Mucoadhesive compositions for local treatment of vaginal disorders

Abstract

The invention relates to compositions in the form of a mucoadhesive hydrogel comprising, or consisting of, lactic acid, glycogen and hyaluronic acid for use in the prevention and treatment of vaginal dysbiosis, prevention and treatment of bacterial vaginal infections caused by dysbiosis, and prevention of recurrences, by restoring the correct balance of the vaginal microbiota.

Description

[0001] MUCOADHESIVE COMPOSITIONS FOR LOCAL TREATMENT OF VAGINAL

[0002] DISORDERS

[0003] The invention relates to compositions in the form of a mucoadhesive hydrogel comprising, or consisting of, lactic acid, glycogen and hyaluronic acid. The compositions are intended for application in the prevention and treatment of vaginal dysbiosis, prevention and treatment of bacterial vaginal infections caused by dysbiosis, and prevention of recurrences, by restoring the correct balance of the vaginal microbiota.

[0004] Prior art

[0005] The human body houses numerous commensal, symbiotic and pathogenic microorganisms that populate its cavities, either continuously or discontinuously with the exterior. Said micro-organisms, collectively called microbiota, generally include bacteria, fungi and viruses. The composition of the microbiota differs not only according to the part of the body concerned, but also many other factors, such as sex, age, ethnic origin, lifestyle, poor or excessive hygiene, etc. (https: / / doi.org / 10.1038 / naturel 1234). and their crucial role in the wellbeing of the individual has long been understood.

[0006] In particular, the microbiota in the vagina consist of a wide range of aerobic and anaerobic micro-organisms, with a strong predominance of various species of Lactobacillus, which are the main species responsible for maintaining the correct balance of the vaginal mucosa. Said balance is essential for well-being at all stages of female life; situations of dysbiosis not only create temporary discomfort or disorders, but can even influence reproductive activity (Amabebe E., Anumba D.O.C.; Front Med, 2018;5:181).

[0007] Under physiological conditions, and therefore in a state of eubiosis, the various strains of lactobacilli can account for up to 90% of the vaginal bacterial flora.

[0008] Lactobacilli perform various functions in the vagina:

[0009] • they are responsible for producing a biofilm that creates a barrier on the vaginal mucosa, which is thus protected against colonisation by pathogens;

[0010] • they produce particular substances called bacteriocins, which are toxic to some pathogenic micro-organisms that may be present;

[0011] • and above all they produce lactic acid, which is essential to maintain the pH of the vaginal environment at around 3.5-4.5. Said pH values are ideal for the growth and activity of lactobacilli, and at the same time strongly counteract the development of pathogens, which cannot generally survive such acidity values.

[0012] In order to perform said functions effectively, the lactobacilli require the presence in the vaginal fluid of substances that nourish them, acting as prebiotics. In particular, the action of the lactobacilli is optimal when there an abundance of glycogen in the vaginal fluid. Glycogen is physiologically present in the vaginal epithelium (Gregoire A T. et al, Fertil Steril, 1971; 22:64-68) together with enzymes, in particular amylase, which facilitate its breakdown, releasing the “building blocks” that will be used as nourishment by the lactobacilli (Mirmosnsef P. et al, Pios ONE; 2014;9:el 02467).

[0013] When the vaginal microbiota is altered for the reasons listed above, a significant depletion of lactobacilli takes place, causing an increase in the vaginal pH to over 4.5 and proliferation of opportunistic micro-organisms which, though part of the vaginal microbiota, act as pathogens when their number increases disproportionately.

[0014] It is clear that an imbalance of the vaginal microbiota, and therefore a situation of dysbiosis, promotes the growth and proliferation of pathogens which can lead to bacterial vaginitis (mainly caused by Gardnerella vaginalis and other anaerobic bacteria), fungal and yeast infections (caused in particular by Candida albicans), and urogenital infections, mainly caused by Escherichia coli.

[0015] The first-line treatment for said disorders is naturally pharmacological, due to the availability of medicaments with an antimicrobial and / or antifungal action. However, it has been demonstrated, and is widely known, that the vaginal microbiota is seriously damaged, and its balance significantly altered, by pharmacological treatment; the vaginal mucosa, which is already weak, is once again in a situation of dysbiosis, and becomes extremely vulnerable because it is not protected against its physiological bacterial flora. This triggers a vicious circle, which leads to frequent recurrences of the disorders described above (Mayer B.T. et al., J Infect Dis, 2015;212:793-802; Sousa L.G.V. et al, Microb Biotechnol, 2023;16:1423-1437).

