Lactoferrin liposome-based eye drops for treating eye diseases

Abstract

An ophthalmic liposomal product for use in a curative or preventive treatment of disorders or diseases of the eye and periocular area, comprises, or consists of, S75 Phospholipids, lactoferrin, Vitamin E TPGS, Stearylamine, EDTA and a viscosifier selected from the Poloxamer 407 / 188 family, is sterilizable by filtration at 0.2, is stable to aging, with pH between 7.0 and 7.5 and is provided with antibacterial activity which falls under efficacy criteria A, as prescribed by the European Pharmacopoeia F.I.XII. The lactoferrin liposomes with an amount of Stearylamine not exceeding 0.0030% of the total weight and 0.07-0.15% of Vitamin E TPGS % are obtained by extrusion at a pressure between 700 and 800 bar. The lactoferrin liposomes are present at 0.5% by weight on the total weight and the ratio between S75 phospholipids and lactoferrin is 1.5 / 0.5 = 3, sufficient for the lactoferrin to be totally encapsulated. The liposomes of lactoferrin, Stearylamine and Vitamin E TPGS have an electrical potential, measured in distilled water, using a particle analyzer by means of a light scattering technique (DLS), less than 0 mV so as to synergistically strengthen the abilities thereof in preventing and / or treating eye disorders, in particular those in which there is an inflammatory condition such as dry eye syndrome and the related complications, such as chalazion, stye (chronic chalazion), conjunctivitis, blepharitis and keratitis and reducing the possible aging of the lactoferrin protein.

Description

[0001] LACTOFERRIN LIPOSOME-BASED EYE DROPS FOR TREATING EYE DISEASES

[0002] DESCRIPTION

[0003] SUMMARY

[0004] The present invention relates to a liposomal composition with antibacterial activity for ophthalmic use, stable to aging, comprising lactoferrin in synergistic combination with a pegylated non-ionic surfactant, and with Stearylamine, and the use thereof for treating disorders or diseases of the eye and periocular area, where:

[0005] 1) the pegylated surfactant is selected from: Kolliphor Solutol HS15, and Vitamin E TPGS, both with an HLB (Hydrophilic-Lipophilic Balance) between 15 and 13.2, and with a linear PEG chain, outside the phospholipid layer of the liposomes, capable of coordinating water molecules, between 1000 and 712 Daltons in length, and where

[0006] 2) a positive charge is added to the lactoferrin liposomes between 0.1 and 0.5% by weight on the total weight, by adding an appropriate amount of Stearylamine therein, demonstrating that this further positive charge, albeit in a minimal amount, not exceeding 0.0030% of the total weight, so as not to create cytotoxicity problems on the eye surface, and not to vary the sign of the total electrical potential of the ophthalmic product (due to the phospholipids containing lactoferrin) which is negative, a) enhancing the antibacterial / antifungal effect of lactoferrin, also resolving any aging of the protein; b) improving filterability (by virtue of the presence of both vitamin E TPGS containing a linear PEG, as well as Solutol HS15), c) surprisingly allowing vitamin E TPGS to inhibit, with a concentration not exceeding 0.1% of the total weight (0.66 MICRO Molar) and with the same efficacy, the microbial growth not only of gram negative bacteria, but also of gram positive bacteria, always maintaining the total charge of the ophthalmic product with a negative sign, so as to strengthen the abilities thereof to prevent and / or treat eye disorders, in particular those in which there is an inflammatory condition such as dry eye syndrome and the related complications, such as chalazion, stye (chronic chalazion), conjunctivitis, blepharitis and keratitis and reducing the possible aging of the lactoferrin protein.

[0007] According to a peculiar feature of the invention, the lactoferrin and surfactant liposomes, with Stearylamine in the phospholipid layer of the liposomes, are produced with an extrusion at a pressure of 700-800 bar, so as to make the liposomes always filterable with 0.2 micron filters, to avoid sterilization with gamma rays which, despite the negative effect on both the product stability of toxicity, is still a recurring practice in the context of current preparations, as we will highlight hereinbelow.

[0008] The liposome sizes were advantageously measured using a particle analyzer by means of a light scattering (DLS) technique, for example by means of the Zetasizer Nano ZS Instrument by Malvern Panalytical.

[0009] Liposomes are known for the soothing and emollient properties thereof, as well as the ability thereof to act as a carrier, increasing the bioavailability of the substances they carry.

[0010] In a preferred embodiment, the phospholipid structure thereof consists of S75 phospholipids, which contain 68-70% phosphatidylcholine (neutral charge) and other phospholipids such as phosphatidylglycerol and phosphatidylinositol (negative charge), which leads to the preparation of negatively charged liposomes (-13) even after having encapsulated the lactoferrin and stearylamine. This indicates a complete encapsulation of the lactoferrin.

[0011] The presence of Vit E TPGS, in the amounts indicated, allows structuring liposomes which have, on the surface of the outer parts, PEG chains, capable of attributing features of pegylated liposomes thereto, thus improving filterability and antibacterial efficacy.

[0012] It is further known that the Stearylamine (positively charged lipophilic amine) carried by the liposomes (interspersed in the lipid film), allows, due to the positive charge thereof, improved adhesiveness on the epithelial surface and contributes to inhibiting microbial growth.

[0013] It is disclosed herein that, in a liposomal composition for ophthalmic use simultaneously comprising lactoferrin, Vitamin E TPGS and Stearylamine, synergistic effects are highlighted, whereby Vitamin E TPGS, which is potentially an antioxidant, and capable of resolving any aging of Lactoferrin, with the presence of Stearylamine, albeit in minimal quantities so as not to create cytotoxicity problems, is also capable of exhibiting an equal microbial inhibition capacity on both gram-negative bacteria and gram-positive bacteria, as experimentally demonstrated, while the ophthalmic product continues to maintain a negative electrical potential.

[0014] FIELD OF THE INVENTION

[0015] Lactoferrin is a protein belonging to the category of transferrins, protein molecules with the ability to bind and transport iron, and is involved in several biological mechanisms. It has an iron-transporting action, it belongs to the transferrin family and is widely studied for the multiple beneficial effects thereof, such as:

[0016] - antioxidant activity;

[0017] - immunostimulant;

[0018] - it regulates iron homeostasis;

[0019] - antimicrobial activity;

[0020] - it restores the intestinal microbiota.

[0021] Lactoferrin also possesses an iron-independent bactericidal activity capable of attacking and lysing the bacterial membrane by removing the lipopolysaccharide component. In combination with lysozyme, which in turn is capable of cleaving the |31- 4 glycosidic bonds of peptidoglycan, it induces the death of the bacterium by cytolysis.

[0022] These abilities derive from the molecular structure thereof which is three- dimensional and consists of a single polypeptide chain of 692 amino acids, structured in two globular lobes, N-terminal lobe and C-terminal lobe, joined by an alpha-helical structure. The two lobes can take an open or closed shape, and switch from one shape to another. The N-terminal lobe or Apo-lactoferrin (iron-depleted form of lactoferrin) takes an open shape, while the C-terminal lobe or Holo-lactoferrin remains closed.

[0023] However, Apo-lactoferrin by its own nature is unstable and is particularly sensitive to the proteolytic action of pepsin at the gastric level and to the proteolytic hydrolysis of pancreatin at the intestinal level.

[0024] This results in protein denaturation, poor absorption and inactivation thereof.

[0025] To prevent such a process of Apo-lactoferrin, liposomes are used. Liposomes are lipid spheres which allow the protection and subsequent release of Apo- lactoferrin. Apo-lactoferrin is more active than the lactoferrin itself (5 times) since the iron binding is carried out in the open form of the apoprotein, allowing easier access to Fe3+ or complexed ions.

[0026] Some bacteria require the presence of iron to proliferate effectively, but the presence of lactoferrin is capable of removing it from the environment, thus preventing or at least reducing cell replication.

[0027] In viruses, however, the mechanism of action is different and is mainly based on the ability to bind to the same membrane proteins which viruses use to penetrate the body's cells, thus competing for the same binding site, but there are earlier studies which highlight a possible RNA destruction activity (genetic material used by some viruses).

[0028] Although these are the most relevant mechanisms, further ones are known which overall broaden and amplify the immune efficacy of lactoferrin, although probably the main role thereof in the body is more properly linked to iron management (with collection and transport functions).

[0029] It is appropriate to provide herein, albeit briefly, the features and properties of lactoferrin hitherto recognized in the literature.

[0030] Properties of lactoferrin

[0031] Lactoferrin possesses iron-binding / transferring, antibacterial, antiviral, antifungal, anti-inflammatory and anticancer properties. These functional properties are intimately dependent on the structural integrity of LF, particularly the higher order shape thereof. LF is mainly extracted from bovine milk and then added to many commercial products such as food supplements, formula, cosmetics and toothpastes. LF is sensitive to temperature-induced denaturation and other physicochemical stresses. (1)

[0032] LF is a single-chain polypeptide glycoprotein with a molecular weight of about 78 kDa. Detailed structural studies have reported that there are 691 and 696 amino acids in human and bovine LF, respectively (Baker et al., 2000; Moore et al., 1997).

[0033] One of the most noteworthy features of the structure of LF is that the surface thereof is positively charged. This facilitates the binding of LF with anionic biocompounds. LF promotes iron absorption in the human body (Paesano et al., 2010). It modulates cell growth, eliminates harmful free radicals, and inhibits the formation of several toxic compounds (Baveye et al., 1999). For these reasons, LF is added to many commercial products including powder infant milk, therapeutic beverages, fermented milk, cosmetics, and toothpastes (Tomita et al., 2009).

[0034] The multiple health-promoting functions of LF and the wide-ranging real-life applications thereof have spurred increased research interest.

[0035] Function and applications of lactoferrin

[0036] The unique structural features of LF provide a variety of nutritional and medicinal values. In terms of nutritional function, LF transports iron and detoxifies free radicals in biological fluids.

[0037] Antibacterial and antiviral activities

[0038] The antibacterial property of intact LF has demonstrated bacteriostatic effects against a variety of Gram-positive (Francesca et al., 2004; Lee et al., 2005; Rodriguez- Franco et al., 2004) and Gram-negative (Beeckman et al., 2007; Ostan et al., 2017; Rogan et al., 2004) bacteria.

