Ophthalmic external composition containing fermentation metabolites of Lactobacillus

Abstract

The present invention relates to a sterile buffered ophthalmic topical composition containing fermentation metabolites of Lactobacillus belonging to the species casei or paracasei, and the ophthalmic use of the composition in preventing or treating microbial eye infections, eye allergies, vernal keratoconjunctivitis and keratoconjunctivitis sicca, and blepharitis.

Description

Field of the Invention

[0001] The present invention relates to a stable topical ophthalmic composition containing a fermentation metabolite of Lactobacillus.

[0002] The present invention originates from the field of ophthalmic products and eye drops.

[0003] In particular, the present invention relates to a composition for external use for ophthalmic administration, which contains a postbiotic as an active ingredient and has a specific formulation that stabilizes the postbiotic in an aqueous solution.

[0004] background

[0005] Probiotics are live microorganisms that, when administered in biologically active amounts, exert beneficial effects on the host. Postbiotics are by-products of the fermentation process carried out by probiotics.

[0006] Postbiotics refer to soluble factors secreted by living bacteria or released after bacterial lysis, that is, metabolites or subproducts, such as enzymes, peptides, teichoic acid, muropeptide, polysaccharides, cell surface proteins and organic acids derived from peptidoglycan.

[0007] Because postbiotics are considered bioactive products with a high safety profile, their use in both the dietary and pharmaceutical fields is increasing.

[0008] The use of probiotics in the form of eye drops is also known, for example from Bonni et al.

[0009] The use of postbiotics in the ophthalmic field is difficult due to their low stability in aqueous solutions.

[0010] Therefore, there is a need for useful ophthalmic formulations containing postbiotics that remain stable.

[0011] One of the objects of the present invention lies in the fact of providing a composition for external use for ocular administration containing postbiotics as biologically active components.

[0012] Overview

[0013] Ophthalmic compositions with high stability containing specific postbiotics as bioactive components have now been identified and form the subject of the present invention.

[0014] Such compositions have a formulation that maintains the postbiotics stable in ophthalmic solution.

[0015] In particular, the composition of the present invention prevents and substantially reduces postbiotic protein components that cause instability phenomena such as protein aggregation or denaturation thereof resulting from changes in the tertiary and quaternary structures of proteins.

[0016] According to a first aspect, there is provided a composition for external ophthalmic use, the composition comprising

[0017] Postbiotics, which are fermentation products of Lactobacillus casei or Lactobacillus paracasei,

[0018] A buffer system comprising sodium dihydrogen phosphate, in particular monohydrate, and disodium hydrogen phosphate, in particular dodecahydrate,

[0019] an isotonic agent comprising magnesium chloride or calcium chloride and a mixture thereof,

[0020] a physiologically acceptable water-based carrier,

[0021] The composition has a pH comprised between 6.7 and 6.9, preferably between 6.8 and 6.9.

[0022] The compositions according to the present invention may also contain any buffering agents, isotonicity agents and preservatives, as described in detail below.

[0023] The ophthalmic composition thus obtained shows a surprising ability to act at the level of the ocular surface and therefore enables, with high efficacy and a wide safety margin, external treatments suitable for the prevention or treatment of diseases of this part of the eye. The new form of therapy can be used in combination with other known therapies for the same diseases.

[0024] Description of the drawings

[0025] Figure 1 : Percent inhibition of microorganisms obtained from anti-adhesion assays performed on two batches at T0 and T6 months under long-term stability conditions at 25° C. and under accelerated conditions at 40° C. Data represent the mean ± SEM of three replicates and were statistically analyzed by one-way ANOVA followed by the Bonferroni test.

[0026] Figure 2 : (AB) Changes in the percentage of cell viability as a function of the scalar concentration of the formulation tested under wash / no wash conditions according to the STRE protocol. Data represent the mean ± SEM of three replicates and were statistically analyzed by one-sample t-test versus a 50% cutoff value: ****P < 0.0001 versus cutoff wash condition; ###P < 0.001 and ####P < 0.0001 versus cutoff no wash condition.