[0016] It is therefore evident that the maintenance and / or restoration of vaginal eubiosis is extremely important, not only under physiological conditions but even more so in situations of dysbiosis, including those resulting from pharmacological treatments, in particular antibacterial and / or antifungal treatments.

[0017] For this purpose, various pharmaceutical and cosmetic compositions for vaginal use have been devised, substantially containing lactic acid, which is required to restore a sufficiently acid vaginal pH. Other active ingredients are generally used in combination with lactic acid, especially prebiotics, namely indigestible substances (fibres) able to nourish the lactobacilli already present, or probiotics, namely living micro-organisms of recognised usefulness for the human body. Said active ingredients have very often been combined with others, such as bacterial lysates, polypeptides, natural extracts, polysaccharides, hyaluronic acid, etc.

[0018] The known compositions also include consistency regulators and viscosity-controlling agents and a buffer system to prevent rapid depletion of the acidity induced in the vaginal environment by lactic acid, for example a buffer based on sodium lactate, which stabilises the pH.

[0019] Compositions containing lactic acid and prebiotics are described in:

[0020] EP257007: describes a composition for vaginal use comprising lactic acid and glycogen in a ratio ranging from 20: 1 to 500: 1. According to said patent, the large amount of lactic acid avoids the use of preservatives, which would also have adverse effects on the lactobacilli. The efficacy of the preparation is demonstrated, but the patent does not in any way indicate whether the composition, as well as maintaining the lactobacillus population, is able to increase it, thereby increasing the production of lactic acid;

[0021] CN115006512: describes a pharmaceutical composition for vaginal use with a highly complex formula which comprises, in addition to excipients, lactobacillus fermentation lysate, lactic acid and sodium lactate, together with other active ingredients. Numerous in vitro tests demonstrate that only the composition containing the entire range of active ingredients listed significantly increases the amount of ATP produced by the lactobacilli, and therefore the metabolic activity of the bacterial cells, but no increase in the lactobacillus population is described.

[0022] The invention described herein overcomes the state of the art due to the invention of a simple composition, containing very few active ingredients and no traditional preservatives, which is perfectly calibrated in terms of excipient content, and is surprisingly able to stimulate proliferation of the lactobacilli present in the vagina, leading to lactic acid production and restoration of the correct acid pH; in this way, the proliferation of opportunistic pathogenic micro-organisms is reduced to a highly significant extent, and the physiological vaginal microbial flora is therefore restored.

[0023] Description of the invention:

[0024] The invention relates to pharmaceutical compositions in the form of a mucoadhesive hydrogel comprising or consisting of hyaluronic acid, glycogen and lactic acid, and pharmaceutically acceptable excipients suitably selected for use in the prevention and treatment of vaginal dysbiosis, prevention and treatment of bacterial and / or fungal vaginal infections caused by dysbiosis, and prevention of recurrences, as a result of restoration of the correct balance of the vaginal microbiota. As stated, the balance of the vaginal bacterial flora is closely associated with specific factors, such as the pH of the vaginal environment and the strong preponderance of some bacterial species, in particular of the genus Lactobacillus, which in turn are responsible for the production of lactic acid. A pH ranging between about 3 and 4.5, in particular between about 3.5 and 4.5, is ideal to prevent the proliferation of opportunistic organisms and thus preserve the integrity of the vaginal environment. In fact, at said pH values the lactobacilli perform their previously described functions as effectively as possible.

[0025] For this reason, lactic acid has long been used in the treatment of dysbiosis situations or to maintain vaginal eubiosis. Lactic acid was used either alone or, more frequently, in combination with a salt thereof, to form a buffer system that keeps the pH value of the final composition stable.

[0026] Compositions containing lactic acid are often combined with other active ingredients, preferably those known as “prebiotics”, which provide the nourishment required for the growth and well-being of the lactobacilli present in the vagina; these are water-soluble fibres belonging to the oligosaccharide group, often derived from fruit or cereals. Examples of prebiotics comprise inulin, fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), lactulose, isomalt-oligosaccharides (IMO), xylo-oligosaccharides (XOS), long-chain beta-glucans, oligosaccharides of glucomannan, and glycogen, a polysaccharide of glucose.

[0027] The combinations between lactic acid and prebiotics used are highly varied in quantitative terms, because imbalances in any one of the ingredients have an adverse effect on the vaginal mucosa. Excessive amounts of lactic acid would give rise to excessively low pH values, which would be highly undesirable in an epithelium already weakened by dysbiosis, and ineffective for the purposes of the invention; excessive amounts of prebiotic would risk providing nourishment not only for the lactobacilli but also for potentially pathogenic species present in the vaginal environment.