[0039] However it has been experimentally demonstrated (Jahani S, Shakiba A, Jahani L. The antimicrobial effect of Lactoferrin on Gram-negative and Gram-positive Bacteria.. Int J Infect. 2015;2(3):e27954, https: / / doi.org / 10.17795 / iii27594) that lactoferrin is more effective against gram positive bacteria than against gram negative bacteria.

[0040] Recent research results show that the bactericidal effect is directly related to the interaction between LF and bacteria (Jenssen & Hancock, 2009; Orsi, 2004; Ostan et al., 2017). Ostan et al. 2017 reported that the C lobe of LF associates with a bilobed outer membrane-bound lipoprotein at two different Gram-negative bacterial cell sites and forms receptor complexes.

[0041] LF binds anionic molecules (e.g., lipoteichoic acid) on the cell surface of Grampositive bacteria. This electrostatic binding reduces the overall negative charge of the cell wall and facilitates the efficacy of antibacterial compounds such as lysozyme and antibiotics (Barbiroli et al., 2012; Gonzalez-Chavez et al., 2009; Leitch & Willcox, 1999). 11

[0042] LF prevents the interaction between bacteria and host cells. It inhibits bacterial adhesion to host cells by occupying the surface of bacterial cells (Francesca et al., 2004; Oho et al., 2002; Valenti & Antonini, 2005).

[0043] Antibacterial peptides of LF

[0044] The bacteriostatic effect of LF is not limited to the structurally intact form thereof. In fact, the peptides obtained from LF still have bacteriostatic efficiency. It has been shown that the bactericidal effect of peptides can be correlated to the ability thereof to disrupt the membrane integrity (permeabilization and depolarization) of bacteria (Sijbra ndij et al., 2017).

[0045] Antiviral activity of LF

[0046] The antiviral effect of LF lies in the early stage of infection. LF not only prevents the infection of host cells by viruses (Beljaars et al., 2004; Hasegawa et al., 1994; Marchetti et al., 1999) but also inhibits the growth of viruses after host cells have been infected (Ikeda et al. al., 2000; Superti et al., 1997).

[0047] The domain in LF which acts against viruses appears to be different from the domain which acts against bacteria but, also in this case, the most widely reported hypothesis for the antiviral activity of LF is that it binds and blocks the viral receptors of glycosaminoglycans, in particular heparan sulfate (Glycans such as SA, GAG). The binding of LF to viral receptors prevents the first contact between virus and host cell, thus preventing infection (Gonzalez-Chavez et al., 2009; Shimizu et al., 1996). It is important to consider that SA and GAG are both negatively charged glycans and contain a carboxylic acid group.

[0048] Other functional properties

[0049] In addition to the iron-transferring, antibacterial and antiviral properties intensively studied, LF also possesses other functional values such as antifungal, antiinflammatory properties

[0050] Antifungal activity

[0051] LF and LF-derived peptides can act effectively on a broad spectrum of fungal species by virtue of the strong iron (Fe3+) scavenging properties thereof. LF has been observed to kill Candida albicans and Candida krusei (Kirkpatrick et al., 1971; Al-Sheikh, 2009). Iron sequestration by apo-LF has been reported as essential for host defense against Aspergillus fumigatus (Zarember et al., 2007). Apart from the effect of iron deprivation, LF binds directly to the negative surface of fungal cells, destroys the surface and causes an increase in membrane permeability leading to the death thereof.

[0052] Anti-inflammatory activity

[0053] LF is secreted in the mucosal environment of the human and animal body. Since many pathogens tend to enter the body through the mucosa, LF plays a key role in the host defense system (Wiesner & Vilcinskas, 2010). The anti-inflammatory effect thereof can be evidenced by the sharp increase in LF content in body fluids during inflammation (Sagel et al., 2009; Pfefferkorn et al., 2010). Clinical studies have shown that LF can help cure or prevent inflammation of lung (Hwang et al., 2016; Valenti et al., 2016), intestine (Brimelow et al., 2017; Drago-Serrano et al., 2017), intestine (MacManus et al., 2017; Nguyen et al., 2016), etc. The anti-inflammatory activity of LF can be attributed to the positively charged surface thereof. LF interacts with negatively charged moieties (e.g., proteoglycans) on the surface of immune cells. This association can trigger signaling pathways which lead to a physiological anti-inflammatory response (Gonzalez-Chavez et al., 2009; Legrand, 2016).

[0054] Some technological notes:

[0055] Denaturation of lactoferrin

[0056] Denaturation occurs when the protein is subjected to harsh environmental conditions such as strong acid / base, high or subzero temperatures and concentrated organic / inorganic salt and / or ultra-high pressure treatment or high pressure homogenization

[0057] These conditions alter the shape of the protein and cause the breakdown of forces (e.g., hydrogen and disulfide bonds) which give rise to and stabilize higher-order structural characteristics (secondary, tertiary and quaternary). Such a change in shape causes, at least in part, the reduction of the biomedical properties of lactoferrin, such as the antibacterial and antifungal effect, combined with the change in biophysical behavior, such as the reduction in solubility.

[0058] The peculiar properties of lactoferrin have had several applications in the pharmaceutical field.

[0059] In particular, the use of lactoferrin for preparing products for ophthalmic use has already been suggested recently, however leaving some problems unresolved related to both the stability and toxicity of the product intended to come into contact with the eye surface.

[0060] BACKGROUND ART

[0061] We briefly report the content of the most significant documents, which allows us to highlight how the peculiar features of the finding described are all new and inventive:

[0062] These are:

[0063] A) International Publication number WO 2018 / 134792 Al with Italian priority dated 20 / 01 / 2017, in the name of ITALDEVICE S.R.L. entitled "COMPOSITION FOR OPTHALMIC USE";

[0064] B) European Patent Application EP 3 603 621 with Italian priority dated 31 / 07 / 2018, in the name of TDC Technology Dedicated to Care S.r.l. entitled: "LIPOSOMES FOR THE TREATMENT OF OCULAR DISEASES"; and

[0065] C) the article "DEVELOPMENT OF LACTOFERRIN-LOADED LIPOSOMES FOR THE MANAGEMENT OF DRY EYE DISEASE AND OCULAR INFLAMMATION" of Lopez- Machado, A.; Diaz-Garrido, N.; Cano, A.; Espina, M. et al in Pharmaceutics, 2021,13,1698, published on October 15, 2021.

[0066] D) application WO 2020 / 135940 Al in the name of Dr. Rolf Lambert Pharma Consulting GmbH of July 2, 2020.

[0067] E) application WO 2019 / 215700 Al in the name of OFFHEALTH SpA dated November 14, 2019.

[0068] F) the article by MAYHEW ET AL: "Toxicity of non drug-containing liposomes for cultured human cells", EXPERIMENTAL CELL RESEARCH, ELSEVIER, vol.171, No.l, July 1, 1987, pages 195-202, XP024857085, ISSN: 0014-4827.

[0069] WO 2018 / 134792 Al (for brevity document A) describes a composition for ophthalmic use for treating disorders or diseases of the eye and periocular area, excluding creams, comprising a mixture of hydroxypropyl cellulose and / or xanthan gum, lactoferrin or a derivative thereof and EDTA or a salt thereof. Such a composition would be capable of simultaneously ensuring an optimal lubrication of the eye together with an effective antibacterial action. As for the eye drops which are the subject matter of the present invention, it is immediately noted that:

[0070] 1) the composition described is not in liposomal form,

[0071] 2) it has no additions of Stearylamine;

[0072] 3) it uses hydroxypropyl cellulose and / or xanthan gum, unlike the eye drops of the present invention which involves the use of a viscosifier chosen in the Poloxamer 407 / 188 family, a synthetic polymer which tends to increase the viscosity thereof with increasing temperature, facilitating the permanence of the eye drops on the surface of the eye which has a temperature of 30-32 °C; and

[0073] 4) lactoferrin is always present in combination with EDTA or a salt thereof, and with HPMC, following, as explicitly stated by the inventors themselves on page 9, the teachings of PAYNE. C and others, who have demonstrated the efficacy of such an association as far back as 1994.

[0074] It is sufficient to compare the results obtained with the eye drops which are the subject matter of the present invention with those obtained with the eye drops of document A, to verify how the antibacterial and antifungal efficacy is increased as well as the stability thereof.

[0075] In European application EP 3 603 621 (for brevity document B), an ophthalmic product is disclosed which is made of liposomes comprising lactoferrin and a component selected from hyaluronic acid and chitosan as active ingredients, useful for preventing, alleviating and / or treating eye diseases. Lactoferrin is encapsulated within the lipid bilayer. The liposomes are sterilized using gamma rays at a dose of 25 kGy.

[0076] Polysorbate 80 is also present and is used, to improve the liposomal structure, during the preparation thereof, according to the dictates of the "lipid film hydration method", without making any reference to an enhancement of antibacterial activity or a technological improvement such as the 0.2 micron filterability of liposomes.

[0077] From a careful analysis of such a patent document, it is easy to notice some critical issues which compromise the production and regular use of the claimed product in the treatment of eye disorders or diseases:

[0078] 1) the presence of hyaluronic acid (which has a negative charge) only has the effect of increasing the viscosity of the eye drops, without however having any effect on the antibacterial and antifungal ability thereof;

[0079] 2) lactoferrin has a positive charge and is thus incompatible with hyaluronic acid;

[0080] 3) the presence of hyaluronic acid is incompatible with that of chitosan (which has a positive charge) because the two molecules interact with each other and, when present in the same formulation, make the eye drops non-filterable at 0.2 microns, so that the use of hyaluronic acid is claimed alternatively to that of chitosan;

[0081] 4) the liposomes are obtained through a double treatment which initially comprises an ultrasound treatment followed by a mechanical extrusion or homogenization at a pressure of 0.8 bar.

[0082] On the other hand, the use of stearylamine, solutol HS 15 and vitamin E TPGS is never mentioned.

[0083] 5) the inventors of B consider all the liposomes they produced as positively charged, both those in the presence of chitosan and those without chitosan.

[0084] Now, the fact that liposomes are positively charged is a real contradiction because the phospholipids used (Lipoid S75) to form liposomes cannot be positively charged. They can be at most neutral, but more easily, as experimentally found, they are negative.

