EA040310B1 - UROGENITAL COMPOSITION FOR MEDICAL DEVICE BASED ON SUITABLE BIOCHEMICAL COMPOSITIONS FOR STABILIZING ACIDITY AND REDOX STATE OF VAGINAL FLUID - Google Patents

Description

The field of technology to which the invention belongs

The invention relates to a biochemical composition of urogenital compositions that stabilize the acidity in the vagina and create suitable conditions in it for inhibiting urogenital pathogens and for the growth of lactobacilli. The invention provides compositions for vaginal or alternatively oral administration and the use of such compositions to stabilize vaginal or alternatively intestinal acidity and create favorable conditions for inhibition of pathogens and growth of lactobacilli in the urogenital or alternatively in the gastrointestinal tract. These compositions are based on chemically defined ingredients, avoid commonly used natural or poorly defined nutritional components, and the compositions may be associated with human vaginal strains with specific antimicrobial and/or anti-inflammatory properties.

In addition, attention is paid to the correct value of the redox potential after vaginal administration of such a composition, in particular in relation to the inhibition of bacterial vaginosis (BV) associated pathogens such as G. vaginalis.

Finally, compositions are also provided containing an agent for further suppressing the growth of opportunistic yeasts such as Candida albicans without prejudice to the growth of lactobacilli.

State of the art

It is well known that beneficial, well-tolerated non-pathogenic bacteria, the so-called human natural microbiota, predominate in the gut, vagina and urethra/bladder in a healthy state, while overgrowth of pathogenic and opportunistic microorganisms such as bacteria, yeasts, viruses and protozoa often leads to dysbiosis infections in these organs.

The terms aerobic bacteria and anaerobic bacteria are generally used to describe, respectively, those microorganisms that require air and those that cannot grow in the presence of air.

Notably, some of the non-pathogenic and pathogenic bacteria that can be found in the vagina can be defined as non-facultative anaerobes, i.e. they can grow in the presence or absence of air and are generally unaffected by oxidative conditions in the growing medium. Escherichia coli (E. coli) and Staphylococcus aureus, which are known to cause a vaginal condition commonly referred to as aerobic vaginitis, are non-limiting examples of such non-facultative anaerobic bacteria.

Other microorganisms that can be found in the vagina are obligate anaerobes, i.e. they do not grow in the presence of free oxygen and may actually die in the presence of oxygen or under oxidizing conditions.

Anaerobic organisms such as Gardnerella vaginalis, an obligate anaerobe migrating from the perineum to the vagina or acquired through sexual intercourse, can cause dysbiosis with few symptoms, followed by full blown bacterial vaginosis (BV), which is the most common vaginal infection. BV poses a serious risk of preterm birth in pregnant women and increases the risk of sexually transmitted infections (ref. 1). In such cases of vaginal dysbiosis or vaginal infections caused by anaerobes, the pH of the vaginal environment increases from physiological values of 3.5-4.5 to values reaching 5-6 or even higher. This increase in pH is a critical clinical parameter used in the diagnosis of BV and one of the main reasons for the risks associated with BV. The enzymatic action of the lactobacilli of the Doderlein microbiota on glycogen metabolites and other carbohydrates present in the vagina and yielding mainly lactic acid and, to a lesser extent, other organic acids, results in a low pH value of vaginal fluids. In addition, some strains of Gram-negative E. coli that originate from the intestine and migrate first to the vagina and then to the urethra/bladder are the most common cause of urethritis/cystitis in sexually active women. This same E. coli can also cause vaginal dysbiosis, a condition called aerobic vaginitis, which is also accompanied by disruption of the vaginal microbiota, decreased vaginal acidity and an increase in the corresponding pH value (ref. 2).

Candida yeast, which is adaptable and often coexists with lactobacilli, is another pathogen that causes vaginal infections, especially during pregnancy.

Candida is the only yeast organism that behaves like a facultative anaerobe, i.e. it is fundamentally independent of the presence or absence of air and oxidizing conditions.

These infections are usually cured by anti-infective treatment directed against pathogens that must be successfully destroyed during such therapy, at least to a certain extent. However, within 3 months after the initial treatment in about 70% of cases

- 1 040310 teas the patient relapses or reinfects (Ref. 3), either because the pathogens were not killed adequately or due to a lack of protection by the reconstituted local vaginal microbiota. This microbiota is actually often damaged by anti-infective therapy. The local vaginal microbiota, historically referred to as the Doderlein microbiota, is sensitive to many anti-infective agents and, in particular, to antibiotics. It is often disrupted or even destroyed by anti-infective therapy and therefore is not able to inhibit the reproduction of opportunistic, often pathogenic, organisms that have ever been present. As a consequence, bacterial vaginosis treated with a narrow-spectrum anaerobe-specific antibiotic such as clindamycin can lead to fungal infection because the lactobacillus-depleted vaginal microbiota cannot suppress opportunistic yeast.

The possibility of using exogenous Lactobacillus directly in the vagina to treat vaginal dysbiosis or infection had already been suggested by the physician who discovered them, Albert Doderlein himself, in his groundbreaking book in 1892 (ref. 4). This concept has since been widely developed in the scientific and patent literature and has recently been comprehensively reviewed (ref. 5).

WO 2009/123982 A2 relates to methods and compositions for Lactobacillus replacement therapy, ie. for colonizing the vaginal mucosa with the desired microbial species by contacting the vaginal wall with a dried composition of live microbial cells.

WO 2016/020861 A1 relates to film or sponge compositions and to a method for preventing and treating vaginal infections, wherein said film or sponge contains at least a mucoadhesive polymer, at least one probiotic and/or prebiotic, and at least one active compound , such as an antifungal or antibiotic agent, in pharmacologically effective amounts.

In recent years, the inventors have conducted thorough research on the properties of previously unknown strains of human Lactobacillus of vaginal origin, related to competition with the most common urogenital pathogens. In particular, the inventors investigated mechanisms involving the interaction of lactic acid, hydrogen peroxide and other agents, which were found to explain the exceptionally strong and specific antibiotic-like activity, for example, Lactobacillus gasseri against pathogenic Gardnerella vaginalis and Prevotella bivia (ref. 6) and the activity of L jensenii and L. crispatus against uropathogenic E. coli (ref. 7). The approach described in the referenced works and patents (ref. 8) has the additional advantage of using strains isolated from the same tract of perfectly healthy women for therapeutic applications directed to the urogenital tract. Thus, potential tolerability problems with exogenously administered strains of Lactobacillus can be greatly reduced when compared with the use of such strains that are not from humans or from the urogenital tract of healthy young women. For non-human strains, the issue of safety and tolerability for therapeutic agents, for example in urogenital applications, is not clear a priori and therefore must be assessed a posteriori.

In order to provide suitable compositions for a pharmaceutically acceptable carrier, the inventors have studied various formulations used in probiotic products and/or described in the literature. In one published approach (ref. 9), the inventors state that the use of skimmed milk, respectively skimmed milk preparations and derivatives called LGF (Lactobacillus Growth Factor) or NGF (Natural Growth Factor), promotes the growth of desired Lactobacillus while inhibiting the growth of unwanted uropathogens. . Such targeted growth factors that can be used for vaginal applications have also been found in microbiological growth media according to the same inventors. In fact, MRS medium (DeMan-Rogosa-Sharp) is such a most applicable growth medium for Lactobacillus strains (ref. 10). In addition to minerals and vitamins, this broth also contains peptones, sources of carbon and, especially nitrogen, of chemically indeterminate composition. Other inventors recommend the use of complex protein substances, such as skimmed milk and albumin, in a matrix for storing a vaginal drug containing strains of Lactobacillus (ref. 11).

In our opinion, although compositions containing natural nutrients such as skim milk or peptones may be useful for specific applications, they are not suitable as ingredients in pharmaceutical compositions containing Lactobacillus for vaginal applications. This is because, firstly, they are of biological (especially animal) origin, and secondly, these natural ingredients are often chemically poorly defined and thus carry an increased risk of side effects in patients as explained below. In addition, in terms of selective efficacy, these NGFs may not achieve the desired effect of inhibiting the growth of uropathogens. Not only strains of Lactobacillus, but also numerous pathogenic microorganisms can grow in such an ingredient-rich (with proteins, carbohydrates, vitamins, minerals, etc.) formulation as skimmed milk after contact and mixing with vaginal fluid. Pathogens such as E. coli sometimes grow faster in milk than in nutrient broth.

Assuming that 2 pathogens commonly found in the urogenital microbiota, S. aureus and E. coli, both also proliferate by orders of magnitude within about 20 hours when inoculated into a Lactobacillus-specific medium such as MRS (Table 1), similar results were also obtained with skim milk (not shown below).

Table 1. Growth rates of two pathogens in MRS broth

pathogen Time watch) OD 600 nm pH cfu/ml E. coli CCOS 492 0 0.05 6.5 Approx. 10$ 20 3.05 5.1 2.3 x 10 ¹⁰ S. aureus CCOS 461 0 0.05 6.5 Approx. 10$ 20 ZD 5.2 1.8 x 10 ¹⁰

Even starting at a relatively high concentration of 10 ⁸ cfu/ml, these pathogens can grow several logs more in the MRS, but stop growing after about 20 hours at the end due to the low pH they generate. Those. the so-called culture medium, highly specific for Lactobacillus, can also promote the growth of dangerous pathogens. In order to inhibit the growth of such pathogens, an effective and selective inhibitor must be added to the composition.

Therefore, if the subject is primarily settled with healthy strains of Lactobacillus present in sufficient amounts in the vagina, then it may be appropriate to administer LGF, since these native Lactobacillus bacteria can metabolize them. However, it is not advisable to administer to a dysbiotic vagina already infected or deficient in beneficial Lactobacillus, LGF of any species, or such non-specific growth factors, as they may stimulate the growth of pathogens present, instead of rare strains of Lactobacillus. Dysbacteriosis or infection may even be exacerbated by this treatment of LGF, the risks of which should be avoided.

In conclusion, skimmed milk preparations or Lactobacillus media components such as MRS may under some circumstances support the growth of eubiotic, local vaginal Lactobacillus. However, they are generally not suitable as therapeutic ingredients in pharmaceutical compositions for use in a dysbiotic vagina because they have low selectivity for Lactobacillus and are not well defined chemically. The present invention is intended to overcome these shortcomings.