[0028] Other important formulation parameters relate, for example, to:

[0029] • the stability of the compositions: the preservatives normally used are antimicrobials which act indiscriminately, preventing contamination of the composition but adversely affecting the vaginal lactobacillus population;

[0030] • mucoadhesion: after application the compositions must remain in situ stably, in order to perform their function as well as possible for the required time;

[0031] • pH values : a buffer system must be created to keep the values stable, thereby preventing the lactic acid from being rapidly neutralised by the vaginal secretions, which are alkaline. The invention described herein overcomes the state of the art due to the development of a composition in the form of a stable, mucoadhesive hydrogel without the classic paraben preservatives, which contains, or consists of, a minimal number of active ingredi ents in specific weight ratios, perfectly balanced with suitable excipients, and is able to:

[0032] ® increase the number of lactobacilli normally present in a healthy vaginal environment, keeping them perfectly viable, and thus

[0033] ® promote the production of lactic acid by the lactobacilli, thereby helping to restore and / or maintain the state of eubiosis of the vaginal mucosa;

[0034] ® rebalance and maintain the vaginal pH at the right degree of acidity, and finally,

[0035] ® perform a significant antimicrobial action towards the pathogenic bacterial flora, when present, in particular Escherichia coli.

[0036] The composition for the use according to the invention essentially comprises lactic acid, glycogen and hyaluronic acid as active ingredients, combined with specific excipients which, as will be discussed below, significantly contribute to the stability and efficacy of the end product.

[0037] Lactic acid plays the crucial role of restoring the pH of the vagina to values between 3 and 4, namely the substantially physiological values, the lactic acid concentration in the vaginal fluid is about 1% w / v, and acidifies the vaginal environment to a pH of about 3.5.

[0038] Glycogen is present in the vagina at intraepithelial level and, in variable amounts depending on age, constitutes an important energy source for the lactobacilli; as stated, amylases active in the vagina break down glycogen and make it available to the lactobacilli, which thus produce lactic acid.

[0039] Finally, hyaluronic acid (HA) is a linear-chain heteropolysaccharide consisting of alternating residues of D-glucuronic acid andN-acetyl-D-glucosamine, with a molecular weight (MW) ranging between 400 and 3 x 106Da, depending on the source from which it is extracted or the preparation methods used. It is ubiquitously present, and plays an important part in the biological organism as the main component of connective tissue in vertebrate organisms, the synovial fluid of the joints, and vitreous humour. HA acts as a mechanical support for the cells of many tissues such as skin, tendons, muscles and cartilage, and is essential fortissue hydration and joint lubrication.

[0040] HA is crucial in the tissue repair process from both the structural and the metabolic standpoint (Weigel P. et al., J Theoretical Biol, 1986:219-234; Abatangelo G. et al., J SurgRes, 1983, 35:410-416; Goa K. et al., Drugs, 1994, 47:536-566); it further acts as an antiinflammatory by modulating the release of inflammatory cytokines, in particular IL-1, and is also able to bond to specific opioid receptors, mimicking an analgesic effect.

[0041] In view of said widely recognised properties, hyaluronic acid or an alkaline salt thereof have long been used to prepare dressings used in the care of superficial or deep skin or mucosal wounds, ulcers and lesions of various origins due to its reparatory and soothing action, and also in the viscosupplementation treatment of osteoarthritis.

[0042] The hyaluronic acid used in the present invention, either as such or in the form of an alkaline salt, can derive from any source, such as rooster combs (EP 138572), fermentation (from Streptococcus equi or zooepidemicus), or biosynthesis from Bacillus (EP2614088, EP2614087), and can be purified by various techniques (EP3491027; EP3655138). Preferably, HA obtained by fermentation from Streptococcus is used, in particular Streptococcus equi sub- sp. equi, 68222, mutant H-l (EP716688).

[0043] The weight-average molecular weight of the polymer for the applications described herein preferably ranges between 100000 and 1000000 Da, even more preferably between 150000 and 250000 Da; the latter is generally abbreviated to “HA with weight-average MW 200 kDa”, and is preferably in the form of a sodium salt.

[0044] “Average molecular weight” (MW) means the weight-average MW, calculated by the “intrinsic viscosity” method (Terbojevich et al., Carbohydr Res, 1986, 363-377).

