Title of Invention	OPHTHALMIC OIL-IN-WATER EMULSIONS CONTAINING PROSTAGLANDINS
Abstract	The present invention refers to an oil-in water emulsion for ophthalmic application comprising at least one prostaglandin as active agent and a surfactant component comprising a combination of at least two non-ionic surfactants. The emulsion is suitable for medical applications particularly for the treatment of glaucoma, and has an increased chemical stability of the prostaglandin active agent so to allow long-term storage also at room temperature.

Title of Invention

OPHTHALMIC OIL-IN-WATER EMULSIONS CONTAINING PROSTAGLANDINS

Abstract

The present invention refers to an oil-in water emulsion for ophthalmic application comprising at least one prostaglandin as active agent and a surfactant component comprising a combination of at least two non-ionic surfactants. The emulsion is suitable for medical applications particularly for the treatment of glaucoma, and has an increased chemical stability of the prostaglandin active agent so to allow long-term storage also at room temperature.

Full Text	Ophthalmic oil-in-water emulsions containing prostaglandins Description The present invention refers to an oif-in-water emuision comprising at least one prostaglandin as active agent and a surfactant component comprising a combination of at least two non-ionic surfactants. The emulsion is suitable for ophthalmic applications, particularly for the treatment of glaucoma and/or ocular hypertension, and has an increased chemical stability of the prostaglandin active agent so to allow long-term storage, e.g. at room temperature. Prostaglandins are chemical moieties, found in tissues or organs of humans, exhibiting a wide range of physiological activities. Some prostaglandin synthetic F 2a analogues have been known to be useful as ophthalmic phannaceutical agents, specifically as ocular hypotensive antigiaucoma agents. For example latanoprost, travoprost, bimatoprost and unoprostone have been introduced in the market under the trade marks respectively of Xalatan, Travatan, Lumigan and Rescula as ophthalmic eye drop solutions for the treatment of ocular hypertension and glaucoma. Problems associated with these prostaglandin analogues are their rather poor water solubility and their chemical instability especially in aqueous solutions. Consequently many different ophthalmic formulations have been proposed to overcome such problems. EP-A-0 435 682 describes the use of inclusion complexes of prostaglandins with cydodextrins which are water soluble complexing agents with an hydrophobic cavity, wherein hydrophobic drugs such as prostaglandins are hosted inside this cavity leading to higher water solubility and higher stability in water. The stability and clinical efficacy of a latanoprost ophthalmic formulation containing cyclodextrin was shown in The Journal of Clinical Phamiacoiogy, 47, 121-126,2007. The use of modifiecl cyclodextrins (i.e. etherised cyctodextrins) to complex and stabUize prostaglandins is proposed in EPA-0 330 511. The stabilization of the aqueous ophthalmic solution of latanoprost by adjusting the pH of the solution to a value in the range 5 - 6.25 or by the addition of e-aminocaproic acid has been described in EP-A-1 532 981. Solubility and stability of prostaglandins are also improved also by the addition of polyethoxylated castor oil to the aqueous solution (US 5.849,792). An ophthalmic fonnuiation of prostaglandins is proposed In US 2004/076678: acrylate, cellulose or other polymers are added to the aqueous solution of prostaglandins to prolong the efficacy when administered to the eyes. An example of an ophthalmic emulsion is given in US 3,608,073 covering a formulation containing pilocarpine, an oil, an aqueous phase and an interface agent. EP-A-0 521 799 teaches the preparation of submicron ophthalmic emulsions using an oil. an amphoteric surfactant and a phospholipid. Oil, aqueous phase, phospholipid are the components subjected to high pressure homogenization to generate submicron emulsions containing flurbiprofen for opthalmic application (US 5171566). The addition of hydrophobic suspending particles to stabilize submicron emulsions is suggested in US 2003/15471. A microemulsion, obtained by high pressure homogenisation, of latanoprost is described in Int. J. Pharm., 305, 176-179. 2005: Stabilization is obtained by the use of polyvinylalcohol as emutsifier. Benzalkonium chloride is one of most used antimicrobial preservatives for ophthalmic formulations but it has been also widely used in the formation of ophthalmic microemulsions (US 5.698,219) thanks to its positive charge which stabilizes the droplets; this positive charge can be provided also by other cationic agents (WO 2006/050838). The use of positively charged microemulsions for the administration of prostaglandins is described in WO 2006/050836 or WO 2007/042262: the interface film Is formed by the combination of a non-ionic surfactant and a cationic agent such as quatemary ammonium compounds (including benzalkonium chloride), amino alcohols, biguanide salts. The resulting emulsions have a zeta potential of at least 16 mV. However the use of cationic agents for ophthalmic use must be carefully checked in order to guarantee ocular tolerability (Eur.J.Pharm.Biopharm., 53, 263-280. 2002). A combination of benzalkonium chloride with specific number of carbon atoms, a surfactant, a tonicity agent is described in EP-A-1 547 599 describing a stable latanoprost ophthalmic solution. EP-A-0 458 588 discloses an ocuto-hypotensively synergistic combination of a 13.14-dihydro-15-keto-20-C1-6 alkyl prostaglandin and a potyoxyethyiene sorbitan unsaturated C10-C24 aliphatic acid monoester for the manufacture of a medicament useful in the treatment of ocular hypertension. EP-A-1 655 021 discloses an oil-in-water emulsion useful as a delivery vehicle of hydrophobic ingredients such as pharmaceutical drugs, wherein the emulsion particles have a net positive charge and comprise a cationic agent. EP-A-1 681 059 describes a pharmnaceutical composition comprising an oii- in-water emulsion containing a prostaglandin F2a derivative, an oil. a water- soluble polymer and water. The water-soluble polymer may be a polyvinyl compound, a water-soluble cellulose compound or a polysaccharide. The oil may e.g. be an animal or vegetable oil and/or medium chain fatty add triglyceride. US 2006/0182781 describes an ophthalmic microparticle composition, wherein the microparticles comprise a polymer matrix and an active ingredient, e.g. a prostaglandin. WO 2004/082625 and US 2007/0036829 describe self-emulsifying ophthalmic compositions comprising oil globules dispersed in an aqueous phase, wherein the globules comprise a surfactant component and a polar oil component. The oil component is present in an amount of up to 1.25% (w/ w) of the total emulsion. US patent 5,827,835 descrit)es a non-toxic emulsion composition comprising a non-ionic cellulose ether having a molecular weight of at least 30 kD, an oil, water and optionally an emulsifying agent. The emulsion may further comprise pharmaceutical drugs such as prostaglandins. WO 02/064166 described a composition comprising at least one monoglyceride, at least one emulsifier, an aqueous solution and at least one organic solvent. KR 2003/0046553 discloses a temperature-sensitive emulsion composition for external use comprising a prostaglandin E1 as an active ingredient. The composition is applied to the skin and forms a gel at body temperature. The formation of a gel, however, is undesirable for ocular applications. It has been now been unexpectedly found that a combination of an oily phase containing a prostaglandin active agent, an aqueous phase, a surfactant component comprising a combination of at least two non-ionic surfactants can spontaneously generate stable sub-micron emulsions for ophthalmic use. These emulsions preferably have a substantially neutral electrochemical charge (zeta potential) and have excellent stability characteristics both in terms of physical properties of the micro-emulsion and the chemical stability of the prostaglandin active agent. The emulsions may be prepared by simply mixing the components, whereby spontaneous micro- emulsification occurs. It is not necessary to apply high energy processes such as high pressure homogenisation and/or sonication. This contributes further to the stability of the system. Thus, the subject-matter of the present invention is an oil-in-water emulsion having a substantially neutral zeta potential for ophthalmic application comprising as components (i) a dispersed oily phase, (ii) at least one prostaglandin as active agent, which is dissolved in the oily phase component (i), (iii) a surfactant comprising a combination of at least two non-ionic surfactants, and (iv) a contiguous aqueous phase, optionally comprising formulation agents. The emulsion of the present invention is suitable for use as a pharmaceutical formulation, particularly as an ophthalmic formulation. Preferably, the oil-in- water emulsion is used for the prevention, alleviation and/or treatment of ocular diseases such as glaucoma and/or ocular hypertension. The emulsion is suitable for single-dose applications or multiple-dose applications. The emulsions of the present invention are characterized by a high chemical stability as measured by determination of the active ingredient recovery after extended storage time, e.g. by HPLC. The recovery of the active ingredient after storage at 25oC for 6 months, more preferably 9 months and most preferably 12 months is at least 80%, more preferably at least 90% and most preferably at least 95%. The recovery of the active ingredient after storage at 45oC for 14 days, preferably for 30 days and more preferably for 45 days is at least 80%, preferably at least 90% and most preferably at least 95%. Further, the formulations of the present invention are characterised by a high physical stability as measured by droplet size determination. Preferably, the emulsions are stable at 25°C for at least 6 months and at 45°C for at least 45 days. It was found in an especially preferred embodiment that the emulsions have a chemical and physical stability of at least 6 months, preferably at least 9 months and more preferably at least 12 months at 25°C. Surprisingly it was found that the chemical and physical stabHity is not decreased by opening the bottles. The emulsion of the present invention Is preferably a micro-emulsion, wherein the