Due to the production of lactic and various aliphatic acids by the physiological (i.e. vaginal) Doderlein microbiota, vaginal fluids have an acidic pH, averaging between 3.5 and 4.5, which appears to be optimal for vaginal homeostasis since Lactobacillus spp. while the pH can still rise, as a result of which most pathogens and opportunistic microorganisms are suppressed. Above pH 4.5, pathogens can easily grow, but below pH 3.5, the growth of Lactobacillus Doderlein itself is increasingly suppressed. This acidic vaginal fluid, resulting from the fermentation of endogenous carbohydrates such as glycogen metabolites by the Doderlein microbiota, is the very biochemical basis of the vaginal defense mechanism against infection and is of paramount importance for women's reproductive health (ref. 12).

Given these factors, it is not surprising that there are references in the literature (eg ref. 13) regarding the inclusion of an unspecified pH buffering agent in a vaginal composition. In another application (ref. 14) a composition is recommended which contains a buffering agent (not chemically defined in said reference) to stabilize the degree of acidity of the vagina in the pH range of 3.5-5.0.

However, the present inventors have found that in all cases, acidic pH alone is not sufficient to prevent the growth of pathogens. Experiments carried out in the inventors' laboratories (described later in this document) show that even at a pH below 5, relevant pathogens such as S. aureus and E. coli can grow rapidly. The medium used contained lactose as a carbon source and 15% MRS as a growth initiator.

In conclusion, the importance of an acidic vaginal pH is not disputed in the scientific community, and some authors have recognized the utility of adding a buffering agent to a pharmaceutical formulation designed for vaginal use. Thus, the pH range should be clearly defined according to physiology in the range of 3.5 to 4.5. The pH value of 4.5, at the upper end of the physiological range, was deliberately chosen as it is tolerated by patients. More acidic pH values around 4 or lower can cause discomfort such as itching and burning, as the vaginal epithelium is more sensitive in cases of dysbiosis and infections.

In addition, the chemical composition and dosage of the buffering agent must be specified in the pharmaceutical prescription, for example, for a capsule. Finally, the buffer must be such that it acts immediately after intravaginal administration, and its effect is maintained at least until the next administration, i.e. from 12 to 24 h.

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In addition to the acidic buffer and nutritional factors outlined in the above section, there are also other factors that play a role in the development of effective pre- or pro- or symbiotic compositions for vaginal use. One such factor is the redox potential of the vaginal fluid after application of the device.

As we will show, this is especially true in the case of bacterial vaginosis (BV), where the pathogens are obligate anaerobes (Gardnerella vaginalis, Prevotella bivia, Atopobium vaginae, ...). The redox potential can indeed influence the growth of these aforementioned pathogens.

The effect of redox potential on the growth and survival of microorganisms in the vaginal flora is shown in FIG. 3 (according to ref. 22, Eschenbach et al 1985).

Finally, the fungus Candida albicans is another potentially dangerous microbe that is very common as an opportunistic component of the vaginal microflora. The authors present a method for controlling fungal growth without inhibiting the growth of vaginal lactobacilli.

The present invention is directed to providing a solution to accomplish these objectives.

Disclosure of invention

In an embodiment, the present invention relates to a composition for use in the treatment or prevention of urogenital infections caused by pathogenic microorganisms in a female subject, in which the composition contains an effective amount of each of the following of the following:

a) a buffer

b) a preservative that is fully compatible with the vaginal microbiota,

c) a pure molecular or ionic biochemical source of nitrogen suitable for consumption by the vaginal microbiota,

d) a prebiotic polysaccharide fermentable by the vaginal microbiota, wherein the treatment or prophylaxis comprises administering the composition intravaginally to a subject to stabilize the acidity of the subject's vaginal fluid at a physiological value of about pH 4.5 or below.

In an embodiment, the present invention relates to a method for treating or preventing urogenital infections caused by pathogenic microorganisms in a female subject, which comprises intravaginally administering to the subject, optionally after anti-infective treatment of the urogenital infection, a composition with an effective amount of each of the following:

a) a buffer

b) a preservative that is fully compatible with the vaginal microbiota,

c) a pure molecular or ionic biochemical source of nitrogen suitable for consumption by the vaginal microbiota,

d) a prebiotic polysaccharide fermentable by the vaginal microbiota, wherein the treatment or prophylaxis comprises intravaginal administration of the composition to a subject.

In another embodiment, the present invention relates to a method of stabilizing the acidity of the vaginal fluid of a subject at a physiological value of about pH 4.5 or below, which includes intravaginally administering to the subject a composition with an effective amount of each of the following:

a) a buffer

b) a preservative that is fully compatible with the vaginal microbiota,

c) a pure molecular or ionic biochemical source of nitrogen suitable for consumption by the vaginal microbiota,

d) a prebiotic polysaccharide fermentable by the vaginal microbiota, wherein the treatment or prophylaxis comprises administering the composition intravaginally to a subject.

In another embodiment, the present invention relates to a composition suitable for oral administration having a pH in the range of 4 to 5, wherein the composition contains an effective amount of each of the following:

a) a buffer

b) a preservative that is fully compatible with the vaginal microbiota,

c) a pure molecular or ionic biochemical source of nitrogen suitable for consumption by the vaginal microbiota,

d) a prebiotic polysaccharide fermented by the vaginal microbiota.

figures

Fig. 1 is a graphical representation of data obtained from growth experiments with Lactobacillus gasseri KS 120.1 CNCM I-3218.

Fig. 2 is a graphical representation of data from multidrug resistant S. aureus CCOS 461 growth experiments.

Fig. 3 is a schematic 2-dimensional representation of the redox potential Eh and acidity pH in the vagina as a function of the composition of the vaginal microbiota (flora) (according to Eschenbach et al. 1985).

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The present invention provides a composition for use in stabilizing the pH of the vaginal fluid at a physiological value of about 4.5 and optionally the redox potential of the vaginal fluid above 150 mV for a sufficiently long time and for selectively stimulating the growth of Lactobacillus spp. against the growth of pathogenic microorganisms, especially anaerobes, as a method of preventing, alleviating or treating vaginal and urogenital infections such as BV caused by pathogenic microorganisms, according to the appended claims.

The composition of the present invention may optionally include a natural, chemically defined substance at a concentration that inhibits Candida, but not Lactobacillus bacteria.

Acidity and nutrients: compositions against facultative anaerobic pathogens.

This method uses specific, biochemically well-defined compositions of pharmaceutical preparations for vaginal use.

The composition in the context of the present invention contains the following biochemical components:

a biochemical buffer to maintain the pH of the composition (in vitro and in vivo) in the desired range of 4.0 to 5.0, preferably 4.5, after application and up to 12-24 hours thereafter;

antipathogenic preservative of biochemical nature, fully compatible with the bacterial components of the Doderlein (vaginal) microbiota;

a biological chemically defined carbon source selected to selectively stimulate the growth of Lactobacillus spp.;

biologically available, otherwise chemically pure, nitrogen source supporting adequate growth and proliferation of Lactobacillus spp. (already present or introduced); and, optionally, an adequate chemically defined source of sulfur assimilable by lactobacilli; redox modulators (oxidants in combination with antioxidants in the correct ratio);

chemical compounds (organic and/or inorganic) selected for their specific and targeted anti-infective activity against pathogenic microorganisms of various species such as parasites and/or protozoa and/or bacteria and/or fungi and/or viruses, and certain minerals, vitamins, and other optional components that may be relevant to the growth of selected species of Lactobacillus spp. or inhibition of unwanted pathogens. Sorbate is a non-limiting example of an antifungal agent, especially against Candida.

These ingredients may be the only ones in the composition of the present invention or may be used in combination with: probiotic strains of Lactobacillus as live cells or lyophilisates thereof, their cell-free culture supernatants or their tyndallisates, selected for their specific properties, including, but not limited to, antibiotic-like activity against urogenital pathogens, adhesion to vaginal/urethral epithelial cells, production of H ₂ O ₂ , lactic acid, as well as the corresponding bacteriocins and enzymes.

The composition itself is in a suitable galenical form in a pharmaceutically acceptable carrier or delivery system. The actual composition may be administered as a lyophilized preparation, cream, paste, liquid gel or suppository, capsule or tablet/stylus for intestinal, urethral or vaginal use. Alternatively, the drug may be a capsule or tablet or suppository, cream or gel (lubricant) for vaginal delivery.

The probiotic Lactobacillus species mentioned above should be practiced as a route of administration as viable whole cells, typically between 105 and 10 ¹⁰ CFU, or as the cell-free supernatant of appropriate cultures in liquid, semi-solid or lyophilized form. Lactobacillus species are preferably selected from the group consisting of L. crispatus, L. jensenii, L. gasseri, L. acidophilus, L. iners, L. plantarum, L. fermentum, L. lactis and L. johnsonii.

In contrast, the method of the present invention does not include the use of media such as natural dairy nutrients or animal components, as previously suggested elsewhere. In addition to the fact that the effectiveness of these growth factors needs further confirmation in clinical studies, such nutrients and components are not suitable for pharmaceutical intravaginal or intraurethral use for the following medical reasons. They may contain known and unknown proteinaceous substances with allergenic or irritant potential for sensitive vaginal and urethral epithelium and/or bacterial spores, viruses, prions or their components/metabolites which may survive sterilization and become a potential health hazard to the patient.

The same considerations detailed above for optimal probiotic formulation apply similarly to the gastrointestinal (GI) environment. Although the gastrointestinal tract is richer in nutrients than the urogenital tract for the resident microbiota as well as for the pathogens living there, the concept

- 5 040310 that the probiotic formulation should help maintain optimal acidity and redox status in the gastrointestinal tract, and at the same time should promote the growth of healthy probiotic bacteria while inhibiting the growth of pathogenic microorganisms and opportunistic species, remains valid .

However, when shifting the focus from the genitourinary system to the gastrointestinal tract as an ecosystem, two major differences must be considered.

First, the acidity in the gastrointestinal tract, due to its nature and length, is not constant (ref. 17). The following pH values are commonly measured in normal people:

A. Stomach: 1.0 to 2.5

b. Proximal small intestine: 6.6

c. Terminal ileum: 7.5

d. Caecum: 6.4

e. Rectum: 7.0 The pH in the intestine varies from 6 to 7, compared to values of 3.5 to 5 in a healthy vagina.

Another difference is that the main product of acidic fermentation of the GI microbiota will not be lactic acid, but a range of short chain fatty acids (SCFAs), including acetate or butyrate, which constitute the main carbon flux from the diet through the GI microbiota to the host and make an important contribution. into host metabolism (ref. 18).

