than about 30 nm, less than about 20 nm, or less than
about 10 nm. In other embodiments of the invention, at least about 50%, at least about
60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at
least about 99% of the nanoparticles of cyclosporine or a derivative thereof can have a
diameter less than the size listed above, e.g., less than about 900 nm, less than about 800
nm, etc.
[0075] In other embodiments of the invention, at least about 60%, at least about 70%, at
least about 80%, at least about 90%, at least about 95%, or at least about 99% of the
particles of cyclosporine or a derivative thereof, or droplets comprising solubilized
cyclosporine or a derivative thereof, have a size less than the mean particle size, less than
about 3 microns, less than about 2900 nm, less than about 2800 nm, etc.
2. Oils
[0076] For both the methods of Route I and Route II and the compositions of the
invention, any suitable oil can be used. Exemplary oils that can be used include, for
example, vegetable oils, nut oils, fish oils, lard oil, mineral oils, squalane, tricaprylin, and
mixtures thereof. Specific examples of oils that may be used include, but are not limited
to, almond oil (sweet), apricot seed oil, borage oil, canola oil, coconut oil, com oil, cotton
seed oil, fish oil, jojoba bean oil, lard oil, linseed oil (boiled), Macadamia nut oil, medium
chain triglycerides, mineral oil, olive oil, peanut oil, safflower oil, sesame oil, soybean oil,
squalene, sunflower seed oil, tricaprylin (1,2,3-trioctanoyl glycerol), wheat germ oil, and
mixtures thereof.
3. Stabilizers or Surfactants
[0077] The stabilizer used in the methods and compositions of the invention associates
with, or adsorbs, to the surface of the nanoparticulate cyclosporine or a derivative thereof,
but does not covalently bind to cyclosporine or a derivative thereof. In addition, the
individual stabilizer molecules are preferably free of cross-linkages. The stabilizer is
preferably soluble in water. One or more stabilizers may be used in the compositions and
methods of the invention. As used herein, the terms "stabilizer", "surface stabilizer", and
"surfactant" are used interchangeably.
[0078] Any suitable nonionic or ionic surfactant may be utilized in the compositions of
the invention, including anionic, cationic, and zwitterionic surfactants. Exemplary
stabilizers or surfactants that may be used in both Routes I and II include, but are not
limited to, non-phospholipid surfactants, such as the Tween (polyoxyethylene derivatives
of sorbitan fatty acid esters) family of surfactants {e.g., Tween 20, Tween 60, and Tween
80), nonphenol polyethylene glycol ethers, sorbitan esters (such as Span and Arlacel),
glycerol esters (such as glycerin monostearate), polyethylene glycol esters (such as
polyethylene glycol stearate), block polymers (such as Pluronics®), acrylic polymers (such
as Pemulen®), ethoxylated fatty esters (such as Cremophor® RH-40), ethoxylated alcohols
(such as Brij®), ethoxylated fatty acids, monoglycerides, silicon based surfactants,
polysorbates, Tergitol NP-40 (Poly(oxy-l,2-ethanediyl), a -(4-nonylphenol)-.omega.-
hydroxy, branched [molecular weight average 1980]), and Tergitol NP-70 (a mixed
surfactant-AQ=70%).
4. Solvents
[0079] Any suitable solvent can be used in the methods and compositions of the
invention. Exemplary solvents include, but are not limited, to isopropyl myristate,
triacetin, N-methyl pyrrolidinone, aliphatic or aromatic alcohols, polyethylene glycols,
propylene glycol. An example of an alcohol useful in the present invention includes, but
is not limited to ethanol. Other short chain alcohols and/or amides may be used. Other
solvents include dimethyl sulfoxide, dimethyl acetamide, and ethoxydiglycol. Mixtures of
solvents can also be used in the compositions and methods of the invention.
5. Water or Buffer
[0080] If the methods and/or compositions of the invention use or comprise water or a
buffer, the aqueous solution is preferably a physiologically compatible solution such as
water or phosphate buffered saline.
6. Preservatives
[0081] The formulations of the invention have anti-microbial properties. The anti-
microbial properties can be associated with the formulation. As an example, the
formulations described herein, in the absence of cyclosporine, can exhibit antimicrobial
activity.
[0082] Antimicrobial agents or preservatives are added to nonsterile dosage forms to
protect them from microbiological growth or from microorganisms that are introduced
inadvertently during or subsequent to the manufacturing process. In the case of sterile
articles used in multi-dose containers, antimicrobial preservatives are added to inhibit the
growth of microorganisms that may be introduced by repeatedly withdrawing individual
doses.
[0083] U.S. Food and Drug Administration guidelines require that antimicrobial
effectiveness, whether inherent in the product (e.g., for an antibiotic agent) or whether
produced because of the addition of an antimicrobial agent, must be demonstrated for all
injections packaged in multiple-dose containers or for other products containing
antimicrobial preservatives. Antimicrobial effectiveness must be demonstrated for
multiple-dose topical and oral dosage forms, and for other dosage forms such as
ophthalmic, otic, nasal, irrigation, and dialysis fluids. See USP 25, Section 51,
"Antimicrobial Effectiveness Testing."
[0084] The formulations of the invention meet the Antimicrobial Effectiveness Test as
described in the United States Pharmacopeia (USP - General Chapter #51). The standard
