AU2012202829B2 - Ophthalmic emulsions containing an immunosuppressive agent - Google Patents

Abstract

Abstract Ophthalmic oil-in-water emulsions, which comprises colloid 5 particles having an oily core surrounded by an interfacial film, said emulsion comprising an immunosuppressive agent, an oil, preferably at least 50 % of which being MCT, and tyloxapol. Use of such an emulsion for the manufacture of medicament for treatment of eye conditions, particularly 10 of dry eye diseases. 3078822_1 (GHMatters) P88392.AU I

Description

AUSTRALIA Patents Act 1990 COMPLETE SPECIFICATION Standard Patent Applicant(s): NOVAGALI PHARMA SA Invention Title: Ophthalmic emulsions containing an immunosuppressive agent The following statement is a full description of this invention, including the best method for performing it known to me/us: - la OPHTHALMIC EMULSIONS CONTAINING AN IMMUNOSUPPRESSIVE AGENT. This invention relates to ophthalmic oil-in-water emulsions comprising an immunosuppressive agent as an active 5 ingredient, in a specific vehicle containing oil and tyloxapol, and being suitable for the treatment of eye conditions, particularly of dry eye diseases. Dry eye disease, in the meaning of this invention, relates to any condition relating to ocular dryness and/or tear 10 deficiency, including but not limited to conditions related to a pathological evaporation of fluid from the cornea surface, or conditions related to defective tear film, or more generally any corneal or conjunctive dryness, which can be due to insufficient production of tears, and the conditions resulting therefrom, 15 such as corneal keratitis or corneal epithelium erosion. For example keratoconjunctivis sicca (KCS) , atopic keratoconjunctivitis sicca (AKC) and vernal keratoconjunctivitis (VKC) are dry eye diseases. Cyclosporines are a large class of peptide compounds 20 having various pharmaceutical applications, such as immunosuppressant and anti-inflammatory applications. Cylosporines include cyclosporine A, B, C and D. The most widely investigated cyclosporin is cyclosporin A and cyclosporine A derivatives. Other common immunosuppressive agents are sirolimus 25 or tacrolimus and their derivatives. Cyclosporine oil-in-water ophthalmic emulsions have been described, and among them the emulsions commercialized by Allergan under the trademark name Restasis* (cyclosporine ophthalmic emulsion 0,05 %). 30 US 4,839,342 describe the use of cyclosporin for the treatment of immune keratoconjunctivis sicca (KCS) , and a method of increasing tear production for a tear-deficient eye, 5838347_1 (GHMatters) P88392.AU.2 JOSHUAF 2 in particular directed to a patient suffering from a immune mediated dysfunction of lacrimal glands. The method disclosed in this patent includes administering cyclosporine, preferably cyclosporine A, with a pharmaceutical excipient being olive 5 oil, arachis oil, castor oil or mineral oil. Shulin Ding and Orest Olejnik have published a poster in the AAPS annual meeting on November 3, 1997 ; this document relates to castor oil-based oil-in-water emulsions using 0.1 to 0.4% cyclosporine and having a weight ratio 10 cyclosporine/oil of 0.08. This poster discloses that the concentration in cyclosporine A in the oil is optimized at the level of 7.4 % w/w which is below the solubility of the cyclosporine in the specific oil vehicle castor oil (10 % w/w). 15 W02005/032577 describes a method for treating an eye of a human comprising administering an emulsion comprising less than 0.1 % cyclosporine and having a weight ratio cyclosporine/ castor oil of less than 0.08. Although this above cited patent application 20 describes further oils, it is not obvious to one skilled in the art that cyclosporine will have the same behaviour in any oily vehicle, because it does not have the same solubility in all oils, and the bioavailability of the cyclosporine in one oily vehicle does not give information on its bioavailability 25 in another oily vehicle. An emulsion is a system consisting of two immiscible liquid phases, one of which, in fine droplets, is dispersed throughout the other, the system being stabilized by a third component, the emulsifying agent. Emulsions are 30 inherently unstable, and emulsifiers are essential for both their initial formation and long-term stability. Due to their small droplet size, submicronic or nano-emulsions may appear transparent, and Brownian motion 3 prevents sedimentation or creaming, hence offering increased stability. The instability of an emulsion will appear as phase separation due to creaming (density differences), Ostwald 5 ripening (disproportionation) flocculation (aggregation