As a consequence, the prebiotic fibers contained in the composition according to the invention for improving the metabolism of Doderlein microbiota strains, when applied in the intestine, will be beneficial not only for Lactobacillus spp., but also, for example, for bifidobacteria and other resident species. Advantageously, the composition according to the invention contains at least two different fibers, acting at different levels of the intestine, since the microbiota of the gastrointestinal tract in the upper and lower intestines, respectively, is somewhat different.

The method of the present invention for urogenital use can be changed without significant changes for oral use. In fact, the optimal pH of an oral drug containing probiotics is the level of yogurt or curdled milk, which is in the region of about 4.5 (ref. 19), the same as that of a healthy vagina.

For this reason, the above considerations and related experiments remain valid when considering a suitable oral formulation containing probiotics. As stated above, the main difference lies in the choice of prebiotic fibers, since it is preferable for the intestines to include at least two fibers.

As used below, the term composition refers to a composition for intravaginal administration as well as a composition for oral administration as defined in the appended claims.

The composition according to the present invention may contain the following biochemical components:

a) a biochemical buffer to maintain the pH of the composition (in vitro and immediately after dissolution in vivo) in the desired range of 4 to 5, preferably 4.5;

b) an antipathogenic preservative of a biochemical nature, fully compatible with the bacterial components of the intestinal lactic microbiota;

c) a biologically acceptable, otherwise chemically pure, source of nitrogen that supports adequate growth and proliferation of Lactobacillus spp. (already present or entered);

d) biological, chemically defined carbon sources selected to selectively stimulate the growth of Lactobacillus spp. and fermentable in lactic acid as well as in SCFA; preferably in combination with

e) a biologically acceptable, otherwise chemically pure, source of sulfur that ensures adequate growth and reproduction of Lactobacillus spp. (already present or entered);

f) a component that modulates the redox potential in the vaginal fluid, which may be contained in the composition in the presence or absence of a source of sulfur,

g) selected minerals and/or vitamins, oxidants/antioxidants and other components that may be relevant to the growth of selected species of Lactobacillus spp. and inhibition of pathogens, and

h) live cell probiotic strains of Lactobacillus (or their lyophilisates, their cell-free culture supernatants, or their tyndallisates) selected for their specific properties, including, without limitation, antibiotic-like activity against urogenital and/or intestinal pathogens, as well as adhesion to intestinal , as well as to vaginal / urethral epithelial cells, the production of H2O2, lactic acid, as well as the corresponding bacteriocins and enzymes.

In the context of the present invention, the terms chemically pure and chemically defined indicate a synthetic medium or substance, the chemical composition of which is precisely known (ref.

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Textbook Basic microbiology ^8th edition, W. A: Volk, J. C, Brown, 1997, pages 39-40).

A complex (indefinite) environment is an environment in which the exact chemical structure of the environment is unknown. Certain environments usually consist of available pure biochemical substances; complex media usually contain complex materials of biological origin, such as blood or milk or yeast extract or beef extract, the exact chemical composition of which is not clearly defined. A defined environment is a minimal environment. The composition itself may be a suitable galenic composition in a pharmaceutically acceptable carrier or delivery system. The actual composition is in liquid (yogurt drinks), semi-solid (yogurt, cheese) or solid form, the latter also including tablets, capsules, sachets and sticks.

The following are preferred embodiments of the invention.

a. Buffer tool.

The purpose of such an ingredient is to help maintain the pH value of the vaginal fluid after application within the physiological pH range of 4 to 5. Acids and their salts having such desirable properties in solution, e.g. organic acids such as aliphatic acids: acetic, propionic , butyric acid, etc. acids, dicarboxylic acids such as oxalic acid, malonic acid, succinic acid, and the like. acids, hydroxy acids such as lactic acid, citric acid, dicarboxylic acids such as malic acid, and the like. Other acids that are found in the human body, such as phosphoric acid, may also be considered.

b. An anti-pathogenic preservative compatible with the Doderlein microbiota.

The specificity of the metabolism of lactic acid bacteria is, as the name itself indicates, in the production of lactic acid. This is the main reason for the use, in both traditional and industrial foods, of lactic acid fermentation to kill potential pathogens present in raw nutrients and to preserve them at the end of the fermentation process through an effective amount of lactic acid (e.g. sauerkraut) . Since Lactobacillus spp., especially the vaginal ones, tolerate acidic environments containing lactic acid well, this latter compound is in fact a compatible preservative with Doderlein. Lactic acid, suitably selected salts thereof (calcium, magnesium, potassium, sodium, ammonium, etc.), are included in the compositions to achieve this effect.

c. Biochemically suitable source of nitrogen for the maintenance and growth of Lactobacillus spp.

Much attention is now being paid to the properties and uses of so-called prebiotics, polysaccharides specifically metabolized by probiotics such as lactobacilli and bifidobacteria (see next paragraph).

Prebiotics are widely used in combination with probiotic bacteria to obtain the so-called symbiotics, which, when taken orally, provide a better reproduction of probiotic bacteria in the gastrointestinal tract. However, in the vaginal environment, the availability of suitable nutrients to pre-existing or exogenously administered probiotic bacteria is more limited, especially with regard to the necessary nitrogen sources (N-sources). The addition of a suitable organic and/or inorganic, chemically defined and pure N source to a pharmaceutically acceptable carrier is essential for the growth and proliferation of Lactobacillus spp. into the vagina.

For example, ammonium salts (eg, ammonium chloride, ref. 15), urea, amino acids such as glutamic acid and the like (arginine, etc.) capable of stimulating the growth of lactobacilli and bifidobacteria are such chemically defined N-sources.

d. Biological, chemically defined carbon source to support the growth of lactobacilli.

Obviously, a source of carbon is also needed for the growth of vaginal lactobacilli (C source). Preferably one such source is a specific nutrient for Lactobacillus spp. and not opportunistic or pathogenic bacteria. Indigestible carbohydrates, especially fructooligosaccharides, galactooligosaccharides, glycooligosaccharides and others, are examples of such a substance. The presence of these so-called prebiotics creates an additional differential competitive advantage in both the vagina and the intestines for the growth of Lactobacillus spp. no relation to the growth of true pathogens or opportunistic pathogens.

Optionally, other components are present in the composition according to the invention, such as a source of sulfur, minerals, redox (redox) modulators and vitamins.

Preferably, the composition of the present invention contains a biologically acceptable, otherwise chemically pure, source of sulfur that supports adequate growth and proliferation of Lactobacillus spp. (already present or administered), which may be, without limitation, an organic/inorganic compound such as sulfate, sulfite, sulfide, thiosulfate, a sulfur-containing amino acid such as cysteine and methionine, cysteine (oxidized cysteine dimer), and the like.

Minimal, chemically defined growth medium for various Lactobacillus spp. was found

- 7 040310 and published (reference 18a). In addition to the components listed above, in conditions of very poor availability of nutrients in the vagina, it may be useful to consider also adding the following components to the preferred formulations: metal ions: for example, Mg ²⁺ and Mn ²⁺ , antioxidants: for example, vitamin C, N- acetylcysteine, thiosulfate, vitamins: eg riboflavin and nicotinic acid; singly or in combination with each other.

Probiotics such as live cells, lyophilisates, tyndallizates, supernatants, dry or liquid, of species and strains with proven activity against infectious or inflammatory conditions of the vagina and preferably of human, urogenital origin are also preferred ingredients. Such species can be found and isolated from the Doderlein microbiota of healthy young women and are represented worldwide by L. gasseri, L. jensenii, L. crispatus, L. acidophilus, L. fermentum, L. plantarum and L. iners.

Compounds having activity against pathogens (bacteria, viruses, fungi, and the like), which, in the intention and understanding of the present invention, such as are generally capable of inhibiting the growth of pathogenic microorganisms without disturbing the metabolism and growth of probiotic bacteria, are also preferred ingredients. . Cysteine, sorbate or thiosulfate, respectively, are examples of such suitable antifungal compounds of organic or inorganic origin.

Preferably, the composition of the present invention contains a biologically applicable redox modulator, i.e. a component that modulates the redox potential in a biological fluid. In the context of the present invention, the term redox modulator means a substance or at least a pair of substances capable of reducing or increasing the redox potential of a particular biological environment. A method for determining the redox potential of the vagina in bacterial vaginosis is given in reference 22.

The specified redox modulator may be contained in the composition of the present invention in the presence or absence of a source of sulfur, as defined above.

If a redox modulator is present, the composition of the present invention is particularly suitable for the treatment or prevention of infections caused by obligate anaerobes, such as bacterial vaginosis (BV).

Preferably, the redox modulator is selected from: sulfur containing redox systems such as sodium thiosulfate/bisulfite and reduced state glutathione (GSH) and oxidized state (GSSG), oxygen based redox systems such as quinone/hydroquinone, nitrogen based redox systems such as nitrite/nitrate salts, selenium based redox systems (selenite/selenate and selenide). For example, the redox potential can be raised to become definitely positive (>150 mV, preferably 200-300 mV) and inhibit anaerobes like Gardnerella.

Preferably, in the context of the present invention, at least one strain of lactobacilli is added to the composition of the invention, said strain being selected from the group consisting of L. gasseri, L. jensenii, L. crispatus, L. acidophilus, L. helveticus, L. plantarum , L. fermentum and L. johnsonii, more preferably said strain is selected from the group consisting of L. gasseri KS 114.1 deposited by Medinova AG at the National Collection of Microorganism Cultures (CNCM) on July 22, 2005 with CNCM accession number I-3482, L. crispatus 116.1 deposited by Medinova AG at the National Collection of Cultures of Microorganisms (CNCM) on July 22, 2005 with accession number CNCM I3483, L. jensenii KS 119.1, deposited by Medinova AG at the National Collection of Cultures of Microorganisms (CNCM) on June 4, 2004 with accession number CNCM I -3217, L. crispatus 119.4, deposited by Medinova AG at the National Collection of Cultures of Microorganisms (CNCM) on July 22, 2005 with CNCM accession number I-3484, L. gasseri 120.1, deposited by Medinova AG at the National Collection of Microorganism Cultures (CNCM) on June 4, 2004 with accession number CNCM I-3218, L. jensenii KS 121.1; deposited by Medinova AG at the National Collection of Cultures of Microorganisms (CNCM) on June 4, 2004 with accession number CNCM I- 3219, L. Gasseri 123.1, deposited by Medinova AG at the National Collection of Cultures of Microorganisms (CNCM) on 22 July 2005 with CNCM accession number I-3485, L. Gasseri 126.2, L. crispatus 127.1, deposited by Medinova AG at the National Collection of Cultures of Microorganisms (CNCM) ) July 22, 2005 with accession number CNCM I-3486, and L. acidophilus KS 400, most preferably said Lactobacillus strain was selected from the group consisting of L. jensenii KS 119.1 CNCM I-3217, L. crispatus 119.4 CNCM I-3484, L. gasseri 120.1 CNCM I-3218 and L. gasseri 124.3 CNCM I-3220.