[0045] It has now surprisingly been found that by mixing the active ingredients described in a precise weight ratio and combining them with suitable excipients, a hydrogel formulation is obtained which, as will be demonstrated below:

[0046] ® directly promotes restoration of the natural vaginal acidity, due to the pH value of the composition according to the invention;

[0047] ® maintains, promotes and restores the viability of the lactobacilli present in the vagina; and above all, unexpectedly

[0048] ® stimulates the proliferation of the lactobacilli, increasing their population;

[0049] ® acts significantly as an antibacterial towards opportunistic pathogens, specifically towards Escherichia coli, without containing substances with a specific effect or the classic preservatives, which in turn are antimicrobi ls.

[0050] Moreover, the presence of hyaluronic acid gives the composition a reparatory and soothing effect towards the mucosa, while mucoadhesion is ensured by the selection of suitable excipients.

[0051] The lactic acid:glycogen:sodium hyaluronate weight ratio that proved exceptionally effective for the purposes of the invention is 10:1:2, which totally contradicts the findings known from the state of the art; as already stated, in EP257007 the lactic acid:glycogen ratio ranges from 20:1 to 500:1, and is preferably 50:1, i.e. significantly unbalanced in favour of lactic acid. Conversely, CN115006512, which also involves the presence of hyaluronic acid, describes a composition wherein the lactic acid:glycogen:HA ratio is 1:10:1, with a definite preponderance of prebiotic which is the source of nourishment for all the species present in the vagina, including the opportunistic bacterial and fungal species, which are highly undesirable.

[0052] The concentrations of the active ingredients of the composition described can range as follows:

[0053] • lactic acid: between 0.5 and 1.5%, preferably 1 wt% of the weight of the final composition;

[0054] • glycogen: between 0.05 and 0.15%, preferably 0.1 wt% of the weight of the final composition

[0055] • hyaluronic acid sodium salt: between 0.1 and 0.3%, preferably 0.2 wt% of the weight of the final composition, maintaining the 10: 1 :2 ratio unchanged.

[0056] In addition to the main active ingredients described above, the composition according to the invention comprises a base such as sodium hydroxide for the creation of a buffer system required to maintain the pH at the specified values, and suitable excipients such as wetting agents and gelling agents which, in the precise proportions detailed below, give the composition adequate stability, viscosity and mucoadhesion and unexpectedly help to promote the proliferation of lactobacilli, increasing their number and maintaining their viability.

[0057] Sodium hydroxide is used as buffer system in variable amounts relative to the lactic acid content, to obtain a pH ranging between 3.5 and 4.5, preferably between 3.7 and 3.9.

[0058] The excipients preferably consist of a mixture of:

[0059] • glycerol;

[0060] • propanediol;

[0061] • a mixture of phenethyl alcohol and ethylhexylglycerin (tradename Sensiva PA 20): this is a wetting agent, possessing deodorant activity because it controls the breakdown of Gram-positive bacteria which cause the odour found in dysbiosis, used in amounts ranging between 0.2 and 0.5% by weight of the final composition, preferably 0.3%; ® a hy droxy ethyl acrylate / acrylyloyldimethyl taurate copolymer (tradename Sepineo DERM): this is a gelling agent and mucoadhesive, stable at wide pH intervals, used in amounts ranging between 3 and 4% by weight of the final composition, preferably between 3.6 and 3.8%

[0062] ® purified water.

[0063] The invention is described in greater detail in the examples below7, purely by 'way of example and not of limitation.

[0064] Example 1: Preparation of a pharmaceutical composition in hydrogel form for vaginal application based on 1% w / w lactic acid, 0.1% w / w glycogen and 0.2% w / w hyaluronic acid sodium salt

[0065] From the total specified amount of purified water, take up an amount 6 times the weight of the NaOH in the formula and transfer it to a suitable container, add the specified amount of NaOH, and stir until completely dissolved. Heat the remaining part of the purified water in a turboemulsifier until about 40°C is reached, and activate stirring. Add the lactic acid and maintain stirring for at least 5 minutes, until a homogeneous solution is obtained. Add the NaOH solution previously prepared and maintain stirring for at least 15 minutes, until a homogeneous solution is obtained. Add the hyaluronic acid sodium salt (weight-average MW of HA= 200 kDa), leaving the system under stirring until a clear, colourless solution, not containing any undissolved material, is obtained (about 45 min). Maintaining the system under stirring, continue by sequentially adding glycerol, propanediol and phenethyl alcohol / ethylhexylglycerin (Sensiva PA20) until a clear, homogeneous solution is obtained. Keeping the stirring and heating system active, introduce the glycogen and wait for complete dissolution of the substance, until a slightly opalescent homogeneous solution is obtained (about 45 min). Start the cooling stage, boosting the stirring system and activating the vacuum system, then add Sepineo DERM until dispersion is complete, and a homogeneous, viscous, opalescent gel not containing any undissolved bodies is consequently obtained. The composition thus prepared has a pH value =3.8.