through interparticle collision) , and coalescence (fusion of separate droplets) . An important distinction is that flocculation is often reversible while coalescence is not. The process of droplet coalescence is the normal way in which an 10 emulsion coarsens with time, i.e. the mean particle size of the droplet increases upon storage. Presence of larger aggregates promotes faster creaming and thus facilitates coalescence. This is especially true for polydisperse systems where different creaming rates 15 produce enhanced droplet encounter rates. As a consequence, the droplet size distribution of an emulsion governs emulsion properties such as long-term stability. Therefore, a monomodal droplet population has the benefits of increased stability besides of the drug content 20 uniformity which is essential in pharmaceutical applications for the administration of a correct drug dose. Even though the prior art formulations, such as Restasis®, are currently useful to treat conditions for which few treatment are available, it is known that the 25 bioavailability of the prior art formulations is not optimal, and one of the known drawbacks of Restasis*, for example, is that it may take several weeks to take full effect. In addition, castor oil-based cyclosporine emulsion such as Restasis* presents a bimodal droplet population (Ding, 30 abstract AAPS 1997). Such formulation is expected to have decreased shelf life as a result of the coalescence of the oil droplets. There is therefore still a need for alternative solutions for treating dry eye conditions, with emulsions - 4 having at least the same physico-chemical stability properties and improved bioavailability of the drug. In the sense of this invention, bioavailability means the percentage of drug that can be detected in the target ocular tissue after its 5 administration. Detection of the drug can be accomplished pharmacodynamically (quantification of a biological response to the cyclosporine) or pharmacokinetically (quantification of actual drug concentration). Moreover, patients suffering from dry eye diseases have 10 very sensitive eyes. It would therefore be advantageous if at least preferred embodiments of the present invention were to provide a composition providing comfort as well as therapy. The emulsions of this invention comprise a combination of ingredients, allowing them to respond to both requirements of 15 stability and bioavailability. In a first aspect, the present invention provides an ophthalmic oil-in-water emulsion, which comprises colloid particles having an oily core surrounded by an interfacial film, said emulsion comprising an immunosuppressive agent, oil and 20 tyloxapol, wherein said emulsion comprises 0.5 to 4% of MCT compared to the total weight of the emulsion, and said emulsion being free of castor oil. In some embodiments, the ophthalmic oil-in-water emulsion can be administered topically. 25 In some embodiments, the immunosuppressive agent is selected from the group consisting of cyclosporine, sirolimus or tacrolimus. In some embodiments, the cyclosporine is or includes cyclosporine A. 30 In some embodiments, the amount of immunosuppressive agent is from 0.01 to 4% w/w, preferably 0.05 to 0.3% w/w, of the emulsion. In some embodiments, the amount of tyloxapol is less than 1%, preferably comprised between 0.01 to 0.6%, by weight of the 35 total weight of the emulsion. 5838347_1 (GHMatters) P88392.AU.2 JOSHUAF - 4a In some embodiments, the ophthalmic oil-in-water emulsion further includes one or more oils selected from the group consisting of, olive, soy, corn, mineral, cottonseed, safflower, sesame. 5 In some embodiments, the emulsion is anionic. In some embodiments, the emulsion is cationic. When the emulsion is cationic, the cationic agent may, for example, be comprised between 0.001 and 0.1% w/w, preferably between 0.002 and 0.05% w/w, and still more preferably between 0.003 and 0.03% 10 w/w. In some embodiments, the cationic agent is benzalkonium chloride. In some embodiments, the ophthalmic oil-in-water emulsion is characterized in that it meets the zeta potential stability Test A requirements. 