The above bacterial strains have been deposited under the terms of the Budapest Treaty and made available for general use.

Preferably, in the context of the present invention, the buffering agent a) is an acid or a salt thereof selected from the groups comprising or alternatively consisting of organic acids such as aliphatic acids, including acetic, propionic, butyric acids, dicarboxylic acids, including oxalic, malonic, succinic acid, hydroxy acids such as lactic acid, citric acid, dicarboxylic acids such as malic acid.

Preferably, in the context of the present invention, the preservative b) is lactic acid or its derivative and/or its salt.

Preferably, in the context of the present invention, the nitrogen source c) is an ammonium salt, a naturally occurring urea or a synthetic amino acid such as glutamic acid, and mixtures thereof.

Preferably, in the context of the present invention, the carbon source d) is an indigestible carbohydrate such as: fructooligosaccharides, galactooligosaccharides, glycooligosaccharides and mixtures thereof. Preferably, the composition according to the present invention contains, for example, the following ingredients per 1 ml solution/suspension of ingredients. The actual composition, such as tablets or capsules, is obtained by multiplying the number of ingredients by 3:

final pH of the solution/suspension and: approx. 4.5 Lactobacillus compatible preservative Ca-lactate pentahydrate 10-30, preferably 20 mg/ml buffer system Dibasic magnesium citrate 5-15, preferably 10 mg/ml carbon source a-glycooligosaccharide 15-30, preferably 20 mg/ml Nitrogen source Glutamic acid 10-30 mg Sulfur source cystine 10-30 mg Excipients q.s. 150-250 mg

The invention includes the following embodiments (E).

E1) A composition for use in the treatment or prevention of urogenital infections caused by pathogenic microorganisms in a female subject, subjects in which the composition contains an effective amount of each of the following:

(a) a buffering agent, (b) a preservative that is fully compatible with the vaginal microbiota, (c) a pure molecular or ionic biochemical nitrogen source suitable for consumption by the vaginal microbiota, (d) a prebiotic polysaccharide fermentable by the vaginal microbiota, and optionally, (e) an antioxidant component wherein the treatment or prophylaxis comprises administering the composition intravaginally to a subject to stabilize the acidity of the subject's vaginal fluid at a physiological value of about pH 4.5 or less.

E2) A composition for use according to E1, wherein the vaginal composition is a lubricating gel for personal use.

E3) Composition for use according to E1 or E2, in which the antioxidant is selected from sulfur-containing compounds such as N-acetylcysteine, cysteine, methionine, sodium thiosulfate.

E4) A composition for use according to any one of E1-E3, wherein one or more strains of lactobacilli are added to a composition from the group consisting of L. gasseri, L. jensenii, L. crispatus, L. acidophilus, L. helveticus, L. plantarum , L. fermentum, L. lactis, L. johnsonii and L. acidophilus KS 400.

E5) A composition for use according to any one of E1-E4, wherein one or more strains of lactobacilli are added to a composition from the group consisting of L. gasseri, L. jensenii, L. crispatus, L. acidophilus, L. helveticus, L. plantarum , L. fermentum, L. lactis and L. johnsonii.

E6) A composition for use according to any of E1-E5, wherein said Lactobacillus strain is selected from the group consisting of L. jensenii KS 109, L. gasseri KS 114.1 deposited by Medinova AG in the National Collection of Microorganism Cultures on July 22, 2005 with accession number CNCM I3482, L. crispatus 116.1, deposited by Medinova AG at the National Collection of Microorganism Cultures (CNCM) on July 22, 2005 with accession number CNCM I-3483, L. jensenii KS 119.1, deposited by Medinova AG at the National Collection of Microorganism Cultures (CNCM) on June 4 2004 with accession number CNCM I-3217, L. crispatus 119.4, deposited by Medinova AG at the National Collection of Cultures of Microorganisms (CNCM) July 22, 2005 with accession number CNCM I-3484, L. gasseri 120.1, deposited with Medinova AG at the National Collection of Cultures Microorganisms (CNCM) June 4, 2004 with accession number CNCM I-3218, L. jensenii KS 121.1, deposited by Medinova AG at the National Collection of Cultures of Microorganisms (CNCM) 4 June 2004 with accession number CNCM I-3219, L. jensenii KS 122.1, L. gasseri 123.1, deposited by Medinova AG in the National Collection of Cultures of Microorganisms (CNCM) on July 22, 2005 with accession number CNCM I-3485, L. gasseri 124.3, deposited by Medinova AG at the National Collection of Microorganism Cultures (CNCM) on June 4, 2004 with accession number CNCM I-3220, L. gasseri 126.2, L. crispatus 127.1, deposited by Me- 9 040310 dinova AG at the National Collection of Microorganism Cultures (CNCM) on July 22 2005 with accession number CNCM I-3486, L. jensenii KS 130.1, L. helveticus KS 300, deposited by Medinova AG in the National Collection of Cultures of Microorganisms (CNCM) on February 10, 2005 with accession number CNCM I3360 and L. acidophilus KS 400.

E7) A composition for use according to any of E1-E6, wherein said Lactobacillus strain is selected from the group consisting of L. jensenii KS 119.1 deposited by Medinova AG at the National Collection of Cultures of Microorganisms (CNCM) on June 4, 2004 with CNCM accession number I- 3217, L. crispatus 119.4, deposited by Medinova AG at the National Collection of Microorganism Cultures (CNCM) on July 22, 2005 with CNCM accession number I-3484, L. gasseri 120.1, deposited by Medinova AG at the National Collection of Microorganism Cultures (CNCM) on June 4, 2004 with accession number CNCM I-3218, L. gasseri 124.3, deposited by Medinova AG with the National Collection of Microorganism Cultures (CNCM) on June 4, 2004 with accession number CNCM I-3220.

E8) A composition for use according to any one of E1-E7, wherein the composition further comprises an effective amount of a therapeutically effective antimicrobial agent capable of inhibiting or killing urogenital pathogens.

E9) A method of treating or preventing urogenital infections caused by pathogenic microorganisms in a female subject, which comprises intravaginally administering to the subject, optionally after anti-infective treatment of the urogenital infection, a composition with an effective amount of each of the following:

(a) a buffering agent, (b) a preservative that is fully compatible with the vaginal microbiota, (c) a pure molecular or ionic biochemical nitrogen source suitable for consumption by the vaginal microbiota, (d) a prebiotic polysaccharide fermentable by the vaginal microbiota, and optionally, (e) antioxidant component.

E10) A method of stabilizing the acidity of a subject's vaginal fluid at a physiological value of about pH 4.5 or below, which comprises intravaginally administering to the subject a composition with an effective amount of each of the following:

(a) a buffering agent, (b) a preservative that is fully compatible with the vaginal microbiota, (c) a pure molecular or ionic biochemical nitrogen source suitable for consumption by the vaginal microbiota, (d) a prebiotic polysaccharide fermentable by the vaginal microbiota, and optionally, (e) antioxidant component.

E11) The method according to E9 or E10, wherein the buffer agent (a) is an acid or salt thereof selected from the groups consisting of or alternatively consisting of organic acids such as aliphatic acids, including acetic, propionic, butyric acid, dicarboxylic acids including oxalic, malonic, succinic acid, hydroxy acids such as lactic acid, citric acid, dicarboxylic acids such as malic acid.

E12) The method according to any one of E9-E11, wherein the preservative (b) is lactic acid or a derivative and/or salt thereof.

E13) The method of E9-E12, wherein the nitrogen source (c) is an ammonium salt, urea, a natural or synthetic amino acid such as glutamic acid, and mixtures thereof.

E14) The method according to any one of E9-E13, wherein the carbon source (d) is an indigestible carbohydrate such as: fructooligosaccharides, galactooligosaccharides, glycooligosaccharides and mixtures thereof.

E15) A composition suitable for oral administration having a pH in the range of 4 to 5, wherein the composition comprises an effective amount of each of the following:

(a) a buffering agent, (b) a preservative that is fully compatible with the gastrointestinal microbiota, (c) a pure molecular or ionic biochemical source of nitrogen, (d) a prebiotic polysaccharide fermentable by the gastrointestinal microbiota, and optionally, (e) an antioxidant component .

E16) Composition according to E15, in which the antioxidant (e) is selected from sulfur-containing compounds such as N-acetylcysteine, cysteine, methionine, sodium thiosulfate.

E17) Composition according to E15 or E16, in which at least one strain of lactobacilli is added to the composition, said strain being selected from the group consisting of L. gasseri, L. jensenii, L. crispatus, L. acidophilus, L. helveticus, L .plantarum, L. fermentum and L. johnsonii.

E18) A composition according to any one of E15-E17, wherein at least one strain is added to the composition, said strain being selected from the group consisting of L. jensenii KS 109, L. gasseri KS 114.1 deposited by Medinova AG in the National Collection of Microorganism Cultures July 22

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2005 with accession number CNCM I-3482, L. crispatus 116.1, deposited by Medinova AG at the National Collection of Microorganism Cultures (CNCM) July 22, 2005 with accession number CNCM I3483, L. jensenii KS 119.1, deposited with Medinova AG at the National Collection of Microorganism Cultures (CNCM) June 4, 2004 with accession number CNCM I-3217, L. crispatus 119.4, deposited by Medinova AG at the National Collection of Cultures of Microorganisms (CNCM) July 22, 2005 with accession number CNCM I-3484, L. gasseri 120.1, deposited with Medinova AG in the National Collection of Microorganism Cultures (CNCM) on June 4, 2004 with accession number CNCM I-3218, L. jensenii KS 121.1, deposited by Medinova AG in the National Collection of Cultures of Microorganisms (CNCM) on June 4, 2004 with accession number CNCM I-3219, L. jensenii KS 122.1, L. gasseri 123.1, deposited by Medinova AG at the National Collection of Cultures of Microorganisms (CNCM) on July 22, 2005 with CNCM accession number I-3485, L. gasseri 124.3, deposited deposited by Medinova AG at the National Collection of Microorganism Cultures (CNCM) on June 4, 2004 with accession number CNCM I-3220, L. gasseri 126.2, L. crispatus 127.1, deposited by Medinova AG at the National Collection of Microorganism Cultures (CNCM) on July 22, 2005 with accession number accession CNCM I-3486, L. jensenii KS 130.1, L. helveticus KS 300, deposited by Medinova AG in the National Collection of Cultures of Microorganisms (CNCM) on February 10, 2005 with accession number CNCM I-3360 and L. acidophilus KS 400.