[0066] Example 2: Evaluation of activity of the composition according to Example 1 on lactobacillus growth (spectrophotometric method) at 18h

[0067] The activity of the composition in question on lactobacillus cultures was evaluated in vitro by the widely used microplate spectrophotometric method; said method is based on the optical density (OD) or absorbance reading of suitably prepared cultures, at precise wavelengths. Optical density measures the turbidity of the samples, which depends on the concentration of the cells they contain; thus the higher the optical density value, the larger the number of cells in the sample. The enzyme amylase, which is physiologically present in the vaginal environment, and provides nouri shment for the lactobacilli by breaking down glycogen, was added to some samples in this specific case.

[0068] Materials and methods:

[0069] Lactobacilli of strain Lactobacillus vaginalis ATCC 49540 were cultured in MRS broth (Oxoid, Italy) with the addition of 0.05% w / v L-cysteine (MRSc, pH 5.5), under conditions of anaerobiosis obtained by adding sterile paraffin to the bacterial suspension and incubating at 37° C for 24 h.

[0070] The composition in question was prepared according to Example 1, and is hereinafter called “SPL”; 500 mg was weighed in 2 mL cryovials, then resuspended directly in sterile broth to obtain a final dilution factor of 1:4. For the test, 160 pl of the sample thus prepared was loaded into the wells of a 96-well microplate.

[0071] For the tests involving the use of the enzyme, a porcine pancreatic a-amylase (A3176- 1MU, Sigma Aldrich Type VLB) was used at the dose of 0.5 mg / ml, solubilised in a sterile medium and, after addition of the enzyme, subjected to further filtration with 0.22 micrometre beaker filters.

[0072] For the inoculum, an aliquot of stock solution of the lactobacillus strain (maintained at -80°C in MRSc and 20% glycerol) was tested for purity, and 50 pl was inoculated into 5 ml of freshly prepared sterile MRSc, and incubated at 37°C for 24 h.

[0073] After 24 hours’ incubation, an aliquot was taken up and diluted 1 : 10 for reading of the ODeoo, adjusting the turbidity of the culture broth with a suitable dilution in sterile 0.9% w / v NaCl to an ODeoo value ranging between 0.16 and 0.2 (corresponding to 1 McFarland); 300 pl was then inoculated into 30 ml (1 : 100) of sterile 0.9% w / v NaCl, so that the amount subsequently loaded into each well (20 pl) contained a final concentration of about 3xl05 CFU / ml.

[0074] For the tests, 20 pl of said bacterial suspension was inoculated into the wells of 96-well microplates containing the SPL samples. For the anaerobiosis, about 10 pl of sterile paraffin was added to each well, to cover the surface in contact with the air; the microplate was then placed in an incubator at 37°C under stirring at 150 rpm. After 18 hours’ incubation, the optical density was read at 630 nm (ODeao).

[0075] The percentage lactobacillus growth induced by the SPL composition was calculated by comparison with the untreated control, namely Lactobacillus vaginalis strain ATCC 49540 grown simply in MRSc (Ctrl+). The results of the spectrophotometric reading are illustrated in Figure 1, which shows the percentage growth of Lactobacillus vaginalis ATCC 49540 (spectrophotometric method) in MRSc medium (18 h)

[0076] It is immediately clear that after only 18 hours’ incubation, the SPL composition gave rise to much higher lactobacillus growth than the control, namely 30% more than that obtained with the control. The figure is even more surprising for the samples containing amylase, for which 88% more growth than the control was recorded; as expected, amylase had no effect on the control, not having a substrate on which to act.

[0077] This test therefore demonstrates that the composition tested is able to stimulate the proliferation of lactobacilli, increasing their concentration compared with the control; the stimulation is even more marked in the presence of amylase, which breaks down glycogen, providing the lactobacilli with an essential source of nourishment.

[0078] The test conducted with the spectrophotometric method provides a quantitative indication; the optical density (OD) value is the result of a turbidimetric reading, which detects everything present in suspension in the samples, whether live or non-viable cells, bacterial lysates or artefacts; a certain influence is even exerted by the shape of the bacteria (e.g. rods or cocci), which can abnormally diffract the incident light, thus altering the final reading. Moreover, said method is unsuitable for experiments conducted over relatively long periods (such as 48 hours or more) because, due to the factors described above, when the samples become particularly turbid, and therefore dense, the optical density values detected are lower than the real growth (Mira P et al., 2022: PLoS ONE 17(10): e0276040. https: / / doi.org / 10.1371 / journal.pone.0276040).