[0085] Instead, such a feature is specifically claimed in claim 4 of document B, where it is specified that the formed liposomes have a positive potential charge Z, preferably greater than or equal to 10 mV, and more preferably greater than or equal to 20 mV.

[0086] We can only assume that the positive charge reported in the cited document B refers only to the liposome solution containing chitosan (0.2%), added after extrusion.

[0087] Therefore, in the example where chitosan is not present, the positive charge declared by the inventors, can only depend on the lactoferrin not completely encapsulated in the liposomes. This is due to the inefficient extrusion technique used, which involves only 0.8 bar, and to the fact that the S75 phospholipids have a concentration (1.5%) which is probably insufficient for encapsulating lactoferrin (0.8%). In fact, the weight ratio thereof is 1.5 / 0.8 = 1.87, while, as will be better seen hereafter, in the eye drops which are the subject matter of the present application, the ratio between S75 phospholipids (1.5%) and lactoferrin (0.5%) is 1.5 / 0.5 =3.

[0088] 5) It should also be considered that chitosan is insoluble in water or buffer with a pH between 6.5-7.5. It is only soluble in an acidic environment at pH 4.5-5.5, a pH which however is not compatible with the surface of the eye.

[0089] This limited solubility in water can explain the need in document B to sterilize the liposomes by gamma radiation, because, while claiming to have liposomes with a size less than 200nm, in practice it was not possible for the authors to subject the liposomes to sterilization by 0.2 micron filtration, since the clogging of the filter due to the presence of chitosan is encountered very easily.

[0090] According to a peculiar feature of the present invention, contrary to what document B teaches, in the eye drops which are the subject matter of the present invention, the lactoferrin liposomes contain Solutol HS15 or Vitamin E TPGS, both in association with Stearylamine, which, as experimentally proven, have an antibacterial efficacy exceeding liposomes containing only lactoferrin and Solutol HS15 or lactoferrin and Vitamin E TPGS.

[0091] In fact, it has surprisingly been verified that the addition of a small amount of Stearylamine to the liposomes, such as not to create cytotoxicity problems on the eye surface, is sufficient to achieve a threefold result: increasing the antibacterial and antifungal effect of the eye drops, with a mechanism other than that of lactoferrin; decreasing, if present, the total negative charge of the ophthalmic product, while maintaining it with a negative sign, in order to strengthen the use thereof in the prevention and / or treatment of eye disorders, in particular those in which there is an inflammatory condition such as dry eye syndrome and the related complications, such as chalazion, stye (chronic chalazion), conjunctivitis, blepharitis, and keratitis; and finally also solving any aging of the protein, lactoferrin, with an increase, although not significant, in the stability of the ophthalmic product obtained.

[0092] The third document, C, is an article published by the journal "Pharmaceutics", "Development of Lactoferrin-Loaded Liposomes for the Management of Dry Eye Disease and Ocular Inflammation." of October 15, 2021, which describes the method of preparing Lactoferrin-loaded liposomes to be used in the treatment of DED (Dry Eye Disease) and eye inflammation, according to which to overcome the reduced stability thereof in water and the high drainage of the tear duct, the lactoferrin is incorporated in liposomes by homogenization at 0.8 bar, thus adding hyaluronic acid to the liposomes.

[0093] The oily (lipophilic) phase is formed by dissolving a certain amount of lipids (lipoid S75) and cholesterol in ethanol.

[0094] The aqueous phase is obtained by dissolving lactoferrin and polysorbate 80 (with HLB 15) in deionized water. The lactoferrin-loaded liposomes show an average size of 90 nm.

[0095] It should be considered that the liposomes described in said article are, by the authors' admission, the same as those which are the subject matter of patent EP 3603621, which is document B mentioned above.

[0096] There is no reference to the use of Stearylamine within liposomes containing lactoferrin and Vit E TPGS, or Solutol HS15, to increase the antibacterial and antifungal ability thereof, without incurring cytotoxicity problems, nor to the use, during liposome extrusion, of a pressure of 700-800 bar, such as that disclosed herein, which is at least 103higher than that indicated in the cited prior art documents; while cholesterol is present in the oily phase.

[0097] Document D discloses a formulation of eye drops comprising liposomes for use in treating dry eye syndrome. The liposomal composition comprises phospholipids and vit. E TGPS , buffered with Tris borate / boric acid. No lactoferrin or stearylamine is described.

[0098] Document E discloses liposomal compositions comprising phospholipids, stearylamine, and borate / boric acid buffer. The compositions exhibit antimicrobial activities and can be used to treat ophthalmic infections. Lactoferrin, solutol HS15, and vit. E TPGS are not described.

[0099] Document F discloses that liposomes not containing drugs comprising stearylamine were found to be cytotoxic. Such a document does not describe lactoferrin, solutol HS 15 and vitamin E TPGS.

[0100] The true novelty of the present invention is that the use disclosed herein of selected surfactants in an ophthalmic compound comprising lactoferrin liposomes added with stearylamine, is aimed at synergistically increasing both the antibacterial effect and improving the filterability of the liposomes themselves, and that to this end the antibacterial activity of liposomes prepared with Solutol HS15 and Vit E TPGS with respect to liposomes prepared with Polysorbate 80, combined with an easier filtration ability through a 0.2 micron filter, has been tested.

[0101] The research led to rationalizing such results by studying the physical chemical profile of each non-ionic surfactant which highlighted the fact that taking into account that the two most effective surfactants selected, such as Vitamin E TPGS and Solutol HS15, have a much longer PEG-free chain than Polysorbate 80, respectively: 1000 Daltons of Vitamin E TPGS, about 712-720 Daltons of Solutol HS15, compared to about 177 Daltons of Polysorbate 80 in which only 3 short PEG ends and not a single much longer chain are bound to sorbitan outside the phospholipid layer. The HLB (Hydrophilic-Lipophilic balance) value of each surfactant shows that Solutol HS15 and Vit. E TPGS have a value (15 and 13.2, respectively), which is substantially similar if not equal to that of Polysorbate 80, also having an HLB value of 15. This indicates that the HYDROPHILIC portion most present in the HLB balance (Griffin Method) expressed by formula below: hydrophilic part molecular weight / total lipophilic part molecular weight.

[0102] HLB = 1 ■ Hydrophilic part MW ■ 100 5 total MW

[0103] Below are some considerations that can explain the greater efficacy of liposomes with Vitamin E TPGS and Kolliphor HS15 (Solutol HS15) as compared to liposomes with Polysorbate 80:

[0104] Kolliphor HS15 or Solutol HS15

[0105] Solutol HS 15 is a non-toxic polyoxyethylene ester of 12-hydroxystearic acid.

[0106] It is a PEGylated fatty acid and can be used as a solubilizer for lipophilic ingredients.

[0107] - HLB Value =15.2

[0108] The structural formula thereof is as follows:

[0109] Structure Solutol HS 15

[0110] CAS No.61909-81-7

[0111] Chemical name: Solutol HS 15

[0112] C20H40O4 ,

[0113] Molecular weight 963.2

[0114] Synonyms: Polyethylene glycol 12-hydroxystearate, Solutol HS 15

[0115] Starting from an HLB value = 15.2 and, taking into account that the Molecular weight thereof is 963.2 g / mol we have:

[0116] Calculation of the hydrophilic part :

[0117] 15.2 = 1 x Hydrophilic part x 100

[0118] 5 963.2

[0119] Hydrophilic part = 15.2 x 5 x 963.2 = 732.03,

[0120] 100 which can be ascribed to the PEG linear chain

[0121] Therefore the % Hydrophilic part = 732 x 100 = 75.99

[0122] 963.2

[0123] Molar Concentration 1.03 pM Vitamin E TPGS

[0124] Vitamin E TPGS (d-alpha-tocopheryl polyethylene glycol 1000 succinate or TPGS) is a water-soluble derivative of natural vitamin E, formed together with polyethylene glycol 1000 by esterification of d-alpha-tocopheryl polyethylene glycol succinate.

[0125] - HLB value of 13.2

[0126] Molecular weight 1513 g / mol

[0127] Calculation of the hydrophilic part :

[0128] 13.2= l x Hydrophilic part x 100

[0129] 5 1513

[0130] Hydrophilic part = 13.2x5x1513 = 1000.56

[0131] 100 which can be ascribed to the PEG linear chain.

[0132] Therefore: the % Hydrophilic part = 1000.16 x 100 = 66

[0133] 1516

[0134] Molar Concentration 0.66 p.M POLYSORBATE 80

[0135] Polysorbate 80, also known as polyoxyethylene sorbitan monooleate, consists of sorbitol, ethylene oxide and oleic acid. It has a molecular formula of C64H124O26, molecular weight 1310.

[0136] It is a polymer consisting of PEGylated sorbitan, where the total number of polyethylene glycol) units is 20 (w + x + y + z = 20) and a single terminal is covered by an oleic group.

[0137] Polysorbate 80 is a non-ionic hydrophilic surfactant with HLB value 15 .

[0138] Molecular weight 1310 g / mol

[0139] Calculation of the hydrophilic part

[0140] 15 = 1 x Hydrophilic part x 100

[0141] 5 1310

[0142] Hydrophilic part = 15x5x1310 = 982.5

[0143] 100 It is the sum of Sorbitan and the 4 PEG chains; moreover considering the molecular weight of sorbitan, the weight of PEG can be calculated = 982.5-164 =718.

[0144] Dividing 718 by 4 (since 4 are the PEG chains) yields 177, which is the length of each PEG chain.

[0145] GENERAL CONSIDERATIONS

[0146] - ALL THREE TESTED SURFACTANTS CONTAIN PEG

[0147] Vitamin E TPGS has d-alpha-tocopherol succinate coupled, through a succinate linker, to polyethylene glycol (PEG) 1000;

[0148] The terminal part of PEG is a single free linear chain of about 1000 Daltons;

[0149] In the Liposomes, d-alpha-tocopherol succinate is interspersed / inserted in the phospholipid layer and the free PEG 1000 chain is outside and coordinates the water molecules.

[0150] Kolliphor HS15 or Solutol HS15 has a single PEG chain, of which one end is esterified with stearic acid, the other end is free.