USP testing requires evaluation in five microorganisms: Aspergillus niger (ATCC
16404), Candida albicans (ATCC 10231), Escherichia coli (ATCC 8739), Pseudomonas
aeruginosa (ATCC 9027) and Staphylococcus aureus (ATCC 6538).
[0085] The formulations of the invention comprise preservatives, e.g., water soluble
quaternary ammonium compounds, such as cetyltrimethylammonium bromide,
cetylpyridinium chloride, benzethonium chloride, and benzalkonium chloride. Preferably,
the preservative is benzalkonium chloride.
[0086] BKC has been used as a preservative in ophthalmic formulations for many years.
The formulation of the present invention contains BKC at a concentration that is
recognized to be sufficient to qualify the USP Preservative Efficacy testing. There is
sufficient literature evidence available to prove preservative efficacy for BKC used in
ophthalmic formulations (Handbook of Pharmaceutical Excipients, 3ri edition (2000),
edited by Arthur H. Kibbe, published by American Pharmaceutical Association and
Pharmaceutical Press, pp. 33-35). Hence, the formulation of the invention is suitable for
multi-dose packaging.
[0087] In the present invention, BKC unexpectedly functions as a cationic agent, which
contributes to the prolonged drug residence time. This property of the compositions of the
invention enable fewer applications of the dosage form to the eye, which is generally
preferred by patient populations. Moreover, the dosage applied is more effective, as the
cationic property of the dosage form results in the drug "adhering" to the eye, rather than
having much of the dosage form run out of the eyes following administration.
[0088] It is believed that the beneficial aspects of the novel opthalmic cyclosporine
compositions of the invention result from a synergistic combination of: (1) particle size of
cyclosporine; (2) potential presence of cyclosporine in both a solubilized and particulate
form in the dosage; (3) the cationic property of the dosage form, and (4) the stable, non-
irritating final product suitable for multi-dose packaging.
[0089] In general, tissues in the body are considered to be negatively charged. As a
result, a positively-charged (cationic) product is considered to have better chance of
improved interaction of the product with the tissue. This improved interaction between
the cationic product and body tissues is not always observed, however.
[0090] Prior art describes that for a biphasic system, oil-soluble cationic lipids, e.g..
stearylamine, has been used to impart a net positive charge onto oil droplet, whereby the
droplet being electrostatically attracted to the anionic surface of the eye. See, for example,
U.S. Patent No. 6,656,460.
[0091] When water-soluble, quaternary ammonium compounds are used, they will not
impart positive charge on the emulsion. Instead, they are likely to be adsorbed or loosely
bound onto the surface and may result in a charge. Typically, when quaternary
ammonium compounds are used, the acidic pH needs to be maintained for protonation of
the groups to impart positive charge. In the present invention, it is unexpectedly found
that BKC induces positive charge, under about neutral pH, in a multi-phasic composition
having a solid particulate phase and an oil phase dispersed in aqueous milieu.
Surprisingly, the cationic emulsion prepared using BKC shows significantly prolonged
residence time in the eye using human cornea mounted on a Franz cell.
[0092] Due to the presence of the preservative, the formulations of the present invention
can be packaged into multi-dose containers. Moreover, as demonstrated in the following
working examples, the presence of cyclosporine was only detected locally and
administration of the formulations of the invention does not cause any irritation to the eye.
Hence, the formulations of the invention are suitable for daily application.
7. Viscosity Modifier
[0093] The ophthalmic formulation may further comprise a viscosity modifier, such as a
cellulose derivative, a polysaccharide or a synthetic polymer. The viscosity of the
formulations of the present invention can be increased without compromising other
properties.
[0094] The following examples are given to illustrate the present invention. It should be
understood, however, that the invention is not to be limited to the specific conditions or
details described in these examples. Throughout the specification, any and all references
to a publicly available document, including U.S. patents, are specifically incorporated by
reference.
Example 1
[0095] The purpose of this example was to prepare formulations according to the
invention comprising cyclosporine and a cationic preservative, benzalkonium chloride.
[0096] The commercial Restasis® standard, which contains cyclosporine A (0.05% w/w)
and inactive ingredients like glycerin, castor oil, polysorbate 80, carbomer 1342, purified
water and sodium hydroxide, was used as a control for the purpose of comparing the
properties of the formulation.
[0097] Cyclosporine is commercially available under the trade names SANDIMMUNE81
and NEORAL®. It is a cyclic polypeptide immunosuppressant agent consisting of 11
amino acids. It is produced as a metabolite by the fungus species Beauveria nlyea.
Chemically, cyclosporine is designated as [R-[R*,R*-(E)]]-cyclic(L-alanyl-D- alanyl-N-
methyl-L-leucyl-N-methyl-L-leucyl-N-methyl-L-valyl-3-hydroxy-N, 4-dimethyl-L-2-
amino-6-octenoyl-L-a-amino-butyryl-N-methylglycyl-N-methyl-L-leucyl-L-valyl-N-
methyl-L-leucyl). The chemical structure of cyclosporine (also known as cyclosporin A)
is:

[0098] Cyclosporine was dissolved in ethanol at room temperature. Polysorbate 80 and
crodamol (Table 1) were then added to the cyclosporine solution and mixed well with a
paddle stirrer. Then benzalkonium chloride solution and water were added and mixing
was continued for about 5 minutes. The coarse emulsion was then fed into a high pressure
homogenizer (APV Invensys, model APV-1000) and homogenized at 10,000 psi for three
passes. The resultant 50X concentrated composition, described below in Table 1,
comprised cyclosporine dissolved in the solvent ethanol and nanoparticulate cyclosporine
particles associated with the surface stabilizer polysorbate 80 present in the water portion
of the emulsion. The concentrated emulsion was collected and diluted with phosphate
buffered saline (PBS), pH 7.2, at the ratio of 1 part of concentrated emulsion with 49 parts
of PBS to obtain the final product, cationic micellar nanoparticle formulation (cMNP) of
cyclosporine. The cMNP and Restasis® have similar appearance as milky white emulsion.
The viscosity of the formulation of the invention can be increased without compromising
otiier properties.
[0099] The resultant particle size and zeta potential of cMNP of cyclosporine were
measured and compared with those of the commercial Restasis® standard, as shown in
Figure 1A and 1B, as well as in Table 2.

Example 2
[01001 The purpose of this example was to prepare formulations comprising
cyclosporine and a permeation enhancer, vitamin E TPGS. The formulations of this
example are used as a comparison to the formulations of the invention prepared according
to Example 1.
[0101] Cyclosporine and Restasis® standard are the same as those in Example 1.
Vitamin E TPGS (d-alpha-tocopheryl polyethylene glycol-1000 succinate, obtained from
Eastman Chemical Company) is a water-soluble form of natural-source vitamin E.
[0102] Cyclosporine was dissolved in ethanol at room temperature. Polysorbate 80 and
crodamol (Table 3) were then added to the cyclosporine solution and mixed well with a
paddle stirrer. Then vitamin E TPGS solution and water were added and mixing was
continued for about 5 minutes. The coarse emulsion was then fed into a high pressure
homogenizer (APV Invensys, model APV-1000) and homogenized at 10,000 psi for three
passes. The resultant 50X concentrated composition, described below in Table 3,
comprised cyclosporine dissolved in the solvent ethanol and nanoparticulate cyclosporine
particles associated with the surface stabilizer polysorbate 80 present in the water portion
of the emulsion. The concentrated emulsion was collected and diluted with phosphate
buffered saline (PBS), pH 7.2, at the ratio of 1 part of concentrated emulsion with 49 parts
of PBS to obtain the final product, neutral micellar nanoparticle formulation (nMNP) of
cyclosporine. The nMNP and Restasis® have similar appearance as milky while emulsion.
The viscosity of the formulation of the invention can be increased without compromising
other properties.
[0103] The resultant particle size and zeta potential of nMNP of cyclosporine were
measured and compared with those of the commercial Restasis® standard, as shown in
Figure 1A and 1C, as well as in Table 4.