E19) A composition according to any one of E15-E18, wherein said Lactobacillus strain is selected from the group consisting of L. jensenii KS 119.1 deposited by Medinova AG at the National Collection of Cultures of Microorganisms (CNCM) on June 4, 2004 with CNCM accession number I-3217, L. crispatus 119.4 deposited by Medinova AG at the National Collection of Microorganism Cultures (CNCM) on July 22, 2005 with accession number CNCM I-3484, L. gasseri 120.1 deposited by Medinova AG at the National Collection of Microorganism Cultures (CNCM) on June 4, 2004 with accession number accession CNCM I-3218, L. gasseri 124.3, deposited by Medinova AG with the National Collection of Microorganism Cultures (CNCM) on June 4, 2004 with accession number CNCM I-3220 and L. acidophilus KS 400.

E20) A composition for use according to any one of E1-E8, or a composition according to any one of E15-E19, wherein the buffering agent (a) is an acid or salt thereof selected from the groups consisting of, or alternatively consisting of, organic acids, such as aliphatic acids including acetic, propionic, butyric acid, dicarboxylic acids including oxalic, malonic, succinic acid, hydroxy acids such as lactic acid, citric acid, dicarboxylic acids such as malic acid.

E21) A composition for use according to any of E1-E8 or E20, or a composition according to any of E15E20, wherein the preservative (b) is lactic acid or a derivative and/or salt thereof.

E22) A composition for use according to any of E1-E8 or E20-E21, or a composition according to any of 15-21, wherein the nitrogen source (c) is an ammonium salt, urea, a natural or synthetic amino acid such as glutamic acid, and their mixtures.

E23) A composition for use according to any of E1-E8 or E20-E22, or a composition according to any of E15-E22, wherein the carbon source (d) is an indigestible carbohydrate such as: fructooligosaccharides, galactooligosaccharides, glycooligosaccharides and mixtures thereof.

E24) A composition for use according to any of E1-E8 or E20-E23, or a composition according to any of E15-E23, having a pH of 4.3 to 4.6, after dissolving/suspending the solids to a volume of 3 ml, and which comprises the following ingredients per dose:

Ca-lactate pentahydrate: 30-90, preferably 60 mg

Dibasic magnesium citrate: 15-45, preferably 30 mg

Prebiotic fiber: 50-150, preferably 100mg

Glutamic acid: 20-60, preferably 40 mg

Cysteine/thiosulfate: 10-50, preferably 30 mg

Auxiliary substances up to 400-1100 mg: q. s.

E25) Composition for use or composition according to E24, additionally comprising: lyophilisates of L. gasseri 120.1 deposited by Medinova AG in the National Collection of Cultures of Microorganisms (CNCM) on June 4, 2004 with accession number CNCM I-3218 and/or L. gasseri 124.3 and/ or L. crispatus 119.4 deposited by Medinova AG at the National Collection of Microorganism Cultures (CNCM) on July 22, 2005 with CNCM accession number I-3484 and/or L. jensenii KS 119.1 deposited by Medinova AG at the National Collection of Microorganism Cultures (CNCM) on June 4 2004 with CNCM accession number I-3217 for a total of 2 to 10 billion CFU, which is 50 to 200 mg.

Examples

Example 1

Composition of various growth media and growth conditions.

Assuming complete dissolution in the vaginal medium (volume: 2-5 ml), a number of formulations are provided to compare the growth of Lactobacillus spp. and pathogens under test conditions.

Growth control (positive) for lactobacilli: MRS.

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Normal MRS (Biolife) medium without pH adjustment ensures optimal growth of Lactobacillus spp. and was chosen for this purpose.

Growth control (positive) for pathogens: TS.

Conventional tryptic soy (TS) broth (BD, 211825) was chosen without pH adjustment.

Baseline control (negative).

A dilute solution of said MRS (15% by volume), buffered or unbuffered, was chosen to ensure the survival of the tested lactobacilli and pathogens at low levels of growth or inhibition, respectively.

Wednesday 17 (according to the invention):

optimized test composition in accordance with the following table, and intended to stabilize the pH, as well as to ensure the growth of Lactobacillus spp. and at the same time to inhibit pathogens.

Wednesday 17 Function Substance Origin Concentration preservative Ca-lactate pentahydrate ""Applichem"", AZ 670 20 mg/ml Buffer Dibasic ammonium citrate Sigma, 09833 10 mg/ml A source of nitrogen (ammonium, found in buffer) Final solution pH: 4.56 carbon source a-glycooligosaccharide BioEcolians, UCIB Solabia Group, AB020 20 mg/ml Contains sulfur compounds mrs broth Biolife 4017292 15% on volume

Growth conditions and counting procedure.

All cultures were grown at 37° C. under anaerobic conditions without agitation.

The first culture was obtained from material stored at -80°C, streaked on agar plates. After incubation for no more than 24 hours, one colony from the plate was used to inoculate 5 ml of liquid culture and incubated for up to 18 hours, and then used to inoculate 25 ml of each of the various media for culture experiments. The density of the original cells was adjusted to correspond to an optical density of 0.05 at 600 nm.

All experiments were performed in duplicate.

Growth of the cultures was monitored by measuring absorbance at 600 nm, pH, plating serial dilutions on agar plates (cfu/ml) and counting the total number of cells using a hematocytometer (improved by Neubauer).

Strains tested: Vaginal Lactobacillus spp. and urogenital pathogens.

The vaginal strains tested include all relevant Lactobacillus species of the physiological vaginal microbiota, i.e. L. gasseri, L. crispatus and L. jensenii and especially L. gasseri 120.1 CNCM I-3218, L. gasseri 124.3 CNCM I-3220, L. crispatus 119.4 CNCM 1-3484 and L. jensenii 119.1 CNCM I-3217.

The following were selected as representative pathogens in these experiments:

a) Gram-negative: uropathogenic B. coli CCOS 492, a multidrug-resistant human clinical isolate from the University Hospital of Zurich, isolated in 1976,

b) Gram-positive: multi-resistant S. aureus, CCOS 461, MRSA, human clinical isolate, Oxford Hospital, UK, isolated in 1960.

Uropathogenic E. coli are the most common strain causing cystitis. S. aureus is associated with vaginal infections of a type called aerobic vaginitis (AV).

A comment.

As mentioned in the title of this section, acidifying, preservative and nutritional factors have been selected for use against facultative anaerobic pathogens. They can produce energy both using oxygen, such as aerobes, or also anaerobically (fermenting sugars to lactic acid). E. coli and S. aureus are representatives of this class of bacteria.

Comparative compositions.

The following are examples of compositions which, although having a composition similar to medium 17, do not equally well inhibit the growth of the pathogens mentioned above.

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Example 2a. The effect of a slight increase in pH.

The following environment 11 is given as a first comparative example:

Wednesday 11 Substance Origin Concentration Ca-lactate in the form pentahydrate Applichem A3670 20 mg/ml Dibasic ammonium citrate Sigma, 09833 10 mg/ml Tribasic ammonium citrate Sigma 10 mg/ml Final pH solution: 5.00 a-glycooligosaccharide Bio-Ecolians, UCIB Solabia Group, AB020 20 mg/ml mrs broth "Biolife", 4017292 15% by volume

In this environment, the increase in the number of pathogens mentioned above was studied under similar experimental conditions.

E. coli CCOS 492:

Wednesday 11 100% MRS Time (h.min) pH Quantity (CFU) pH Quantity (CFU) 00.00 5.00 1*10 ^l5 6.35 1*1 ₀ a5 06.10 4.98 8*yu ^a5 5.53 5*1 ₀ a ⁷ 20.20 4.98 4* ^10L 6 5.01 >1*10 ^l9

S. aureus CCOS 461:

Wednesday 11 100% MRS Time (h.min) pH Quantity (CFU) pH Quantity (CFU) 00.00 5.00 P1QA5 6.35 1*1qa5 06.10 4.99 1*10 ^l6 5.69 6*10 ^l7 20.20 4.98 4*1qa6 5.05 >1*1qa ⁹

Therefore, increasing pH 4.5 by half a unit, to a final value of 5.0, was sufficient to allow more than one log of pathogen growth, pathogens that would otherwise be inhibited at pH 4.5.

Example 2b. The effect of reducing lactate concentration by 50%.

As an embodiment, the following medium 12 was prepared and the change in the amount of E. coli in the same medium was observed.

Wednesday 12 Substance Origin Concentration Ca-lactate in the form pentahydrate Applichem A3670 10 mg/ml Dibasic ammonium citrate Sigma, 09833 10 mg/ml Tribasic ammonium citrate Sigma 10 mg/ml Final solution pH: 5.18 a-glyco-oligosaccharide Bio-Ecolians, UCIB Solabia Group, AB020 20 mg/ml mrs broth "Biolife", 4017292 15% by volume

In this medium, as well as in TSA, taken as a positive control medium, the change in the number of the above-mentioned pathogenic E. coli CCOS 492 was studied under similar experimental conditions as in example 2a.

E. coli CCOS 492:

Wednesday 12 TSA Time(hours. min) pH Quantity (CFU) pH Quantity (CFU) 00.00 5.18 1*10 ^l5 6.7 1*10 ^l5 06.10 5.21 4*Yu ^l6 6.1 5*10 ^l7 20.20 5.23 8*10 ^l6 5.5 >1*10 ^l9

It turned out that a decrease in the concentration of lactate, as in medium 12, to half the value of medium 17, is accompanied by a significant decrease in the inhibitory ability compared to the latter, which leads to an increase in the number of pathogens by almost 2 log.

Thus, it appears that the exact concentrations of the active components (citrate, lactate and

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etc.), given as an example for medium 17, are preferred to ensure their high inhibitory activity.

Example 2 Detailed discussion of results for L. gasseri 120.1 CNCM I-3218.