[0151] In the Liposomes, stearic acid is interspersed / inserted in the phospholipid layer and the free PEG linear chain, about 712-720 Daltons, is outside and coordinates the water molecules;

[0152] Polysorbate 80: It is a composite polymer, a 4-point PEGYLATED sorbitan, the PEG chains are short, one of these ends is esterified with oleic acid. The total number of PEG units is 20 (4X5), each chain is bound to sorbitan and has a length of about 177 Daltons.

[0153] In the liposomes, the oleic acid is interspersed / inserted in the phospholipid layer while only 3 short ends of sorbitan-bound PEGs are on the outside.

[0154] It is thus the difference in linear length of PEG which at least partly induces a greater fluidity of the liposomal solution and an unexpected enhancement of the antibacterial effect, which is synergistically increased by the presence, in the liposomes, of stearylamine which, in addition to contributing, as known, due to the positive charge thereof, to a better adhesiveness on the epithelial surface, inhibiting microbial growth, in the experiments carried out it has surprisingly demonstrated the ability to make the Vit. E TPGS equally effective against both gram-positive and gram- negative bacteria.

[0155] The inventors then focused their attention on identifying the amount of stearylamine preferably to be added within liposomes having a phospholipid structure, consisting of S75 phospholipids, which contain 68-70% phosphatidylcholine (neutral charge) and other phospholipids such as phosphatidylglycerol and phosphatidylinositol (negative charge), which would have allowed obtaining positively charged liposomes, even after encapsulating lactoferrin (Zeta potential: -13).

[0156] AIM OF THE INVENTION

[0157] The presence of a pegylated surfactant selected from Solutol HS15 (also referred to as Kollifor HS15) and Vitamin E TPGS, which have a PEG free chain outside the phospholipid layer between 1000 and 710 Dalton, much longer than Polysorbate 80 which has 4 short PEG chains of only 177 Dalton, and the addition of a very small amount of Stearylamine, not exceeding 0.0030% of the total weight, within the phospholipid structure, consisting of S75 phospholipids, which contain 68-70% phosphatidylcholine (neutral charge) and other phospholipids such as phosphatidylglycerol and phosphatidylinositol (negative charge), which should lead to the preparation of positively charged liposomes, even after encapsulating lactoferrin, surprisingly gives the structure a negative final electrical potential (-13). This feature is also maintained with the inclusion of lactoferrin, despite the positive charge thereof. The following experimental data show that the positive charge of lactoferrin is totally cancelled once it is included in liposomes with 0.0025% stearylamine and 0.1% Vitamin E TPGS.

[0158] Preferably, the surfactant is Vit. E TPGS, by virtue of the fact that: such a Vitamin, potentially being an antioxidant, is also capable of solving any aging of Lactoferrin, and that with the presence of Stearylamine, the microbial inhibition ability thereof also extends to gram positive bacteria.

[0159] According to a peculiar feature of the present invention, this depends on the extrusion technique, which involves a well-defined much higher pressure than that indicated in the cited documents (as already mentioned 700-800 bar and not 0.8 bar as in the previous cited documents of the known art) and the right ratio between phospholipids and lactoferrin, in our case 3 versus 1.87, which lead to the formation of liposomes which completely include lactoferrin.

[0160] Following measurements made on the different components used, the relative zeta potentials are shown in the following Table 1 which allows an immediate comparison.

[0161] 5 The sizes were measured using a particle analyzer by means of a light scattering

[0162] (DLS) technique, for example by means of the Zetasizer Nano ZS Instrument by Malvern Panalytical.

[0163] Note that the electrical potentials were measured with solutions in distilled water.

[0164] 10 The degree of dispersibility in the size is indicated with PDL, in intensity mode.

[0165] TABLE 1

[0166] DETERMINATION OF THE ZETA POTENTIAL OF SOME PREPARATIONS: This Table 1 allows us to verify, before analyzing the data related to the electrical potential, the dispersibility (PDI) of the liposomes containing lactoferrin, stearylamine and Vit E TPGS, which has a value of 0.212, as also seen in Fig. 12, which is excellent.

[0167] Returning to the analysis of the data related to the electrical potential, from the same Table 1, it can be noted that: a) the liposomes consisting of S75 phospholipids have a negative charge; and b) the lactoferrin has a positive charge.

[0168] Therefore, it can be considered that, if the final charge is of a negative value, all, or a good part, of the lactoferrin is included in the hydrophilic interior of the liposomes.

[0169] As already mentioned, this result is completely contrary to what is disclosed in European patent EP 3 603 621 of TDC Sri, where it is stated (see paragraph 0027 and claim 4) that liposomes with lactoferrin and components selected from hyaluronic acid and chitosan have a positive charge.

[0170] It is a further object of the present invention to provide an ophthalmic product comprising lactoferrin liposomes in which the liposomes can be obtained by conventional techniques, in particular prepared by the known thin-film hydration method, which includes preparing an organic phase containing the lipids forming the liposomes, together with Stearylamine and Vitamin E TPGS, and preparing an aqueous phase comprising the components which will form the core of the aqueous solution, such as lactoferrin, in which the components themselves are added in the aqueous phase.

[0171] These and further objects and advantages of the invention, which will become apparent from the detailed description below, are obtained from the composition having the specific features of the appended claims.

[0172] DETAILED DESCRIPTION OF THE INVENTION

[0173] Following an intensive research program, the Applicant initially provided for the following:

[0174] 1) developing a product which can prevent, alleviate and / or treat eye disorders, consisting of liposomes comprising only lactoferrin and a surfactant, such as Vitamin E TPGS (D-a-Tocopherol polyethylene glycol succinate), which, selected from other nonionic surfactants such as Solutol HS15, and Polysorbate 80, has experimentally shown to provide the best product, from the point of view of antibacterial activity, that comprising lactoferrin liposomes and vitamin E TPGS;

[0175] 2) the possibility of adding a positive charge to the liposomes then occurred, by adding an appropriate amount of Stearylamine, demonstrating that the presence of this further positive charge, albeit in a minimal amount, so as not to create cytotoxicity problems on the eye surface, even in the presence of a negative total net charge due to the phospholipids containing lactoferrin, can: a) enhance the antibacterial / antifungal effect of lactoferrin, also resolving any aging of the protein; b) improve filterability; and c) surprisingly allow vitamin E TPGS to inhibit, with the same efficacy, the microbial growth not only of gram negative, but also of gram positive bacteria, a property so far never experimentally ascertained.

[0176] In the present application, the term "liposomes" means a vesicle consisting of at least one lipid bilayer and a core of aqueous solution encapsulated within the lipid bilayer.

[0177] The liposomes comprise other compounds of a lipophilic nature in the lipid bilayer thereof.

[0178] The lactoferrin comprised in the liposomes is encapsulated within the lipid bilayer (i.e., in the hydrophilic core of the liposome) as it can be demonstrated by the negative potential which derives mainly from liposomes consisting of S75 phospholipids.

[0179] In light of the above, the Inventors of the present invention focused their interest on a sterile topical application, specifically eye drops, capable of overcoming the drawbacks of current products and capable of ensuring an effective antibacterial action even several months from the production thereof, by virtue of its stability.

[0180] Bearing in mind the structural profile of Lactoferrin, summarily described above, and the presence on the market of some eye drops containing Lactoferrin, the Applicant sought to optimize a specific (Rationale) sterile EYE Drops product through experimental work which was carried out following two concurrent and competing main guidelines: a) Lactoferrin is a hydrophilic molecule which, in order to properly interact with the eye surface to obtain good bioavailability, requires a transport system such as liposomes; and b) Lactoferrin has "a positive charge" which regulates the biochemical activity thereof.

[0181] LIST OF FIGURES

[0182] The description of the invention will be better followed with reference to the accompanying drawings which show, by way of non-limiting example, the graphs of the logarithmic trend of the presence of microorganisms which survived over time in relation to the different formulations tested, and the stability tests carried out thereon as well as graphs showing the mean Gaussian distributions of the dimensions of extruded S75 phospholipids and a liposomal preparation according to the invention with lactoferrin, stearylamine, and Vit. E TPGS.

[0183] In the drawings:

[0184] Fig. 1 is a diagram showing the logarithmic trend of the concentration of the microorganisms which survived over time using a formulation of lactoferrin liposomes, EDTA and Polysorbate 80; referred to as the Formulation 1;

[0185] Fig. 2 is a diagram showing the logarithmic trend of the concentration of microorganisms which survived over time using a formulation of lactoferrin liposomes with EDTA, and Kolliphor SOLUTOL SH15, referred to as the Formulation 2;

[0186] Fig. 3 is a diagram showing the logarithmic trend of the concentration of microorganisms which survived over time using a formulation of lactoferrin liposomes with EDTA, and Vitamin E TPGS, referred to as the Formulation 3;

[0187] Fig. 4 is a diagram showing the logarithmic trend of the concentration of microorganisms which survived over time using a formulation of lactoferrin liposomes, EDTA, stearylamine and vitamin E TPGS and HPMC, referred to as the Formulation 4:

[0188] Fig. 5 is a diagram showing the logarithmic trend of the concentration of microorganisms which survived over time using a formulation of lactoferrin liposomes, EDTA, Kolliphor (SOLUTOL) HS15 and HPMC, referred to as the Formulation 5;

[0189] Fig. 6 is a diagram showing the logarithmic trend of the concentration of microorganisms which survived over time using a formulation of 0.5% lactoferrin liposomes and 0.1% Vitamin E TPGS by weight on the total weight of the composition; referred to as the Formulation 6;

[0190] Figs. 7 to 11 are graphs resulting from stability tests at 40°C for 2 months carried out by the Applicant, in particular:

[0191] Fig. 7 is a diagram showing the logarithmic trend of the concentration of surviving microorganisms in hours using a formulation of 0.5% lactoferrin liposomes, and 0.1% Vitamin E TPGS kept for one month at 40°C;

[0192] Fig. 8 shows the logarithmic trend of the concentration of surviving microorganisms in hours using the same formulation of lactoferrin liposomes in Fig. 7, after keeping it for 2 months at 40°C;

[0193] Figs. 9, 10 and 11 compare the values found in the logarithmic reduction of the microorganisms tested in the samples at To, Timonth and T2mOnths of 0.5% lactoferrin liposomes, 0.0025% Stearylamine and 0.1% Vitamin E TPGS, where it is highlighted that there are no substantial variations, the composition remaining stable after 60 days at 40°C;

[0194] Fig. 12 depicts the mean Gaussian size distribution (in nanometers) of liposomes containing Lactoferrin, Stearylamine and Vit E TPGS in intensity mode, having negative electrical potential equal to -13.1;

[0195] Fig. 13 refers to S75 phospholipid-only liposomes, which have a DPI equal to 0.669, and an electrical potential equal to -35.3;

[0196] Fig. 14 diagrammatically shows the liposomal structure of the eye drops according to the invention, highlighting how the PEG chains are outside the phospholipid wall.