[0027] Figure 3 : (AE) pH, size, osmolality, pZ and PDI values ​​shown on the graph as a function of study time under 40±2°C / 75±5% RH accelerated stability conditions and 25±2°C / 60±5% RH long-term stability conditions.

[0028] Figure 4 : (AE) pH, size, osmolality, pZ and PDI values ​​are shown as a function of study time under accelerated stability conditions of 40±2°C / 75±5% RH and long-term conditions of 25±2°C / 60±5% RH.

[0029] Figure 5 A: illustrates a photographic reproduction of a preparation according to Example 2 containing postbiotics obtained from fermentation of Lactobacillus paracasei CNCM 1-5220 in water and NaCl, maintained at 40° C. for three weeks;

[0030] Figure 5 B: illustrates a photographic reproduction of a formulation according to Example 2 containing postbiotics obtained from the fermentation of Lactobacillus paracasei CNCM 1-5220 in water and NaCl, maintained at 40° C. for one week.

[0031] Detailed description

[0032] The present invention arises from the discovery that specific conditions enable the postbiotic-containing composition / aqueous solution defined herein to remain stable and prevent or substantially reduce denaturation of its protein components.

[0033] These conditions are a) the pH of the solution is selected in the range of 6.7 to 6.9, preferably 6.8 to 6.9, using a suitable buffer or buffer system, and b) the presence of divalent calcium salts and divalent magnesium salts, such as magnesium chloride and calcium chloride, in the solution.

[0034] Applicants have also observed how to obtain effective adjustment of the pH to the desired range using buffers of sodium dihydrogen phosphate and disodium hydrogen phosphate.

[0035] The bioactive components of the composition include postbiotics, in particular fermentation products from bacterial strains belonging to the species Lactobacillus casei or Lactobacillus paracasei, preferably Lactobacillus paracasei.

[0036] According to a preferred embodiment, the prebiotic is the Lactobacillus paracasei strain CNCM I-1390, filed on July 26, 2017 under the Budapest Treaty under the CNCM (Collection Nationale de Cultures de Microorganismes, Institut Pasteur, Paris, FR) number I-5220.

[0037] According to some embodiments, the composition contains postbiotics in an amount of 0.05% to 5%, 0.1% to 1.9%, 0.2% to 1% weight / volume (g / 100 ml).

[0038] The composition of the present invention contains a buffer or a buffer system comprising sodium dihydrogen phosphate, especially monohydrate, and disodium hydrogen phosphate, especially dodecahydrate.

[0039] Preferably, the ratio between sodium dihydrogen phosphate monohydrate / sodium hydrogen phosphate dodecahydrate is 16 / 41=0.39.

[0040] According to some embodiments, the composition comprises an additional buffer comprising sodium citrate.

[0041] The composition of the present invention comprises at least one isotonic agent selected from magnesium chloride, calcium chloride, and mixtures thereof. In a preferred embodiment, the isotonic agent comprises magnesium chloride and calcium chloride. Preferably, the magnesium chloride is magnesium chloride hexahydrate and the calcium chloride is dihydrate. Preferably, the ratio of magnesium chloride hexahydrate to calcium chloride dihydrate is 10 / 7 = 1.42.

[0042] According to some embodiments, the composition of the invention comprises an additional isotonicity agent comprising glycerol.

[0043] Typically, the compositions of the present invention contain a physiologically acceptable water-based carrier in which the postbiotics described herein are dispersed. Preferably, the water is purified.

[0044] The composition of the present invention has a pH comprised between 6.7 and 6.9, preferably between 6.8 and 6.9. Therefore, the components of the composition: buffer, isotonicity agent are present in the composition of the present invention in an amount capable of obtaining a pH comprised between 6.7 and 6.9, preferably between 6.8 and 6.9.

[0045] According to one embodiment, the composition of the invention has the following formula:

[0046]

[0047]

[0048] According to a preferred form, the composition has the formulation of Example 3.

[0049] As defined herein, the compositions of the present invention are stable.

[0050] Furthermore, in the composition, a combination of a sodium dihydrogen phosphate buffer (preferably monohydrate) and a sodium hydrogen phosphate (preferably dodecahydrate) in an amount capable of providing a pH of 6.7 to 6.9 and an isotonic agent based on magnesium chloride and calcium chloride stabilizes the composition.

