IT202300026142A1 - MUCOADHESIVE COMPOSITIONS FOR THE LOCAL TREATMENT OF VAGINAL CONDITIONS - Google Patents

Description

Description of the patent for an industrial invention entitled:

?MUCOADHESIVE COMPOSITIONS FOR THE LOCAL TREATMENT OF VAGINAL CONDITIONS?

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

State of the art

The human body is home to numerous microorganisms—commensal, symbiotic, and pathogenic—that populate its cavities, whether or not they are connected to the outside world. This collection of microorganisms is called the microbiota and generally includes bacteria, fungi, and viruses. The composition of the microbiota varies not only depending on the body area considered but also on many other factors, such as gender, age, ethnicity, lifestyle, poor or excessive hygiene, etc. (https://doi.org/10.1038/nature11234), and its fundamental role in individual well-being has long been understood.

In particular, at the vaginal level, the microbiota consists of a wide range of aerobic and anaerobic microorganisms, with a clear predominance of various species of Lactobacillus, primarily responsible for maintaining the correct balance of the vaginal mucosa. This balance is essential for well-being at every stage of a woman's life: situations of dysbiosis can not only create discomfort or temporary pathologies, but even affect reproductive activity ( ; Front Med, 2018;5:181).

Under physiological conditions, therefore in a state of eubiosis, the different strains of lactobacilli can represent up to 90% of the vaginal bacterial flora.

Lactobacilli perform various functions at the vaginal level:

? are responsible for the production of a biofilm that creates a barrier on the vaginal mucosa, which is thus protected from colonization by pathogens;

? they are able to produce particular substances, bacteriocins, which are toxic to some pathogenic microorganisms that may be present;

Above all, they produce lactic acid, which is essential for maintaining the vaginal pH at around 3.5-4.5. These pH values are optimal for the growth and activity of lactobacilli and at the same time strongly discourage the development of pathogens, which generally do not survive at these acidic levels.

To effectively perform these functions, lactobacilli require the presence of substances in the vaginal fluid that nourish them, acting as prebiotics. In particular, the action of lactobacilli is optimal when the vaginal fluid is rich in glycogen, physiologically present in the vaginal epithelium (Fertil Steril, 1971; 22:64-68) along with enzymes, particularly amylase, which facilitate its degradation, releasing the "building blocks" that will be used as nourishment by the lactobacilli (Mirmosnsef P. et al, Plos ONE; 2014;9:e102467).

When, for the reasons listed above, the vaginal microbiota is altered, a clear depletion of lactobacilli is observed, a consequent increase in vaginal pH above 4.5 and the proliferation of opportunistic microorganisms which, although part of the vaginal microbiota, behave like pathogens when their number increases excessively.

It is clear that an imbalance in the vaginal microbiota, and therefore a situation of dysbiosis, favors the attachment and proliferation of pathogens that can lead to bacterial vaginitis (caused mainly by Gardnerella vaginalis and other anaerobes), fungal and yeast infections (in particular, by Candida albicans), and urogenital infections, mostly caused by Escherichia coli.

The treatment of choice for these pathologies is naturally pharmacological, possible thanks to the availability of drugs with antimicrobial and/or antifungal action. However, it has been demonstrated and is widely known that following pharmacological treatment, the vaginal microbiota is severely damaged and its balance profoundly altered: the vaginal mucosa, already weak, then finds itself once again in a situation of dysbiosis and becomes extremely vulnerable because it is not protected by its physiological bacterial complement. This triggers a vicious circle that results in frequent recurrences of the pathologies described above (J Infect Dis, 2015;212:793-802; Microb Biotechnol, 2023;16:1423-1437).

It is therefore clear that maintaining and/or restoring vaginal eubiosis is extremely important both in physiological conditions and, even more so, in situations of dysbiosis, even following pharmacological treatments, particularly antibacterial and/or antifungal ones.