[0079] To overcome the limitations of the spectrophotometric method and be certain of the efficacy of the composition claimed herein, cell count tests were therefore performed, to evaluate the real viability of the bacteria present in the cultures. Schematically, by seeding suitable dilutions of the culture broth whose absorbance has been read in an adequate agar culture medium suitable for the growth of that strain, the colonies can be counted to determine the viable bacteria count, even over relatively long periods.

[0080] Example 3: Evaluation of the activity of the composition according to Example 1 on the growth of lactobacilli (culture method) at 24 h

[0081] To confirm the quantitative data described in Example 2 and verify the real viable bacteria count, the samples were tested by the culture method.

[0082] To obtain even more precise and significant data, the growth test was performed by culturing the lactobacilli in simulated vaginal fluid, prepared in the laboratory as described below. The vaginal fluid culture provides a picture very close to the real situation.

[0083] Materials and methods:

[0084] The composition to be tested (SPL) was prepared as described in Example 1, and 500 mg, accurately weighed, was resuspended in 2 ml of sterile purified water.

[0085] For the inoculum, an aliquot of stock solution of the strain prepared according to Example 2 (maintained at -80°C in a liquid culture medium to which 20% glycerol was added) was tested for purity, and 50 pl was inoculated into 5 ml of freshly prepared sterile liquid culture medium, and incubated at 37°C for 24 h.

[0086] After incubation an aliquot was taken up and diluted 1 : 10 for reading of the ODeoo; the turbidity of the culture broth was adjusted with a suitable dilution in sterile 0.9% w / v NaCl to a value ranging between 0.16 and 0.2 (corresponding to 1 McFarland), and 300 pl of said suspension was inoculated into 30 ml of sterile 0.9% w / v NaCl (1 : 100), to obtain a final lactobacillus concentration of about 3xl05CFU / ml.

[0087] The simulated vaginal fluid was prepared as described by Owen, D.H.; Katz, D.F., 1999: Contraception, 59, 91-95, with the following composition

[0088] - 3.5 g / L of NaCl,

[0089] - 1.4 g / L ofKCl,

[0090] - 0.02 g / L of bovine serum albumin,

[0091] - 2.0 g / L of lactic acid,

[0092] - 1.0 g / L of acetic acid,

[0093] - 0.2 g / L of glycerol,

[0094] - 0.4 g / L of urea,

[0095] - 5.0 g / L of glucose

[0096] - 0.2 g / L of Ca(OH)2adjusted to pH=4.2. 250 mg / mL of the SPL composition, prepared as described above, was added to said fluid.

[0097] The lactobacilli precultured in standard growth medium were inoculated at the ratio of 1 : 1 into the vaginal fluid containing the SPL composition, thus obtaining a concentration of the composition amounting to 125 mg / mL; each sample was left under incubation at 37°C for 24 h.

[0098] The assay was performed in:

[0099] - chemically simulated vaginal fluid containing the SPL composition;

[0100] - simulated vaginal fluid containing the SPL composition with the further addition of porcine pancreatic alpha-amylase type VLB (0.5 mg / mL - Sigma Aldrich A3176-1MU, Italy), and then filtered through 0.22 pm filters.

[0101] To count the number of colony -forming units (CFU) per millilitre, 100 pL of each of the samples tested in vaginal fluid, after suitable dilution, was seeded in duplicate in plates prepared with agar and incubated anaerobically, according to the method known to the skilled person for the growth of lactobacillus (obtained by using a specific jar and a sachet of AnaeroGen - Oxoid, Italy) which gave rise to an O2 concentration < 0.1% in 2.5 hours, and a CO2 concentration ranging between 7 and 15% in 24 h. The plates were incubated at 37°C for 24 hours. The results are expressed as the mean CFU / ml and illustrated in Figure 2, which shows the growth of Lactobacillus vaginalis ATCC 49540 (culture method) in simulated vaginal fluid (24 h) in the presence of SPL, with and without a-amylase enzyme.

[0102] Analysis of the data in Figure 2 unequivocally demonstrates that the quantitative findings obtained with the spectrophotometric method are confirmed from the qualitative standpoint; the bacterial cultures in simulated vaginal fluid clearly demonstrate that after only 24 h the lactobacilli treated with the composition according to the invention are viable and proliferating, to an even more marked extent when the amylase enzyme is present in the culture. This result is extremely important, because the chemically simulated vaginal fluid represents an extreme condition for bacterial growth, due to the low presence of carbon sources usable as nourishment by the strains tested; the fact that the viability and proliferation of the lactobacilli was observed after only 24 h, especially when in the presence of the enzyme, is an outstanding result, confirming the efficacy of the composition in question.

