Ophthalmic topical composition containing lactic acid bacteria fermentation metabolites

Abstract

The present invention relates to a sterile buffered topical ophthalmic composition for ophthalmic use in the prevention or treatment of microbial eye infections, ocular allergies, vernal conjunctivitis, keratoconjunctivitis sicca and blepharitis, comprising fermentation metabolites of lactic acid bacteria belonging to the species Casei or Paracasei.

Description

[Technical Field]

[0001] The present invention relates to a stable topical ophthalmic composition containing a lactic acid bacteria fermentation metabolite.

[0002] The present invention comes from the field of ophthalmology and eye drops.

[0003] In particular, the present invention relates to a composition for topical use for administration to the eye that contains a postbiotic as an active ingredient and has a specific formulation that keeps the postbiotic stable in aqueous solution. [Background technology]

[0004] Probiotics are live microorganisms that exert a beneficial effect on the host when administered in biologically active amounts. Postbiotics are by-products of the fermentation process carried out by probiotics.

[0005] Postbiotics refer to soluble factors, i.e., metabolic products or by-products, secreted by live bacteria or released after bacterial lysis, such as enzymes, peptides, teichoic acids, muropeptides, polysaccharides, cell surface proteins, and organic acids derived from peptidoglycan.

[0006] Postbiotics are being used in ever increasing numbers in nutritional and pharmaceutical fields due to their biological activity and their perceived safety.

[0007] The use of probiotics in the form of eye drops is also known, for example by Bonni et al.

[0008] Postbiotics are difficult to use in the ophthalmic field due to their poor stability in aqueous solutions.

[0009] Therefore, there is a need to provide available ophthalmic formulations that contain postbiotics that remain stable.

[0010] It is an object of the present invention to provide a topical ophthalmic composition that contains a postbiotic as a biologically active ingredient. Summary of the Invention

[0011] Highly stable ophthalmic compositions containing specific postbiotics as biologically active ingredients have now been identified and form the subject of the present invention.

[0012] Such compositions have formulations that maintain the stability of postbiotics in ophthalmic solutions.

[0013] In particular, the compositions of the present invention prevent or substantially reduce the protein components of postbiotics from causing instability phenomena such as protein aggregation resulting from changes in the tertiary and quaternary structure of proteins or their denaturation.

[0014] According to a first aspect, there is provided a topical ophthalmic composition comprising: A postbiotic, which is a fermentation product of Lactobacillus casei or paracasei species, a buffer system comprising sodium hydrogen phosphate, in particular the monohydrate, and disodium phosphate, in particular the dodecahydrate; tonicity agents including magnesium chloride, calcium chloride and mixtures thereof; a physiologically acceptable aqueous carrier; The composition has a pH of 6.7 to 6.9, preferably 6.8 to 6.9. The composition of the present invention may further comprise an optional buffering agent, an isotonicity agent and a preservative, as described in detail below.

[0015] The ophthalmic compositions thus obtained exhibit a surprising ability to act at the surface level of the eye, making it possible to carry out topical treatments with high efficacy and a wide margin of safety, adapted to prevent or cure diseases of this ocular segment. This new form of treatment can be used in combination with other known treatments for the same disease. [Brief explanation of the drawings]