15 In some embodiments, the colloid particles of the emulsion of the invention have an average particle size of equal or less than 1 pim, and its size population distribution is monomodal. In some embodiments, the colloid particles of the emulsion of the invention have an average particle size of equal or less 20 than 1 rim, advantageously equal or less than 300 nm, more advantageously in the range of 100 to 250 nm. In a second aspect, the present invention provides a process of preparation of an ophthalmic oil-in-water emulsion containing an immunosuppressive agent and oil and tyloxapol, 25 comprising the steps of adding the aqueous phase to the oily phase followed by shear mixing and then high pressure homogeneization. In a third aspect, the present invention provides use of an ophthalmic oil-in-water emulsion according to the first 30 aspect for the manufacture of medicament for treatment of eye conditions. In a fourth aspect, the present invention provides a method for the treatment of eye conditions, comprising ocular 5838347_1 (GHMatters) P88392.AU.2 JOSHUAF - 4b topical administration of an ophthalmic oil-in-water emulsion according to the first aspect. In some embodiments, the eye condition is a dry eye disease. In some embodiments, the eye condition is KCS, AKC or 5 VKC. Also described herein are ophthalmic emulsions which comprise colloid particles having an oily core surrounded by an interfacial film, said emulsion comprising at least one immunosuppressive agent, preferably selected from the group 10 consisting of cyclosporine, sirolimus, tacrolimus, in a vehicle comprising an oil and tyloxapol. It has been surprisingly found by the inventors that the use of MCT, a vegetal oil selected among all, provides unexpected stability and bioavailability to cyclosporine 15 containing ophthalmic emulsions. MCT has also been found by the inventors to better solubilize cyclosporine, which may play a role in the observed improved bioavailability of cyclosporine in the emulsions of the invention (Examples 1-6) . According to an embodiment of the invention, the amount of MCT is at least 50 % 20 in weight of MCT by weight of the total oil content of the emulsion. According to a preferred embodiment, the oil content of the emulsion is 100% MCT. 5838347_1 (GHMatters) P88392.AU.2 JOSHUAF - 5 The amount of MCT is from 0.5 to 4 %, preferably 0.9 to 3 %, more preferably 0.9 to 2 % w/w of the emulsion. According to a preferred embodiment of the invention the amount of tyloxapol is less than 1%, preferably comprised 5 between 0.01 to 0.6% by weight of the total weight of the emulsion. Even more surprisingly, the inventors have found that MCT, when combined with tyloxapol, provide even better results in terms of bioavailability of an immunosuppressive agent (Example 10 7) . The inventors have demonstrated a synergistic effect of tyloxapol and MCT in an ophthalmic emulsion of the invention containing an immunosuppressive agent, especially cyclosporine, sirolimus or tacrolimus. Finally, W02005/032577 describes 1.25% castor-oil based 15 emulsions for which increasing the amount of cyclosporine from 0.05% to 0.1% (and therefore the CsA/oil ratio from 0.04 to 0.08) does not improve the overall efficacy in treating dry eye disease. Contrary to this teaching, the Inventors have unexpectedly found that, when increasing the amount of 20 immunosuppressive agent in the emulsions of the invention, an increasing amount of drug is transferred into the ocular target (see example 8) ; this means that the emulsions of the invention are not limited to a specific ratio of immunosuppressive agent/oil. Thus, the emulsions of the invention make it possible 25 to increase the therapeutic amounts of drugs to be delivered of the target site, thus increasing the therapeutic values of the used immunosuppressive agent. According to an embodiment of the invention, the emulsion may further include any other suitable oily components, 30 especially one or more oils selected from the group consisting of, olive, soy, corn, mineral, cottonseed, safflower, sesame. The ophthalmic oil-in-water emulsion of the invention is free of castor oil. 5838347_1 (GHMatters) P88392.AU.2 JOSHUAF 6 In a first embodiment of the invention, the emulsion is anionic. According to a preferred embodiment, the emulsion of the invention includes one or more components selected from the group consisting of phospholipids, cholic 5 acid and its derivatives, metal carboxymethylcelluloses, metal carboxymethylhydroxyethylcelluloses, metal carboxymethylstarchs metal, carboxy methylhydroxyethylstarchs hydrolyzed polyacrylamides and polyacrylonitriles heparin gucoaminoglycans hyaluronic acid chondroitin sulphate, 10 dermatan sulphate, peptides and polypeptides alginic acid, metal alginates homopolymers and copolymers of one or more of: acrylic and methacrylic acids metal acrylates and methacrylates vinylsulfonic acid, metal vinylsulfonate, amino acids, such as aspartic acid, glutamic acid and the like metal 15 salts of amino acids p-styrenesulfonic acid metal p styrenesulfonate, 2-methacryloyloxyethylsulfonic acids, metal 2 -methacryloyloxethylsulfonates, 3 -methacryloyloxy- 2 hydroxypropylsulonic acids metal 3-methacryloyloxy-2 hydroxypropylsulfonates, 2-acrylamido-2-methylpropanesulfonic 20 acids metal 2-acrylamido-2-methylpropanesulfonates allylsulfonic acid metal allylsulfonate and the like. In a second embodiment of the invention, the emulsion is cationic. In this embodiment, it is preferred that the concentration of the cationic agent is comprised between 25 0.001 and 0.1% w/w, preferably between 0.002 and 0.05% w/w and still more preferably between 0.003 and 0.03% w/w. This second embodiment is particularly preferred, because it has been found that the cationic charge of the emulsion improves the bioavailability of the cyclosporine contained in the emulsion. 30 Advantageously, the cationic cyclosporine-containing ophthalmic emulsion of the invention is an oil-in-water type emulsion, which comprises colloid particles having an oily core surrounded by an interfacial film, said emulsion comprising at least one cationic agent, at least one non ionic 7 tensioactive, said emulsion having a positive zeta potential. According to a preferred embodiment, the cationic ophthalmic emulsion of the invention meets the zeta potential stability Test A requirements as described below: 5 Test A consists in measuring the stability of the emulsion zeta potential under thermal stress conditions. Zeta potential of the emulsion is measured at T=0, i.e. as soon as the emulsion has been prepared, the obtained value being named Zo. Glass vials (Type I) of 10ml effective capacity 10 containing between 5-10ml of emulsion and sealed under nitrogen atmosphere (without bubbling) are stored at 80 0 C. Then at T=7 days the zeta potential Z7days is measured. Then at T=15 days the zeta potential Z1sdays is measured. The value 5A = Z 7 h-ZO or Zish-Zo is then calculated. 15 For each measurement of the zeta potential, it is operated as follows: The zeta potential of the emulsion droplet surface is determined by electrophoretic mobility in an apparatus such as a Malvern Zetasizer 2000 (Malvern Instruments, UK) equipped 20 with suitable software and calibrated with the supplied standard. The emulsion is diluted in double distilled water if needed in order to obtain the scattering intensity allowing optimal particle detection. The sample count rate should be 25 between 100 to 1000 KCps, in homodyne detection (if heterodyne detection is used, the contribution of the reference beam should be deduced) . Three consecutive measurements are performed at 25 0 C using a constant cell drive of 150mV. The electrophoretic mobility is converted into zeta potential 30 values through the Smoluchowsky equation, using the dielectric constants and viscosity of water. The measured value corresponds to the average of the 3 obtained values. It is considered that the emulsion meets zeta potential stability Test A if 5A is less than the standard error of 8 measurements, preferably less than 10mV, and even more preferably less than 5mV. In a preferred embodiment of the invention, the emulsion of the invention contains cyclosporine A. .5 Advantageously, the emulsion of the invention contains from 0.01 to 0.4 % w/w, preferably 0.05 to 0.3 % of immunosuppressive agent, preferably cyclosporine, more preferably cyclosporine A. According to another embodiment of the invention, the emulsion comprises an amount of tacrolimus 10 of 0.01 to 0.3 % w/w, preferably 0.05 to 0.2% w/w of the emulsion. According to still another embodiment of the invention, the emulsion comprises an amount of sirolimus of 0.01 to 0.3 % w/w, preferably 0.05 to 0.2% w/w of the emulsion. 15 In another preferred embodiment of the invention, the weight ratio of immunosuppressive agent to oil is from 0.0125 to 0.1. In a particular embodiment of the invention, the emulsion of the weight ratio of immunosuppressive agent to 20 oil is from 0.083 to 0.1. In another particular embodiment of the invention, the emulsion of the weight ratio of immunosuppressive agent to oil is from 0.0125 to 0.05. Advantageously, the emulsion of the invention is submicronic, and in a very preferred embodiment, monomodal submicronic, 25 which means that the colloid particles of the invention are very homogeneous in their size equal or less than 1 Rm, in that they have a droplet size polydispersity index close to 0.2, generally between 0.1 and 0.15. The calculation of the polydispersity index is defined in the ISO standard document 30 13321 :1996 E. Preferably, colloid particles of the emulsion of the invention have an average particle size of equal or less than 1 im, advantageously equal or less than 300 nm, more advantageously in the range of 100 to 250 nm.