Example 3
[0104] The purpose of this example was to test the formulations prepared in Example 1
and Example 2 for drug residence on cornea, for possibility of once-daily application, and
for systemic absorption, using an in vitro Franz cell-human cornea assembly.
[0105J The protocol is as follows: isolated human cornea was obtained from an eye
bank. The corneas were collected from donors and refrigerated in a suitable preservation
medium. The preserved cornea was allowed to reach room temperature for about 20
minutes, and then rinsed with PBS to remove the preservation medium. The corneas were
mounted horizontally between the donor and receptor halves of the Franz diffusion cells.
The surface area of the cornea exposed to the formulation in the donor chamber is 0.64
cm2, and the receptor cell volume was 5.0 ml. The receptor compartment was filled with
0.01M PBS, pH 7.4, and ethanol at a ratio of 9:1. A double water circulation jacket at
37°C surrounds the receptor cell in order to have the cornea temperature maintained at
physiologic level. The cMNP and nMNP formulations prepared according to Example 1
and Example 2, as well as the Restasis® standard, were applied over the surface of the
cornea gravimetrically using a syringe. Periodic samples of 1.0 ml each were taken from
the receptor cell to measure the amount of drug transporting across the cornea at time
points of 0, 2,4, 8, and 24 hours, respectively. At each time point, the donor sample was
discarded and fresh donor sample was replenished. At the end of 24 hours, the cornea
were collected, washed and homogenized in PBS:ethanol (9:1) and assayed for drug
residence.
[0106] No drug presence in the receptor compartment was detected at any time point.
Hence, both formulations are suitable for once-daily application and the systemic
availability of cyclosporine for both formulations is expected to be at comparable level to
that of Restasis®. Moreover, Figure 2 illustrates that significantly high level of
cyclosporine was retained in the cornea for the cMNP formulation prepared according to
Example 1. Therefore, the cMNP formulation is expected to have a longer retention
within the cul-de-sac via electrostatic attraction with ocular tissue.
Example 4
[0107] The purpose of this example was to test the formulations prepared in Example 1
and Example 2 for biocompatibility.
[0108] To determine the in vivo irritant and/or corrosive effects, the cMNP and nMNP
formulations prepared according to Example 1 and Example 2 were instilled into the rabbit
eye upon once-a-day application for 7 consecutive days.
[0109] Six healthy New Zealand White rabbits were assigned to two groups of three
animals per group. One group was dosed with the cMNP formulation, while the other was
dosed with the nMNP formulation. All animals were dosed daily for seven consecutive
days with 0.1 ml of the formulation into the conjunctival sac of the left eye of each rabbit.
The right eye served as the untreated control. The treated eyes were examined for
irritation using the Draize technique, pretest, prior to each subsequent dose and at the time
points of 24, 48 and 72 hours, respectively, following the final dose. Observations for
mortality, toxicity and pharmacologic effects were made prior to each ocular observation
period. Body weights were recorded pretest. The animals were humanely sacrificed using
CO2, following the final observation.
[0110] The results show that there was no evidence of ocular irritation noted in any eye
dosed with either the cMNP or the nMNP formulation and that there were no abnormal
physical signs noted during the study. Hence, both the cMNP and the nMNP formulations
are well-tolerated.
Example 5
[0111] The purpose of this example was to test the formulations prepared in Example 1
and Example 2 for stability.
[0112] Both formulations were subjected to an in-house or informal non-ICH stability
study. The storage temperatures and time points taken for the stability test are as follows:
1. 25°C: 0, 1, 2, 4, 12, and 40 weeks;
2. 40°C: 1, 2, 4 and 12 weeks;
3. 60°C: 1week
[0113] The stability data, in terms of the potency of cyclosporine and the mean particle
size, is depicted in Figure 3 and Figure 4, for the cMNP formulation and the nMNP
formulation, respectively. For the cMNP formulation, the results show that there was no
change in physical appearance of the emulsion for any storage condition at any time point
tested. pH was stable at 7.3±0.3 across all storage conditions and time pointes tested. The
cyclosporine content dropped by approximately 10% for 60°C storage condition at 1-week
time point. However, the 25°C and 40°C storage conditions did not cause any significant
decrease in potency during the test period. The mean particle size did not change during
the test period for any storage condition tested. Hence, the cMNP formulation of
cyclosporine will likely be stable for two years at room temperature. Figure 4 shows that
the nMNP formulation is stable for up to 12 weeks at 25°C or at 40°C.
Example 6
[0114] The purpose of this example was to manufacture the cyclosporine formulation.
[0115] 50X concentrated cyclosporine formulation containing 2.5% w/w cyclosporine is
manufactured aseptically according to the description in Example 1 or Example 2. The
particles of cyclosporine in the concentrated product have a mean particle size of less than