[0016] [Figure 1] Percent microbial inhibition obtained from anti-adhesion assays performed on two batches at T0 and T6 months under long-term stability conditions at 25°C and growth conditions at 40°C. Data represent the mean ± SEM of three replicates, and statistical analysis was performed by one-way ANOVA followed by Bonferroni's test. [Figure 2] (A-B) Cell viability as a function of scalar concentration of formulations tested according to the STRE protocol under washed / non-washed conditions. Data represent the mean ± SEM of triplicates. Statistical analysis was performed by one sample t-test with a 50% cutoff (****p<0.0001 for the washed condition, ###p<0.001 and ####p<0.0001 for the non-washed condition). [Figure 3] (A-E) pH, size, osmolality, pZ, and PDI values ​​graphed as a function of test time under growth stability conditions of 40±2°C / 75±5% RH and long-term stability conditions of 25±2°C / 60±5% RH. [Figure 4] (A-E) pH, size, osmolality, pZ, and PDI values ​​graphed as a function of test time under growth stability conditions of 40±2°C / 75±5% RH and long-term stability conditions of 25±2°C / 60±5% RH. [Figure 5A] 1 shows a photographic reproduction of a formulation containing postbiotics obtained from the fermentation of Lactobacillus paracasei CNCM I-5220 in water and NaCl according to Example 2, kept at 40° C. for 3 weeks. [Figure 5B]1 shows a photographic reproduction of a formulation containing postbiotics obtained from the fermentation of Lactobacillus paracasei CNCM I-5220 in water and NaCl according to Example 2, kept at 40° C. for 1 week. DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention stems from the discovery of specific conditions that allow compositions / aqueous solutions containing postbiotics as defined herein to retain their stability and prevent or significantly reduce denaturation of the protein components of the postbiotics.

[0018] These conditions include a) the selection of a pH of the solution in the range of 6.7 to 6.9, preferably in the range of 6.8 to 6.9, using an appropriate buffer or buffer system, and b) the presence in the solution of divalent calcium and magnesium salts, such as magnesium chloride and calcium chloride.

[0019] Applicant has also observed how effective control of pH in the desired range can be obtained using sodium hydrogen phosphate and disodium phosphate buffers.

[0020] The subject of the present invention is an ophthalmic composition as defined in claim 1.

[0021] Further embodiments of the composition are defined in the attached dependent claims 2 to 9.

[0022] The biologically active ingredients of the composition include postbiotics, in particular fermentation products from bacterial strains belonging to the species Lactobacillus casei or Lactobacillus paracasei, preferably Lactobacillus paracasei.

[0023] In a preferred embodiment, the prebiotic (probiotic) is the strain Lactobacillus casei CNCMI-1390, deposited on July 26, 2017, in accordance with the Budapest Treaty at the CNCM (Collection Nationale de Cultures de Microorganismes, Institut Pasteur, Paris, FR) under number I-5220.

[0024] In some embodiments, the composition comprises postbiotics in an amount of 0.05-5%, 0.1-1.9%, 0.2-1% weight / volume (g / 100ml).

[0025] The compositions of the present invention comprise a buffer or buffer system comprising sodium hydrogen phosphate, particularly the monohydrate, and disodium phosphate, particularly the dodecahydrate.

[0026] Preferably, the ratio of sodium hydrogen phosphate monohydrate to disodium phosphate dodecahydrate is 16 / 41=0.39.

[0027] In some embodiments, the composition comprises an additional buffering agent comprising sodium citrate.

[0028] The composition of the present invention comprises at least one tonicity adjusting agent selected from magnesium chloride, calcium chloride, and a mixture thereof. In a preferred embodiment, the tonicity adjusting 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.

[0029] In some embodiments, the compositions of the present invention comprise an additional tonicity agent, including glycerol.

[0030] Typically, the compositions of the present invention comprise a physiologically acceptable water-based carrier in which the postbiotics described herein are dispersed. Preferably, the water is purified.

[0031] The composition of the present invention has a pH of 6.7 to 6.9, preferably 6.8 to 6.9. Therefore, the buffer and tonicity agent components of the composition are present in the composition of the present invention in amounts that adjust the pH to 6.7 to 6.9, preferably 6.8 to 6.9.

[0032] In one embodiment, the composition of the invention has the following formulation:

[0033] [Table 1]

[0034] According to a preferred form, the composition has the formulation of Example 3. The compositions of the present invention are stable, as defined herein.