An in vitro model for testing the effect of a pharmaceutical composition in the vagina used in this application is a liquid medium containing the ingredients of the composition at approximately the same concentration as after dissolution in the vagina. The aim of the in vitro experiments, which will be described below, was to find a nutrient medium composition that allows a physiological pH of 4.0-4.5 to be set and maintained over a one-day period. In addition, said environment should allow lactobacilli to quickly colonize and grow in it, while at the same time urogenital pathogens should be suppressed.

The number of L. gasseri KS 120.1 CNCM I-3218 cells grown for 19.4 hours on MRS medium reached 1x10 ⁸ CFU/ml, the highest value among all 3 tested media. As expected, a clear decrease in pH from 6.4 to 4.0 was observed. This control experiment shows the good suitability of the inoculum used and the correctness of the growth curves observed in other media.

Comparing the growth obtained in medium 17 with the MRS control, a slightly reduced growth (0.4 log units) was observed. Since medium 17, with its relatively simple composition, contained only a small amount of trace elements, this effect was expected.

Comparing growth in medium 17 with growth in 15% MRS medium, a decrease in the final number of cells between these media was observed at a level of 4.9 log. This clearly shows that the additional nutrients and components present in medium 17 do promote the growth of the Lactobacillus strain tested.

Acidity: The pH values remained surprisingly stable at about 4.5 from the start of the experiment until 20 hours thereafter (changing from 4.58 to 4.41), thus confirming the sufficient strength of the buffer system.

Conclusion: The present medium 17, formulated in accordance with the description of the present invention, caused a significant growth of inoculated Lactobacillus gasseri, slightly less than for pure MRS, but definitely stronger than for diluted (15%) MRS. The pH of the system remains constant with an optimal target value of approximately 4.5.

Example 3 Detailed discussion of results for S. aureus CCOS 461.

In this case, the hypothesis was tested that the selected medium 17 is capable of inhibiting the growth of the selected multidrug-resistant pathogen.

The number of S. aureus CCOS 461 cells grown for 21.1 h on TS medium reached the highest value, 2x10 ⁸ cfu/ml, among all 3 media tested, starting with a value of 9x10 ^L 6 at t=0 h. Also in In this case, a clear drop in pH from 7.0 to 4.8 was observed. This control experiment indicates the good suitability of the inoculum used and the correctness of the growth curves observed in other media.

Comparing the increase in the number of cells obtained in medium 17 with the positive control TS, a strong inhibition of S. aureus CCOS 461 to -5.2 logarithmic units was observed. For dilute MRS, inhibition was also high, at -4.4 logs.

Acidity: Even in the presence of a live pathogen, the pH values again remain surprisingly stable at about 4.5 from the start of the experiment until 20 hours thereafter, with a slight decrease (varying from an initial 4.59 to a final 4.40).

Conclusion: The present medium 17, formulated according to the description of the present invention, causes a significant reduction in the amount of inoculated pathogen S. aureus CCOS 461: in relation to the positive control of TS medium, but even in relation to diluted MRS. Wednesday 17 demonstrates antimicrobial activity against this pathogen.

Example 4. Summary of experimental series.

The results of the remaining strains mentioned above in Example 2 will be tabulated along with the last two examples and commented.

Preliminary remarks: All 4 lactobacilli tested showed the same trend: good growth in medium 17, reduced growth in 15% MRS broth, and excellent growth in control MRS broth medium. A drop in pH was also observed in the non-buffered medium (15% MRS and pure MRS).

All 4 strains of Lactobacillus and 2 pathogens were pH adjusted to maintain a pH of 4.5, measurements confirm excellent medium acidity stability for up to 20 hours or longer reaction times.

The pathogens S. aureus and E. coli also clearly could not grow in medium 17. The corresponding cell concentrations in this medium decreased sharply with time.

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Table Exp.4. pH values in medium 17 and log (decrease or increase) in the number of cells of test organisms grown on medium 17 (according to the invention) compared to the reference medium

Change in CFU/ml on medium 17 vs. pH value, medium 17 Strain Time 15%MRS MRS TSB original final KS 119.4 CNCM 1-3484 28.6 h 0.57 -0.49 4.57 4.33 KS 119.1 CNCM 1-3217 18.6 h 1D2 -2.15 4.59 4.37 KS 120.1 CNCM 1-3218 19.4 h 4.94 -0.40 4.58 4.44 KS 124.3 CNCM 1-3220 28.6 h 0.44 -0.90 4.57 4.33 CCOS 461 21.1 h -4.44 -5.19 4.59 4.40 CCOS 492 22.1 h -5.70 -8.13 4.49 4.42

Table Exp.4 summarizes the differences in logarithmic numbers at the end of the respective growth experiments. The largest growth promotion effect was observed in Lactobacillus gasseri KS 120.1 CNCM I-3218 with an almost 5 log increase in levels compared to 15% MRS, while the weakest effect was found in Lactobacillus gasseri KS 124.3 CNCM I-3220 with a log increase of only 0.44.

Overall conclusion: during the fermentation process, medium 17 did indeed stabilize the pH at 4.5 to 4.3 for all strains tested. As for the Lactobacillus strains, they grew better on medium 17 than on 15% MRS medium. In this particular medium, the addition of ammonium citrate as an additional nitrogen source and calcium lactate as a buffer, as well as the use of a selective carbon source (in this particular case, glycooligosaccharide) for probiotics, was sufficient to differentially stimulate the growth of the tested lactobacilli. On the other hand, 2 pathogens tested (E. coli and S. aureus) did not grow on Medium 17 but were inhibited by it.

Example 5

From systematic experiments leading to suitable media such as Medium 17 described above, it is possible to calculate the quantitative composition of a pharmaceutical dosage form (capsule, tablet, ...) for intravaginal use. As mentioned above, this requires a quantification of the volume of vaginal fluid, the amount of which obviously varies depending on the age and health of the woman. Studies have shown that the volume of such a liquid averages about 3 ml (ref. 16).

Table Exp.5.1. Quantitative composition of the prebiotic vaginal capsule according to the invention

Capsule #6 Substance Quantity Ca-lactate pentahydrate 60 mg Dibasic ammonium citrate 30 mg a-glyco-oligosaccharide 100 mg Target pH during vagina: 4.5 Glutamic acid 40 mg cysteine or cystine 20 mg Auxiliary substances as needed 200 mg total weight 450 mg

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Table Exp.5.2. Quantitative composition of the vaginal capsule according to the invention

Tablet #8 Substance Quantity Lyophilisates of vaginal strains L. gasseri 120.1 and 124.3 at 10^ cfu/g 100 mg Ca-lactate pentahydrate 60 mg Dibasic ammonium citrate 30 mg arabinogalactan 100 mg Target pH in the vagina: approximately 4.5 thiosulfate 30 mg Auxiliary substances as needed 270 mg total weight 600 mg

Table Exp.5.3. Quantitative composition of the vaginal capsule according to the invention

Tablet #9 Substance Quantity Ca-lactate pentahydrate 60 mg Dibasic magnesium citrate 30 mg arabinogalactan 100 mg Target pH in vaginal preparation: approximately 4.5 thiosulfate 30 mg Aspartic acid 60 mg Auxiliary substances as needed 270 mg The total mass of auxiliary substances/additives 550 mg Lyophilizate L. gasseri KS 120.1 CNCM 1-3218 50 mg Lyophilisate L. jensenii 119.1 CNCM 1-3484 50 mg Z. crispatus KS 119.4 lyophilisate CNCM 1-3217 50 mg Total weight including lyophilizates 700 mg

Table Exp.5.4. Quantitative composition of the oral symbiotic preparation (sachet) according to the invention

Sachet #9 Substance Quantity Ca-lactate pentahydrate 100 mg Dibasic magnesium citrate 50 mg Alpha-glycooligosaccharide 1000 mg Target pH in oral preparation when dissolved in 150 ml of water: 4.5 arabinogalactan 1000 mg thiosulfate 30 mg Aspartic Acid Alanine 60 mg 60 mg Auxiliary substances as needed 350 mg Total weight of excipients/additives 2,650 mg Lyophilizate L. gasseri KS 120.1 CNCM 1-3218 50 mg Lyophilizate L. jensenii 119.1 CNCM 1-3217 50 mg Lyophilisate L. crispatus KS 119.4 CNCM 1-3484 50 mg Total weight including lyophilizates 2,850 mg

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Table Exp.5.5. The quantitative composition of the prebiotic vaginal lubricant according to the invention

Lubricating Gel #3 Substance Quantity Lactic acid 50 mg Target pH of the finished gel - 4.5 Lemon acid 30 mg Glycerol 80 mg Carbomer 5 mg Short chain fructo-oligosaccharides 50 mg thiosulfate 10 mg Glutamic acid 10 mg NaOH 0.1 mol or HC1 0.1 mol for pH=4.5 q.s. Deionized water up to 1000 mg

Optimization of the redox potential of the vaginal fluid.

In Example 5.1 above, the sulfur source added to M17 is cysteine or cystine, in Example 5.2 it is sodium thiosulfate. All 3 compositions provided above lead, for example, to a pH value in 2-5 ml of the respective solution of about 4.5. In terms of acidity, nutrients, preservatives, they look very similar.

The question we are now raising is whether they are equivalent in terms of their task, which is to stimulate the growth of lactobacilli in the vagina and inhibit the growth of pathogens. To refine this question, we must focus on specific pathogens; we must select the most common vaginal infection that occurs, as mentioned above, the so-called bacterial vaginosis or BV associated with pathogens such as Gardnerella vaginalis or Prevotella bivia. In 1985, David Eschenbach and colleagues published a research paper (ref. 22) in which they studied the correlation between bacterial vaginosis and vaginal redox according to pH.

On the one hand, they confirmed, as expected, that in 15 patients, pH in the range of 4.0 to 4.5 was mainly associated with healthy vaginal microbiota, while in the pH range of 4.5 to 5.5 patients showed a significant presence of anaerobes with few lactobacilli and symptoms of BV. The novelty was that they were also able to show a strong dependence of the redox potential Eh in the vagina in vivo on the state of the microbiota: while the Eh values in patients with BV range from +50 to -250 mV, the Eh values of healthy vaginas is in the range above 100-300 mV. They also found that this does not represent a characteristic of individual women, but only depends on the state of the microbiota. After successful use of metronidazole, all women with a negative redox potential returned to a positive potential (and in addition, the pH also became more acidic).

Interestingly, no attempt has been made to therapeutically apply this fact to the production of vaginal pharmaceutical products for the treatment of BV, which guarantee a higher redox potential.