[0051] In this document, the term "stable" means that the composition has an average particle dispersion (size) of ≤ 200 nm at 40°C and 25°C for 6 months, in particular with respect to postbiotics. Thus, under the conditions tested, no significant changes occurred attributable to the formation of aggregates of material derived from the postbiotics, i.e., an increase in size of more than 200 nm at 25°C or 40°C.

[0052] In the context of the present invention, the term postbiotic means fermented species Lactobacillus casei or Lactobacillus paracasei or a mixture thereof or fermentation products of species Lactobacillus casei or Lactobacillus paracasei or a mixture thereof, and in particular products of fermentation of Lactobacillus paracasei CNCM 1-5220.

[0053] It has been observed that when the composition according to any of the aforementioned embodiments has a barrier effect on epithelial cells, the biosurfactants, glycoproteins, and mixtures of organic acids and peptides present in the postbiotics prevent pathogenic bacteria from binding to the ocular mucosa. Thus, the composition has a preventive effect or treatment on ocular infections.

[0054] Furthermore, the composition has immunomodulatory activity and can be used to prevent and / or treat ocular allergies and / or inflammation.

[0055] The composition may be in single or multiple dose form.

[0056] In particular, the buffer component is present in the composition at a concentration capable of achieving / maintaining a pH comprised between 5 and 8, which is compatible with ocular tissues and the active ingredients carried.

[0057] Preferably, the composition is isotonic with tear fluid and has a value comprised between 270 mOsm / Kg and 310 mOsm / Kg.

[0058] Preferably, the composition of the present invention is an eye wash or eye gel.

[0059] The compositions according to the present invention formulated in multiple doses may also contain antimicrobial preservatives such as, for example, parabens, quaternary ammonium salts, polyhexamethylene biguanide (PHMB) and other antimicrobial preservatives that may be included in compositions intended for ophthalmic use. The solvent used in the composition is preferably water or an aqueous solution of one or more components compatible with topical ophthalmic use.

[0060] For administration purposes, the above composition may be in the form of eye drops or liquid or gel.

[0061] Another subject of the present invention is the external ophthalmic use of a composition as defined above for the preparation of a medicament for the treatment or prevention of ocular inflammation and / or allergies.

[0062] The composition of the present invention can be used to treat ocular allergies, vernal keratoconjunctivitis and keratoconjunctivitis sicca, and blepharitis.

[0063] The composition of the present invention can also be used to prevent foreign matter or microorganisms from adhering to the surface of the eye, as experimentally demonstrated in Example 1 below.

[0064] The following examples further illustrate the present invention but do not constitute any limitation of the present invention.

[0065] Example 1

[0066] In this experimental study, the formulation of Example 3 was used, which contains postbiotics derived from the fermentation process of Lactobacillus paracasei CNCM I-5220, hereinafter referred to as Different assays were developed to evaluate the two batches of (R&D1907, R&D1908) Anti-adhesion effect, safety and stability of the composition.

[0067] method:

[0068] The anti-adhesion properties of formulations R&D1907 and R&D1908 against Staphylococcus aureus (Sa) strain ATCC 43300 were evaluated using an anti-adhesion assay relative to vehicle (VHC). Specifically, 200 μl of each formulation was added to each well and the plates were incubated at 4°C for 18 h. After this time, the test articles were removed and the same volume of 3×10 8The plates were replaced with a bacterial suspension of SaATCC 43300 at a density of 10 ufc / ml, and the plates were incubated at 4°C for 4 hours to promote microbial adhesion. After 4 hours, two washes were performed in phosphate-buffered saline (PBS) to remove any bacterial suspension that had not adhered to the wells, and the adhered microorganisms were fixed by adding 200 μl of 99% methanol / well and incubating the plates at room temperature (RT) for 15 minutes. Then, the methanol was removed and the plates were incubated at 37°C for 5 minutes to evaporate any residual methanol. Finally, the adhered microorganisms were stained by adding 200 μl of 2% crystal violet to each well for 5 minutes at room temperature. Then, the excess dye was removed and the dye bound to the adhered microorganisms was dissolved by adding 200 μl of 33% glacial acetic acid / well. The optical density of each well was measured by reading at 595 nm on a spectrophotometer. Anti-adhesion assays were performed on all batches at 25°C and 40°C at two times: time zero (T0) and 6 months (T6). The data represent the mean ± SEM of three replicates and were statistically analyzed by one-way ANOVA followed by Bonferroni test.