9 According to a preferred embodiment of the invention the emulsions of the invention are cationic submicronic emulsions, and do not contain any substances capable of generating a sufficient amount of negative charge 5 for affecting the positive zeta potential of the emulsion. In this embodiment, advantageously, the emulsion does not contain phospholipids. The invention also relates to a process of preparation of the emulsions containing at least one 10 immunosuppressive agent, preferably chosen in the group consisting of cyclosporine, preferably cyclosporine A, sirolimus or tacrolimus and MCT and tyloxapol according to the invention, comprising one step of shear mixing followed by a high pressure homogenization. 15 The best mode of making and using the present invention are described in the following examples. These examples are given .only to provide direction and guidance in how to make and use the invention, and are not intended to limit the scope of the invention in any way. 20 Examples In the following examples, the following abbreviations are used: 25 MCT: Medium chain triglycerides (Soci6t6 des Ol6agineux, France) BAK: Benzalkonium chloride CsA: Cyclosporin A Cremophor: Cremophor EL (BASF, Germany) 30 Lipoid E80 (Lipoid GMBH, Germany) Lutrol: Lutrol F68 (BASF, Germany) Pemulen TR-2 (Noveon, US) Phospholipon 90G (Natterman, Germany) 10 Example 1: Preparation of the emulsions of this invention The oily phase components are successively weighed and then stirred under a slight heating until a limpid and slightly 5 viscous phase is obtained. Aqueous phase components are successively weighed and then magnetically stirred under a slight heating until a transparent, limpid and fluid phase is obtained. Both phases are heated and the coarse emulsion is formed by rapid addition of the aqueous phase in the oily 10 phase and then rapidly heated to 75 0 C. The emulsion is white and slightly transparent. The emulsion droplet size is then decreased by high shear mixing by using for example a POLYTRON PT 6100, followed by its cooling down. Final emulsion is obtained by high pressure homogenization in 15 suitable equipment such as microfluidizer (CS, Avestin) using several continuous cycles. Final emulsion is milky and very fluid. The emulsion temperature is then decreased to 25 0 C, its pH was measured and then adjusted to 6.0, 7.0, 8.0 using a 0.1 M HCl or 0.1 M NaOH solution. Sterilization can be done by 20 autoclave 20 minutes at 121*C or by filtration through a 0.22 um filter. Example 2: Characterization of the emulsions of this invention 25 The mean particle size of the emulsions droplets is determined by quasi-elastic light scattering after dilution in water using for example a High Performance Particle Sizer (Malvern Instruments, UK). This instrumentation is also used to 30 determine the polydispersity index. The electrophoretic mobility is measured at 25*C in an appropriate apparatus such as Malvern Zetasizer 2000 (Malvern Instruments, UK) following a 1:200 dilution in double distilled water and converted into zeta potential through the Smoluchowski equation.