average size of the oil droplets is less than 1 µm. More preferably, the average droplet size of the oil droplets Is 700 nm or less. It is further preferred that the emulsion of the present invention does not change its physical state from 4 ° - 45 °C, and particulariy does not form a gel. Preferably the emulsion is characterised by having a substantially neutral zeta potential, i.e. a zeta potential between -10 mV and +10 mV, preferably between -4 mV and +4 mV and more preferably between -2mV and +2mV. The oily phase (i) is preferably present in an amount of at least 3% (w/w), more preferably at least 5% (w/w) based on the total weight of the emulsion. The upper amount of the oily phase is preferably 25% (w/w) and more preferably 20% (w/w) based on the total weight of the emulsion. The oily phase component (i) is selected from pharmaceuticaity acceptabie oils, e.g. animal oils, vegetable oils, synthetic oils or mixtures thereof. Preferably, the oily phase comprises pharmaceutically acceptable fatty acid esters, e.g. fetty triglycerides or fatty acid monoesters. More preferably, the components of the oily phase are chosen on the basis of four factors: (1) acceptability for application to the eye (2) a good solubilisation degree of at least 0.1 mg/ml, preferably at least 2 mg/ml, and more preferably at least 10 mg/ml for the prostaglandin component (ii); (3) chemical stabilisation of the prostaglandin component (ii) as described above, (4) a strong oil-water partitioning effect in favour of the oil, preferably at least log P = 0.5 and more preferably at least log P = 2. Specific examples of suitable olly phase components are ethyl oleate, Migtyol812, i.e. a mixture of the C8-10 fatty acid triglycerides, ricinus oil, com oil or mixtures thereof. The oil-in-water emulsion of the invention comprises at least one prostaglandin as active agent. Preferably, the prostaglandin is a lipophilic prostaglandin, e.g. a prostaglandin F2a analogue such as latanoprost, travoprost, bimatoprost, unoprostone or mixtures of two or more thereof. More preferably, the prostaglandin component is latanoprost. The prostaglandin is preferably present in an amount of 0.001-5% (w/w), more preferably 0.002-0.1% (w/w) based on the total weight of the emulsion. This surfactant component (ii) comprises a combination of at least two non- Ionic surfactants. The choice of the combination of the two surfactants is preferably done on the basis of the following considerations: (1) only non-ionic surfactants acceptable for ocular application (eye tolerability) are used; (2) the combination and amounts of surfactants is chosen such that the first non-ionic surfactant is added either to the oil or water phase, the oil and water phase are mixed and the second non-ionic surfactant is added to the mixture in a quantity sufficint to generate homogeneous oil/water emulsions without phase separation or formation of large visible droplets and wherein the average droplet size determined by laser light scattering analysis is preferably less than 1 pm and more preferably 700 pm or less. The non-ionic surfactants of the surfactant component (ii) may be selected from lipophilic non-ionic surfactants, hydrophilic non-ionic surfactants, or combinations thereof. Preferably, the non-ionic surfactants of the surfactant component (iii) have a combined total HLB value of at least 10. more preferably of at least 13 and preferably up to 20 and more preferably up to 18. The surfactants are present in an amount which promotes spontaneous emulgation. Preferably, the surfactant component comprises first and second non-ionic surfactants which are present each in amounts of 0.1-10% (w/w) based on the total weight of the emulsion. The combined amount of non- ionic surfactants is preferably from 1-20% (w/w), more preferably from 2-12% (w/w) based on the total weight of the emulsion. The non-ionic surfactants are preferably chosen from polyoxyethylene fatty acid esters, e.g. polyoxyethylene sorbitan, mono- or polyesters and/or polyoxyethylene fatty alcohol ethers. Preferably, the non-ionic surfactants of the surfactant component (iii) are selected from the group consisting of polyoxyethylene (20) sorbitan monooleate (Tween 80) , polyoxyethylene (20) sorbitan monolaurate (Tween 20), polyoxyethylene (2) cetyiether (Brij 52), polyoxyetylene (10) cetyiether (Brij 56), polyoxyettiylene (20) cetyiether (Brij 58). More preferably, the combination of non-ionic surfactants of the surfactant component (iii) is selected from the combinations polyoxyethylene (20) sorbitan monooleate (Tween 80) / polyoxyethylene (20) sorbitan monolaurate (Tween 20), polyoxyethylene (20) sorbintan monooleate (Tween 80) / polyoxyl (2) cetyiether (Brij 52), polyoxyl (2) cethylether (Brij 52) / polyoxyl (20) cethylether (Brij 58) and polyoxyl (20) cethylether (Brij 58) / polyoxyl (10) cethylether (Brij 56). The oil-in-water emulsion of the invention is preferably free from cationic surfactants, anionic surfactants, short-chain, e.g. C1-4 monohydric alcohols, fatty acids, e.g. C4-8 fatty acids or from the class of lecithins or/and phospholipids. Such compounds may present problems of eye compatibility or physical/chemlcai instability. The emulsion may, however, comprise other agents commonly used in ophthalmic formulations, e.g. buffer agents such as phosphate salts, citrate salts etc., isotonic agents such as glycerol, sorbitol, glucose, sodium chloride etc., viscosity-increasing compounds such as hydroxypropylcellulose or other water-soluble cellulose derivatives, polymethylmethacrylate or other poiyacryiic add derivatives, chitosan, hyaluronic acid, poiyvinytpyrrolidone etc., antimicrobial preservatives, particulariy chlorobutanol, antioxidants or stabilizers. The emulsion may comprise the prostaglandin as the only active agents. In different embodiments, the emulsion may comprise one or more further active agents, particulariy hydrophilic active agents which are preferably present in the aqueous phase. Preferred examples of further active agents are as follows: - Beta blodcers, e.g. Timolol; Levobunotol; Betaxolol etc. - Anti-inflammatory agents, e.g. Ketorolac, Beta- or Dexamethasone.etc. - Anti-viral agents, e.g. aciclovir, etc. - Topical ocular anaesthetic agents, e.g. Oxybuprocaine, etc. - Anti-allergic agents, e.g. Olopatadine, Azelastine, Epinastine, Lodoxamide, etc. - Anti-Dry Eye agents, e.g. hyaluronic add, acetylcysteine, polyvinyl alcohol, etc. - Other 10P Reducing Drugs, e.g. Brimonidine, Brinzoiamide, Dorzolamide, etc. - Agents for emergency treatment of glaucoma, e.g.Pilocarpine The aqueous phase (iv) of the emulsion is preferably a pharmaceutically acceptable aqueous phase, which is preferably selected from the group consisting of sterilized water, purified water or any other type of water suitable for ophthalmic application. The aqueous phase is preferably present in an amount of 30-95% (w/w), more preferably in an amount of 50-95% (w/ w) based on the total weight of the emulsion. The amount of the aqueous phase also Includes the weight of standard ophthalmic agents, buffers, preservatives, isotonic agents etc., which are optionally added. In an especially preferred embodiment, the components (i)-(iv) are present in the following amounts: (i) 5.0 - 20.0% (w/w) of the oily phase, (ii) 0.001 - 5.0% (w/w) of the prostaglandin active agent, (ill) 0.1 - 10.0% (w/w) of the first non-ionic surfactant and 0.1 -10.0% (w/w) of the second non-ionic surfactant, and (iv) 50.0 - 95.0% (w/w) of the aqueous phase; based on the total weight of the emulsion. The above-described components, when mixed, spontaneously generate stable sub-micron emulsions without the need of high energy shear procedures. For example, the emulsions may be prepared by a method comprising the steps: (a) optionally solubilising formulation agents in the aqueous phase, (b) solubilising the first non-ionic surfactant either in the aqueous or oily phase, (c) solut>iiising the second non-ionic surfactant either in the aqueous or oily phase, (d) solubilising the prostaglandin active agent in the oily phase, and (e) mixing the oily phase with the aqueous phase. The mixing step is preferably canied out with standard mixing procedures, e.g. using paddle mixers, magnetic stirres, homogenizers etc. The use of high energy mixing procedures such as high pressure homogenisation or sonication can be avoided, However, it is possible to use high energy mixing procedures such as high pressure homogenisation or sonication. For pharmaceutical purposes, the emulsion is preferably prepared using sterile components and devices. All steps of the manufacturing process are preferably performed under aseptic conditions and the final formulations are tested following the official pharmacopeial requirements. If the emulsion is formulated as a multiple-dose preparation, an antimicrobial preservative such as chlorobutanol is added. The invention is further described by the following examples, which should in no way be considered as limiting. EXAMPLE 1 An o/w submicron emulsion was prepared by mixing with a paddle mixer an oil/surfactants solution (Ethyl oleate:Tween 80:Tween 20* 1:1:0.5 w:w:w) containing Latanoprost dissolved at a concentration of 0.44mg/ml with a physiological aqueous phase (0.9%NaCI/ pH 7.4). The ratio oil phase to water phase was approximately 1:20 (wt./wt.). The composition of the resulting submicron emulsion was: Latanoprost 0005g Ethyl oleate 4.51 g Polyoxyethytene (20) sorbitan monooleate (Tween 80) 4.51 g Polyoxyethylene (20) sorbitan monoolsurate (Tween 20) 2.30g Physiological Solution (0.9%NaCI, pH=7.4) 88.7g EXAMPLE 2 An o/w submicron emulsion was prepared by mixing with a magrtetic stirrer an oil/surfactants solution (Miglyol 812:Tween 80:Tween 20 1:1.5:1) containing Latanoprost dissoved at a concentration of 0.33mg/ml with a phoshphate buffer aqueous solution (sodium dihydrogen/sodium monohydrogenphosphate) containing an isotonic agent (sorbitol). The ratio between the oil phase and the water phase was approximately 1:20 (wt./wt.). The composition of the resulting submicron emulsion was: Latanoprost 0.005g Miglyol 812 4.28g Polyoxyethylene (20) sorbitan monooleate (Tween 80) 6.42g Polyoxyethylene (20) sorbitan monoolaurate (Tween 20) 4.28g Phosphate buffer aqueous solution 85.02g (sodium dihydrogen/sodium monohydrogenphosphate, sorbitol) EXAMPLES A fluid o/w submicron emulsion was prepared by mixing using a paddle mixer a surfactant /oil solution (Ricinus oil:Brij 56* 1:0.036) containing Latanoprost dissolved at a concentration of 039 mg/ml with a physiological aqueous phase (0.9%NaCI, pH 7.4) containing Brij 58* at the percentage of 4% (wt./wt.)