[0069] The safety profile of the formulations was evaluated on the human corneal epithelial cell line HCE using a cytotoxicity protocol modified from the standard protocol, which envisages repeated exposure to the test article for a short treatment duration (STRE). The cells were treated with two formulations, R&D1907 and R&D1908, at different concentrations (2.5%, 5%, 10%, 20%, 100%) for 5 minutes in duplicate (6X). After each treatment, the protocol followed two different conditions:

[0070] Washing: After 5 minutes of contact on HCE, the treatment was removed by washing with PBS. This condition represents what occurs in vivo when the eyelids are closed, where a portion of the eyewash solution is washed away by the tear film.

[0071] No wash: After 5 minutes on HCE, the eyewash solution was removed without any washing. This condition represents the "exasperated" situation before (washing) and can be compared to the situation in which tear film volume changes were present.

[0072] In both protocols described, between one treatment and the next there was a 90-minute "recovery" period in complete medium at 37° C. After the indicated times, cell viability was assessed by MTT assay.

[0073] Cell viability values ​​less than 50% indicate cytotoxicity (DB-ALM: protocol no. 17).

[0074] To evaluate the stability profile of the formulation, two different temperature conditions were considered: 25 ± 2 ° C / 60 ± 5% RH for long-term stability studies and 40 ± 2 ° C / 75 ± 5% RH for accelerated conditions. During the stability study, the following chemical / physical parameters were evaluated: pH, osmolality, and average particle distribution (size), zeta potential (pZ), and polydispersity index (PDI) at different time intervals (0, 1, 2, 3, and 6 months).

[0075] result:

[0076] Anti-adhesion test: All batches of the formulations studied showed anti-adhesion activity against SaATCC 43300 at both test temperatures, with microbial inhibition values ​​as high as 32.3% ( Figure 1 Furthermore, the anti-adhesion effect appeared to remain constant for up to 6 months. In fact, there were no statistically significant differences between T0 and T6 months for either formulation, or between the formulations themselves (R&D 1907, R&D 1908).

[0077] Safety evaluation: The results of the cell viability assay performed on HCE have shown that batches R&D1907 and R&D1908 were not cytotoxic at any concentration tested, either with or without washing, after treatment according to the STRE protocol ( Figure 2 A- Figure 2 B).

[0078] Stability study: The stability study data of two batches of preparations were Figure 3 (AE) and Figure 4 (AE) are provided as a graph over the study time. During the stability study, no significant differences in the chemical-physical parameters (pH, size, PDI, pZ and osmolality) were apparent under either accelerated stability conditions or long-term stability conditions. The product can be considered stable.

[0079] in conclusion:

[0080] The results showed that the ophthalmic topical formulation was safe, stable and characterized by anti-adhesion activity due to the presence of a mixture of biosurfactants, proteins and organic acids. These results suggest that such a formulation may be useful for preventing diseases in which the conjunctival epithelial barrier function is impaired, such as eye infections, allergies and keratoconjunctivitis.

[0081] Example 2

[0082] Tests were performed to verify how the selection of a pH in the interval of 6.7 to 6.9, in particular 6.8 to 6.9, is very important to keep the composition stable and prevent precipitation of the protein fraction from the bioactive postbiotic component.

[0083] The preferred pH of the preparations tested was equal to 6.8-6.9 (formulation 1 of Example 3), while the pH of the solution containing postbiotics obtained from the fermentation of Lactobacillus paracasei CNCM 1-5220, hereinafter referred to as postbiotics, in the absence of any buffer was equal to 6.3.

[0084] The compositions of the present invention have a pH that helps maintain the tertiary and quaternary three-dimensional structure of the postbiotic proteins so that they are not denatured and do not form aggregates. The average particle dispersion (size) data of batches R&D 1907 and R&D 1908 are optimal as they have a size of ≤200 nm and did not show any significant changes during the stability study at both 40°C and 25°C up to 6 months, i.e. no increase in size due to aggregate formation.