To this end, various pharmaceutical and cosmetic formulations for vaginal use have been developed, essentially containing lactic acid, which is necessary for restoring a sufficiently acidic vaginal pH. Generally, other active ingredients are used in combination with lactic acid, particularly prebiotics, i.e., non-digestible substances (fibers) that nourish the lactobacilli already present, or probiotics, i.e., live microorganisms recognized as beneficial to our bodies. Very often, these active ingredients are combined with other active ingredients, such as bacterial lysates, polypeptides, natural extracts, polysaccharides, hyaluronic acid, etc.

Known compositions also contain consistency and viscosity agents and, to prevent the rapid depletion of lactic acid-induced acidity in the vaginal environment, a buffer system, for example based on sodium lactate, which stabilizes the pH.

Compositions containing lactic acid and prebiotics are described in:

- EP257007: describes a composition for vaginal use comprising lactic acid and glycogen in a ratio of 20:1 to 500:1. According to this patent, the high amount of lactic acid avoids the use of preservatives, which would also negatively affect the lactobacilli. The effectiveness of the preparation is demonstrated, but it is not at all clear whether the composition, in addition to maintaining the lactobacilli population, is capable of increasing it, and consequently increasing the production of lactic acid;

- CN115006512: describes a pharmaceutical composition for vaginal use with a highly complex formula, which includes, in addition to the excipients, lactobacteria fermentation lysate, lactic acid, and sodium lactate, along with other active ingredients. Numerous in vitro tests have shown that only the composition containing the full range of listed active ingredients is capable of significantly increasing the amount of ATP produced by lactobacilli, and therefore the metabolic activity of bacterial cells, but no increase in the lactobacilli population is described.

The invention described here goes beyond the state of the art thanks to the development of a simple composition, containing very few active ingredients, free of traditional preservatives and perfectly calibrated in terms of excipients, capable, surprisingly, of stimulating the proliferation of lactobacilli present in the vagina, with consequent production of lactic acid and restoration of the correct acid pH: in this way, the proliferation of opportunistic pathogenic microorganisms is highly reduced, and therefore the physiological vaginal microbial complement is rebalanced.

Description of the invention:

The invention relates to pharmaceutical compositions in the form of mucoadhesive hydrogels comprising or consisting of hyaluronic acid, glycogen, and lactic acid, and appropriately selected pharmaceutically acceptable excipients. The compositions are used in the prevention and treatment of vaginal dysbiosis, in the prevention and treatment of bacterial and/or fungal vaginal infections caused by dysbiosis, and in the prevention of recurrences by restoring the correct balance of the vaginal microbiota. As mentioned, the balance of the vaginal bacterial flora is strongly linked to specific factors, such as the pH of the vaginal environment and the clear preponderance of certain bacterial species, particularly the Lactobacillus genus, which in turn are responsible for the production of lactic acid. A pH between approximately 3 and 4.5, particularly between approximately 3.5 and 4.5, is in fact optimal for preventing the proliferation of opportunistic organisms and thus preserving the integrity of the vaginal environment. In fact, at these pH values, lactobacilli best perform their functions, previously described.

For this reason, the use of lactic acid in the treatment of dysbiosis or to maintain vaginal eubiosis has been practiced for a long time. Lactic acid has been used alone or, more frequently, in combination with one of its salts, to form a buffer system that keeps the pH value of the final composition stable.

Other active ingredients, preferably so-called "prebiotics," are often added to lactic acid-containing formulations to provide the necessary nutrients for the growth and well-being of the lactobacilli present in the vagina. These are water-soluble fibers belonging to the oligosaccharide group, often derived from fruit or cereals. Examples of prebiotics include inulin, fructooligosaccharides (FOS), galactooligosaccharides (GOS), lactulose, isomaltooligosaccharides (IMO), xylooligosaccharides (XOS), long-chain beta-glucans, glucomannan oligosaccharides, and glycogen, a glucose polysaccharide.

The combinations of lactic acid and prebiotics used are extremely varied, in quantitative terms, because imbalances in either component can have a detrimental effect on the vaginal mucosa. Excessive amounts of lactic acid would result in excessively low pH values, which are certainly undesirable in an epithelium already suffering from dysbiosis and ineffective for the purposes of the invention; excessive amounts of prebiotics risk providing nourishment not only to lactobacilli but also to potentially pathogenic species present in the vaginal environment.