200 nm. Subsequently, the concentrated composition is diluted with sterile PBS/saline to
achieve 0.05% w/w cyclosporine in the final product, which has similar appearance to
Restasis®. The final product is packaged and sealed in 0.4 ml per dose.
[0116] It will be apparent to those skilled in the art that various modifications and
variations can be made in the methods and compositions of the present invention without
departing from the spirit or scope of the invention. Thus, it is intended that the present
invention cover the modifications and variations of this invention provided they come
within the scope of the appended claims and their equivalents.
WHAT IS CLAIMED IS:
1. An ophthalmic formulation comprising:
(a) cyclosporine or a derivative thereof,
(b) at least one solvent,
(c) at least one oil,
(d) at least one surfactant,
(e) at least one preservative, and
(f) water or buffer.
2. The opthalmic formulation of claim 1, wherein the cyclosporine is
cyclosporine A.
3. The ophthalmic formulation of claim 1, wherein cyclosporine or a
derivative thereof is in a solid particulate state and in a soluble state.
4. The ophthalmic formulation of claim 3, wherein the formulation is a
mixture of particles of cyclosporine or a derivative thereof suspended in emulsion droplets
and sterically stabilized particulate cyclosporine or a derivative thereof in the water or
buffer.
5. The ophthalmic formulation of claim 1, wherein the formulation comprises
globules of oil comprising dissolved cyclosporine or a derivative thereof, wherein the
globules have a diameter of less than about 10 microns.
6. The ophthalmic formulation of claim 5, wherein the globules have a
diameter selected from the group consisting of less than about 9 microns, less than about 8
microns, less than about 7 microns, less than about 6 microns, less than about 5 microns,
less than about 4 microns, less than about 3 microns, less than about 2 microns, less than
about 1000 nm, less than about 900 nm, less than about 800 nm, less than about 700 nm,
less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about
300 nm, less than about 290 nm, less than about 280 nm, less than about 270 nm, less than
about 260 nm, less than about 250 nm, less than about 240 nm, less than about 230 nm,
less than about 220 nm, less than about 210 nm, less than about 200 nm, less than about
190 nm, less than about 180 nm, less than about 170 nm, less than about 160 nm, less than
about 150 nm, less than about 140 nm, less than about 130 nm, less than about 120 nm,
less than about 110 nm, less than about 100 nm, less than about 90 nm, less than about 80
nm, less than about 70 nm, less than about 60 nm, less than about 50 nm, less than about
40 nm, less than about 30 nm, less than about 20 nm, or less than about 10 nm.
7. The formulation of claim 1, wherein the oil is selected from the group
consisting of sweet almond oil, apricot seed oil, borage oil, canola oil, coconut oil, corn
oil, cotton seed oil, fish oil, jojoba bean oil, lard oil, boiled linseed oil, Macadamia nut oil,
medium chain triglycerides (Crodamol GTCC), mineral oil, olive oil, peanut oil, safflower
oil, sesame oil, soybean oil, squalene, sunflower seed oil, tricaprylin (1,2,3-trioctanoyl
glycerol), and wheat germ oil.
8. The formulation of claim 1, wherein the solvent is selected from the group
consisting of isopropyl myristate, triacetin, N-methyl pyrrolidinone, aliphatic and aromatic
alcohols, ethanol, dimethyl sulfoxide, dimethyl acetamide, ethoxydiglycol, polyethylene
glycols, and propylene glycol.
9. The formulation of claim 1, wherein the surfactant is selected from the
group consisting of sorbitan esters, glycerol esters, polyethylene glycol esters, block
polymers, acrylic polymers, ethoxylated fatty esters, ethoxylated alcohols, ethoxylated
fatty acids, monoglycerides, silicon based surfactants, and polysorbates.
10. The formulation of claim 9, wherein the surfactant is Polysorbate 80.
11. The formulation of claim 9, wherein the sorbitan ester surfactant is Span or
Arlacel, wherein the glycerol ester is glycerin monostearate, wherein the polyethylene
glycol ester is polyethylene glycol stearate, wherein the block polymer is a Pluronic,
wherein the acrylic polymer is Pemulen, wherein the ethoxylated fatty ester is Cremophor
RH-40, wherein the ethoxylated alcohol is Brij, and wherein the ethoxylated fatty acid is
Tween 20.
12. The formulation of claim 1, wherein formulation is cationic.
13. The formulation of claim 1, wherein the preservative is cationic.
14. The formulation of claim 1, wherein the preservative is a quaternary
ammonium compound.
15. The formulation of claim 1, wherein the preservative is selected from the
group consisting of cetyltrimethylammonium bromide, cetylpyridinium chloride,
benzethonium chloride, and benzalkonium chloride.
16. The formulation of claim 1, wherein the preservative is benzalkonium
chloride.
17. The formulation of claim 1, further comprising a viscosity modifier.
18. The formulation of claim 17, wherein the viscosity modifier is selected
from the group consisting of a cellulose derivative, a polysaccharide and a synthetic
polymer.
19. An ophthalmic formulation comprising:
(a) cyclosporine A,
(b) ethanol,
(c) medium chain triglycerides,
(d) Polysorbate 80,
(e) benzalkonium chloride, and
(f) phosphate buffer.
20. A method of treating a subject in need comprising administering the