- The ratio between the oily phase and the aqueous phase was approximately 1:10 (wt./wt.). The composition of the resulting submicron emulsion was: Latanoprost 0.001g Ricinus oil 17.9g Polyoxyl (20) cetyl ether (Brij 58) 6.98g Polyoxyl (10) cetyl ether (Brij 56) 0.64g Physiological aqueous phase (0.9%NaCI, pH=7.4) 174.5g EXAMPLE 4 An o/w submicron emulsion was prepared by mixing with a paddle mixer an oil/surfactants solution (Ethyl oieate: Tween 80:Tween 20 1:1:0.5) containing Latanoprost dissolved at a concentration of 0.45 mg/mi with a physiological aqueous phase (0.9%NaCI, pH 7.4) containing an antimicrobiai agent (Chiorobutanoi 0.5%). The ratio between the oil phase and the water phase was approximately 1:20 (wt:wt.). The composition of the resulting submicron emulsion was: Latanoprost 0.0075g Ethyl oieate 6.62g Polyoxyethylene (20) sorbitan monooleate (Tween 80) 6.62g Polyoxyethylene (20) sorbitan monoolaurate (Tween 20) 3.31 g Chiorobutanoi 0.80g Physiological aqueous phase (0.9%NaCI, pH=7.4) 132.6g EXAMPLE 5 An o/w submicron emulsion was prepared by nnixing with a shear mixer an oit/surfactant solution (Ethyl oteate:Brij 52* 1:0.045) containing Latanoprost dissolved at a concentration of 0.59 mg/mi with a citrate buffer solution (citric acid, sodium citrate) contahiing Brij* 58 at the percentage of 4% (wt./wt.). The citric buffer solution had sorbitol as isotonic agent dissolved. The ratio between the oily phase and the aqueous phase was approximately 1:20 (wt./ wt.). The composition of the resulting submicron emulsion was: Latanoprost 0.0050g Ethyt oleate 4.65g Polyoxyl (20) cetyl ether (Brij 58) 3.66g Polyoxyl (2) cetyl ether (Brij 52) 0.21 g Citrate buffer (citric acid, sodium citrate, sorbitol) 91.5g EXAMPLE 6 A fluid o/w submicron emulsion was prepared by mixing with a paddle mixer a surfactant /oil solution (Ricinus oil:Brij52® 1:0.05) containing Latanoprost dissolved at a concentration of 0.40mg/ml with a citrate buffer (citric add, sodium citrate) solution containing Brij 58* at the percentage of 4% (wt./wt.). The citric buffer solution had glycerol as isotonic agent dissolved. The ratio between the oily phase to the aqueous phase was approximately 1:10 (wt./wt.). The composition of the resulting submicron emulsion was: Latanoprost 0.01 g Ricinus oil 17.3g Polyoxyl (20) cetyl ether (Brij 58) 7.0g Polyoxyl (2) cetyl ether (Brij 52) 0.84g Citrate buffer (citric acid, sodium citrate, glycerol) 174.8g EXAMPLE 7 A o/w subinicroemulsion was prepared by mixing using a shear mixer an oil/ surfactant solution (Ethyl oleate: Tween 80:Brij 52 1:1:0.04) containing Travoprost dissolved at a concentration of 0.41mg/mi with a physiological solution (pH 7.4, 0.9% NaCI). The ratio of oil phase:water phase was approximately 1:20 (wt./wt.). The composition of the resulting submicron emulsion was: Travoprost 0.0040g Ethyl oleate 4.51 g Poiyoxyethytene (20) sorbitan monooleate (Tween 80) 4.48g Polyoxyl (2) cetyl ether Brij 52) 0.18g Physiological Solution (0.9%NaCI, pH=7.4) 90.3g EXAMPLE 8 A fluid o/w submicron emulsion was prepared by mixing vtnth a paddle mixer an oil/surfactants phase (Mygliol 812:Tween80:Tween20* 1:1.5:0.5) containing Bimatoprost dissolved at a concentration of 2.3mg/ml with a physiological aqueous phase (0.9%NaCI, pH 7.4) containing an antimicrobial agent (Chlorobutanol 0.5%). The ratio oil/water was approximately 1:20 (wt.:wt.). The composition of the resulting submicron emulsion was: Bimatoprost 0.06g Mygliol 812® 8.46g Polyoxyethylene (20) sorbitan monooleate (Tween 80) 12.89g Polyoxyethylene (20) sorbitan monooleate (Tween 20) 4.40g Chlorobutanol 1 .Og Physiological aqueous phase (0.9%NaCI, pH=7.4) 173.1 g The following example has been prepared for comparison reasons with a low oil/water ratio to show the effect of a specific range of oil/water ratios on the prostaglandin stability. COMPARATIVE EXAMPLE A An o/w submicron emulsion was prepared by mixing with a paddle mixer an oil/surfactants solution (Ethyl oleater:Tweeh 80:Tween 20 1:1:0.5) containing Latanoprost dissolved at a concentration of 0.79 mg/ml with a physiological aqueous phase (0.9%NaCI, pH 7.4). The ratio of the oil phase to the water phase was 1:40 (wt.:wt.). The composition of the resulting submlcron emulsion was: Latanoprost 0.0125g Ethyl oleate 6.37g Poiyoxyethytene (20) sorbitan monooleate (Tween 80) 6.37g Polyoxyethylene (20) sorbitan monoolaurate (Tween 20) 3.15g Physiological Solution (0.9%NaCI. pH=7.4) 234.1 g EXAMPLE 9 CHARACTERiZATiON TESTS The o/w submicron emulsions prepared as described in the above examples were characterized from a physico-chemical point of view (the size and the electrochemical charge of the sub-micron emulsions, the chemical stability of prostaglandin at different storage conditions, the antimicrobial efficacy). The resulting data are reported here as shown in the following paragraphs and tables. 9.1 Size and electrochemical charge determination of the droplets of the submicron emulsions of the invention The size and electrochemical charge (zeta potential) of the droplets of the submicron emulsions prepared as shown in the Examples 1-8 and Comparative Example A were detemnined by Dynamic Laser Light Scattering (Malvem Instalments. Zetasizer Nano ZS). Zeta potential measuremente were carried out with the Zetasizer NanoZS of the Malvem Instruments Ltd (UK). The sample (0.75 ml) to be analyzed was placed, by usir^g a syringe to avoid bubbles, in a folded capillary cell fitted with electrodes. The cell was inserted in the instrument and the measurement sequence was started automatically; all the individual measurement runs were accumulated together and then summed to give the final Zeta potential result. It was not necessary to dilute the samples for the analysis and consequently there was no risk to artificially change the characteristics of the sample. The data are reported in Table 1. Table 1: Droplest size and Zeta potential of the submicron emulsions of the invention All the formulations prepared resulted to be homogeneous fluid dispersions, with no separation of phases or formation of visible large droplets. Actually the data reported in Table 1 confirm the formation of sub-micron emulsions with droplets size at least below 700 nm; the electrochemical charge (zeta potential) values were all approximately equal to 0, indicating an almost neutral charge as a result of the use of non-Ionic surfactants as interface agents. 9.2 Chemical Stability studies of Latanoprost Stability studies at the storage conditions of 45°C, 25°C and 4°C were carried out on the Latanoprost submicron emulsion of Example 1. The sample was preserved in a glass container and a commercial Latanaprost ophthalmic aqueous solution (Xalatan) was used as the reference product. At 45°C the submicron emulsion of Example 1 was placed not only in a glass vial but also in two types of plastic bothes. The Xaiatan formulation is a simple buffered isotonic aqueous solution made of the following components: Latanoprost (0.005 g in 100 ml) Sodium Chloride Sodium Phosphate monobasic Sodium Phosphate dibasic Benzalkonium chloride Water for injectables This composition is totally different from the microemulsion of the present invention: there is no oil component such as ethyl oleate and there is a strong cationic preservative such as benzylalkonium chloride, whereas the present formulations only comprise non-ionic surfactants such as Tween 80 and Tween 20. Latanoprost content was determined with an HPLC (Agilent series 1100) equipped with an UV detector and using a mixture of two mobile phases. The data reported in the following Tables 2a, 2b, 2c are expressed as Latanoprost concentration per mi of liquid formulation. Table 2a: Stability studies at 4°C Notes: a. all the latanoprost concentration data are expressed in µg/ml b. bottle 1 is made of polyethylene c. bottte 2 is made of low density polyethylene The data reported prove a dearly higher stability of the sub-micron emulsion of the invention in comparison to the marketed aqueous solution Xalatan. It is particularly interesting to stress the very good stability at 25°C, which can lead to room temperature storage indications avoiding the necessity of low temperature conditions. Furthermore, the data In Table 2c at 45°C show that the microemulsion of the invention is very much more stable than Xalatan both in glass vial and in plastic containers. Thus, it was found that the formulation of Example 1 remained fluid and homogeneous, with no separation of phases, at all tested conditions. The potential influence of the oil/water ratio of the sub-micron emulsion on the chemical stability of the prostaglandin was studied by comparing the stabHity of the sutMnicron emulsion of the invention (Example 1) wnth the sub-mioon emulsion of referem^ of example a, which differs only for a more diluted oil In water ratio. Latanoprost concentrations are determined with the HPLC method described before. Table 3: Stability studies at 25°C of submieron emulsion with diffierent o/w ratios The data reported in Table 3 clearly show that the sub-micron emulsion with higher oil/water ratio (Example 1, o/w ratio 1/ 20) is more stable than the one with lower oil/water ratio (Comparative Example A, o/w ratio 1/40). 9.3 Physico-chemical stability of tits sub-micron emulsions The formulations of the invention stored at different temperatures were also characterized in terms of the size of the droplets by Laser Light Scattering (Malvern Zetasizer Nano ZS). Data are reported in Table 4. As dearly evidentiated by the data in Table 4, the prostaglandin ophthalmic formulations of the invention maintained the sub-micron size of the droplets also at prolonged times at high temperature. 