We explored this possibility with the following experimental procedure.

Brief description of the testing procedure.

A medium was prepared consisting of 20 mg/ml Ca-lactate, 10 mg/ml dibasic ammonium citrate, 20 mg/ml Ecolians and 15% MRS broth (corresponding to M17 described above). After measuring the redox potential and pH, several compounds (sources of nitrogen and sulfur) were added to this medium and their effect on pH and redox potential was determined.

Brief description of experiments.

A redox electrode (Mettler Toledo LE 501 ORP connected to an MP230 pH meter, Mettler Toledo) was used to measure the redox potential of the medium composition before and after the addition of test compounds. Redox values were recorded after the signal was stable for at least 30 s. In addition, pH was measured using a standard pH electrode connected to an MP225 pH meter (Mettler Toledo).

Before each series of measurements, the redox electrode was calibrated with a reference solution (Mettler Toledo, no. 51350060, 220 mV, pH 7 (UH=427 mV)).

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Analysis results:

Compound Concentration Substance pH Oxidation reduction potential Value comparisons 1 20 mg/ml Sa-lactate 4.67 235.0 mV 236.2 mV 10 mg/ml Ammonium citrate, dibasic 15% mrs broth 20 mg/ml Ecolians 10 mg/ml L-Glutamic acid 4.48 310.8 mV 2 20 mg/ml Sa-lactate 4.67 235.0 mV 236.2 mV 10 mg/ml Ammonium citrate, dibasic 15% mrs broth 20 mg/ml Ecolians 10 mg/ml L-Glutamic acid 4.41 265.2 mV 10 mg/ml cystine 3 20 mg/ml Sa-lactate 4.67 235.0 mV 236.2 mV 10 mg/ml Ammonium citrate, dibasic 15% mrs broth 20 mg/ml Ecolians 10 mg/ml L-Glutamic acid 4.30 -103.0 mV 10 mg/ml Cysteine 4 20 mg/ml Sa-lactate 4.67 235.0 mV 233.0 mV 10 mg/ml Ammonium citrate, dibasic 0% mrs broth 20 mg/ml Ecolians 10 mg/ml L-Glutamic acid 4.26 284.2 mV 20 mg/ml Na-thiosulfate 4.21 -36.5 mV 5 20 mg/ml Sa-lactate 4.48 237.8 mV 234.9 mV 15 mg/ml Ammonium citrate, dibasic 0% mrs broth 20 mg/ml Ecolians 10 mg/ml L-Glutamic acid 4.27 301.3 mV 20 mg/ml Dimethylsulfone 4.26 273.3 mV

Comments.

Whether or not 15% MRS was added to the solution had little to no effect on pH or Eh.

The addition of a nitrogen source, glutamic acid, makes the environment slightly more acidic and more electropositive/oxidizing, reaching a value of 300 mV.

Sulfur compounds have in this particular series of experiments the most pronounced effect on redox potential and little effect on acidity: compare, for example, composition 2 and composition 3, where cysteine and cystine are interchanged. With cysteine, Eh decreases to 103.0 mV, while with the same amount of cysteine, it increases to 265.2 mV. The pH level in this case changes only minimally, from 4.30 to 4.41.

Both cystine (the oxidized dimeric form of cysteine) and MSM (dimethyl sulfone) both maintain a high positive redox potential, while cysteine and thiosulfate cause reduction instead of oxidation of the solution, reaching negative values of -103 and -36 mV.

The conclusion is that by adding, for example, cysteine or thiosulfate, an environment favorable for the growth of anaerobes is obtained, while the addition of cystine or dimethyl sulfone helps to create an environment favorable for the development of lactobacilli. This is of therapeutic interest, as we can choose in this way not only which pH, but also which redox potential is best suited for local control of a particular pathogen. Thus, by using an appropriate redox modulator, the effectiveness of the composition against BV according to the invention can be increased.

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The above considerations apply against BV pathogens (Gardnerella, Prevotella, Atopobium, which are by nature obligate anaerobes, i.e. microbes that cannot defend themselves against oxidants such as, for example, hydrogen peroxide or other oxidizing agents. Against these pathogens , increased redox potential is an additional therapeutic weapon that works without damaging or inhibiting vaginal lactobacilli: the reason is that lactobacilli, although anaerobes, are so-called aerotolerant bacteria, i.e. oxygen and, more generally In this case, an environment with a high redox potential (Eh) does not prevent their growth.

The choice of redox modulator.

Preferably, in the context of the present invention, the term redox modulator means a substance or at least a couple of substances capable of reducing or increasing the redox potential of a particular biological environment. Preferably, the redox modulator e) is selected from sulfur-containing compounds of the cysteine/cystine pair, such as N-acetylcysteine, cysteine, methionine, quinone/hydroquinone, a redox system based on selenium (selenite/selenate, for example in the form of sodium salt), sodium thiosulfate /bisulfite, reduced glutathione (GSH) and oxidized state (GSSG). For example, if it is necessary to increase the redox potential so that it becomes definitely positive (> 150 mV) and suppresses anaerobes such as Gardnerella, then cystine (the oxidized form) will be chosen for this purpose, not cysteine.

Specific examples of an anti-BV composition with increased redox potential.

Example 6.1. prebiotic composition.

Table Exp.6.1. Quantitative composition of the anti-BV prebiotic vaginal capsule according to the invention

Capsule #10 Substance Quantity Target Eh in vaginal fluid: > 200 mV Ca-lactate pentahydrate 60 mg Dibasic magnesium citrate 30 mg a-glycooligosaccharide 100 mg Target pH in the vagina: 4.5 Glutamic acid 40 mg Redox modulator and source S cystine 20 mg Auxiliary substances as needed 200 mg total weight 450 mg

Example 6.2. Symbiotic composition against anaerobic vaginal infection according to the invention

Table Exp.6.2. Quantitative composition of the symbiotic vaginal capsule against BV

Tablet #11 Substance Quantity Strains with proven, pronounced activity against Gardnerella Lyophilizates of vaginal strains L. gasseri 120.1 and 124.3 at concentrations of 10 CFU/g Every 100 mg Target oxidative potential in the vagina: Ca-lactate pentahydrate 60 mg Dibasic magnesium citrate 30 mg

> 200 mV Alanine 40 mg arabinogalactan 100 mg Target pH in the vagina: approx. 4.5 Dimethylsulfone (MSM) 30 mg Auxiliary substances as needed 140 mg total weight 600 mg

eradication of candida albicans.

As mentioned above, Candida is a microbe frequently found in the vagina, either as an opportunistic agent or as a pathogen, for example emerging after anti-infective treatment of vaginal infections such as bacterial vaginosis. A feature of Candida is that, unlike the anaerobic pathogens involved in BV, they can coexist with lactobacilli, thereby affecting only the composition of the vaginal microbiota. The composition of the Doderlein flora in these three different states (no infection, BV, or candidiasis) has recently been studied in detail and published by G. Donders and co-workers (ref. 23).

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For the above reasons, the authors consider that it would be useful, if possible, to formulate a composition designed to stimulate lactobacilli, but at the same time, to inhibit pathogenic microorganisms, for example, to have an inhibitory effect also on yeast in general and on Candida albicans. , especially.

It would be tempting to use a classic and safe antifungal drug such as clotrimazole for this purpose, but this molecule also has bactericidal activity against gram-positive bacteria such as lactobacilli (ref. 24). While looking for a suitable molecule for our intended purpose, we found a naturally occurring organic acid (sorbic acid) that is widely used as a safe preservative against yeast and mold in the food and pharmaceutical industries. Therefore, the challenge was to test whether sorbic acid (usually available as sodium or potassium sorbate) was able to inhibit Candida at concentrations that did not inhibit vaginal lactobacilli. The optimum pH for sorbate activity is around 4.5, which is ideal for the selected range. The concentration that is used in practice for preservation against yeast and mold is 0.1% (ref. 25).

For this purpose, the following experiments were carried out.

A summary of the testing procedure.

A fresh overnight culture of the test strains was used to inoculate the test medium supplemented with various concentrations of potassium sorbate. Growth was determined by measuring absorbance and pH at baseline, 6 hours later, and 24 hours later.

growth conditions:

Environment for night culture: Bouillon MRS with Tween Tested environment: MRS broth with Tween, diluted to 30%, pH adjusted to 4.5 with lactic acid Tested volume: 5 ml Incubation: 37°C, anaerobic, with shaking (120 rpm) Cell density for inoculations: approximately 10^ cfu/ml Potassium Sorbate Concentrations: 0.00%, 0.01%, 0.05%, 0.1%, 0.5%, 1.0%, 1.5% (w/v) Measurement parameters: pH, OD at 590 nm Measurements at: t= 0 h, 6 h and 24 h

Tested bacterial cultures.

Lactobacillus gasseri CCOS 960, Lactobacillus gasseri KS 121.1, Lactobacillus crispatus KS 116.1.

Getting cultures.

All cultures were reactivated from cryopreserved (-80° C.) stock solutions by culturing on MRS agar for 1-3 days. A single colony from these dishes was used to inoculate 5 ml of MRS broth. After overnight incubation, 10 ⁷ cfu/ml was used for experiments.

Analysis results:

Strain Potassium sorbate concentration OD pH Interpretation 0h 6 h : 24 hours 0h 6 4 24 hours Lactobacillus gasseri.. KS 120.1 0.00% 0.51 JJ: .1.26 . 4.71 4.,57. 4.27 0 4:00% 0.54 0.78 0.97 5.16 5 5.07 1 1:50% 0.53 0.73 0.72 5.31 5.26 ::.5::26:: 1 · KS119.1 0.10% 0.08 0.23 0.44' 4.5 4.5 4.5 0 0.50% 0.07 0.11 0.124 4.5 4.5 4.85 1 1.00% 0.18 0.26 0.16 5.03 5.01 5.09 2 1.50% 0.17 0.24 0.13 5.32 5.18 5.25 2 ·: Lactobacillus crispatus KS 119.4 D.00% . 0.72 1.36 - 1.36 4.73 4.5 4.37 θ • 0.01% · ::.:O./ .. 1.star 1.47 471 .::4.42.: 4.35 about SU . 0.05%. 0.6 1.16 : 1.24. 4.69 . 4.43. 432 0. . • 0.10% · 0.68 1.34 5 1.24 4.7 4.48 444 ό 0.50% About? v.24 1.15 ,4.97, 4.73 .4.77 . . 0 1.00% 0.65 ι.νι 006 5.8 5.01 5.08 1 1.50% 0.64 ο.99 0.76 5.32 5.2 5.26 1

Interpretation: 0 - no inhibition, 1 - weak inhibition, 2 - strong inhibition.