11 CsA in the emulsion is determined by a validated HPLC-UV method. Example 3: Compositions of this invention containing 5 CsA/oil/Tyloxapol Ingredient EM047 EM048 EM049 EM050 EM051 EM052 EM053 CsA 0.025 0.025 0.05 0.05 0.2 0.2 0.1 MCT 0.75 2 1 2 2 4 2 BAK 0.02 0.02 0.02 0.02 0.02 0.02 0.02 Tyloxapol 0.12 0.3 0.16 0.3 0.3 0.3 0.3 Poloxamer 0.1 0.1 0.1 0.1 0.1 0.1 0.1 Vitamin E 0.01 0.01 0.01 0.01 0.01 0.01 0.01 Glycerin 2.25 2.25 2.25 2.25 2.25 2.25 2.25 Purified qs qs qs qs qs qs qs water Example 4: Stability of compositions of this invention Methods: 10 Stability of the emulsions is assessed by measuring physicochemical parameters such oil droplet size, zeta potential and CsA content after sterilization by autoclave or filtration and during an accelerated stability test at 80 0 C. Results: 15 Emulsion EM047 EM048 EM049 EM050 EM051 EM052 EM053 Emulsion TO 172 182 151 211 188 170 204 globule size T7 188 193 173 212 195 201 211 (nm) T14 194 200 177 221 206 195 226 Polydispersity TO 0.155 0.144 0.148 0.116 0.078 0.138 0.108 index Zeta potential TO 20.9 19.6 25.0 17.9 20.1 28.4 23.5 (mV) T7 18.9 19.6 24.7 20.3 21.9 24.5 23.2 12 T14 16.9 19.4 19.2 18.5 19.0 23.0 ND CsA (% of TO 100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 100.0% initial T7 96.4% 97.5% 97.8% 95.8% 92.8% 96.0% 97.4% content) T14 94.6% 96.4% 96.0% 94.2% 88.2% 96.6% 94.8% ND: Not determined Conclusions: MCT/Tyloxapol-based emulsions of CsA produced by the process 5 of the invention including a shear mixing step followed by a high pressure homogenisation step are stable following preparation and for at least two weeks at 80 0 C. Example 5: Comparative example - Compositions containing 10 CsA/castor oil EMCIC007 EMCIC003 Restasis* Ingredient % w/w Ingredient % w/w Ingredient % w/w CsA 0.2 CsA 0.2 CsA 0.05 Castor oil 2.5 Castor oil 2.5 Castor oil 1.25 Oleylamine 0.12 Stearylamine 0.12 Phospholipon 0.5 Lipoid E80 0.5 Polysorbate 1 90G 80 Lutrol F68 0.42 Lutrol F68 0.42 Pemulen TR- 0.05 2 Glycerin 2.25 Glycerin 2.25 Glycerin 2.2 Purified qs Purified qs Purified qs water water water Example 6: Stability of emulsions containing CsA/castor oil Methods: 15 Similar to Example 1-4 Results: 13 Emulsion EMCIC003 Restasis* EMCIC007 Emulsion TO Broken 279 Broken globule emulsion emulsion size (nm) T7 ND 514 (56%) and ND 2467 (43%) T14 ND Broken ND emulsion Zeta TO ND -43.7 ND potential T7 ND -41.6 ND (mV) T14 ND ND ND CsA (% of TO ND 100 . 0% ND initial T7 ND 55.3% ND content) T14 ND ND ND ND: Not determined. Percentages indicate the relative populations as measured by scattered light. 5 Conclusions: Castor oil-emulsions of CsA produced by homogenisation techniques as described above are not stable following preparation or after one week at 80 0 C. 10 Example 7: Efficacy of emulsions of this invention as evaluated by their pharmacokinetic parameters. Methods: Ninety-six (96) pigmented rabbits from the HYRNZ104 strain 15 were randomly divided into two (2) treatment groups of forty eight (48) animals, each group being sub-divided into eight (8) subgroups of six (6) animals corresponding to eight (8) time-points (0.33, 0.66, 1, 2, 4, 8, 12 and 24 hrs). The animals received a single instillation into right eyes of the 20 above described EM050 or Restasis* (Allergan, US) . At the 14 corresponding time-points, animals were euthanized and the conjunctiva