[0197] Before a detailed description thereof, we consider it appropriate to briefly summarize the various activities carried out so as to reach, according to the present invention, an ophthalmic product consisting of lactoferrin liposomes in synergistic combination with a non-ionic surfactant such as Vitamin E TPGS, and with Stearylamine.

[0198] A) Verification of the filterability of Lactoferrin at 0.5% by weight in liposomes to avoid sterilization with gamma rays, as is the case for the products already on the market (product of TDC Sri);

[0199] B) Verification of the filterability of Lactoferrin in liposomes with the addition of a surfactant such as Solutol HS15, Vitamin E TPGS, and Polysorbate 80;

[0200] Verification of the effect of such surfactants on antibacterial activity.

[0201] Once such an effect was verified, we set ourselves the task of developing the most effective product from an antibacterial point of view, moving on to:

[0202] C) Verification of the possibility of adding a positive charge to the liposomes by adding a very small amount of Stearylamine in the hypothesis that the presence thereof could: a) enhance the antibacterial / antifungal effect of lactoferrin, also resolving any aging of the protein; and b) improve filterability; to then

[0203] D) Verify the possible enhancement of the antibacterial / antifungal activity of the lactoferrin liposomes and lactoferrin liposomes and Stearylamine.

[0204] The following experiments are thus shown:

[0205] • Comparison of lactoferrin liposomes with different surfactants;

[0206] • verification of lactoferrin liposome efficacy with and without Vitamin E TPGS;

[0207] • verification of liposome efficacy with Vitamin E TPGS with and without Stearylamine after two months at 40°C.

[0208] The tests carried out showed that:

[0209] • the two liposome products with: lactoferrin and Vitamin ETPGS, and lactoferrin, Vitamin ETPGS and Stearylamine, were both effective as antibacterials; and

[0210] • the Liposomes plus lactoferrin, Vitamin E TPGS and Stearylamine product was the most effective and the most stable.

[0211] Preparation of Lactoferrin-containing liposomes (5 liters)

[0212] The charged lactoferrin liposomes were produced using the known lipid film hydration method according to which: the lipophilic phase was formed by dissolving 75g of non-hydrogenated Natural Phospholipids from Soy (Lipoid S75) in 300ml of ethanol, obtaining a clear solution; The lipid film was obtained by removing the organic solvent of said lipophilic phase, under reduced pressure, using the rotary evaporation method. (Rotavapor® R- 210 / 215 Buchi, Flawil, Switzerland).

[0213] The aqueous phase was obtained by dissolving 25g of Lactoferrin in 5 liters of deionized water.

[0214] Then, the lactoferrin-containing aqueous phase was added to the dry lipid film (dry powder), the mixture was then mixed using a Silverson high shear mixer (AX5), thereby obtaining a slightly cloudy homogeneous solution.

[0215] To obtain the liposomes, the solution was subjected to high pressure homogenization at 700-800 bar at room temperature (5-6 cycles).

[0216] Filtration by means of a 0.2 micron filter was applied to sterilize the 5-liter liposomal solution. However, the system did not work properly because after little time the filter was completely clogged.

[0217] The tested Formulation is reported below: FORMULATION 1 This result did not surprise the Inventors, considering the fragility of lactoferrin under mechanical pressure with degradation of the molecular structure of lactoferrin and probable loss of solubility.

[0218] On the other hand, it was known that to obtain lactoferrin liposomes, other researchers used a very bland approach, such as ultrasonic technology plus extrusion at very low pressure (0.8 bar), but even with such a bland method they had to sterilize their liposomal solution by gamma irradiation of 10- 20 KGy, and not by 0.2 micron filtration.

[0219] Such an approach is very time consuming, because as soon as the liposomal solution of lactoferrin is extruded / produced, it must be sterilized by gamma radiation by a specialized company which is certainly not always present in the same place where the liposomal solution is produced. Such a difficulty is especially felt when production occurs on an industrial scale, for example on 200-400 liters in bulk.

[0220] Another consideration to be made is the possible production of toxic intermediate substances due to the effect of the gamma radiation on all the components of the liposomal solution, even if the structures of the liposomes seem stable or unmodified.

[0221] To mitigate such a problem, the Applicant prepared three new Formulations, each containing a specific surfactant to increase the fluidity of the liposomes so as to improve the 0.2 micron filtration and slightly lowered the pressure to 700 - 800 bar during the extrusion process which is repeated 5 times.

[0222] The choice of non-ionic surfactants was made taking into account the HLB (Hydrophilic-Lipophilic Balance) thereof; the equation used is indicated below assuming that this parameter could especially influence the fluidity and deformability of the liposomes.

[0223] Preparation of liposomes containing Lactoferrin plus a SURFACTANT (5 liters)

[0224] The liposomes loaded with lactoferrin and surfactants were always produced using the lipid film hydration method.

[0225] The lipophilic phase was formed by dissolving 75g of Non-Hydrogenated Natural Phospholipids from Soy (for example the Lipoid S75 product currently marketed by Lipoid Kosmetik AG Company (DE) 5, and 5 grams of Surfactant (respectively Polysorbate 80, Kolliphor HS15 (SOLUTOL HS15) and Vitamin E TPGS) in 300ml of ethanol, obtaining a clear solution.

[0226] The lipid film was obtained by removing the organic solvent from the lipophilic phase of phospholipids, using a Rotavapor Buchi under vacuum, obtaining a dry phospholipid powder.

[0227] The aqueous phase was obtained by dissolving 15 g of Lactoferrin in 5 liters of deionized water.

[0228] Then, the aqueous phase containing the lactoferrin was added to the dry phospholipid powder, the mixture was homogenized using a Silverson high shear mixer (Ax5), obtaining a slightly cloudy, homogeneous solution containing phospholipids and lactoferrin. This solution, mixed with a homogenizer (Silverson-inline), was subjected to high-pressure extrusion. In particular, to obtain the liposomes (less than 200 nm in size), such a solution was extruded by high pressure homogenization at 800 bar, at room temperature (5-6 cycles). Filtration through a 0.2 micron filter was applied to sterilize the 5-liter liposomal solution.

[0229] The three tested formulations are as follows :

[0230] Lactoferrin plus Polysorbate 80 FORMULATION 2

[0231] Lactoferrin plus Kolliphor HS 15 FORMULATION 3

[0232]

[0233] Lactoferrin plus Vitamin ETPGS - FORMULATION 4 Such formulations were evaluated for the antibacterial effect thereof, as prescribed by the European Pharmacopoeia F.L XII

[0234] E.coli ATCC 8739 blue color

[0235] Escherichia coli is a Gram-negative bacterium (they turn red after being subjected to Gram staining, a chemical process used for the classification thereof), which is normally found in the bacterial flora of our intestine

[0236] Pseudomonas aeruginosa ATCC 9027 red

[0237] Pseudomonas aeruginosa is a Gram-negative bacterium, an opportunistic pathogen which mainly affects people with compromised immune defenses or physical barriers (skin or mucous membranes). It is the pathogen isolated most often in patients hospitalized for more than a week and one of the microbes involved in the phenomenon of multi-drug resistance.

[0238] Staphylococcus aureus ATCC 6538 gray

[0239] Staphylococci are aerobic Gram-positive microorganisms. Staphylococcus aureus is the most pathogenic germ; it typically causes skin infections and sometimes pneumonia, endocarditis and osteomyelitis. It often leads to abscess formation

[0240] Candida albicans ATCC 10231 light greenish color

[0241] Candida albicans is a saprophytic fungus. Candida albicans is a diploid fungus which can take the unicellular (yeast) or multicellular (hyphae, pseudo-hyphae) form. A unique feature of this microbe is that it can switch from one phenotype to another. The change between the two phenotypes can occur several times and is spontaneous.

[0242] Aspergillus nigerATCC 16404 brown color

[0243] Aspergillus niger is a haploid filamentous fungus .

[0244] LACTOFERRIN LIPOSOMES WITH POLYSORBATE 80 and EDTA (Formulation 2)

[0245] The results reported below in Table 2 describe the time-dependent logarithmic reduction in the number of microorganisms (on day 14).

[0246] TABLE 2

[0247] Reference values: F.LXII ed. Inoculum microorganisms 10A5 - 10A6 CFU / ml

[0248] NOTE: Definition of efficacy criteria A and B

[0249] To meet criteria A, the bacteria must decrease by at least 2 logarithmic units at 6 hours and 3 units at 24 hours and there must be no recovery at 28 days

[0250] To meet criteria A, the fungi must decrease by at least 2 units at 7 days and have no increase at 28 days

[0251] Note that criteria A represent the recommended efficacy to be achieved. In justified cases where criteria A cannot be met, for example due to an increased risk of undesirable reactions, criteria B apply: Logarithmic reduction.

[0252] Nl** : no increase NR* : no recovery

[0253] The graphical presentation of the logarithmic trend of the concentration of the surviving microorganisms as a function of time using formulation 1 is shown in Fig. 1.

[0254] As it can be seen, the product tested up to 14 g falls within the criteria B recommended by the F.U.I. XII ed.

[0255] The result is positive. Comments: The tested product falls within criteria B recommended by F:U :l XII ed.

[0256] Notes: The product highlights an average reduction of 1.6 Log units for all the microorganisms tested, thus falling under criteria B of the F:U : I and corr. However, it is also observed that at 24 h the bacteria tested in the trial have a greater reduction of 3.7 log units on average, thus falling under criteria A of the F.U.I, while for yeasts and molds the log reduction is equal to an average of 2.2, the latter also falling under criteria A of F:U:I and corr., and with no recovery at T7 d.