[0103] In view of the results obtained with the tests according to Examples 2 and 3, it was decided to evaluate the effect of the composition in question on the proliferation and viability of lactobacilli over a longer period, namely 48 h, as described in the example below. For this purpose, it was decided to use as culture broth the MRSc medium already used in Example 1, which is particularly suitable for the type of lactobacillus in question, and not to conduct the test for samples also containing amylase, as the amplifying effect of the enzyme had been extensively demonstrated in the preceding tests.

[0104] Example 4: Evaluation of the activity of the composition according to Example 1 on the growth of lactobacilli (culture method) at. 48 h

[0105] The experiment was conducted by the procedure illustrated in Example 3, suitably modified as follows:

[0106] • the composition prepared according to Example 1 (SPL) was accurately weighed (500 mg) and directly resuspended in 2 ml of sterile broth (not in sterile water) to obtain a final dilution factor of 1 :4 (equal to a final concentration of 125 mg / ml of the SPL under test);

[0107] • the lactobacilli (Lactobacillus vaginalis ATCC 49540) were always cultured and treated in the sole presence of MRS broth (Oxoid, Italy) added to 0.05% w / v L- cysteine (MRSc);

[0108] • the tests on the samples, treated with SPL or untreated, were conducted after 48 hours’ incubation, while maintaining the standard temperature conditions (37°C) and the unit of measurement considered (mean CFU / ml).

[0109] The test results are set out in Figure 3: after 48 hours the lactobacilli present in the sample treated with SPL had grown enormously compared with the control, and were perfectly viable. Figure 3 shows the growth of Lactobacillus vaginalis ATCC 49540 (culture method) in agar:MRSc medium at 48 hours in the presence of SPL vs the control.

[0110] This test confirms once again that the composition in question stimulates the proliferation of lactobacilli and is able to maintain their viability, in both the short term (24 h) and long term (48 h).

[0111] As already stated, in situations of dysbiosis the vaginal environment is characterised by alterations that promote the proliferation of pathogenic micro-organisms, with the consequences already described.

[0112] It was therefore deemed important to evaluate the effect of the composition in question on pathogenic bacterial species known to be present in the vaginal environment. The choice fell on the bacterium Escherichia coli. which is not only extensively represented in the dysbiotic vaginal environment, but is also Gram negative.

[0113] Example 5: Measurement of activity of the composition according to Example 1 on growth Escherichia coli ATCC 9637 (spectrophotometric method).

[0114] The procedure used to conduct the experiments described follows the method illustrated in Example 2, adapted to the different micro-organism used.

[0115] The composition to be tested (SPL) was prepared according to Example 1, and 500 mg was accurately weighed in 2 mL cryovials, then directly resuspended in sterile broth to obtain a final dilution factor of 1 :4.

[0116] An aliquot of the stock solution of strain Escherichia coli ATCC 9637 in Mueller Hinton broth (Oxoid, Italy), maintained at -80°C in liquid culture medium with the addition of 20% glycerol, was tested for purity, and 50 pl was inoculated into 5 ml of freshly-prepared sterile liquid culture medium, and incubated at 37°C for 18-24h.

[0117] After 24 hours’ incubation, an aliquot was taken up and diluted 1 : 10 for reading of the ODeoo, adjusting the turbidity of the culture broth by suitable dilution with sterile 0.9% w / v NaCl to an ODeoo value ranging between 0.16 and 0.2 (corresponding to 1 McFarland); 300 pl was inoculated into 30 ml (1 : 100) of sterile 0.9% w / v NaCl, to give a final concentration of about 3xl05CFU / ml in each well.

[0118] The assays were conducted in culture medium as is, and with the addition of porcine pancreatic a-amylase (A3176-1MU, Sigma Aldrich Type VI-B) at the dose of 0.5 mg / ml, then filtered through 0.22 pm beaker filters.

[0119] The culture broths of Escherichia coli strain ATCC 9637 grown in the culture medium without samples, with or without enzyme, were used as positive control.

[0120] For the assay, about 160 pl of each SPL sample diluted with broth was loaded into the wells of a 96-well plate. The inoculation was performed by adding 20 pl of the bacterial suspension prepared as already described to obtain a final concentration of about 3xl05CFU / ml per well.

[0121] The microplate was then placed in the incubator at 37°C, under stirring at 150 rpm. The absorbance was read after 24 hours’ incubation.