[0085] The presence of calcium and magnesium salts in the formulation aids stabilization, and glycerol, in addition to its isotonicity, also acts as a viscosity enhancer / suspending agent. The presence of divalent calcium and magnesium salts is crucial because they can form weak interactions with the (-COO-) groups present in proteins, stabilizing their structure.

[0086] Testing of formulations in the absence of a buffer system of sodium dihydrogen phosphate and sodium hydrogen phosphate (optionally in hydrated form, e.g. the first as a monohydrate and the second as a dodecahydrate), but in the presence of an isotonic agent, did not result in stable formulations, since the formation of precipitates was already observed in the formulations after one week of study at 40°C, e.g. Figure 5 As shown in B.

[0087] Preparation 2

[0088] Components Function %w / v Postbiotics Main ingredients 0.280 NaCl Isotonic agents 0.720 purified water solvent Replenish to 100

[0089] Additional tests were performed on the following buffers with pH values ​​of 5.6-5.8 (Formulations 3 and 4), but unsatisfactory results were not obtained, as in both cases, at lower postbiotic concentrations (0.1%), the average particle dispersion (size) values ​​obtained were greater than 200 nm in size. The sample sizes were high and not very uniform. These factors are indicative of protein aggregation and formulation instability.

[0090] Preparation 3

[0091]

[0092] Table 1. Formulation 3, studied at T = 1 week

[0093]

[0094]

[0095] Preparation 4

[0096] Components Function %w / v Postbiotics Main ingredients 0.100 <![CDATA[Na2HPO4 12H2O]]> buffer 0.415 citric acid buffer 0.880 purified water solvent Make up to 100mL

[0097] Table 2. Formulation 4, studied at T = 1 week

[0098] <![CDATA[pH 1 ]]> - 5.78 <![CDATA[Osmolality, weight 1 > Osmol / Kg 0.239 size nm 745.60 PDI - 0.689

[0099] Example 3

[0100] Preparation of solutions for ophthalmic use

[0101]

[0102]

Claims

1. A stable ophthalmic composition comprising Postbiotics, the postbiotics are fermentation products of Lactobacillus paracasei strain CNCM I-1390, which was submitted on July 26, 2017, under the CNCM number I-5220 of the National Collection of Microorganisms of the Pasteur Institute in Paris, France, a liquid carrier, said liquid carrier being water, a buffer system comprising sodium dihydrogen phosphate and sodium hydrogen phosphate in an amount producing a pH of 6.7 to 6.9, An isotonic agent comprising magnesium chloride and calcium chloride.

2. The composition of claim 1, wherein the buffer system comprises sodium dihydrogen phosphate and sodium hydrogen phosphate in an amount to produce a pH of 6.8 to 6.

9.

3. The composition according to claim 1 or 2, comprising an additional isotonic agent selected from sodium chloride, potassium chloride and mixtures thereof.

4. The composition according to claim 1, wherein the composition has the following composition 5. The composition according to claim 4, further comprising a glycerol-based suspending-isotonic agent.

6. The composition of claim 5, comprising an additional buffering agent which is sodium citrate.

7. The composition according to claim 6, having the following formula 8. The composition according to claim 4, characterized in that The following ratios: magnesium chloride hexahydrate / calcium chloride dihydrate = 1.42 and / or sodium dihydrogen phosphate monohydrate / sodium hydrogen phosphate dodecahydrate = 0.

39.

9. The composition according to claim 8, characterized in that At 40°C, after six months, the average dispersion size of the particles is ≤200nm.

10. The composition according to claim 9, for use in preventing or treating microbial eye infections or for treating eye allergies, vernal keratoconjunctivitis, keratoconjunctivitis sicca and / or blepharitis.

11. The composition according to claim 9, for preventing foreign objects or microorganisms from adhering to the ocular surface.

12. The composition according to claim 1, wherein the sodium dihydrogen phosphate is a monohydrate, the disodium hydrogen phosphate is a dodecahydrate, the magnesium chloride is a hexahydrate, and the calcium chloride is a dihydrate.

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