[0257] As mentioned above, the product under test falls under criteria B only for the logarithmic reduction after 6h and then continues with a good reduction at T24h and with no recovery at T7.

[0258] FORMULATION 3 was then tested, i.e.:

[0259] LACTOFERRIN LIPOSOMES WITH Kolliphor (SOLUTOL HS15) and EDTA

[0260] Again, as seen in Fig. 2, the logarithmic reduction of the concentration of microorganisms is time-dependent.

[0261] Table 3 below contains all the products, contact times and the average Log decrease.

[0262] TABLE 3

[0263] Reference values: F. XII ed. Inoculum microorganisms 10A5 - 10A6 CFU / Logarithmic reduction

[0264] Nl** : no increase NR* : no recovery

[0265] Comments: The tested product highlights an average reduction of 2.4 Log units for all the microorganisms tested (therefore sufficiently respecting the criterion at 6h as set out by the F.U.I and corr.) while for Pseudomonas aeruginosa a much higher reduction is noted at 6h, equal to 4.1. It is further observed that at 24h all strains have a greater reduction than the criteria recommended by the F.U.I and corr., equal to an average of 4 Log units and with no recovery at T7 d. This data is not found for the product with Polysorbate 80, which means that this liposomal product with Kolliphor HS15 is more active than the liposomal product with Polysorbate 80.

[0266] The last test was carried out with

[0267] FORMULATION 4: LACTOFERRIN LIPOSOMES WITH VITAMIN E TPGS and EDTA which, as shown in Fig. 3, always shows a time-dependent logarithmic reduction of microorganisms, as shown below in

[0268] TABLE 4

[0269] Reference values: F.LXII ed. Inoculum microorganisms 10A5 - 10A6 CFU / ml Logarithmic reduction

[0270] Nl** : no increase NR* : no recovery

[0271] Comments: The tested product falls within criteria A recommended by the F.U.I. XII ed.

[0272] The graphical presentation of the test is shown in Fig. 3. The product tested falls within criteria A recommended by the F.U.I. XII ed.

[0273] The tested product highlights a reduction of 2 Log units for all the tested microorganisms (therefore sufficiently respecting the 6h criterion as set out by the F.U.I and corr), while for yeasts and molds, even if the control is not set out by the F.U.I and corr.) it is highlighted that Aspergillus niger is slightly lower than 2 Log units. It is further observed that at 24 h, all strains have an average reduction which is 5 log units higher than the criteria recommended by the F.U.I and corr and with no recovery at T7C|. This result indicates that the Lactoferrin liposomes containing Vit. E TPGS are more effective than the Lactoferrin liposomes with Kolliphor HS15, albeit slightly.

[0274] THE RESULT IS EXCELLENT

[0275] CONCLUSIONS:

[0276] The liposomes prepared with Kolliphor (SOLUTOL) HS15 and Vitamin E TPGS exert greater antibacterial activity (criterion A) as compared to the liposomes prepared with Polysorbate 80 (criterion B), and this unexpected property was combined with easier filterability through a 0.2 micron filter.

[0277] From this point on, the Applicant decided to interrupt formulation development with Polysorbate 80 and instead develop the other two formulations, increasing the viscosity of both to improve the permanence of the eye drops on the eye surface.

[0278] The following formulations were thus prepared: Preparation of a viscous liposome solution containing Lactoferrin plus a SURFACTANT (5 liters)

[0279] The Lactoferrin liposomes and surfactants were again produced using the lipid film hydration method.

[0280] The lipophilic phase was formed by dissolving 75g of Non-Hydrogenated Natural Phospholipids from Soy (Lipoid S75 / Lipoid S80), and 5 grams of Surfactant (respectively: Kolliphor HS15 and Vitamin E TPGS) in 300 ml of ethanol, obtaining a clear solution.

[0281] The lipid film was obtained by removing the organic solvent of the lipophilic phase, under reduced pressure, using the rotary evaporation method (Rotovapor).

[0282] The aqueous phase was obtained by dissolving 25 g of Lactoferrin in 5 liters of deionized water.

[0283] Then, the lactoferrin-containing aqueous phase was added to the dry lipid film, the mixture was then mixed using a Silverson high shear mixer (AX5), and an opalescent homogeneous solution was obtained.

[0284] To obtain the liposomes, such a solution was extruded by high pressure homogenization at 800 bar at room temperature (5-6 cycles). Then the viscosity of each liposomal solution (Lactoferrin + Kolliphor HS15, or Lactoferrin + Vitamin E TPGS ) was increased by respectively adding:

[0285] - 5 g of hyaluronic acid

[0286] - 5 g of HPMC,

[0287] - 250 g of Poloxamer 188.

[0288] After mixing with Shear Mixer (AX5), 3 viscous liposomal solutions were obtained, one for each Surfactant (6 formulations).

[0289] All the solutions were sterilized by 0.2 micron filter filtration.

[0290] The formulations are listed hereinbelow:

[0291] Lactoferrin LIPOSOMES plus Kolliphor SH 15 and Hyaluronic acid FORMULATION 5

[0292] Lactoferrin LIPOSOMES plus Kolliphor SH 15 and HPMC

[0293] FORMULATION 6

[0294]

[0295] Lactoferrin LIPOSOMES plus Kolliphor SH 15 and Poloxamer 188

[0296] FORMULATION 7

[0297] Lactoferrin LIPOSOMES plus VITAMIN E TPGS and Hyaluronic Acid

[0298] FORMULATION 8 Lactoferrin LIPOSOMES plus Vitamin E TPGS and HPMC

[0299] FORMULATION 9

[0300] Lactoferrin LIPOSOMES + Vitamin E TPGS and Poloxamer 188

[0301] FORMULATION 10

[0302] CONCLUSIONS:

[0303] The formulations with surfactants (Kolliphor SH15, Vitamin E TPGS), containing HPMC and Poloxamer 188 were easily sterilized by 0.2 micron filter filtration. Unfortunately, it was impossible to sterilize the formulations containing

[0304] Hyaluronic Acid because after little time the filter became clogged, which indicates a specific (complex) interaction between the positive charge of Lactoferrin, even if included in the liposomes, and the negative charge of hyaluronic acid. This result indicates the possibility of an interaction between lactoferrin and the medium outside the liposomes.

[0305] Therefore, the Applicant decided to interrupt the development of:

[0306] Lactoferrin Liposomes plus Kolliphor HS15 and Hyaluronic Acid; and of Lactoferrin Liposomes plus Vitamin E TPGS and Hyaluronic Acid, as such compositions are clearly unsuitable for industrial development given the impossibility of being sterilized by 0.2 micron filtration.

[0307] The Applicant thus continued developing: Lactoferrin Liposomes plus viscous Kolliphor HS15 of HPMC,

[0308] Lactoferrin Liposomes plus viscous Vitamin E TPGS of HPMC, and

[0309] Lactoferrin Liposomes plus viscous Vitamin E TPGS of Poloxamer 188.

[0310] Starting from the peculiar presence of a positive charge on the lactoferrin molecule as reported in the literature, the present inventors tried to strengthen such an aspect, so important for the antibacterial properties of Lactoferrin, by inserting Stearylamine (positively charged lipophilic amine) in the phospholipid layer of liposomes.

[0311] Therefore, further formulations were developed:

[0312] "PREPARATION OF LACTOFERRIN LIPOSOMES ENRICHED WITH A POSITIVE CHARGE, WITH THE ADDITION OF STEARYLAMINE, PLUS A SURFACTANT"

[0313] Liposomes loaded with Lactoferrin and Vitamin E TPGS containing a positive charge by the addition of Stearylamine, were again produced using the lipid film hydration method, namely: the lipophilic phase was carried out by dissolving 75g of Non-Hydrogenated Natural Phospholipids from Soy (Lipoid S75), 5 grams of Vitamin E TPGS and 125 mg of Stearylamine in 300 ml of ethanol, obtaining a clear solution.

[0314] The lipid film was obtained by removing the organic solvent of the lipophilic phase, under reduced pressure, using a Rotavapor.

[0315] The aqueous phase was obtained by dissolving 25 g of Lactoferrin in 5 liters of deionized water.

[0316] Then the aqueous phase containing the lactoferrin was added to the dry lipid film; the mixture consisting of phospholipids, stearylamine, Vitamin E TPGS and lactoferrin was then mixed using a Silverson high shear mixer (AX5), obtaining the solution to be extruded.

[0317] To obtain the liposomes, the solution was extruded by high pressure homogenization at 800 bar at room temperature (5-6 cycles).

[0318] Then the viscosity of the liposomal solution (lactoferrin plus Vitamin E TPGS and Stearylamine) was increased by adding 10 g of HPMC.

[0319] After mixing with Shear Mixer (AX5), a liposomal solution (Phospholipids Stearylamine Vitamin E TPGS) and HPMC was obtained. The liposomal solution of lactoferrin, Stearylamine and Vitamin E TPGS was finally sterilized by 0.2 micron filter filtration.

[0320] A first tested formulation is shown below:

[0321] LIPOSOMES OF LACTOFERRIN, STEARYLAMINE, PLUS VITAMIN E TPGS AND HPMC FORMULATION 11

[0322] PREPARATION OF LACTOFERRIN LIPOSOMES ENRICHED WITH A POSITIVE CHARGE

[0323] WITH THE ADDITION OF STEARYLAMINE, PLUS KOLLIPHOR SH15

[0324] These liposomes were again produced using the lipid film hydration method. The lipophilic phase was carried out by dissolving:

[0325] - 75g of Natural Non-Hydrogenated Phospholipids from Soy (Lipoid S75),

[0326] - 5 grams of Kolliphor HS15,

[0327] - 125 mg of stearylamine in 300 ml of ethanol, obtaining a clear solution.

[0328] The lipid film was obtained by removing the organic solvent of the lipophilic phase, under reduced pressure, using Rotavapor.

[0329] The aqueous phase was obtained by dissolving 25 g of Lactoferrin in 5 liters of deionized water.

[0330] Then, the aqueous phase containing the lactoferrin was added to the dry lipid film, the mixture consisting of the phospholipids, the Stearylamine, the Kolliphor HS15 and the lactoferrin was then mixed using a Silverson high shear mixer (AX5): a solution was obtained, consisting of lactoferrin, phospholipids, Stearylamine and Kolliphor HS15, slightly opalescent.