9.4 Antlmicrobiai efficacy of the sub-micron emulsion In the case of ophthalmic formulations it is mandatory to prove the antimiorobial efficacy of the preparation, showing that during storage and use of the formulation microbial contamination is prevented. Consequently we introduced in the aqueous phase of the sub-microemulsion the mild non-irritant preservative chlorobutanoi (Example 4). This formulation was tested following the requirements of the Italian Pharmacopeia (11th edition, pp 533-534,2002), which is in line with the European Pharmacopeia. This official test is based on the inoculation into the formulation under examination of controlled concentrations of two species of bacteria (Pseudomonas aeruginosa, Staphylicoccus aureus) and one spedes of fungi (Candida albicans). At predetermined times up to one month adequate sample of inoculated formulations were analyzed in terms of live micro- organisms. Data are reported in the following Table 5. TABLE 5: Logarithmic reduction of number of micro-organisms inoculated into formulation of example 4 Micro-organism The data reported in Table 5 show a very good antimicrobial efficacy of the sub-microemuision tested. Actually the offcial criteria of acceptance A are satisfied. Further, it was found that a formulation of the invention (Example 4) containing chlorobutanol as preservative is stable even after long-term storage at high temperatures. TABLE 6: Stability studies of latanoprost microemuision of Example 4 stored in glass vial The stability was tested as described under Section 9.2 by an HPLC analytical procedure. EXAMPLE 10 DETERMINATION OF OCULAR IRRITATION POTENTIAL An o/w microemulsion of the invention (without latanoprost) was tested with regard to its eye inritation potential using the SkinEthic Reconstituted Human Corneal Epitheliai (RHCE) model (Nguyen D. H., Beuerman R. W., De Wever B. and Rosdy M. Three-dimensional construct of the human corneal epithelium for in vitro toxicology. In: H. Salem and S. A. Katz, Editors, Aitemative Toxicological Methods, CRC Press (2003). pp. 147-159). The principle of the assay is based on the measurement of cycotoxicity in recortstituted human corneal epithelium cultures after topical exposure to the test material by means of the colourimetric MTT (3-[4,5-dimethylthiazol-2- yl]-2,5-diphenyl-tetrazoiiumbromide) reduction assay. The test material was classified based on MTT viability analysis according to the following prediction model after a 60-minute exposure period and 16- hour post exposure incubation period: i) The test material was considered to be non-irritant to the eye if the tissue viability was >50%. ii) The test material was considered to be irritant to the eye if the tissue viability was =0%. Compared to the negative control tissues, the MTT relative viability of the test material treated tissues after a 60-minute exposure period and a 16- hour post exposure Incubation period was 104.3%. in conclusion, under the conditions of the test, the test material was considered to be non-irritant. EXAMPLE 11 IN VIVO TEST An o/w microemulsion of the invention (without latanoprost) was tested with regard to its eye initation potential in an in vivo rabbit test model. The test was performed on 3 New Zealand White Rabbits, in a single application, 0.1 ml of the microemulsion was applied with readings taken 1, 2 and 3 days thereafter. The test method was according to ISO 10993-1: 2003, ISO 10993-10: 2002 and ISO 10993-12: 2007. The mean value of eye inritation scores are shown in Table 7. TABLE 7 It can be stated that the microemulsion produced slightly irritating effects after the application, whidi were fully reversible within 24 hours post instillation. Based on these results, the microemulsion is considered to be a non-eye-irritant. WE CLAIM: 1. An oil-in-water emulsion having a substantially neutral zeta potential for ophthalmic use comprising as components (i) a dispersed oily phase, (ii) at least one prostaglandin as active agent, which is dissolved in the oily phase component (i), (iii) a surfactant comprising a combination of at least two non-ionic surfactants, and (iv) a contiguous aqueous phase, optionally comprising formulation agents. 2. The oil-in-water emulsion of daim 1, wherein the oily phase (i) Is present in an amount of at least 5% (w/w) based on the total weight of the emulsion. 3. The oil-in-water emulsion of claim 1 or 2, wherein the oily phase component (i) is selected firom pharmaceutically acceptable oils, such as animal or vegetable oils, synthetic oils and mixtures thereof. 4. The oil-in-water emulsion of any of claims 1 to 3, wherein the oily phase component (i) is selected from the group consisting of ethyl oleate, a mixture of C8 to C10 fatty acid triglycerides, ricinus oil, corn oil or mixtures thereof. 5. The oil-in-water emulsion of any one of claims 1-4, wherein the oily phase (i) has a solubilization degree of at least 0.1 mg/ml for the prostaglandin component (ii). 6. The oil-in-water emulsion of claim 5, wherein the oily phase (i) has a solubilization degree of at least 10 mg/ml for the prostaglandin component (ii). 7. The oil-in-water emulsion of anyone of claims 1-6, wherein the oil/water partition coefficient of the prostaglandin component (ii) Is at least logP=0.5 in favor of the oily phase (i). 8. The oil-in-water emulsion of claim 7, wherein the oil/water partition coefficient of the prostaglandin component (ii) is at least iogP=2 in favor of the oily phase (i). 9. The oil-in-water emulsion of any one of the preceding claims, wherein the prostaglandin component (ii) is a prostaglandin F2a analogue. 10. The oil-in-water emulsion of claim 9, wherein the prostaglandin component (ii) is selected from the group consisting of latanoprost travoprost, bimatoprost and unoprostone and mixtures of two or more thereof. 11. The oil-in-water emulsion of claim 10, wherein the prostaglandin component (ii) is latanoprost. 12. The oil-in-water emulsion of any one of the preceding claims, wherein the at least two non-ionic surfactants of the surfactant component (iii) are selected from lipophilic non-ionic surfactants, hydrophilic non-ionic surfactants or combinattons thereof. 13. The oil-in-water emulsion of anyone of the preceding claims, wherein the non-ionic surfactants of the surfactant component (iii) have a combined total HLB value of at least 10, preferably of at least 13 and preferably up to 18. 14. The oil-in-water emulsion of anyone of the preceding claims, wherein the at least two surfactants of the surfactant component (iii) are present in a self-emulgation promoting amount. 15. The oil-in-water emulsion of anyone of the preceding claims, wherein the non-ionic surfactants of the surfactant component (lii) are selected from the group consisting of polyoxyethylene (20) sorbitan monooleate (Tween 80) , polyoxyethylene (20) sorbitan monolaurate (Tween 20), polyoxyethylene (2) cetylether (Brij 52»). polyoxyetylene (10) cetylether (Brij 56), polyoxyethylene (20) cetylether (Brij 58»). 16. The oil-in-water emulsion of claim 15, wherein the combination of non- ionic surfactants of the surfactant component (iii) is selected from the combinations polyoxyethylene (20) sorbitan monooleate (Tween 80) / polyoxyethylene (20) sorbitan monolaurate (Tween 20), polyoxyethylene (20) sorbitan monooleate (Tween 80) / polyoxyl (2) cetylether (Brij 52), polyoxyl (2) cethylether (Brij 52) / polyoxyl (20) cethylether (Brij 58) and polyoxyl (20) (»thylether (Brij 58) / polyoxyl (10) cethylether (Brij 56*). 17. The oil-in-water emulsion of any one of the preceding claims, wherein the aqueous phase component (iv) is a pharmaceuticaliy acceptable aqueous phase, preferably selected from the group consisting of sterilized water, purified water or of any other type of water suitable for ophthalmic application. 18. The oil-in-water emulsion of anyone of the preceding claims, wherein the aqueous phase component (iv) optionally comprises further additives such as buffer agents, isotonic agents, viscosity-increasing compounds, antimicrobial preservatives. antioxidants, stabilizers. 19. The oil-in-water emulsion of claim 18, werein the antimicrobial preservative is chilorobutanol. 20. The oil-in-water emulsion of anyone of the preceding claims, which has a zeta potential between -10mV and +10mV, preferably between -4mV and +4mV. 21. The oll-in-water emulsion of anyone of the preceding daims, which is a microemuision wherein the average size of the oil droplets is less than 1 pm. 22. The oil-in-water emulsion of any one of the preceding claims, wherein the average droplet size of the oily droplets of the emulsion is 700 nm or less. 23. The oil-in-water emulsion of anyone of the preceding claims, which has a stability of at least 6 months, preferably at least 9 months, more preferably at least 12 months at 25°C. 24. The oil-in-water emulsion of any one of the preceding claims, wherein the components (i) to (iv) are present in the following amounts: (i) 5.0 - 20.0% (w/w) of the oily phase, (ii) 0.001 - 5.0% (w/w) of the prostaglandin active agent, (iii) 0.1 -10.0% (w/w) of the first non-ionic surfactant and 0.1 -10.0% (w/ w) of the second non-ionic surfactant, and (iv) 50.0 - 95.0% (w/w) of the aqueous phase; based on the total weight of the emulsion. 25. The oil-in-water emulsion of any one of the preceding claims characterized in that the emulsion is free from cosurfactant components, in particularly selected from short chain alcohols, mono carboxylic adds, cationic and/or anionic surfactants, lecithins and/or phospholipids. 26. The oil-in-water emulsion of any one of claims 1-25 for use as an ophthalmic formulation for the prevention, alleviation and/or treatment of glaucoma and/or ocular hypertension. 27. A process for the preparation of the oil-in-water emulsion of any one of the preceding claims, comprising the steps (a) optionally solubilising formulation agents in the aqueous phase, (b) solubilising the first non-ionic surfactant either in the aqueous or oily phase, (c) solubilising the second non-ionic surfactant either in the aqueous or oily phase, (d) solubilising the prostaglandin active agent in the oily phase, and (e) mixing the oily phase with the aqueous phase. 28. The process of claim 27, wherein the mixing step (e) is performed without the use of high energy shear processes such as high pressure homogenisation or sonication. 29. The process of claim 27 or 28, wherein an oil-in-water emulsion is spontaneously formed in the mixing step (e). 30. The process of claim 27, wherein the mixing step (e) is performed with the use of high energy shear processes such as high pressure homogenisation or sonication. The present invention refers to an oil-in water emulsion for ophthalmic application comprising at least one prostaglandin as active agent and a surfactant component comprising a combination of at least two non-ionic surfactants. The emulsion is suitable for medical applications particularly for the treatment of glaucoma, and has an increased chemical stability of the prostaglandin active agent so to allow long-term storage also at room temperature.