Other important formulation parameters concern for example:

? stability of the compositions: the preservatives normally used are antimicrobials that act indiscriminately, avoiding contamination of the composition but negatively interfering with the population of vaginal Lactobacilli;

? mucoadhesiveness?: after application the compositions must remain stably in situ, in order to perform their function in the best possible way and for the established time;

pH values: It is necessary to create a buffer system that maintains stable values, so as to prevent lactic acid from being rapidly neutralized by alkaline vaginal secretions.

The invention described herein goes beyond the state of the art thanks to the development of a composition in the form of a hydrogel, stable, mucoadhesive and free of classic paraben-type preservatives, containing, or consisting of, a minimum number of active ingredients, in specific weight ratios and perfectly balanced with appropriate excipients and capable of:

? increase the number of lactobacilli normally present in a healthy vaginal environment, keeping them perfectly vital, and therefore

? promote the production of lactic acid by the lactobacilli themselves, contributing to the restoration and/or maintenance of the state of eubiosis of the vaginal mucosa;

? rebalance and maintain the vaginal pH at the right level of acidity, and finally

? exert a significant antimicrobial action against pathogenic bacterial flora, when present, and in particular against Escherichia coli.

The composition of the invention essentially comprises lactic acid, glycogen and hyaluronic acid as active ingredients, to which specific excipients are associated which, as will be discussed below, contribute significantly to the stability and efficacy of the final product.

Lactic acid plays a fundamental role in restoring the vaginal pH to values between 3 and 4, which, as is known, are essentially physiological: the concentration of lactic acid in vaginal fluid is approximately 1% w/v, and acidifies the vaginal environment to a pH of approximately 3.5.

Glycogen is present in the vagina at the intraepithelial level and, although in quantities that vary depending on age, constitutes an important source of energy for lactobacilli: as mentioned, in fact, amylases are active in the vagina that degrade glycogen and make it available to lactobacilli, which then produce lactic acid.

Finally, hyaluronic acid (HA) is a heteropolysaccharide composed of alternating residues of D-glucuronic acid and N-acetyl-D-glucosamine, with a linear chain, with a molecular weight (MW) that can vary between 400 and 3 x 10<6 >Da, depending on the extraction source or the preparation method used. Ubiquitously present, it plays an important role in the biological organism, as the main component of the connective tissue of vertebrates, the synovial fluid of joints, and the vitreous humor. HA serves as a mechanical support for the cells of many tissues such as skin, tendons, muscles, and cartilage, and is essential for tissue hydration and joint lubrication.

HA is crucial in the tissue repair process from both a structural and metabolic point of view (J Theoretical Biol, 1986:219-234;

J Surg Res, 1983, 35:410-416; Drugs, 1994, 47:536-566); it also acts as an anti-inflammatory, modulating the release of inflammatory cytokines, particularly IL-1, and is also able to bind to specific opioid receptors mimicking an analgesic effect.

The combination of these widely recognized properties has meant that hyaluronic acid or its alkaline salts have long been used in the preparation of dressings used to treat wounds, ulcers, and skin and mucosal lesions of various origins, both superficial and deep, thanks to their restorative and soothing action, and also in the viscosupplementation therapy of osteoarthritis.

The hyaluronic acid used in the present invention, as such or in the form of an alkaline salt, can be derived from any source, for example, from rooster combs (EP138572), from fermentation (from Streptococcus equi or Zooepidemicus), or from biosynthesis by Bacillus (EP2614088, EP2614087) and can be purified according to different techniques (EP3491027; EP3655138). Preferably, HA from fermentation by Streptococcus is used, in particular Streptococcus equi sub-sp. equi, 68222, mutant H-1 (EP716688).

The weight-average molecular weight of the polymer for the applications described here is preferably between 100,000 and 1,000,000 Da, even more preferably between 150,000 and 250,000 Da; the latter, for brevity, is generally defined as "HA with a weight-average molecular weight of 200 kDa" and is preferably in the form of a sodium salt.