[0331] To obtain the liposomes, the solution was extruded by high pressure homogenization at 800 bar at room temperature (5-6 cycles).

[0332] Then the viscosity of the liposomal solution (Lactoferrin plus Kolliphor HS15 and Stearylamine) was increased by adding 10 g of HPMC.

[0333] After mixing with Shear Mixer (AX5), a liposomal solution of lactoferrin with Stearylamine plus Kolliphor HS15 and HPMC was obtained.

[0334] The liposomal solution Lactoferrin Stearylamine plus Kolliphor SH15 and HPMC, after the addition of the borate buffer, EDTA and Trehalose, was sterilized by filtration with a 0.2 m filter.

[0335] Such a formulation is shown hereinbelow:

[0336] LIPOSOMES OF LACTOFERRIN, STEARYLAMINE PLUS KOLLIPHOR SH 15 AND HPMC

[0337] FORMULATION 12

[0338] Both formulations:

[0339] - Formulation 11:

[0340] Lactoferrin liposomes containing Stearylamine and Vitamin E TPGS and HPMC ; and - Formulation 12:

[0341] Lactoferrin Liposomes containing Stearylamine and Kolliphor SH15 and HPMC were tested to define antibacterial efficacy.

[0342] LIPOSOMES OF LACTOFERRIN, STEARYLAMINE AND VITAMIN E TPGS and HPMC FORMULATION 11

[0343] TABLE 5

[0344] Conclusions: Logarithmic reduction in the number of micro-organisms as a function of time

[0345]

[0346] Reference values: F.LXII ed. Inoculum microorganisms 10A5 - 10A6 CFU / ml

[0347] Logarithmic reduction

[0348] Nl** : no increase NR* : no recovery

[0349] Comments: As it can be seen in Fig. 4, the product tested up to 14 days falls within the criteria A recommended by the F.U.L XII ed.

[0350] Excellent result, and better than the previous Formulation 9 without stearylamine.

[0351] Criterion A WITHOUT STEARYLAMINE

[0352] The comparison between the two Formulations 9 and 11 is shown below.

[0353] Such results demonstrate that:

[0354] 1) A Lactoferrin Liposome Formulation containing Stearylamine and Vitamin E TPGS and HPMC exerts a stronger antibacterial effect as compared to Lactoferrin Liposomes plus Vitamin E TPGS and HPMC;

[0355] 2) Stearylamine enhances the antibacterial effects of lactoferrin. This is linked to the fact that the antibacterial effect increases by increasing the positive charge due to Stearylamine and Lactoferrin, despite the fact that the net charge of the entire liposomal structure including Lactoferrin is negative. This is due to the fact that the negative charge of the phospholipids is predominant with respect to the rest (stearylamine plus lactoferrin). LACTOFERRIN LIPOSOMES PLUS VITAMIN E TPGS and HPMC

[0356] FORMULATION 9

[0357] TABLE 6

[0358] Conclusions: Logarithmic reductions in the number of microorganisms as a function of time.

[0359] LIPOSOMES OF LACTOFERRIN, STEARYLAMINE AND VITAMIN E TPGS and HPMC

[0360] FORMULATION 11

[0361] TABLE 7 Conclusions: Logarithmic reduction in the number of micro-organisms as a function of time From the tables above it is apparent that the formulation containing lactoferrin, stearylamine, Vitamin E TPGS and HPMC, No. 11, has greater antifungal antibacterial power than the formulation containing lactoferrin, vitamin E TPGS and HPMC, No. 9.

[0362] At this point, the following were compared:

[0363] Liposomes of lactoferrin, stearylamine, Kollifor SH15 and HPMC No. 12; and Lactoferrin liposomes, Kollifor SH15 and HPMC, No. 9.

[0364] LIPOSOMES OF LACTOFERRIN, STEARYLAMINE AND KOLLIPHOR HS15 and HPMC FORMULATION 12

[0365] TABLE 8

[0366] Conclusions: Logarithmic reduction of microorganisms as a function of time

[0367] Reference values: F. XII ed. Inoculum microorganisms 10A5 - 10A6 CFU / ml

[0368] Logarithmic reduction

[0369] Nl** : no increase NR* : no recovery

[0370] Comments: The product tested up to 14 g falls within the recommended criteria A

[0371] LACTOFERRIN LIPOSOMES with Kolliphor HS15 and HPMC

[0372] FORMULATION 9 TABLE 8

[0373] Conclusions: Logarithmic reduction in the number of micro-organisms as a function of time

[0374] Reference values : F.LXII ed. Inoculum microorganisms 10A5 - 10A6 CFU / ml

[0375] Logarithmic reduction

[0376] Nl** : no increase NR* : no recovery

[0377] Conclusions: a) Formulation No.12, Lactoferrin Liposomes containing Stearylamine and Kolliphor HS15 and HPMC, exerts a stronger antibacterial effect as compared to Formulation No. 9, that of Lactoferrin Liposomes plus Kolliphor HS15 and HPMC. b) Excellent result, but slightly lower than the formulations containing Vitamin ETPGS;

[0378] Such results demonstrate that:

[0379] 1) The Formulation: Lactoferrin Liposomes containing Stearylamine and Kolliphor HS15 and HPMC exerts a stronger antibacterial effect as compared to Lactoferrin Liposomes plus Kolliphor HS15 and HPMC.

[0380] 2) Stearylamine enhances the antibacterial effects of lactoferrin: this is linked to the fact that the antibacterial effect increases by increasing the positive charge due to stearylamine and lactoferrin, despite the fact that the net charge of the entire liposomal structure including lactoferrin is negative. This is because the negative charge of the phospholipids is preponderant with respect to the rest (stearylamine plus lactoferrin). See the following tables:

[0381] LACTOFERRIN LIPOSOMES with Kolliphor HS15 and HPMC FORMULATION 9

[0382] TABLE 9

[0383] Conclusions: Logarithmic reduction in the number of micro-organisms as a function of time

[0384] Reference values : F.LXII ed. Inoculum microorganisms 10A5 - 10A6 CFU / ml

[0385] LIPOSOMES OF LACTOFERRIN, STEARYLAMINE and Kolliphor HS15 and HPMC

[0386] FORMULATION 11

[0387] TABLE 10

[0388] Conclusions: Logarithmic reduction in the number of micro-organisms as a function of time

[0389] Final conclusion

[0390] If we compare the formulations:

[0391] - LIPOSOMES OF LACTOFERRIN, STEARYLAMINE and VITAMIN E TPGS and HPMC, and - LIPOSOMES OF LACTOFERRIN, STEARYLAMINE and KOLLIPHOR HS15 and HPMC, the presence of Stearylamine is seen to enhance the antibacterial effect of the lactoferrin of both formulations but, more specifically, the formulation containing Vitamin E TPGS exerts an even stronger antibacterial effect. See the following tables :

[0392] LIPOSOMES OF LACTOFERRIN, STEARYLAMINE and VITAMIN E TPGS and HPMC

[0393] FORMULATION 11

[0394] TABLE 11

[0395] Conclusions: Logarithmic reduction in the number of micro-organisms as a function of time

[0396] LIPOSOMES OF LACTOFERRIN, STEARYLAMINE and KOLLIPHOR HS15 and HPMC

[0397] FORMULATION 12

[0398] TABLE 12 Conclusions: Logarithmic reduction in the number of micro-organisms as a function of time

[0399] Further considerations a) Liposomes containing lactoferrin can also be suitably prepared on an industrial scale (200-400 liters) with a high pressure extrusion homogenizer (700-800 bar), by adding a specific surfactant which allows the sterilization thereof by means of a 0.2 micron filter; b) In particular, the inclusion of Vitamin E TPGS, or Kolliphor HS15 within the liposomal structure, improves the filterability of the Lactoferrin liposomes; c) the presence of Vitamin E TPGS, or Kolliphor HS15 within the phospholipid structure of the Lactoferrin liposomes, strengthens the antibacterial effect of the Lactoferrin liposomes and, unexpectedly, such an improvement of the antibacterial effect is stronger than the formulation containing Polysorbate 80, a known surfactant used in the preparation of liposomes; d) Vitamin E TPGS and Kolliphor SH15 (or Solutol HS15) have a specific HLB, respectively 13.5 and 15, substantially similar to that of Polysorbate 80 ( 15 ): therefore, it is not such a parameter that can justify, or explain, at least in part, the unexpected fluidity and antibacterial results, but a characteristic thereof deriving from PEG.

[0400] In fact, the two surfactants Solutol HS15 and Vitamin E TPGS having a high HLB (15 and 13, respectively), have the lipophilic part thereof (minority) interspersed within (the fatty acids of) the phospholipids, while they have the hydrophilic part thereof (majority, consisting of PEG) outside the liposomes, as shown in Fig. 14, without interfering with the electrical potential of the entire liposomal structure.

[0401] This feature linked to PEG is the basis of the greater fluidity and activity of the preparations and further differentiates the preparations from what has been described so far.

[0402] To increase the antibacterial effects, the further development of lactoferrin liposomes containing specific surfactants (vitamin E TPGS, Kolliphor SH15) led to the preparation of liposomes having, interspersed in the phospholipids, Stearylamine, therefore with an increase in the positive charge consisting of Lactoferrin and Stearylamine, while in the end, the entire liposomal structure has a negative net charge due to the preponderance of phospholipids which include all the lactoferrin.

[0403] It is useful to emphasize once again for clarity and specificity that the addition of Stearylamine, at low concentrations, can modify the charge distribution phospholipids that are negatively charged, without however leading to a positive net charge. This feature becomes a peculiarity of formulations containing Stearylamine, both from a chemical-physical structural point of view, and for the antibacterial and antifungal efficacy. In particular, it is important to also consider the percentage W / W ratio between the amount of lactoferrin and S75 phospholipids, which is 3:1.

[0404] In conclusion, the antibacterial effect of the new formulations:

[0405] Liposomes of Lactoferrin, Stearylamine and Vitamin E TPGS and HPMC, and Liposomes of Lactoferrin, Stearylamine and Kolliphor HS15 and HPMC, was much stronger than the same lactoferrin liposomes without Stearylamine.