[0035] Furthermore, the combination of sodium hydrogen phosphate buffer, preferably the monohydrate, and disodium phosphate, preferably the dodecahydrate, in an amount to bring the pH to 6.7-6.9 in the composition with a tonicity agent based on magnesium chloride and calcium chloride makes the composition stable.

[0036] As used herein, the term "stable" means that the composition, particularly with respect to postbiotics, has a mean particle dispersion (size) of ≦200 nm at 6 months at 40° C. and 25° C. Thus, under the test conditions, no significant variation occurs due to the formation of aggregates of material derived from the postbiotic, i.e., an increase in size of more than 200 nm at 25° C. or 40° C.

[0037] Within the scope of the present invention, the term postbiotics refers to the fermentation products of the species Lactobacillus casei, Lactobacillus paracasei or mixtures thereof, in particular the fermentation products of Lactobacillus paracasei CNCMI-5220.

[0038] It has been observed that while the composition according to any of the previous embodiments has a barrier effect on epithelial cells, due to the presence of a mixture of biosurfactants, glycoproteins, organic acids and peptides in the postbiotic, these components of the postbiotic can prevent the binding of pathogenic bacteria to the ocular mucosa, thereby providing the composition with a prophylactic or therapeutic effect against ocular infections.

[0039] Furthermore, the composition has immunomodulatory activity and can be applied in the prevention and / or treatment of ocular allergies and / or inflammations.

[0040] The compositions may be in single or multidose form.

[0041] In particular, the buffer component is present in the composition at a concentration such as to obtain / maintain a pH between 5 and 8 that is compatible with ocular tissue and the carried active ingredient.

[0042] Preferably, the composition is isotonic with tear fluid, having a viscosity of 270 to 310 mOsm / Kg.

[0043] Preferably, the compositions of the present invention are eye washes or ophthalmic gels. Multi-dose compositions of the present invention may contain antimicrobial preservatives, such as parabens, quaternary ammonium salts, polyhexamethylene biguanide (PHMB), and other ingredients that may be included in ophthalmic compositions. The solvent used in the compositions is preferably water or an aqueous solution of one or more ingredients compatible with topical ophthalmic use.

[0044] For purposes of administration, the aforementioned compositions may be presented as eye drops or in the form of a liquid or gel.

[0045] A further subject of the present invention is the topical ophthalmic use of a composition as defined above in the manufacture of a medicament for the treatment or prevention of ocular inflammations and / or allergies.

[0046] The compositions of the present invention can be used to treat ocular allergies, vernal conjunctivitis, keratoconjunctivitis sicca, and blepharitis.

[0047] The compositions of the present invention may also be applied to prevent the adhesion of foreign bodies or microorganisms to the ocular surface, as experimentally demonstrated in Example 1 below.

[0048] The following examples further illustrate the invention without limiting it. [Example]

[0049] In this experimental study, the formulation of Example 3, hereafter referred to as EyeLac®, containing postbiotics derived from the fermentation process of Lactobacillus paracasei CNCMI-5220, was used. Various assays were developed to evaluate the anti-adhesion efficacy, safety, and stability of two batches of EyeLac® (R&D1907, R&D1908) compositions.

[0050] method: The anti-adhesion properties of formulations R&D1907 and R&D1908 relative to the carrier (VHC) were evaluated against the strain Staphylococcus aureus (Sa) ATCC 43300 using an anti-adhesion assay. Specifically, 200 μl of each formulation was added to each well and the plates were incubated at 4° C. for 18 hours. After that period, the test substances were removed and 3×10 8The plate was then replaced with the same volume of SaATCC43300 bacterial suspension at a density of ufc / ml and incubated at 4°C for 4 hours to promote microbial adhesion. After 4 hours, the wells were washed twice with phosphate-buffered saline (PBS) to remove any unattached bacterial suspension. Meanwhile, the attached microorganisms were fixed by adding 200 μl of 99% methanol per well and incubating the plate at room temperature (RT) for 15 minutes. The methanol was then removed, and the plate was incubated at 37°C for 5 minutes to evaporate any remaining methanol. Finally, the attached microorganisms were stained by adding 200 μl of 2% crystal violet to each well for 5 minutes at RT. The excess dye was then removed, and the dye bound to the attached microorganisms was dissolved by adding 200 μl of 33% glacial acetic acid per well. The optical density per well was measured by reading at 595 nm on a spectrophotometer. Anti-adhesion assays were performed on all batches at time zero (T0) and 6 months (T6) at 25°C and 40°C. Data represent the mean ± SEM of three replicates, and statistical analysis was performed by one-way ANOVA followed by Bonferroni test.