Full Text

Ophthalmic oil-in-water emulsions containing prostaglandins
Description
The present invention refers to an oif-in-water emuision comprising at least
one prostaglandin as active agent and a surfactant component comprising a
combination of at least two non-ionic surfactants. The emulsion is suitable
for ophthalmic applications, particularly for the treatment of glaucoma and/or
ocular hypertension, and has an increased chemical stability of the
prostaglandin active agent so to allow long-term storage, e.g. at room
temperature.
Prostaglandins are chemical moieties, found in tissues or organs of humans,
exhibiting a wide range of physiological activities. Some prostaglandin
synthetic F 2a analogues have been known to be useful as ophthalmic
phannaceutical agents, specifically as ocular hypotensive antigiaucoma
agents. For example latanoprost, travoprost, bimatoprost and unoprostone
have been introduced in the market under the trade marks respectively of
Xalatan, Travatan, Lumigan and Rescula as ophthalmic eye drop solutions
for the treatment of ocular hypertension and glaucoma.
Problems associated with these prostaglandin analogues are their rather
poor water solubility and their chemical instability especially in aqueous
solutions. Consequently many different ophthalmic formulations have been
proposed to overcome such problems.
EP-A-0 435 682 describes the use of inclusion complexes of prostaglandins
with cydodextrins which are water soluble complexing agents with an
hydrophobic cavity, wherein hydrophobic drugs such as prostaglandins are
hosted inside this cavity leading to higher water solubility and higher stability
in water.
The stability and clinical efficacy of a latanoprost ophthalmic formulation
containing cyclodextrin was shown in The Journal of Clinical Phamiacoiogy,
47, 121-126,2007.
The use of modifiecl cyclodextrins (i.e. etherised cyctodextrins) to complex
and stabUize prostaglandins is proposed in EPA-0 330 511.
The stabilization of the aqueous ophthalmic solution of latanoprost by
adjusting the pH of the solution to a value in the range 5 - 6.25 or by the
addition of e-aminocaproic acid has been described in EP-A-1 532 981.
Solubility and stability of prostaglandins are also improved also by the
addition of polyethoxylated castor oil to the aqueous solution
(US 5.849,792).
An ophthalmic fonnuiation of prostaglandins is proposed In US 2004/076678:
acrylate, cellulose or other polymers are added to the aqueous solution of
prostaglandins to prolong the efficacy when administered to the eyes.
An example of an ophthalmic emulsion is given in US 3,608,073 covering a
formulation containing pilocarpine, an oil, an aqueous phase and an
interface agent.
EP-A-0 521 799 teaches the preparation of submicron ophthalmic emulsions
using an oil. an amphoteric surfactant and a phospholipid.
Oil, aqueous phase, phospholipid are the components subjected to high
pressure homogenization to generate submicron emulsions containing
flurbiprofen for opthalmic application (US 5171566).
The addition of hydrophobic suspending particles to stabilize submicron
emulsions is suggested in US 2003/15471.
A microemulsion, obtained by high pressure homogenisation, of latanoprost

is described in Int. J. Pharm., 305, 176-179. 2005: Stabilization is obtained
by the use of polyvinylalcohol as emutsifier.
Benzalkonium chloride is one of most used antimicrobial preservatives for
ophthalmic formulations but it has been also widely used in the formation of
ophthalmic microemulsions (US 5.698,219) thanks to its positive charge
which stabilizes the droplets; this positive charge can be provided also by
other cationic agents (WO 2006/050838).
The use of positively charged microemulsions for the administration of
prostaglandins is described in WO 2006/050836 or WO 2007/042262: the
interface film Is formed by the combination of a non-ionic surfactant and a
cationic agent such as quatemary ammonium compounds (including
benzalkonium chloride), amino alcohols, biguanide salts. The resulting
emulsions have a zeta potential of at least 16 mV.
However the use of cationic agents for ophthalmic use must be carefully
checked in order to guarantee ocular tolerability (Eur.J.Pharm.Biopharm., 53,
263-280. 2002).
A combination of benzalkonium chloride with specific number of carbon
atoms, a surfactant, a tonicity agent is described in EP-A-1 547 599
describing a stable latanoprost ophthalmic solution.
EP-A-0 458 588 discloses an ocuto-hypotensively synergistic combination of
a 13.14-dihydro-15-keto-20-C1-6 alkyl prostaglandin and a potyoxyethyiene
sorbitan unsaturated C10-C24 aliphatic acid monoester for the manufacture of
a medicament useful in the treatment of ocular hypertension.
EP-A-1 655 021 discloses an oil-in-water emulsion useful as a delivery
vehicle of hydrophobic ingredients such as pharmaceutical drugs, wherein
the emulsion particles have a net positive charge and comprise a cationic
agent.

EP-A-1 681 059 describes a pharmnaceutical composition comprising an oii-
in-water emulsion containing a prostaglandin F2a derivative, an oil. a water-
soluble polymer and water. The water-soluble polymer may be a polyvinyl
compound, a water-soluble cellulose compound or a polysaccharide. The oil
may e.g. be an animal or vegetable oil and/or medium chain fatty add
triglyceride.
US 2006/0182781 describes an ophthalmic microparticle composition,
wherein the microparticles comprise a polymer matrix and an active
ingredient, e.g. a prostaglandin.
WO 2004/082625 and US 2007/0036829 describe self-emulsifying
ophthalmic compositions comprising oil globules dispersed in an aqueous
phase, wherein the globules comprise a surfactant component and a polar
oil component. The oil component is present in an amount of up to 1.25% (w/
w) of the total emulsion.
US patent 5,827,835 descrit)es a non-toxic emulsion composition comprising
a non-ionic cellulose ether having a molecular weight of at least 30 kD, an
oil, water and optionally an emulsifying agent. The emulsion may further
comprise pharmaceutical drugs such as prostaglandins.
WO 02/064166 described a composition comprising at least one
monoglyceride, at least one emulsifier, an aqueous solution and at least one
organic solvent.
KR 2003/0046553 discloses a temperature-sensitive emulsion composition
for external use comprising a prostaglandin E1 as an active ingredient. The
composition is applied to the skin and forms a gel at body temperature. The
formation of a gel, however, is undesirable for ocular applications.
It has been now been unexpectedly found that a combination of an oily
phase containing a prostaglandin active agent, an aqueous phase, a

surfactant component comprising a combination of at least two non-ionic
surfactants can spontaneously generate stable sub-micron emulsions for
ophthalmic use. These emulsions preferably have a substantially neutral
electrochemical charge (zeta potential) and have excellent stability
characteristics both in terms of physical properties of the micro-emulsion and
the chemical stability of the prostaglandin active agent. The emulsions may
be prepared by simply mixing the components, whereby spontaneous micro-
emulsification occurs. It is not necessary to apply high energy processes
such as high pressure homogenisation and/or sonication. This contributes
further to the stability of the system.
Thus, the subject-matter of the present invention is an oil-in-water emulsion
having a substantially neutral zeta potential for ophthalmic application
comprising as components
(i) a dispersed oily phase,
(ii) at least one prostaglandin as active agent, which is dissolved in the oily
phase component (i),
(iii) a surfactant comprising a combination of at least two non-ionic
surfactants, and
(iv) a contiguous aqueous phase, optionally comprising formulation agents.
The emulsion of the present invention is suitable for use as a pharmaceutical
formulation, particularly as an ophthalmic formulation. Preferably, the oil-in-
water emulsion is used for the prevention, alleviation and/or treatment of
ocular diseases such as glaucoma and/or ocular hypertension. The emulsion
is suitable for single-dose applications or multiple-dose applications.
The emulsions of the present invention are characterized by a high chemical
stability as measured by determination of the active ingredient recovery after
extended storage time, e.g. by HPLC. The recovery of the active ingredient
after storage at 25oC for 6 months, more preferably 9 months and most
preferably 12 months is at least 80%, more preferably at least 90% and most
preferably at least 95%. The recovery of the active ingredient after storage at

45oC for 14 days, preferably for 30 days and more preferably for 45 days is
at least 80%, preferably at least 90% and most preferably at least 95%.
Further, the formulations of the present invention are characterised by a high
physical stability as measured by droplet size determination. Preferably, the
emulsions are stable at 25°C for at least 6 months and at 45°C for at least 45
days.
It was found in an especially preferred embodiment that the emulsions have
a chemical and physical stability of at least 6 months, preferably at least 9
months and more preferably at least 12 months at 25°C. Surprisingly it was
found that the chemical and physical stabHity is not decreased by opening
the bottles.
The emulsion of the present invention Is preferably a micro-emulsion,
wherein the average size of the oil droplets is less than 1 µm. More
preferably, the average droplet size of the oil droplets Is 700 nm or less. It is
further preferred that the emulsion of the present invention does not change
its physical state from 4 ° - 45 °C, and particulariy does not form a gel.
Preferably the emulsion is characterised by having a substantially neutral
zeta potential, i.e. a zeta potential between -10 mV and +10 mV, preferably
between -4 mV and +4 mV and more preferably between -2mV and +2mV.
The oily phase (i) is preferably present in an amount of at least 3% (w/w),
more preferably at least 5% (w/w) based on the total weight of the emulsion.
The upper amount of the oily phase is preferably 25% (w/w) and more
preferably 20% (w/w) based on the total weight of the emulsion. The oily
phase component (i) is selected from pharmaceuticaity acceptabie oils, e.g.
animal oils, vegetable oils, synthetic oils or mixtures thereof. Preferably, the
oily phase comprises pharmaceutically acceptable fatty acid esters, e.g. fetty
triglycerides or fatty acid monoesters.
More preferably, the components of the oily phase are chosen on the basis