[0406] STABILITY TESTS

[0407] The Applicant carried out a series of tests at 40°C for 2 months to evaluate the stability of the different formulations.

[0408] Test on :

[0409] SAMPLE A: Niolip Liposomes with 0.5% Lactoferrin, 0.1% Vit E TPGS and 0.2% HPMC and 0.1% EDTA at To.

[0410] BATCH: NLL-207-356 / 03-22

[0411] TABLE 13

[0412] Conclusions: Logarithmic reduction in the number of micro-organisms as a function of time

[0413] SAMPLE B: Niolip Liposomes with 0.5% Lactoferrin, 0.1% Vit E TPGS * 0.2% HPMC and 0.1% EDTA at Tx month at 40°C BATCH: NLL-207-356 / 03-22

[0414] TABLE 14

[0415] Conclusions: Logarithmic reduction of the number of microorganisms as a function of

[0416] Time Comments:

[0417] From the comparison of the values found in the logarithmic reduction of the microorganisms tested in the samples at Toand Timonth, no substantial changes were observed, remaining stable after 1 month at 40°C. SAMPLE: Niolip liposomes with 0.5% lactoferrin with 0.1% Vit E TPGS and 0.2% HPMC and 0.1% EDTA at T2 mOnth at 40°C BATCH: NLL-207-356 / 03-22

[0418] TABLE 15

[0419] Conclusions: Logarithmic reduction in the number of micro-organisms as a function of time

[0420] Comments:

[0421] From the comparison of the values found in the logarithmic reduction of the microorganisms tested in the samples at To, Timonth and T2month, no substantial changes were observed, remaining stable after 2 months at 40°C.

[0422] LACTOFERRIN LIPOSOMES PLUS VITAMIN E TPGS AND HPMC and EDTA FORMULATION 9

[0423] CONCLUSIONS

[0424] Formulation 9 shows good stability after 60 days at 40°C.

[0425] NEW SUMMARY TABLE 15

[0426] SAMPLE: Niolip liposomes with 0.5% lactoferrin and 0.0025% Stearylamine with 0.1%

[0427] Vit E TPGS and 0.1% EDTA at To

[0428] BATCH: NLL-209-374 / 06-22

[0429] TABLE 16 Conclusions: Logarithmic reduction in the number of micro-organisms as a function of time

[0430]

[0431] SAMPLE: Niolip liposomes with 0.5% lactoferrin and 0.0025% Stearylamine with 0.1%

[0432] Vit E TPGS and 0.1% EDTA at Timonth at 40°C

[0433] BATCH: NLL-209-374 / 06-22

[0434] TABLE 17

[0435] Conclusions: Logarithmic reduction in the number of micro-organisms as a function of time Comments:

[0436] From the comparison of the values found in the logarithmic reduction of the microorganisms tested in the samples at Toand Timonth, no substantial changes were observed, remaining stable after aging at 40°C. SAMPLE: Niolip liposomes with 0.5% lactoferrin and 0.0025% stearylamine with 0.1% Vit E TPGS and EDTA = 1% at T2 month at 40°C

[0437] BATCH: NLL-209-374 / 06-22 TABLE 18

[0438] Conclusions: Logarithmic reduction in the number of micro-organisms as a function of time Comments:

[0439] From the comparison of the values found in the logarithmic reduction of the microorganisms tested in the samples at ToTimonth and T2month, no substantial changes were observed, remaining stable after 60 days at 40°C. SAMPLE: Niolip liposomes with 0.5% lactoferrin and 0.0025% Stearylamine with 0.1% Vit E TPGS and 0.1% EDTA at T2months at 40°C BATCH: NLL-209-374 / 06-22

[0440] TABLE 19

[0441] Conclusions: Logarithmic reduction in the number of micro-organisms as a function of time

[0442] SAMPLE: Niolip liposomes with 0.5% lactoferrin with 0.1% Vit E TPGS and 0.1% EDTA at

[0443] T2month at 40 C

[0444] BATCH: NLL-207-356 / 03-22 TABLE 20

[0445] Conclusions: Logarithmic reduction in the number of micro-organisms as a function of time

[0446] Comments:

[0447] From the comparison of the values found in the logarithmic reduction of the microorganisms tested in the samples at To, Timonth and T2month, no substantial changes were observed, remaining stable after aging at 40°C. The best formulation because it is most effective and stable over time is the one corresponding to Batch: N 11-209-374 / 06-22, therefore

[0448] NIOLIP LIPOSOMES WITH 0.5% LACTOFERRIN and 0.0025% STEARYLAMINE WITH 0.1%

[0449] Vit E TPGS and 0.1% EDTA

[0450] BATCH: NLL-209-374 / 06-22

[0451] BEST FORMULATION because MORE EFFECTIVE:

[0452] FORMULATION 13 where EDTA only has the common function of stabilizing negatively charged phospholipids since it complexes calcium.

[0453] The above formulation 13 was then improved by replacing the HPMC with a synthetic polymer of the Poloxamer family, in particular Poloxamer 188 which, with the same stability and efficacy on the tested microorganisms, in addition to ensuring a good viscosity, being a thermosensitive polymer, has the advantageous feature of increasing the viscosity thereof with increasing temperature.

[0454] This resulted in a final formulation 14 of eye drops based on lactoferrin liposomes with Vit. E TPGS, Stearylamine, EDTA and Poloxamer 188 for treating eye diseases, which is sterilizable by 0.2 filtration, stable to aging, and with antibacterial activity which falls under efficacy criteria A, as prescribed by the European pharmacopoeia F.I.XII.

[0455] The final formulation is given below in detail :

[0456] NIOLIP LIPOSOMES WITH 0.5% LACTOFERRIN and 0.0025% STEARYLAMINE WITH

[0457] 0.1% Vit E TPGS and 0.1% EDTA and 5% Poloxamer 188

[0458] FINAL FORMULATION 14

[0459] Table 21 below shows the values found in the logarithmic reduction of the microorganisms tested in the sample of Final formulation 13. SAMPLE: Niolip liposomes with 0.5% lactoferrin and 0.0025% Stearylamine with 0.1%

[0460] Vit E TPGS and 0.1% EDTA and 5% Poloxamer 188

[0461] BATCH: NLL-209-374 / 06-22

[0462] TABLE 21 Conclusions: Logarithmic reduction in the number of micro-organisms as a function of time

[0463] An ophthalmic liposomal product has thus been defined for use in a curative or preventive treatment of disorders or diseases of the eye and periocular area, which, in mutual synergistic combination, comprises, or consists of, S75 phospholipids, lactoferrin, Vit. E TPGS, Stearylamine, EDTA, and a viscosifier selected from the Poloxamer 407 / 188 family; it is sterilizable by filtration at 0.2, is stable to aging, with pH between 7.0 and 7.5, and is provided with antibacterial activity which falls under efficacy criteria A, as prescribed by the European pharmacopoeia F.I.XII, in which the lactoferrin liposomes with an amount of Stearylamine not exceeding

[0464] 0.0030% of the total weight and 0.07-0.15% of Vitamin ETPGS are obtained by extrusion at a pressure between 700 and 800 bar; the lactoferrin liposomes are present at 0.5% by weight on the total weight and the ratio between S75 phospholipids and lactoferrin is 1.5 / 0 .5 =3, sufficient for the lactoferrin to be totally encapsulated; and the liposomes of lactoferrin, Stearylamine and Vit. E TPGS have an electrical potential, measured in distilled water, using a particle analyzer by means of a light scattering technique (DLS), less than 0 mV, this to synergistically strengthen in said ophthalmic liposomal product the ability to prevent and / or treat eye disorders, in particular those in which there is an inflammatory condition such as dry eye syndrome and the related complications, such as chalazion, stye (chronic chalazion), conjunctivitis, blepharitis and keratitis, and reducing the possible aging of the lactoferrin protein.

Claims

AMENDED CLAIMS received by the International Bureau on May 28, 2025 (28.05.2025)CLAIMS1) A liposomal product with antibacterial activity, comprising lactoferrin, for use in a curative or preventive treatment of disorders or diseases of the eye and periocular area, characterized in that:I) in an amount between 0.1 and 0.5% by weight on the total weight, a pegylated non-ionic surfactant with an HLB (Hydrophilic-Lipophilic Balance) greater than 13, and with a linear PEG chain, outside the phospholipid layer of the liposomes, capable of coordinating water molecules, of length between 1000 and 712 Daltons, for an amount by weight on the total weight of the composition equal to 0.07-0.15%, is added to the lactoferrin liposomes, where the pegylated non-ionic surfactant is selected from: Kolliphor, Macrogol 15 Hydroxy stearate and Vitamin E TPGS; and whereII) a positive charge is added to the lactoferrin liposomes and said pegylated non-ionic surfactant, adding therein, interspersed in the lipid film of the same liposomes, an amount of Stearylamine not exceeding 0.0030% of the total weight, such as not to create cytotoxicity problems on the eye surface, and to however maintain the total charge of the ophthalmic product with a negative sign, said lactoferrin liposomes, non-ionic surfactant, Stearylamine, being obtained by extrusion at a pressure between 700 and 800 bar.2) The liposomal product according to claim 1, characterized in that the pegylated non-ionic surfactant is Vitamin E TPGS.3) The liposomal product according to claim 1, characterized in that the pegylated non-ionic surfactant is Macrogol 15 Hydroxystearate.4) The liposomal product according to claim 2, characterized in that it comprises Vitamin E TPGS for an amount by weight on the total weight of the composition equal to 0.07-0.15%, preferably equal to 0.1%.5) The liposomal product according to any one of the preceding claims, characterized in that the liposomes of lactoferrin, Stearylamine and Vit. E TPGS have an electrical potential, measured in distilled water, using a particle analyzer by means of a light scattering technique (DLS) by means of a Zetasizer Nano ZS Instrument fromMalvern Panalytical, less than 0 mV.6) The liposomal product according to any one of the preceding claims, characterized in that the liposomes of lactoferrin, stearylamine and Vit. E TPGS are obtained by the thin-film hydration method.7) The liposomal product according to claim 1, characterized in that it has the following formulation:8) The liposomal product according to claim 1, characterized in that the lactoferrin liposomes with 0.0025% stearylamine and 0.1% Vitamin E TPGS are obtained by extrusion at a pressure between 700 and 800 bar.

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