was sampled. Content of CsA was determined by HPLC-MS. 5 Results: 350 --- 0.05% CsA in MCT/Tyloxapol emulsion 300 ---- 0.05% CsA in Castor oil emulsion 0 250 * 200 0 150 to 100 50 0 0 4 8 12 16 20 24 Time (hr) Conclusions: The emulsions described in this invention show tissue 10 concentrations at least as good as the castor oil-based marketed emulsion Restasis*. Example 8: Impact of the CsA/oil ratio in the emulsions of this invention on their efficacy as evaluated by their 15 pharmacokinetic parameters. Methods: One hundred and forty-four (144) pigmented rabbits from the HYRNZ104 strain were randomly divided into three (3) treatment 20 groups of forty eight (48) animals, each group being sub divided into eight (8) subgroups of six (6) animals corresponding to eight (8) time-points (0.33, 0.66, 1, 2, 4, 8, 12 and 24 hrs) . The animals received a single instillation into right eyes of the above described EM048, EM050 or EM053 15 (0.025, 0.05 and 0.1% w/w CsA in 2% MCT emulsions) . At the corresponding time-points, animals were euthanized and the conjunctiva was sampled. Content of CsA was determined by HPLC-MS. The area under the curve (AUC) indicating the animal 5 exposure to the compound was calculated using the trapezoidal rule. Results: 3,000 -Z 2,000 C-) M 1,500- 1,000 c 2 500 0 , 0 0.01 0.02 0.03 0.04 0.05 0.06 CsA/MCT ratio 10 Conclusions: The emulsions described in this invention show a linear correlation between the CsA/oil ratio and the conjunctival concentration. As the clinical efficacy is correlated to the 15 CsA concentration, it can be assumed that this particular vehicle will provide increasing therapeutic value for rising amounts of CsA Example 9: Ocular tolerance of the emulsions of this 20 invention The aim of this study was to determine the ocular tolerance of the emulsions of this invention (EM048, EM050 and EM053; see composition in previous examples) after multiples daily ocular 16 topical administrations for 28 consecutive days into the right eye of albino rabbits. Methods: 5 Ten (10) New Zealand White albino rabbits per group (5 males and 5 females) were involved in this study. Treatments (50 01 ocular topical administrations) were given four times a day for 28 consecutive days. General tolerance (body weight, food and water consumptions, general aspect, clinical signs, 10 hematology and blood biochemistry), ocular tolerance (observations with an ophthalmoscope, slit lamp examinations and ocular histology) and necropsy (gross macroscopic examination, main organ weights) were investigated. A statistical analysis (MANOVA LSD test) was also performed on 15 body and organ weights, on food and water consumption data, and on haematological and biochemical parameters Results: General behaviour, food consumption and water consumption, 20 body weight, organ weights were unaffected by treatments. There were no remarkable observations at necropsy due to treatment. Ophthalmologic observations and microscopic examinations of the eyes and adnexa revealed no adverse effects. Ocular reactions were confined to slight conjunctival 25 redness that were observed in all animals in the study and are commonly observed in rabbits after multiple instillations of ophthalmic products Conclusions: 30 The emulsions described in this invention are well tolerated following chronic topical administration.