of four factors:
(1) acceptability for application to the eye
(2) a good solubilisation degree of at least 0.1 mg/ml, preferably at least 2
mg/ml, and more preferably at least 10 mg/ml for the prostaglandin
component (ii);
(3) chemical stabilisation of the prostaglandin component (ii) as described
above,
(4) a strong oil-water partitioning effect in favour of the oil, preferably at least
log P = 0.5 and more preferably at least log P = 2.
Specific examples of suitable olly phase components are ethyl oleate,
Migtyol812, i.e. a mixture of the C8-10 fatty acid triglycerides, ricinus oil, com
oil or mixtures thereof.
The oil-in-water emulsion of the invention comprises at least one
prostaglandin as active agent. Preferably, the prostaglandin is a lipophilic
prostaglandin, e.g. a prostaglandin F2a analogue such as latanoprost,
travoprost, bimatoprost, unoprostone or mixtures of two or more thereof.
More preferably, the prostaglandin component is latanoprost. The
prostaglandin is preferably present in an amount of 0.001-5% (w/w), more
preferably 0.002-0.1% (w/w) based on the total weight of the emulsion.
This surfactant component (ii) comprises a combination of at least two non-
Ionic surfactants. The choice of the combination of the two surfactants is
preferably done on the basis of the following considerations:
(1) only non-ionic surfactants acceptable for ocular application (eye
tolerability) are used;
(2) the combination and amounts of surfactants is chosen such that the first
non-ionic surfactant is added either to the oil or water phase, the oil and
water phase are mixed and the second non-ionic surfactant is added to
the mixture in a quantity sufficint to generate homogeneous oil/water
emulsions without phase separation or formation of large visible droplets
and wherein the average droplet size determined by laser light scattering
analysis is preferably less than 1 pm and more preferably 700 pm or
less.
The non-ionic surfactants of the surfactant component (ii) may be selected
from lipophilic non-ionic surfactants, hydrophilic non-ionic surfactants, or
combinations thereof. Preferably, the non-ionic surfactants of the surfactant
component (iii) have a combined total HLB value of at least 10. more
preferably of at least 13 and preferably up to 20 and more preferably up to
18. The surfactants are present in an amount which promotes spontaneous
emulgation. Preferably, the surfactant component comprises first and second
non-ionic surfactants which are present each in amounts of 0.1-10% (w/w)
based on the total weight of the emulsion. The combined amount of non-
ionic surfactants is preferably from 1-20% (w/w), more preferably from
2-12% (w/w) based on the total weight of the emulsion.
The non-ionic surfactants are preferably chosen from polyoxyethylene fatty
acid esters, e.g. polyoxyethylene sorbitan, mono- or polyesters and/or
polyoxyethylene fatty alcohol ethers. Preferably, the non-ionic surfactants of
the surfactant component (iii) are selected from the group consisting of
polyoxyethylene (20) sorbitan monooleate (Tween 80*) , polyoxyethylene
(20) sorbitan monolaurate (Tween 20*), polyoxyethylene (2) cetyiether (Brij
52*), polyoxyetylene (10) cetyiether (Brij 56*), polyoxyettiylene (20)
cetyiether (Brij 58*). More preferably, the combination of non-ionic
surfactants of the surfactant component (iii) is selected from the
combinations polyoxyethylene (20) sorbitan monooleate (Tween 80*) /
polyoxyethylene (20) sorbitan monolaurate (Tween 20*), polyoxyethylene
(20) sorbintan monooleate (Tween 80*) / polyoxyl (2) cetyiether (Brij 52*),
polyoxyl (2) cethylether (Brij 52*) / polyoxyl (20) cethylether (Brij 58*) and
polyoxyl (20) cethylether (Brij 58*) / polyoxyl (10) cethylether (Brij 56*).
The oil-in-water emulsion of the invention is preferably free from cationic
surfactants, anionic surfactants, short-chain, e.g. C1-4 monohydric alcohols,
fatty acids, e.g. C4-8 fatty acids or from the class of lecithins or/and