By weight average molecular weight (MW) we mean the weight average MW calculated using the ?intrinsic viscosity? method (Carbohydr Res, 1986, 363-377).

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

? directly promotes the restoration of natural vaginal acidity, thanks to the pH value of the composition which is the subject of the invention;

? maintains, promotes, restores the vitality of the lactobacilli present in the vagina; but above all and unexpectedly

? stimulates the proliferation of lactobacilli, increasing their population; ? acts significantly as an antibacterial against opportunistic pathogens, specifically Escherichia coli, without containing substances with a specific effect or classic preservatives, which are themselves antimicrobials.

Furthermore, the presence of hyaluronic acid gives the composition a repairing and soothing effect on the mucosa, while mucoadhesiveness is ensured by the choice of appropriate excipients.

The weight ratio of lactic acid:glycogen:sodium hyaluronate that has proven extraordinarily effective for the purposes of the invention is 10:1:2, and therefore is completely contrary to what is known in the state of the art: as already mentioned, in EP257007 the lactic acid:glycogen ratio ranges from 20:1 to 500:1, and is preferably 50:1, clearly unbalanced in favor of lactic acid. On the contrary, CN115006512, which also contemplates the presence of hyaluronic acid, describes a composition in which the lactic acid:glycogen:HA ratio is 1:10:1, with a clear preponderance of prebiotic which is a source of nourishment for all species present in the vagina, including opportunistic bacterial and fungal ones, which are completely unwanted.

As regards the concentrations of the active ingredients of the composition described, they can vary as follows:

? lactic acid: ? between 0.5 and 1.5%, preferably equal to 1% by weight with respect to the weight of the final composition;

? glycogen: ? between 0.05 and 0.15%, preferably equal to 0.1% by weight with respect to the weight of the final composition

? hyaluronic acid sodium salt: ? between 0.1 and 0.3%, preferably equal to 0.2% by weight with respect to the weight of the final composition, maintaining the 10:1:2 ratio unchanged.

In addition to the main active ingredients described above, the composition of the invention includes a base such as sodium hydroxide to create a buffer system necessary to maintain the pH at the established values, and suitable excipients such as wetting and gelling agents which, in the precise proportions detailed below, give the composition adequate stability, viscosity and mucoadhesiveness and unexpectedly contribute to promoting the proliferation of lactobacilli, increasing their number and maintaining their vitality.

Sodium hydroxide is used as a buffer system in quantities varying according to the lactic acid content, in order to obtain a pH between 3.5 and 4.5, preferably between 3.7 and 3.9.

The excipients preferably consist of a mixture of:

? glycerol;

? propanediol;

It is a mixture of phenethylalcohol and ethylhexylglycerin (trade name, Sensiva PA 20): it is a humectant, with deodorant activity because it controls the degradation of Gram-positive bacteria, the cause of the odor that accompanies dysbiosis, used in quantities varying between 0.2 and 0.5% by weight with respect to the final composition, preferably equal to 0.3%;

? a hydroxyethyl acrylate/acryloyldimethyl taurate copolymer (trade name Sepineo DERM): ? a gelling and mucoadhesive agent, stable at wide pH ranges, used in quantities varying between 3 and 4% by weight with respect to the final composition, preferably between 3.6 and 3.8%.

? purified water.

For purely indicative purposes, the invention is described in greater detail in the following examples.

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

From the total amount of purified water required, take a quantity equal to 6 times the weight of NaOH in the formula and transfer it to a suitable container. Add the required amount of NaOH and stir until completely dissolved. Heat the remaining purified water in the turboemulsifier to approximately 40°C and begin stirring. Add the lactic acid and continue stirring for at least 5 minutes, until a homogeneous solution is obtained. Add the previously prepared NaOH solution and continue stirring for at least 15 minutes, until a homogeneous solution is obtained.