[0122] Figure 4 shows the percentage growth of strain Escherichia coli ATCC 9637 compared with the untreated control; after 24 hours’ incubation in the presence of SPL, the growth of strain E. coli ATCC 9637 was much lower (- 46%) than the untreated control whereas, as expected, there were no significant differences due to the presence of the enzyme.

[0123] In particular, Fig. 4 shows the growth of Escherichia coli ATCC 9637 (spectrophotometric method) at 24 hours in the presence of SPL compared with the control.

[0124] As the composition contains no preservatives and the only detectable antibacterial activity is performed towards Gram-positive bacteria, it can readily be deduced that the composition as a whole exercises a marked antibacterial action towards the Gram-negative bacterium most widely represented in situations of dysbiosis in the vaginal environment; this surprising result was wholly unexpected.

[0125] These findings, taken as a whole, unequivocally demonstrate that the compositions according to the invention represent a significant advance on the state of the art because, due to the minimal number of active ingredients in a precise weight ratio combined with specific excipients, they are surprisingly able to:

[0126] ® promote the proliferation of lactobacilli in the short term, as demonstrated in Example 2;

[0127] ® preserve the viability of lactobacilli in the vaginal fluid, as demonstrated in Example 3;

[0128] • promote the proliferation of lactobacilli for lengthy periods of time, maintaining their viability unchanged, as demonstrated in Example 4;

[0129] • act as a selective antibacterial even without the aid of specific active ingredients or preservatives.

[0130] The compositions according to the invention are intended for application in the prevention and treatment of vaginal dysbiosis, prevention and treatment of bacterial vaginal infections caused by dysbiosis, and prevention of recurrences, by restoring the correct balance of the vaginal microbiota.

[0131] They also represent a further industrial advantage, because their preparation, due to the small number of substances used, is particularly simple, with favourable repercussions in terms of costs for both the manufacturer and the patient, and in environmental terms.

Claims

CLAIMS1. Pharmaceutical compositions in mucoadhesive hydrogel form comprising: a) a combination of active ingredients comprising or consisting of lactic acid, glycogen and hyaluronic acid in a weight ratio of 10: 1 :2 respectively, and b) appropriate excipients for use in the prevention and treatment of vaginal dysbiosis, prevention and treatment of vaginal bacterial and / or fungal infections caused by dysbiosis, and prevention of recurrences by restoring the correct balance of the vaginal microbiota.

2. Compositions for use according to claim 1 wherein the bacterial infections are caused by Gram-negative bacteria.

3. Compositions for use according to claim 2 wherein the Gram-negative bacterium is Escherichia coli.

4. Compositions for use according to claim 3 for use in the treatment of vaginal bacterial infections caused by E. coli.

5. Compositions for use according to any one of claims 1 to 4 wherein the excipients are a. glycerol; b. propanediol; c. a mixture of phenethyl alcohol and ethylhexylglycerin; d. a hydroxyethylacrylate / acryloyldimethyl taurate copolymer; e. sodium hydroxide; f. water.

6. Compositions for use according to any one of claims 1 to 5 wherein the weight percentages of the ingredients of combination (a) relative to the total weight of the composition are: lactic acid: between 0.5 and 1.5%, preferably 1; glycogen: between 0.05 and 0.15%, preferably 0.1%; hyaluronic acid sodium salt: between 0.1 and 0.3%, preferably 0.2%, the lactic acid, glycogen and hyaluronic acid weight ratio being 10: 1 :2 respectively.

7. Compositions for use according to any one of claims 1 to 6 wherein the hyaluronic acid has a weight-average MW of 200000 Da.

8. Compositions according to claim 5, 6 or 7 wherein the mixture of phenethyl alcohol and ethylhexylglycerin is present in the amount of 0.2 to 0.5% by weight of the total weight of the composition, preferably 0.3%.

9. Compositions for use according to any one of claims 1 to 8 wherein the hydroxyethylacrylate / acryloyldimethyl taurate copolymer is present in the amount of 3 to 4% by weight of the total weight of the composition, preferably between 3.6 and 3.8%.

10. Compositions for use according to any of claims 1 to 9 adjusted to a pH ranging between 3.5 and 4.5, preferably between 3.7 and 3.9.

11. Compositions for use according to any one of claims 1 to 10 consisting of:Lactic acid 1.00% by weightGlycogen 0.10% by weightHyaluronic acid sodium salt weight average MW 200000 Da 0.20% by weight - Glycerol 4.00% by weightPropanediol 3.00% by weightPhenethyl alcohol / ethylhexylglycerin 0.30% by weightHydroxyethyl acrylate / sodium acryloyldimethyl taurate copolymer 3.70% by weight Sodium hydroxide 0.230% by weight - Purified water q.s. to 100.

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