phospholipids. Such compounds may present problems of eye compatibility
or physical/chemlcai instability.
The emulsion may, however, comprise other agents commonly used in
ophthalmic formulations, e.g. buffer agents such as phosphate salts, citrate
salts etc., isotonic agents such as glycerol, sorbitol, glucose, sodium chloride
etc., viscosity-increasing compounds such as hydroxypropylcellulose or
other water-soluble cellulose derivatives, polymethylmethacrylate or other
poiyacryiic add derivatives, chitosan, hyaluronic acid, poiyvinytpyrrolidone
etc., antimicrobial preservatives, particulariy chlorobutanol, antioxidants or
stabilizers. The emulsion may comprise the prostaglandin as the only active
agents. In different embodiments, the emulsion may comprise one or more
further active agents, particulariy hydrophilic active agents which are
preferably present in the aqueous phase.
Preferred examples of further active agents are as follows:
- Beta blodcers, e.g. Timolol; Levobunotol; Betaxolol etc.
- Anti-inflammatory agents, e.g. Ketorolac, Beta- or Dexamethasone.etc.
- Anti-viral agents, e.g. aciclovir, etc.
- Topical ocular anaesthetic agents, e.g. Oxybuprocaine, etc.
- Anti-allergic agents, e.g. Olopatadine, Azelastine, Epinastine, Lodoxamide,
etc.
- Anti-Dry Eye agents, e.g. hyaluronic add, acetylcysteine, polyvinyl alcohol,
etc.
- Other 10P Reducing Drugs, e.g. Brimonidine, Brinzoiamide, Dorzolamide,
etc.
- Agents for emergency treatment of glaucoma, e.g.Pilocarpine
The aqueous phase (iv) of the emulsion is preferably a pharmaceutically
acceptable aqueous phase, which is preferably selected from the group
consisting of sterilized water, purified water or any other type of water
suitable for ophthalmic application. The aqueous phase is preferably present
in an amount of 30-95% (w/w), more preferably in an amount of 50-95% (w/
w) based on the total weight of the emulsion. The amount of the aqueous
phase also Includes the weight of standard ophthalmic agents, buffers,
preservatives, isotonic agents etc., which are optionally added.
In an especially preferred embodiment, the components (i)-(iv) are present in
the following amounts:
(i) 5.0 - 20.0% (w/w) of the oily phase,
(ii) 0.001 - 5.0% (w/w) of the prostaglandin active agent,
(ill) 0.1 - 10.0% (w/w) of the first non-ionic surfactant and 0.1 -10.0% (w/w)
of the second non-ionic surfactant, and
(iv) 50.0 - 95.0% (w/w) of the aqueous phase;
based on the total weight of the emulsion.
The above-described components, when mixed, spontaneously generate
stable sub-micron emulsions without the need of high energy shear
procedures. For example, the emulsions may be prepared by a method
comprising the steps:
(a) optionally solubilising formulation agents in the aqueous phase,
(b) solubilising the first non-ionic surfactant either in the aqueous or oily
phase,
(c) solut>iiising the second non-ionic surfactant either in the aqueous or oily
phase,
(d) solubilising the prostaglandin active agent in the oily phase, and
(e) mixing the oily phase with the aqueous phase.
The mixing step is preferably canied out with standard mixing procedures,
e.g. using paddle mixers, magnetic stirres, homogenizers etc. The use of
high energy mixing procedures such as high pressure homogenisation or
sonication can be avoided, However, it is possible to use high energy mixing
procedures such as high pressure homogenisation or sonication.
For pharmaceutical purposes, the emulsion is preferably prepared using
sterile components and devices. All steps of the manufacturing process are
preferably performed under aseptic conditions and the final formulations are
tested following the official pharmacopeial requirements. If the emulsion is
formulated as a multiple-dose preparation, an antimicrobial preservative
such as chlorobutanol is added.
The invention is further described by the following examples, which should in
no way be considered as limiting.
EXAMPLE 1
An o/w submicron emulsion was prepared by mixing with a paddle mixer an
oil/surfactants solution (Ethyl oleate:Tween 80*:Tween 20* 1:1:0.5 w:w:w)
containing Latanoprost dissolved at a concentration of 0.44mg/ml with a
physiological aqueous phase (0.9%NaCI/ pH 7.4). The ratio oil phase to
water phase was approximately 1:20 (wt./wt.). The composition of the
resulting submicron emulsion was:
Latanoprost 0005g
Ethyl oleate 4.51 g
Polyoxyethytene (20) sorbitan monooleate (Tween 80*) 4.51 g
Polyoxyethylene (20) sorbitan monoolsurate (Tween 20*) 2.30g
Physiological Solution (0.9%NaCI, pH=7.4) 88.7g
EXAMPLE 2
An o/w submicron emulsion was prepared by mixing with a magrtetic stirrer
an oil/surfactants solution (Miglyol 812:Tween 80*:Tween 20* 1:1.5:1)
containing Latanoprost dissoved at a concentration of 0.33mg/ml with a
phoshphate buffer aqueous solution (sodium dihydrogen/sodium
monohydrogenphosphate) containing an isotonic agent (sorbitol). The ratio
between the oil phase and the water phase was approximately 1:20 (wt./wt.).
The composition of the resulting submicron emulsion was:
Latanoprost 0.005g
Miglyol 812 4.28g
Polyoxyethylene (20) sorbitan monooleate (Tween 80*) 6.42g
Polyoxyethylene (20) sorbitan monoolaurate (Tween 20*) 4.28g
Phosphate buffer aqueous solution 85.02g
(sodium dihydrogen/sodium monohydrogenphosphate, sorbitol)
EXAMPLES
A fluid o/w submicron emulsion was prepared by mixing using a paddle
mixer a surfactant /oil solution (Ricinus oil:Brij 56* 1:0.036) containing
Latanoprost dissolved at a concentration of 039 mg/ml with a physiological
aqueous phase (0.9%NaCI, pH 7.4) containing Brij 58* at the percentage of
4% (wt./wt.)- The ratio between the oily phase and the aqueous phase was
approximately 1:10 (wt./wt.).
The composition of the resulting submicron emulsion was:
Latanoprost 0.001g
Ricinus oil 17.9g
Polyoxyl (20) cetyl ether (Brij 58*) 6.98g
Polyoxyl (10) cetyl ether (Brij 56*) 0.64g
Physiological aqueous phase (0.9%NaCI, pH=7.4) 174.5g
EXAMPLE 4
An o/w submicron emulsion was prepared by mixing with a paddle mixer an
oil/surfactants solution (Ethyl oieate: Tween 80*:Tween 20* 1:1:0.5)
containing Latanoprost dissolved at a concentration of 0.45 mg/mi with a
physiological aqueous phase (0.9%NaCI, pH 7.4) containing an antimicrobiai
agent (Chiorobutanoi 0.5%). The ratio between the oil phase and the water
phase was approximately 1:20 (wt:wt.).
The composition of the resulting submicron emulsion was:
Latanoprost 0.0075g
Ethyl oieate 6.62g
Polyoxyethylene (20) sorbitan monooleate (Tween 80*) 6.62g
Polyoxyethylene (20) sorbitan monoolaurate (Tween 20*) 3.31 g
Chiorobutanoi 0.80g
Physiological aqueous phase (0.9%NaCI, pH=7.4) 132.6g
EXAMPLE 5
An o/w submicron emulsion was prepared by nnixing with a shear mixer an
oit/surfactant solution (Ethyl oteate:Brij 52* 1:0.045) containing Latanoprost
dissolved at a concentration of 0.59 mg/mi with a citrate buffer solution (citric
acid, sodium citrate) contahiing Brij* 58 at the percentage of 4% (wt./wt.).
The citric buffer solution had sorbitol as isotonic agent dissolved. The ratio
between the oily phase and the aqueous phase was approximately 1:20 (wt./
wt.).
The composition of the resulting submicron emulsion was:
Latanoprost 0.0050g
Ethyt oleate 4.65g
Polyoxyl (20) cetyl ether (Brij 58*) 3.66g
Polyoxyl (2) cetyl ether (Brij 52*) 0.21 g
Citrate buffer (citric acid, sodium citrate, sorbitol) 91.5g
EXAMPLE 6
A fluid o/w submicron emulsion was prepared by mixing with a paddle mixer
a surfactant /oil solution (Ricinus oil:Brij52® 1:0.05) containing Latanoprost
dissolved at a concentration of 0.40mg/ml with a citrate buffer (citric add,
sodium citrate) solution containing Brij 58* at the percentage of 4% (wt./wt.).
The citric buffer solution had glycerol as isotonic agent dissolved. The ratio
between the oily phase to the aqueous phase was approximately 1:10
(wt./wt.).
The composition of the resulting submicron emulsion was:
Latanoprost 0.01 g
Ricinus oil 17.3g
Polyoxyl (20) cetyl ether (Brij 58*) 7.0g
Polyoxyl (2) cetyl ether (Brij 52*) 0.84g
Citrate buffer (citric acid, sodium citrate, glycerol) 174.8g
EXAMPLE 7
A o/w subinicroemulsion was prepared by mixing using a shear mixer an oil/
surfactant solution (Ethyl oleate: Tween 80*:Brij 52* 1:1:0.04) containing
Travoprost dissolved at a concentration of 0.41mg/mi with a physiological
solution (pH 7.4, 0.9% NaCI). The ratio of oil phase:water phase was
approximately 1:20 (wt./wt.). The composition of the resulting submicron
emulsion was:
Travoprost 0.0040g
Ethyl oleate 4.51 g
Poiyoxyethytene (20) sorbitan monooleate (Tween 80*) 4.48g
Polyoxyl (2) cetyl ether Brij 52*) 0.18g
Physiological Solution (0.9%NaCI, pH=7.4) 90.3g
EXAMPLE 8
A fluid o/w submicron emulsion was prepared by mixing vtnth a paddle mixer
an oil/surfactants phase (Mygliol 812*:Tween80*:Tween20* 1:1.5:0.5)
containing Bimatoprost dissolved at a concentration of 2.3mg/ml with a
physiological aqueous phase (0.9%NaCI, pH 7.4) containing an antimicrobial
agent (Chlorobutanol 0.5%). The ratio oil/water was approximately 1:20
(wt.:wt.).
The composition of the resulting submicron emulsion was:
Bimatoprost 0.06g
Mygliol 812® 8.46g
Polyoxyethylene (20) sorbitan monooleate (Tween 80*) 12.89g
Polyoxyethylene (20) sorbitan monooleate (Tween 20*) 4.40g
Chlorobutanol 1 .Og
Physiological aqueous phase (0.9%NaCI, pH=7.4) 173.1 g
The following example has been prepared for comparison reasons with a low
oil/water ratio to show the effect of a specific range of oil/water ratios on the
prostaglandin stability.
COMPARATIVE EXAMPLE A
An o/w submicron emulsion was prepared by mixing with a paddle mixer an
oil/surfactants solution (Ethyl oleater:Tweeh 80*:Tween 20* 1:1:0.5)
containing Latanoprost dissolved at a concentration of 0.79 mg/ml with a
physiological aqueous phase (0.9%NaCI, pH 7.4). The ratio of the oil phase
to the water phase was 1:40 (wt.:wt.). The composition of the resulting
submlcron emulsion was:
Latanoprost 0.0125g
Ethyl oleate 6.37g
Poiyoxyethytene (20) sorbitan monooleate (Tween 80*) 6.37g
Polyoxyethylene (20) sorbitan monoolaurate (Tween 20*) 3.15g
Physiological Solution (0.9%NaCI. pH=7.4) 234.1 g
EXAMPLE 9
CHARACTERiZATiON TESTS
The o/w submicron emulsions prepared as described in the above examples
were characterized from a physico-chemical point of view (the size and the
electrochemical charge of the sub-micron emulsions, the chemical stability of
prostaglandin at different storage conditions, the antimicrobial efficacy). The
resulting data are reported here as shown in the following paragraphs and
tables.
9.1 Size and electrochemical charge determination of the droplets of
the submicron emulsions of the invention
The size and electrochemical charge (zeta potential) of the droplets of the
submicron emulsions prepared as shown in the Examples 1-8 and
Comparative Example A were detemnined by Dynamic Laser Light Scattering
(Malvem Instalments. Zetasizer Nano ZS).
Zeta potential measuremente were carried out with the Zetasizer NanoZS of
the Malvem Instruments Ltd (UK). The sample (0.75 ml) to be analyzed was
placed, by usir^g a syringe to avoid bubbles, in a folded capillary cell fitted
with electrodes. The cell was inserted in the instrument and the
measurement sequence was started automatically; all the individual
measurement runs were accumulated together and then summed to give the
final Zeta potential result. It was not necessary to dilute the samples for the
analysis and consequently there was no risk to artificially change the
characteristics of the sample.
The data are reported in Table 1.
Table 1: Droplest size and Zeta potential of the submicron emulsions
of the invention
All the formulations prepared resulted to be homogeneous fluid dispersions,
with no separation of phases or formation of visible large droplets. Actually
the data reported in Table 1 confirm the formation of sub-micron emulsions
with droplets size at least below 700 nm; the electrochemical charge (zeta
potential) values were all approximately equal to 0, indicating an almost
neutral charge as a result of the use of non-Ionic surfactants as interface
agents.
9.2 Chemical Stability studies of Latanoprost
Stability studies at the storage conditions of 45°C, 25°C and 4°C were
carried out on the Latanoprost submicron emulsion of Example 1.
The sample was preserved in a glass container and a commercial
Latanaprost ophthalmic aqueous solution (Xalatan*) was used as the
reference product. At 45°C the submicron emulsion of Example 1 was
placed not only in a glass vial but also in two types of plastic bothes.
The Xaiatan* formulation is a simple buffered isotonic aqueous solution
made of the following components:
Latanoprost (0.005 g in 100 ml)
Sodium Chloride
Sodium Phosphate monobasic
Sodium Phosphate dibasic
Benzalkonium chloride
Water for injectables
This composition is totally different from the microemulsion of the present
invention: there is no oil component such as ethyl oleate and there is a
strong cationic preservative such as benzylalkonium chloride, whereas the
present formulations only comprise non-ionic surfactants such as Tween 80
and Tween 20.
Latanoprost content was determined with an HPLC (Agilent series 1100)
equipped with an UV detector and using a mixture of two mobile phases.
The data reported in the following Tables 2a, 2b, 2c are expressed as
Latanoprost concentration per mi of liquid formulation.
Table 2a: Stability studies at 4°C
Notes: a. all the latanoprost concentration data are expressed in µg/ml
b. bottle 1 is made of polyethylene
c. bottte 2 is made of low density polyethylene
The data reported prove a dearly higher stability of the sub-micron emulsion
of the invention in comparison to the marketed aqueous solution Xalatan*. It
is particularly interesting to stress the very good stability at 25°C, which can
lead to room temperature storage indications avoiding the necessity of low
temperature conditions. Furthermore, the data In Table 2c at 45°C show that
the microemulsion of the invention is very much more stable than Xalatan*
both in glass vial and in plastic containers.
Thus, it was found that the formulation of Example 1 remained fluid and
homogeneous, with no separation of phases, at all tested conditions.
The potential influence of the oil/water ratio of the sub-micron emulsion on
the chemical stability of the prostaglandin was studied by comparing the
stabHity of the sutMnicron emulsion of the invention (Example 1) wnth the
sub-mioon emulsion of referem^ of example a, which differs only for a more
diluted oil In water ratio.
Latanoprost concentrations are determined with the HPLC method described
before.
Table 3: Stability studies at 25°C of submieron emulsion with diffierent
o/w ratios
The data reported in Table 3 clearly show that the sub-micron emulsion with
higher oil/water ratio (Example 1, o/w ratio 1/ 20) is more stable than the one
with lower oil/water ratio (Comparative Example A, o/w ratio 1/40).
9.3 Physico-chemical stability of tits sub-micron emulsions
The formulations of the invention stored at different temperatures were also
characterized in terms of the size of the droplets by Laser Light Scattering
(Malvern Zetasizer Nano ZS). Data are reported in Table 4.
As dearly evidentiated by the data in Table 4, the prostaglandin ophthalmic
formulations of the invention maintained the sub-micron size of the droplets
also at prolonged times at high temperature.
9.4 Antlmicrobiai efficacy of the sub-micron emulsion
In the case of ophthalmic formulations it is mandatory to prove the
antimiorobial efficacy of the preparation, showing that during storage and
use of the formulation microbial contamination is prevented.
Consequently we introduced in the aqueous phase of the sub-microemulsion
the mild non-irritant preservative chlorobutanoi (Example 4). This formulation
was tested following the requirements of the Italian Pharmacopeia (11th
edition, pp 533-534,2002), which is in line with the European Pharmacopeia.
This official test is based on the inoculation into the formulation under
examination of controlled concentrations of two species of bacteria
(Pseudomonas aeruginosa, Staphylicoccus aureus) and one spedes of
fungi (Candida albicans). At predetermined times up to one month adequate
sample of inoculated formulations were analyzed in terms of live micro-
organisms. Data are reported in the following Table 5.

TABLE 5: Logarithmic reduction of number of micro-organisms
inoculated into formulation of example 4
Micro-organism
The data reported in Table 5 show a very good antimicrobial efficacy of the
sub-microemuision tested. Actually the offcial criteria of acceptance A are
satisfied.
Further, it was found that a formulation of the invention (Example 4)
containing chlorobutanol as preservative is stable even after long-term
storage at high temperatures.
TABLE 6: Stability studies of latanoprost microemuision of Example 4
stored in glass vial
The stability was tested as described under Section 9.2 by an HPLC
analytical procedure.
EXAMPLE 10
DETERMINATION OF OCULAR IRRITATION POTENTIAL
An o/w microemulsion of the invention (without latanoprost) was tested with
regard to its eye inritation potential using the SkinEthic Reconstituted Human
Corneal Epitheliai (RHCE) model (Nguyen D. H., Beuerman R. W., De
Wever B. and Rosdy M. Three-dimensional construct of the human corneal
epithelium for in vitro toxicology. In: H. Salem and S. A. Katz, Editors,
Aitemative Toxicological Methods, CRC Press (2003). pp. 147-159). The
principle of the assay is based on the measurement of cycotoxicity in
recortstituted human corneal epithelium cultures after topical exposure to the
test material by means of the colourimetric MTT (3-[4,5-dimethylthiazol-2-
yl]-2,5-diphenyl-tetrazoiiumbromide) reduction assay.
The test material was classified based on MTT viability analysis according to
the following prediction model after a 60-minute exposure period and 16-
hour post exposure incubation period:
i) The test material was considered to be non-irritant to the eye if the tissue
viability was >50%.
ii) The test material was considered to be irritant to the eye if the tissue
viability was =0%.
Compared to the negative control tissues, the MTT relative viability of the
test material treated tissues after a 60-minute exposure period and a 16-
hour post exposure Incubation period was 104.3%.
in conclusion, under the conditions of the test, the test material was
considered to be non-irritant.