Add the hyaluronic acid sodium salt (average weight of HA = 200 kDa) and stir until a clear, colorless solution is obtained, free of undissolved matter (approximately 45 min). Keeping the system stirred, then continue adding glycerol, propanediol, and phenethyl alcohol/ethylhexylglycerin (Sensiva PA20) sequentially until a clear, homogeneous solution is obtained. Keeping the stirring and heating system active, add the glycogen and wait for the substance to dissolve completely, obtaining a homogeneous, slightly opalescent solution (approximately 45 min). Begin the cooling phase by increasing the stirring system and activating the vacuum system, then add Sepineo DERM until it is completely dispersed, resulting in a homogeneous, viscous, opalescent gel free of undissolved matter. The composition thus prepared has a pH value of 3.8.

Example 2: Evaluation of the activity of the composition of Example 1 on the growth of lactobacilli (spectrophotometric method) at 18h

The activity of the composition under examination on lactobacilli cultures was evaluated in vitro using the widely used microplate spectrophotometric method: this is based on reading the optical density (OD) or absorbance of appropriately prepared cultures at precise wavelengths. The optical density measures the turbidity of the samples, which is a function of the concentration of cells they contain; therefore, the higher the number of cells in the sample, the higher the optical density value. In this specific case, the enzyme amylase, which is physiologically present in the vaginal environment and which, by degrading glycogen, provides nourishment for the lactobacilli, was added to some samples.

Materials and methods:

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

The composition under examination was prepared as in Example 1 and will hereafter be referred to as SPL; 500 mg were weighed into 2 mL cryovials and then resuspended directly in sterile broth to obtain a final dilution factor of 1:4. For the test, 160 μL of the sample thus prepared were loaded into the wells of a 96-well microplate.

For tests involving the use of the enzyme, a porcine pancreatic β-amylase (A3176-1MU, Type VI-B) was used at a dose of 0.5 mg/ml, solubilized in sterile medium, subjected after the addition of the enzyme to further filtration with 0.22 micrometer glass filters.

For inoculation, an aliquot of the lactobacilli strain stock (maintained at -80°C in MRSc and 20% glycerol) was checked for purity and 50 μl was inoculated into 5ml of freshly prepared sterile MRSc and incubated at 37°C for 24h.

After 24 hours of incubation, an aliquot was taken and a 1:10 dilution was made to read the OD600, adjusting the turbidity of the broth culture with an appropriate dilution in sterile 0.9% w/v NaCl to an OD600 value between 0.16 and 0.2 (corresponding to 1 McFarland); 300 ?l were then inoculated in 30 ml (1:100) of sterile 0.9% w/v NaCl, so that the quantity subsequently loaded into each well (20 ?l) contains a final concentration of approximately 3x10<5 >CFU/ml.

For the tests, 20 μL of this bacterial suspension was inoculated into the wells of 96-well microplates containing the SPL samples. For anaerobiosis, approximately 10 μL of sterile paraffin was added to each well, covering the surface in contact with air; the microplate was then placed in an incubator at 37°C and shaken at 150 rpm. After 18 h of incubation, the optical density at 630 nm (OD630) was read.

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

It is immediately evident that after just 18 hours of incubation, the SPL composition resulted in a very significant growth of lactobacilli compared to the control, exceeding 30% growth compared to the control. This finding is even more striking for the samples containing amylase, which recorded an 88% growth compared to the control, on which, as expected, amylase had no effect, having no substrate on which to act.

This test therefore demonstrates that the tested composition is capable of stimulating the proliferation of lactobacilli, increasing their concentration compared to the control; the stimulation is even more marked in the presence of amylase, which degrades glycogen, offering the lactobacilli a fundamental source of nutrition.

The test performed 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, regardless of whether they are live or non-viable cells, bacterial lysates, or artifacts. The shape of the bacteria (e.g., rods or cocci) even has a certain influence, as they can diffract the incident light in an anomalous way, altering the final reading. This method is also not very suitable for experiments over relatively long time periods (for example, 48 hours or more) precisely because, due to the above-described conditions, when the samples become particularly turbid, and therefore dense, the optical density (OD) values decrease. detected optical growth are lower than the real growth (Mira P, et al., 2022: PLoS ONE 17(10): e0276040. https://doi.org/10.1371/journal.pone.0276040).