formulation of claim 1 or claim 19 to the eye of a subject.
21. The method according to claim 20, wherein the subject suffers from a
condition selected from the group consisting of an aqueous deficient dry-eye state,
phacoanaphylaxis endophthalmitis, and uveitis.
22. A method for preparing particles of cyclosporine or a derivative thereof,
comprising:
(a) forming an emulsion base by suspending cyclosporine or a derivative
thereof in a mixture of oil, solvent, surfactant, preservative, and water or buffer to form an
emulsion base, and
(b) homogenizing the emulsion base to form particles of cyclosporine or a
derivative thereof, droplets comprising solubilized cyclosporine or a derivative thereof, or
a combination thereof.
23. The method of claim 22, wherein the homogenizing step is performed via a
high-pressure system at 1,000 to 40,000 psi.
24. The method of claim 22, wherein the cyclosporine or a derivative thereof,
droplets have an average or mean particle size selected from the group consisting of less
than about 10 microns, less than about 9 microns, less than about 8 microns, less than
about 7 microns, less than about 6 microns, less than about 5 microns, less than about 4
microns, less than about 3 microns, less than about 2 microns, and about 1 micron or
greater.
25. The method of claim 22, wherein the cyclosporine or derivative thereof,
droplets have a mean particle size selected from the group consisting of less than about 1
micron, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than
about 500 nm, less than about 400 nm, less than about 300 nm, less than about 290 nm,
less than about 280 nm, less than about 270 nm, less than about 260 nm, less than about
250 nm, less than about 240 nm, less than about 230 nm, less than about 220 nm, less than
about 210 nm, less than about 200 nm, less than about 190 nm, less than about 180 nm,
less than about 170 nm, less than about 160 nm, less than about 150 nm, less than about
140 nm, less than about 130 nm, less than about 120 nm, less than about 110 nm, less than
about 100 nm, less than about 90 nm, less than about 80 nm, less than about 70 nm, less
than about 60 nm, less than about 50 nm, less than about 40 nm, less than about 30 nm,
less than about 20 nm, or less than about 10 nm.
26. The method of claim 22, wherein the emulsion base of step (a) further
comprises a viscosity modifier.
27. The method of claim 26, wherein the viscosity modifier is selected from the
group consisting of a cellulose derivative, a polysaccharide and a synthetic polymer.
28. A method for preparing particles of cyclosporine or a derivative thereof,
comprising:
(a) dissolving cyclosporine or a derivative thereof in a mixture of (i) at least
one oil, (ii) at least one solvent, and (iii) at least one surfactant to form an emulsion pre-
mix,
(b) adding at least one preservative, and water or buffer to the emulsion pre-
mix, and
(c) homogenizing or vigorously stirring the mixture of step (b), whereby
cyclosporine A or a derivative thereof is precipitated into particles.
29. The method of claim 28, wherein the homogenizing step is performed via a
high-pressure system at 1,000 to 40,000 psi.
30. The method of claim 28, wherein the cyclosporine or a derivative thereof,
droplets have an average or mean particle size selected from the group consisting of less
than about 10 microns, less than about 9 microns, less than about 8 microns, less than
about 7 microns, less than about 6 microns, less than about 5 microns, less than about 4
microns, less than about 3 microns, less than about 2 microns, and about 1 micron or
greater.
31. The method of claim 28, wherein the cyclosporine A or derivative thereof,
droplets have a mean particle size selected from the group consisting of less than about 1
micron, less than about 800 run, less than about 700 nm, less than about 600 nm, less than
about 500 nm, less than about 400 nm, less than about 300 nm, less than about 290 nm,
less than about 280 nm, less than about 270 nm, less than about 260 nm, less than about
250 nm, less than about 240 nm, less than about 230 nm, less than about 220 nm, less than
about 210 nm, less than about 200 nm, less than about 190 nm, less than about 180 nm,
less than about 170 nm, less than about 160 nm, less than about 150 nm, less than about
140 nm, less than about 130 nm, less than about 120 nm, less than about 110 nm, less than
about 100 nm, less than about 90 nm, less than about 80 nm, less than about 70 nm, less
than about 60 nm, less than about 50 nm, less than about 40 nm, less than about 30 nm,
less than about 20 nm, or less than about 10 nm.
32. The method of claim 28, wherein the mixture of step (a) or the mixture of
step (b) further comprises a viscosity modifier.
33. The method of claim 32, wherein the viscosity modifier is selected from the
group consisting of a cellulose derivative, a polysaccharide and a synthetic polymer.
34. A method for preparing particles of cyclosporine or a derivative thereof,
comprising:
(a) dissolving cyclosporine or a derivative thereof in ethanol to form a
solution,
(b) adding Polysorbate 80 and medium chain triglyceride to the solution of step
(a),
(c) adding benzalkonium chloride dissolved in water to the solution of (b), and
(d) subjecting the solution of (c) to high-pressure homogenization to produce
particles of cyclosporine or a derivative thereof.