[0051] The safety profile of the formulations was evaluated on the human corneal epithelial cell line HCE using a modified cytotoxicity protocol, which simulates short-term repeated exposure (STRE), to the standard protocol. Cells were treated repeatedly (6x) for 5 minutes with different concentrations (2.5, 5, 10, 20, and 100%) of both formulations, R&D1907 and R&D1908. After each treatment, the protocol followed two different conditions. Washing: After 5 minutes of exposure to HCE, the treatment was removed by washing with PBS, a condition that represents what occurs in vivo, where a portion of the eyewash is washed away by the tear film when the eyelids are closed. No wash: After 5 minutes of treatment with HCE, the eyewash was removed without rinsing. This condition represents an "exasperated" state of the previous (rinse) and can be compared to a state where the tear film volume changes.

[0052] In both described protocols, there is a 90-minute "recovery" period between treatments in complete culture medium at 37°C. After the indicated times, cell viability was assessed by MTT assay.

[0053] A cell viability value below 50% indicates cytotoxicity (DB-ALM: Protocol No. 17). To evaluate the stability profile of the formulation, two different temperature conditions were considered: long-term stability test condition of 25±2°C / 60±5%RH and growth stability condition of 40±2°C / 75±5%RH. During the stability test, the following chemical / physical parameters were evaluated at different time intervals (0, 1, 2, 3 and 6 months): pH value, osmolality, mean particle distribution (size), zeta potential (pZ) and polydispersity index (PDI).

[0054] result: Anti-adhesion tests: All tested batches of formulations showed anti-adhesion activity against SaATCC43300 at both temperatures tested, with microbial inhibition values ​​of up to 32.3% for VHC (Figure 1). Furthermore, the anti-adhesion effect appears to remain unchanged up to 6 months. In fact, no statistically significant differences were detected between TO and T6 months for any of the formulations or between the formulations themselves (R&D1907, R&D1908).

[0055] Safety assessment: The results related to the cell viability assays performed on HCE showed that batches R&D1907 and R&D1908 after treatment performed according to the STRE protocol were not cytotoxic at any of the tested concentrations, either in washed or non-washed conditions (Figure 2A-B).

[0056] Stability testing: The stability testing data for the two batches of formulation are shown graphically as a function of testing time in Figures 3 (A-E) and 4 (A-E), respectively. No significant differences in chemical and physical parameters (pH, size, PDI, pZ, and osmolality) were observed during the stability testing under either growth stability or long-term stability conditions. The product can be considered stable.

[0057] Conclusion: The results suggest that the topical ophthalmic formulation is safe, stable, and characterized by anti-adhesion activity due to the presence of a mixture of biosurfactants, proteins, and organic acids. These results indicate that such formulations may be used to prevent diseases in which the barrier function of the conjunctival epithelium is impaired, such as ocular infections, allergies, and keratoconjunctivitis. [Example]

[0058] Tests were carried out to confirm that the selection of a pH in the range of 6.7-6.9, especially 6.8-6.9, is crucial for the purpose of keeping the composition stable and preventing the protein fraction from precipitating from the biologically active postbiotic components.