To overcome the limitations of the spectrophotometric method and to ensure the effectiveness of the composition claimed here, cell count tests were performed, which allow us to evaluate the actual viability of the bacteria present in the cultures. Schematically, by inoculating appropriate dilutions of the broth culture whose absorbance has been read into a suitable agar culture medium suited to the growth of that strain, the colonies can be counted and therefore the viable bacterial load can be determined, even over relatively long time intervals.

Example 3: Evaluation of the activity of the composition of Example 1 on the growth of lactobacilli (culture method) at 24 h

To confirm the quantitative data described in Example 2 and verify the actual viable bacterial load, the samples were tested using the culture method.

To obtain even more precise and significant data, the growth test was performed by culturing the lactobacilli in a simulated vaginal fluid, prepared in the laboratory as described below. Culture in vaginal fluid provides a completely realistic picture.

Materials and methods:

The test composition (SPL) was prepared as per Example 1 and 500 mg of accurately weighed solution was resuspended in 2 ml of sterile purified water.

For inoculation, an aliquot of the strain stock prepared as in Example 2 (maintained at -80°C in liquid culture medium supplemented with 20% glycerol) was checked for purity and 50 μl inoculated into 5 ml of freshly prepared sterile liquid culture medium, and incubated at 37°C for 24 h.

After incubation, an aliquot was taken and a 1:10 dilution was made to read the OD600; the turbidity of the broth culture was adjusted with an appropriate dilution in sterile 0.9% w/v NaCl to a value between 0.16 and 0.2 (corresponding to 1 McFarland) and 300 ?l of this suspension were inoculated in 30 ml of sterile 0.9% w/v NaCl (1:100), so as to have a final concentration of lactobacilli equal to approximately 3x10<5>CFU/ml.

The simulated vaginal fluid was prepared as described by

1999: Contraception, 59, 91?95, with the following composition

- 3.5 g/L of NaCl,

- 1.4 g/L of KCl,

- 0.02 g/L of bovine serum albumin,

- 2.0 g/L of lactic acid,

- 1.0 g/L of acetic acid,

- 0.2 g/L of glycerol,

- 0.4 g/L of urea,

- 5.0 g/L glucose

- 0.2 g/L of Ca(OH)2

adjusted to a pH=4.2.

To this fluid was added 250mg/mL of the SPL composition, prepared as described above.

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

The test was carried out in:

- chemically simulated vaginal fluid containing the SPL composition; - simulated vaginal fluid containing the SPL composition and further added with porcine pancreatic alpha-amylase type VI-B (0.5 mg/mL ? Sigma Aldrich A3176-1MU, Italy) and subsequently filtered with 0.22 ?m filters.

To count the number of colony-forming units (CFU) per milliliter, 100?L of each of the tested samples in vaginal fluid, after appropriate dilution, were seeded in duplicate on plates prepared with agar and incubated anaerobically, according to the technique known to the person skilled in the art for the growth of lactobacillus (obtained using a special jar and a bag of ) which brought the O2 concentration to < 0.1% in 2.5 hours and the CO2 concentration between 7 and 15% in 24 hours. The plates were incubated at 37?C for 24 hours. The results are expressed as the mean CFU/ml and are 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 without and with the enzyme ?-amylase.

Analysis of the data in Figure 2 unequivocally demonstrates that the quantitative findings obtained with the spectrophotometric method are confirmed qualitatively: bacterial cultures in simulated vaginal fluid clearly show that after just 24 hours, the lactobacilli treated with the composition under consideration are viable and proliferating, even more so when the enzyme amylase is present in the culture. This result is extremely important because chemically simulated vaginal fluid represents an extreme condition for bacterial growth, given the scarcity of carbon sources that can be used as nourishment for the tested strains: having highlighted the viability and proliferation of lactobacilli after just 24 hours, especially when the enzyme is present, is an extraordinary result, which attests to the efficacy of the composition under examination.