The present invention provides ophthalmic formulations containing cyclosporine, methods for preparing the formulation,
and methods for using the formulation.

Documents:

http://ipindiaonline.gov.in/patentsearch/GrantedSearch/viewdoc.aspx?id=jsYwfUwJqP455eNklvASRQ==&loc=wDBSZCsAt7zoiVrqcFJsRw==

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Patent Number

279458

Indian Patent Application Number

2575/KOLNP/2010

PG Journal Number

04/2017

Publication Date

27-Jan-2017

Grant Date

23-Jan-2017

Date of Filing

14-Jul-2010

Name of Patentee

NEWGEN BIOPHARMA CORPORATION

Applicant Address

210 JACOBS CREEK ROAD, TITUSVILLE, NEW JERSEY 08560 U.S.A.

Inventors:

#	Inventor's Name	Inventor's Address
1	LEE, ROBERT	C/O NOVAVAX, INC, 508 LAPP ROAD, MALVERN, PA 19355 UNITED STATES OF AMERICA
2	SHENOY, DINESH	C/O NOVAVAX, INC, 508 LAPP ROAD, MALVERN, PA 19355 UNITED STATES OF AMERICA

PCT International Classification Number

A61K 9/00

PCT International Application Number

PCT/US2009/031111

PCT International Filing date

2009-01-15

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	12/007,902	2008-01-16	U.S.A.