- 16a In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or 5 "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention. It is to be understood that, if any prior art publication 10 is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art, in Australia or any other country. 5838347_1 (GHMatters) P88392.AU.2 JOSHUAF

Claims (25)

1. An ophthalmic oil-in-water emulsion, which comprises colloid particles having an oily core surrounded by an 5 interfacial film, said emulsion comprising an immunosuppressive agent, oil and tyloxapol, wherein said emulsion comprises 0.5 to 4% of MCT compared to the total weight of the emulsion, and said emulsion being free of castor oil.

2. The ophthalmic oil-in-water emulsion according to 10 claim 1, which can be administrated topically.

3. The ophthalmic oil-in-water emulsion according to claim 1 or 2, wherein the immunosuppressive agent is selected from the group consisting of cyclosporine, sirolimus or tacrolimus. 15

4. The ophthalmic oil-in-water emulsion according to any one of claims 1-3 wherein the cyclosporine is or includes cyclosporine A.

5. The ophthalmic oil-in-water emulsion according to any one of claims 1-4 wherein the amount of immunosuppressive agent 20 is from 0.01 to 4% w/w of the emulsion.

6. The ophthalmic oil-in-water emulsion according to claim 5 wherein the amount of immunosuppressive agent is from 0.05 to 0.3% w/w of the emulsion.

7. The ophthalmic oil-in-water emulsion according to any 25 one of claims 1-6 wherein the amount of tyloxapol is less than 1% by weight of the total weight of the emulsion.

8. The ophthalmic oil-in-water emulsion according to claim 7 wherein the amount of tyloxapol is comprised between 0.01 to 0.6% by weight of the total weight of the emulsion. 30

9. The ophthalmic oil-in-water emulsion according to any one of claims 1-8, further including one or more oils selected from the group consisting of, olive, soy, corn, mineral, cottonseed, safflower, sesame.

10. The ophthalmic oil-in-water emulsion according to any 35 one of claims 1-9 wherein the emulsion is anionic. 5838347_1 (GHMatters) P88392.AU.2 JOSHUAF - 18

11. The ophthalmic oil-in-water emulsion according to any one of claims 1-10 wherein the emulsion is cationic.

12. The ophthalmic oil-in-water emulsion according to claim 11 wherein the cationic agent is comprised between 0.001 5 and 0.1% w/w.

13. The ophthalmic oil-in-water emulsion according to claim 12 wherein the cationic agent is comprised between 0.002 and 0.05% w/w.

14. The ophthalmic oil-in-water emulsion according to 10 claim 13 wherein the cationic agent is comprised between 0.003 and 0.03% w/w.

15. The ophthalmic oil-in-water emulsion according to any one of claim 11-14 wherein the cationic agent is benzalkonium chloride. 15

16. The ophthalmic oil-in-water emulsion according to any one of claim 11-15, characterized in that it meets the zeta potential stability Test A requirements.

17. The ophthalmic oil-in-water emulsion according to any one of claims 1-16, wherein the colloid particles of the 20 emulsion have an average particle size of equal or less than 1 rim, and its size population distribution is monomodal.

18. The ophthalmic oil-in-water emulsion according to any one of claims 1-17 wherein the colloid particles of the emulsion have an average particle size of equal or less than 1 p.m. 25

19. The ophthalmic oil-in-water emulsion according to claim 18 wherein the colloid particles of the emulsion have an average particle size of equal or less than 300 nm.

20. The ophthalmic oil-in-water emulsion according to claim 19 wherein the colloid particles of the emulsion have an 30 average particle size in the range of 100 to 250 nm.

21. Process of preparation of an ophthalmic oil-in-water emulsion containing an immunosuppressive agent and oil and tyloxapol, comprising the steps of adding the aqueous phase to the oily phase followed by shear mixing and then high pressure 35 homogenization. 5838347_1 (GHMatters) P88392.AU.2 JOSHUAF - 19

22. Use of an ophthalmic oil-in-water emulsion according to any one of claims 1-20 for the manufacture of medicament for treatment of eye conditions.

23. A method for the treatment of eye conditions, 5 comprising ocular topical administration of an ophthalmic oil in-water emulsion according to any one of claims 1-20.

24. Use according to claim 22 or the method according to claim 23, wherein the eye condition is a dry eye disease.

25. Use according to claim 22 or the method according to 10 claim 23, wherein the eye condition is KCS, AKC or VKC. 5838347_1 (GHMatters) P88392.AU.2 JOSHUAF