In light of the results obtained with the tests in Examples 2 and 3, we wanted to evaluate the effect of the composition under examination on the proliferation and viability of lactobacilli over a longer time span, specifically 48 hours, as described in the following example. To this end, we decided to use the MRSc medium already used in Example 1 as the culture broth, as it is particularly suitable for the type of lactobacillus under examination, and we chose not to perform the test on samples also containing amylase: the amplifying effect of the enzyme has in fact been amply demonstrated in previous tests.

Example 4: Evaluation of the activity of the composition of Example 1 on the growth of lactobacilli (culture method) at 48 h

The experiment was conducted according to the procedure illustrated in Example 3, appropriately modified as follows:

? the composition prepared as in Example 1 (SPL) ? was accurately weighed (500 mg) and resuspended directly in 2 ml of sterile broth (and not in sterile water) to obtain a final dilution factor of 1:4 (equal to a final concentration of the SPL to be tested of 125 mg/ml);

? lactobacilli (Lactobacillus vaginalis ATCC 49540) were grown and treated always and only in the presence of MRS broth (Oxoid, Italy) added with L-cysteine 0.05% w/v (MRSc);

? the tests on the samples, treated with SPL and not, were carried out after 48 hours of incubation, maintaining the standard temperature conditions (37?C) and the unit of measurement considered (average CFU/ml).

The test results are presented in Figure 3: after 48 hours, the lactobacilli present in the SPL-treated sample had increased enormously compared to 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 versus the control.

This test confirms once again that the composition under examination stimulates the proliferation of lactobacilli and is able to maintain their vitality, both in the short (24 h) and long term (48 h).

As repeatedly stated, in situations of dysbiosis, the vaginal environment is characterized by alterations that give free rein to the proliferation of pathogenic microorganisms, with the consequences already described.

It was therefore deemed important to evaluate the effect of the composition under examination on pathogenic bacterial species known to be present in the vaginal environment. The choice fell on the bacterium Escherichia coli, which is not only highly represented in the dysbiotic vaginal environment but is also a Gram-negative bacterium.

Example 5: Evaluation of the activity of the composition of Example 1 on the growth of Escherichia coli ATCC 9637 (spectrophotometric method).

The procedure followed to carry out the experiments described follows the method specified in Example 2, adapted to the different microorganism used.

The test composition (SPL) was prepared as in Example 1 and 500 mg were accurately weighed into 2 mL cryovials, then resuspended directly in sterile broth to obtain a final dilution factor of 1:4.

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

After 24 hours of incubation, an aliquot was taken and a 1:10 dilution was made to read the OD600, adjusting the turbidity of the broth culture with an appropriate dilution with sterile 0.9% w/v NaCl to an OD600 value between 0.16 and 0.2 (corresponding to 1 McFarland); 300 μl were inoculated in 30 ml (1:100) of sterile 0.9% w/v NaCl, so as to have a final concentration of approximately 3x10<5>CFU/ml in each well.

The tests were carried out in the culture medium as is and added with porcine pancreatic β-amylase (A3176-1MU, Sigma Aldrich Type VI-B) at a dose of 0.5 mg/ml and subsequently filtered with 0.22 μm glass filters.

Broth cultures of Escherichia coli strain ATCC 9637 grown in the culture medium without samples, with or without enzyme, were considered as positive controls.

For the assay, approximately 160 μL of each broth-diluted SPL sample was loaded into the wells of a 96-well plate. Inoculation was performed by adding 20 μL of the bacterial suspension prepared as described above to reach a final concentration of approximately 3 x 10<5>CFU/ml per well.

The microplate was then placed in an incubator at 37°C and shaken at 150 rpm. The absorbance reading was taken after 24 hours of incubation.

Figure 4 shows the percentage of growth of the Escherichia coli ATCC 9637 strain compared to the untreated control: after 24 h of incubation in the presence of SPL, the growth of the E. coli ATCC 9637 strain is significantly lower (- 46%) than the untreated control, while, as expected, there are no significant differences due to the presence of the enzyme.