EXAMPLE 11
IN VIVO TEST
An o/w microemulsion of the invention (without latanoprost) was tested with
regard to its eye initation potential in an in vivo rabbit test model. The test
was performed on 3 New Zealand White Rabbits, in a single application, 0.1
ml of the microemulsion was applied with readings taken 1, 2 and 3 days
thereafter. The test method was according to ISO 10993-1: 2003, ISO
10993-10: 2002 and ISO 10993-12: 2007. The mean value of eye inritation
scores are shown in Table 7.
TABLE 7
It can be stated that the microemulsion produced slightly irritating effects
after the application, whidi were fully reversible within 24 hours post
instillation. Based on these results, the microemulsion is considered to be a
non-eye-irritant.
WE CLAIM:

1. An oil-in-water emulsion having a substantially neutral zeta potential for
ophthalmic use comprising as components
(i) a dispersed oily phase,
(ii) at least one prostaglandin as active agent, which is dissolved in the
oily phase component (i),
(iii) a surfactant comprising a combination of at least two non-ionic
surfactants, and
(iv) a contiguous aqueous phase, optionally comprising formulation
agents.
2. The oil-in-water emulsion of daim 1, wherein the oily phase (i) Is present
in an amount of at least 5% (w/w) based on the total weight of the
emulsion.
3. The oil-in-water emulsion of claim 1 or 2, wherein the oily phase
component (i) is selected firom pharmaceutically acceptable oils, such as
animal or vegetable oils, synthetic oils and mixtures thereof.
4. The oil-in-water emulsion of any of claims 1 to 3, wherein the oily phase
component (i) is selected from the group consisting of ethyl oleate, a
mixture of C8 to C10 fatty acid triglycerides, ricinus oil, corn oil or mixtures
thereof.
5. The oil-in-water emulsion of any one of claims 1-4, wherein the oily
phase (i) has a solubilization degree of at least 0.1 mg/ml for the
prostaglandin component (ii).
6. The oil-in-water emulsion of claim 5, wherein the oily phase (i) has a
solubilization degree of at least 10 mg/ml for the prostaglandin
component (ii).
7. The oil-in-water emulsion of anyone of claims 1-6, wherein the oil/water
partition coefficient of the prostaglandin component (ii) Is at least
logP=0.5 in favor of the oily phase (i).
8. The oil-in-water emulsion of claim 7, wherein the oil/water partition
coefficient of the prostaglandin component (ii) is at least iogP=2 in favor
of the oily phase (i).
9. The oil-in-water emulsion of any one of the preceding claims, wherein
the prostaglandin component (ii) is a prostaglandin F2a analogue.
10. The oil-in-water emulsion of claim 9, wherein the prostaglandin
component (ii) is selected from the group consisting of latanoprost
travoprost, bimatoprost and unoprostone and mixtures of two or more
thereof.
11. The oil-in-water emulsion of claim 10, wherein the prostaglandin
component (ii) is latanoprost.
12. The oil-in-water emulsion of any one of the preceding claims, wherein
the at least two non-ionic surfactants of the surfactant component (iii) are
selected from lipophilic non-ionic surfactants, hydrophilic non-ionic
surfactants or combinattons thereof.
13. The oil-in-water emulsion of anyone of the preceding claims, wherein the
non-ionic surfactants of the surfactant component (iii) have a combined
total HLB value of at least 10, preferably of at least 13 and preferably up
to 18.
14. The oil-in-water emulsion of anyone of the preceding claims, wherein the
at least two surfactants of the surfactant component (iii) are present in a
self-emulgation promoting amount.
15. The oil-in-water emulsion of anyone of the preceding claims, wherein the
non-ionic surfactants of the surfactant component (lii) are selected from
the group consisting of polyoxyethylene (20) sorbitan monooleate
(Tween 80*) , polyoxyethylene (20) sorbitan monolaurate (Tween 20*),
polyoxyethylene (2) cetylether (Brij 52»). polyoxyetylene (10) cetylether
(Brij 56*), polyoxyethylene (20) cetylether (Brij 58»).
16. The oil-in-water emulsion of claim 15, wherein the combination of non-
ionic surfactants of the surfactant component (iii) is selected from the
combinations polyoxyethylene (20) sorbitan monooleate (Tween 80*) /
polyoxyethylene (20) sorbitan monolaurate (Tween 20*),
polyoxyethylene (20) sorbitan monooleate (Tween 80*) / polyoxyl (2)
cetylether (Brij 52*), polyoxyl (2) cethylether (Brij 52*) / polyoxyl (20)
cethylether (Brij 58*) and polyoxyl (20) (»thylether (Brij 58*) / polyoxyl
(10) cethylether (Brij 56*).
17. The oil-in-water emulsion of any one of the preceding claims, wherein
the aqueous phase component (iv) is a pharmaceuticaliy acceptable
aqueous phase, preferably selected from the group consisting of
sterilized water, purified water or of any other type of water suitable for
ophthalmic application.
18. The oil-in-water emulsion of anyone of the preceding claims, wherein the
aqueous phase component (iv) optionally comprises further additives
such as buffer agents, isotonic agents, viscosity-increasing compounds,
antimicrobial preservatives. antioxidants, stabilizers.
19. The oil-in-water emulsion of claim 18, werein the antimicrobial
preservative is chilorobutanol.
20. The oil-in-water emulsion of anyone of the preceding claims, which has a
zeta potential between -10mV and +10mV, preferably between -4mV
and +4mV.
21. The oll-in-water emulsion of anyone of the preceding daims, which is a
microemuision wherein the average size of the oil droplets is less than
1 pm.
22. The oil-in-water emulsion of any one of the preceding claims, wherein
the average droplet size of the oily droplets of the emulsion is 700 nm or
less.
23. The oil-in-water emulsion of anyone of the preceding claims, which has a
stability of at least 6 months, preferably at least 9 months, more
preferably at least 12 months at 25°C.
24. The oil-in-water emulsion of any one of the preceding claims, wherein
the components (i) to (iv) are present in the following amounts:
(i) 5.0 - 20.0% (w/w) of the oily phase,
(ii) 0.001 - 5.0% (w/w) of the prostaglandin active agent,
(iii) 0.1 -10.0% (w/w) of the first non-ionic surfactant and 0.1 -10.0% (w/
w) of the second non-ionic surfactant, and
(iv) 50.0 - 95.0% (w/w) of the aqueous phase;
based on the total weight of the emulsion.
25. The oil-in-water emulsion of any one of the preceding claims
characterized in that the emulsion is free from cosurfactant components,
in particularly selected from short chain alcohols, mono carboxylic adds,
cationic and/or anionic surfactants, lecithins and/or phospholipids.
26. The oil-in-water emulsion of any one of claims 1-25 for use as an
ophthalmic formulation for the prevention, alleviation and/or treatment of
glaucoma and/or ocular hypertension.
27. A process for the preparation of the oil-in-water emulsion of any one of

the preceding claims, comprising the steps
(a) optionally solubilising formulation agents in the aqueous phase,
(b) solubilising the first non-ionic surfactant either in the aqueous or oily
phase,
(c) solubilising the second non-ionic surfactant either in the aqueous or
oily phase,
(d) solubilising the prostaglandin active agent in the oily phase, and
(e) mixing the oily phase with the aqueous phase.
28. The process of claim 27, wherein the mixing step (e) is performed
without the use of high energy shear processes such as high pressure
homogenisation or sonication.
29. The process of claim 27 or 28, wherein an oil-in-water emulsion is
spontaneously formed in the mixing step (e).
30. The process of claim 27, wherein the mixing step (e) is performed with
the use of high energy shear processes such as high pressure
homogenisation or sonication.

The present invention refers to an oil-in water emulsion for
ophthalmic application comprising at least one prostaglandin
as active agent and a surfactant component comprising
a combination of at least two non-ionic surfactants.
The emulsion is suitable for medical applications
particularly for the treatment of glaucoma, and has an increased
chemical stability of the prostaglandin active agent so
to allow long-term storage also at room temperature.

Documents:

http://ipindiaonline.gov.in/patentsearch/GrantedSearch/viewdoc.aspx?id=DBUEZaX/grW0nGj6rqSi4A==&loc=wDBSZCsAt7zoiVrqcFJsRw==

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Patent Number

272366

Indian Patent Application Number

3566/KOLNP/2009

PG Journal Number

14/2016

Publication Date

01-Apr-2016

Grant Date

30-Mar-2016

Date of Filing

12-Oct-2009

Name of Patentee

AZAD PHARMA AG

Applicant Address

BAHNHOFSTR. 9, CH-3125 TOFFEN, SWITZERLAND

Inventors:

#	Inventor's Name	Inventor's Address
1	BARONIAN, MIHRAN	LINDENWEG 14, CH-3125 TOFFEN, SWITZERLAND
2	CARLI, FABIO	SALITA DI CEDASSAMMARE 3/1, I-34136 TRIESTE ITALY
3	SCHMID, RENE	REBWEG 30, CH-8203 SCHAFFHAUSEN, SWITZERLAND
4	CHIELLINI, ELISABETTA	VIA BUIE DISTRIA 11, I-34126 TRIESTE, ITALY

PCT International Classification Number

A61K 9/00,A61K 47/10

PCT International Application Number

PCT/EP2008/003317

PCT International Filing date

2008-04-24

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	07008357.1	2007-04-24	EUROPEAN UNION
2	60/942,691	2007-06-08	EUROPEAN UNION