[0059] The preferred pH of the tested formulations was equal to 6.8–6.9 (formulation 1 in Example 3), while the pH of the solution containing the postbiotic obtained from the fermentation of Lactobacillus paracasei CNCM I-5220 (hereafter referred to as postbiotic) was equal to 6.3 in the absence of a buffer.

[0060] The compositions of the present invention have a pH that contributes to maintaining the tertiary and quaternary 3D structure of the postbiotic proteins without denaturing and forming aggregates. The mean particle dispersion (size) data is optimal for batches R&D 1907 and 1908, with sizes of ≦200 nm for batches R&D 1907 and 1908, and no significant variation due to aggregate formation, i.e., size increase, was found during stability studies at 40° C. and 25° C. for up to 6 months.

[0061] The presence of calcium and magnesium salts in the formulation contributes to stabilization, and glycerol not only has a tonicity function but also a thickening / suspending agent function. The presence of divalent calcium and magnesium salts is very important because they can form weak interactions with the (-COO-) groups present in proteins, stabilizing the protein structure.

[0062] As shown in Figure 5B, tests on formulations without the buffer system of sodium hydrogen phosphate and disodium phosphate in the hydrate form as required (e.g., sodium hydrogen phosphate is monohydrate and disodium phosphate is dodecahydrate) but with the presence of a tonicity agent did not result in a stable formulation, as precipitate formation was observed in the formulation after a one-week study at 40°C.

[0063] [Table 2]

[0064] Other tests were carried out on the following buffers (formulations 3 and 4) with pH values ​​equal to 5.6-5.8, but failed to produce satisfactory results at low concentrations of postbiotics (0.1%), and the mean particle size values ​​obtained were >200 nm in both cases. The samples were large and not very uniform in size. These factors are indicative of protein aggregation and formulation instability.

[0065] [Table 3]

[0066] [Table 4]

[0067] [Table 5]

[0068] [Table 6]

[0069] Example 3 Ophthalmic solution preparations [Table 7]

Claims

1. a postbiotic which is a fermentation product of Lactobacillus casei species, Lactobacillus paracasei species, or a mixture thereof; a liquid carrier; a buffer system comprising sodium hydrogen phosphate monohydrate and disodium phosphate dodecahydrate in amounts to provide a pH of 6.8 to 6.9; and a tonicity agent comprising magnesium chloride salt, which is a hexahydrate, and calcium chloride, which is a dihydrate, wherein the postbiotic is a fermentation product of Lactobacillus casei strain CNCMI-1390, deposited on July 26, 2017, with the CNCM (Collection Nationale de Cultures de Microorganismes, Institut Pasteur, Paris, France) number I-5220.

2. The topical ophthalmic composition of claim 1 , wherein the liquid carrier is water.

3. 3. The composition according to claim 1 or 2, comprising a further tonicity agent selected from sodium chloride, potassium chloride and mixtures thereof.

4. The composition of any one of claims 1 to 3, having the following composition: Table 1

5. 3. The composition of claim 1 or 2, further comprising a glycerol-based suspension tonicity agent.

6. A composition according to any one of claims 1 to 3, comprising a further buffering agent which is sodium citrate.

7. The composition of any one of claims 1 to 4, having the following formulation: Table 2

8. 8. The composition according to claim 1, characterized in that the ratio of magnesium chloride hexahydrate / calcium chloride dihydrate is 10 / 7=1.42 and / or the ratio of sodium hydrogen phosphate monohydrate / disodium phosphate dodecahydrate is 16 / 41=0.

39.

9. 9. A composition according to any one of claims 1 to 8, characterized in that after 6 months at 40°C the particle mean distribution (size) is ≦200 nm.

10. The composition according to any one of claims 1 to 9, which is used for the prevention or treatment of microbial eye infections, or for the treatment of eye allergies, vernal conjunctivitis, keratoconjunctivitis sicca, or blepharitis.

11. The composition according to any one of claims 1 to 9, which is used to prevent foreign bodies or microorganisms from adhering to the surface of the eye.

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