More specifically, Fig. 4 shows growth of Escherichia coli ATCC 9637 (spectrophotometric method) at 24 hours in the presence of SPL versus control.

Considering that the composition does not contain preservatives and that the only detectable antibacterial activity is directed against Gram-positive bacteria, it can be easily deduced that the composition as a whole exerts a clear antibacterial action against the Gram-negative bacteria most represented, in situations of dysbiosis, in the vaginal environment: this surprising result was completely unexpected.

The sum of these data unequivocally demonstrates that the compositions of the invention represent a clear improvement over the state of the art, since, thanks to a minimum number of active ingredients in a precise weight ratio associated with specific excipients, they are surprisingly capable of:

? promote the proliferation of Lactobacilli in the short term, as demonstrated in Example 2;

? maintain the viability of Lactobacilli in vaginal fluid, as demonstrated in Example 3;

? promote the proliferation of Lactobacilli for prolonged periods of time, maintaining their vitality unchanged, as demonstrated in Example 4;

? act as a selective antibacterial even without the aid of specific active ingredients or preservatives.

The compositions of the invention find application in the prevention and treatment of vaginal dysbiosis, in the prevention and treatment of bacterial vaginal infections caused by dysbiosis, and finally in the prevention of recurrences thanks to the restoration of the correct balance of the vaginal microbiota.

They also represent a further industrial advantage, as their preparation, thanks to the small number of substances used, is particularly simple, with a consequent positive impact both in terms of costs for the manufacturer and for the patient, and on the environment.

Claims (12)

1. Pharmaceutical compositions in the form of mucoadhesive hydrogels 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. 2. Compositions according to claim 1 wherein the excipients are a. glycerol; b. propanediol; c. a mixture of phenethylalcohol and ethylhexylglycerin; d. a hydroxyethyl acrylate/acryloyldimethyl taurate copolymer; e. sodium hydroxide; f. water. 3. Compositions according to claim 1 or 2 wherein the percentages by weight of the components of the combination a) with respect 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 weight ratio of lactic acid, glycogen and hyaluronic acid being 10:1:2 respectively. 4. Compositions according to any of claims 1-3 wherein the hyaluronic acid has a weight average PM equal to 200,000 Da. 5. Compositions according to claim 2, 3 or 4 wherein the mixture of phenethylalcohol and ethylhexylglycerin is present in an amount of 0.2 to 0.5% by weight with respect to the total weight of the composition, preferably 0.3%. 6. Compositions according to any of claims 2-5 wherein the hydroxyethyl acrylate/acryloyldimethyl taurate copolymer is present in an amount of 3 to 4% by weight based on the total weight of the composition, preferably between 3.6 and 3.8%. 7. Compositions according to any of claims 1-6 adjusted to pH between 3.5 and 4.5, preferably between 3.7 and 3.9. 8. Compositions according to any of claims 1-7 consisting of: - lactic acid 1.00% by weight - Glycogen 0.10% by weight - -Hyaluronic acid sodium salt Average weight PM 200000 From 0.20% by weight - Glycerol 4.00% by weight - Propanediol 3.00% by weight - Phenethyl alcohol/ethylhexylglycerin 0.30% by weight - Hydroxyethyl acrylate/sodium acryloyldimethyl taurate copolymer 3.70% by weight - Sodium Hydroxide 0.230% by weight - Purified water q.s. at 100 9. Compositions according to claims 1-8 for use in the prevention and treatment of vaginal dysbiosis, in the prevention and treatment of bacterial and/or fungal vaginal infections caused by dysbiosis and in the prevention of recurrences thanks to the restoration of the correct balance of the vaginal microbiota. 10. Compositions for use according to claim 9 wherein the bacterial infections are caused by Gram-negative bacteria. 11. Compositions for use according to claim 10 wherein the Gram-negative bacterium is Escherichia coli. 12. Compositions according to claim 8 for use in the treatment of vaginal bacterial infections caused by E. coli.