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Conclusions.

A minimum concentration of potassium sorbate of 1% (or 10 mg/ml) was required for a strong inhibitory effect on the growth of the tested lactobacilli, for a slight inhibition of 0.5% or higher.

According to Cramer and Assadian (ref. 25), the microbiostatic concentration to inhibit the growth of pathogenic bacteria and fungi is much lower (S. aureus, E. coli, K. pneumoniae: 50-100 µg/ml, Penicillium notatum, Aspergillus niger. 200-500 µg /ml, Saccharomyces cerevisiae: 50-100 µg/ml).

The lactobacilli tested (gasseri, crispatus and jensenii) represent the standard Doderlein flora and should therefore be able to sustain their growth in the presence of potassium sorbate at concentrations up to 0.1-0.2% (1.5 to 3 mg/day). ml).

Converted to a capsule formula that will dissolve in about 3 ml of vaginal fluid, this corresponds to the addition of 3 to 6 mg of potassium sorbate to the composition (see below).

Example 7.1. Composition against obligate anaerobic pathogens, which also has activity against Candida.

Table Exp.7.1. Quantitative composition of the prebiotic vaginal capsule according to the invention without lyophilisates and containing sorbate as an antifungal ingredient

Capsule #12 Substance Quantity Ca-lactate pentahydrate 60 mg Dibasic magnesium citrate 30 mg a-glycooligosaccharide 100 mg Target pH in the vagina: 4.5 Glutamic acid 40 mg cystine 30 mg K-sorbate 5 mg Auxiliary substances as needed 185 mg total weight 450 mg

Table Exp.7.2. Quantitative composition of the vaginal capsule according to the invention containing lyophilizates, as well as potassium sorbate as an antifungal agent

Tablet #11 Substance Quantity Lyophilisates of vaginal strains L.gasseri 120.1 and 124.3 at concentrations of ¹⁰ CFU/g 100 mg each Ca-lactate pentahydrate 60 mg Dibasic ammonium citrate 30 mg K-Sorbat 5 mg Arginine 20 mg arabinogalactan 100 mg Target pH in the vagina: 4.5 cystine 30 mg Auxiliary substances as needed 155 mg total weight 600 mg

Example 7.3. Lubricant prebiotic composition with activity against BV and Candida.

Table Exp.7.3. quantitative composition of the prebiotic vaginal lubricant according to the invention

Lubricating Gel #3 Substance Quantity Lactic acid (D- or racemic) 50 mg The target pH of the final gel is 4.5 Lemon acid 30 mg glycerol 80 mg Carbomer 5 mg Short chain fructo-oligosaccharides 50 mg MSM 20 mg Target oxidative potential: >150 mV Glutamic acid 20 mg NaOH 0.1 mol or HO 0.1 mol For pH=4.5 q.s. Deionized water ad 1000 mg

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Cited links

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2. Donders G.G., Vereecken A, Bosmans E., Dekeersmaecker A, Salembier G., Spitz B. 2002. Definition of a type of abnormal vaginal microbiota that is distinct from bacterial vaginosis: aerobic vaginitis. BJOG. 109(1):34-43.

3. http://www.patient.co.uk/doctor/bacterial-vaginosis-pro (05/21/2015). Up to 70% of patients have a relapse within three months of successful treatment.

4. Doderlein A 1892. Das Scheidensekret und seine Bedeutung fur das Puerperalfteber. Zbl. Bacteriol. 11:699.

5. Spurbeck R.R., Arvidson C.G. 2011. Lactobacilli at the front line of defense against vaginally acquired infections. Future Microbiology 6(5): 567-582.

6. Atassi F., Brassart D., Grob P., Graf F., Servin AL. 2006. Lactobacillus strains isolated from the vaginal microbiota of healthy women inhibit Prevotella bivia and Gardnerella vaginalis in coculture and cell culture. FEMS Immunol Med Microbio 48 (3) 424-432.

7. Atassi F., Brassart D., Grob P., Graf F., Servin AL. 2006. Vaginal Lactobacillus isolates inhibit uropathogenic Escherichia coli. FEMS Microbiol Lett 257(1): 132-135.

8. EP 1812023 Bl, F. Graf 6/2004, EP 2 561 880 Bl, F. Graf 6/2004.

9. Reid et al, USP 5,645,830, 7/1997.

10. De Man J.C., Rogosa M., Sharpe M.E. 1960. A medium for the cultivation of lactobacilli. Journal of Applied Bacteriology 23(1): 130-135.

11. Chrisope G et al, Gynelogix Inc WO 9846261 www.disknet.com/indiana_biolab/b028.htm_(21.05.2015).

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13. ELLEN AB, Darviczky K et al, 2010, WO2010023222.

14. Procter & Gamble Co, Carella AM et al, 1997 WO9729763.

15. Laxmi N.P., Mutamed M.A., Nagendra P. S 2011. Effect of carbon and nitrogen sources on growth of Bifidobacterium animalis BB12 and Lactobacillus delbrueckii ssp. bulgaricus ATCC 11842 and production of β-galactosidase under different culture condition. International Food Research Journal 18: 373-380.

16. Eschenbach D.A., Thwin S.S., Patton D.L., Hooton T.M., Stapleton A.E., Agnew K., Winter C., Amalia Meier A, Walter E. Stamm W.E. 2000. Influence of the Normal Menstrual Cycle on Vaginal Tissue, Discharge, and Microbiota. Clinical Infectious Diseases 30(6): 901-907.

17. Gut. 1988Aug;29(8): 1035-41.

Measurement of gastrointestinal pH profiles in normal ambulant human subjects.

Evans DF, Pye G, Bramley R, Clark AG, Dyson TJ, Hardcastle JD.

18. Gut Microbes Volume 7, Issue 3, 2016 Special Issue: Gut Microbiota Metabolites in Health and Disease, Douglas J. Morrison & Tom Preston, pages 189-200.

18a. Maria Elli et al, EP1038951 Al, Societe Des Produits Nestle S.A.

19. https://de. wikipedia.org/wiki/Joghurt.

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21. Use and procurement of additional lubricants with male and female condoms: WHO/UNFPA/FH1360 - Advisory Note,

Claims (11)

CLAIM 1. Use of a composition having a pH of 4.3 to 4.6 after dissolution/suspension of the solids in a volume of 3 ml and comprising the following ingredients per dosage: i) Ca-lactate pentahydrate: 30-90 mg, ii) dibasic magnesium citrate: 15-45 mg, iii) prebiotic fiber: 15-150 mg, iv) glutamic acid: 20-60 mg, v) cystine or thiosulfate: 10-50 mg, and vi) excipients up to 400-1100 mg: q.s. in the manufacture of an intravaginal medicament for the treatment or prevention of urogenital infections caused by pathogenic microorganisms in a female subject, for stabilizing the acidity of the subject's vaginal fluid at a physiological value of about pH 4.5 or lower. 2. The use of the composition according to claim 1, in which the composition includes these ingredients in the following amounts per dosage: i) Ca-lactate pentahydrate 60 mg, ii) dibasic magnesium citrate: 30 mg, iii) prebiotic fiber: 100 mg, iv) glutamic acid: 40 mg, v) cystine or thiosulfate: 30 mg, and vi) excipients up to 400-1100 mg: q.s. 3. Use of a composition according to claim 1, wherein the composition is a lubricating gel for personal use or a capsule, tablet, suppository, cream or gel. 4. Use of a composition according to any one of claims 1 to 3, wherein the composition further comprises one or more strains of lactobacilli selected from the group consisting of L. gasseri, L. jensenii, L. crispatus, L. acidophilus, L. helveticus, L. plantarum, L. fermentum, L. lactis, L. johnsonii and L. acidophilus KS 400. 5. Use of a composition according to any one of claims 1 to 4, wherein said Lactobacillus strain is selected from the group consisting of L. jensenii KS 109, L. gasseri KS 114.1 deposited by Medinova AG in the National Collection of Microorganism Cultures on July 22, 2005 with the number accession CNCM I-3482, L. crispatus 116.1, deposited by Medinova AG at the National Collection of Microorganism Cultures (CNCM) on July 22, 2005 with accession number CNCM I-3483, L. jensenii KS 119.1, deposited by Medinova AG at the National Collection of Microorganism Cultures (CNCM) ) June 4, 2004 with accession number CNCM I-3217, L. crispatus 119.4, deposited by Medinova AG at the National Collection of Microorganism Cultures (CNCM) July 22, 2005 with accession number CNCM I-3484, L. gasseri 120.1, deposited with Medinova AG at National Collection of Cultures of Microorganisms (CNCM) on June 4, 2004 with accession number CNCM I-3218, L. jensenii KS 121.1, deposited by Medinova AG with the National Collection of Cultures of Microorganisms (CNCM) on June 4 2004 with accession number CNCM I-3219, L. jensenii KS 122.1, L. gasseri 123.1, deposited by Medinova AG in the National Collection of Cultures of Microorganisms (CNCM) on July 22, 2005 with accession number CNCM I-3485, L. gasseri 124.3, deposited by Medinova AG at the National Collection of Microorganism Cultures (CNCM) on June 4, 2004 with accession number CNCM I-3220, L. gasseri 126.2, L. crispatus 127.1, deposited by Medinova AG at the National Collection of Microorganism Cultures (CNCM) on July 22, 2005 with accession number accession CNCM I-3486, L. jensenii KS 130.1, L. helveticus KS 300, deposited by Medinova AG in the National Collection of Cultures of Microorganisms (CNCM) on February 10, 2005 with accession number CNCM I-3360 and L. acidophilus KS 400. 6. Use of a composition according to claim 5, wherein said Lactobacillus strain is selected from the group consisting of L. jensenii KS 119.1 deposited by Medinova AG at the National Collection of Cultures of Microorganisms (CNCM) on June 4, 2004 with CNCM accession number I-3217, L crispatus 119.4 deposited by Medinova AG at the National Collection of Cultures of Microorganisms (CNCM) on 22 July 2005 with accession number CNCM I-3484, L. gasseri 120.1 deposited by Medinova AG at the National Collection of Cultures of Microorganisms (CNCM) on 4 June 2004 with accession number CNCM I-3218 and L. gasseri 124.3 deposited by Medinova AG with the National Collection of Microorganism Cultures (CNCM) on June 4, 2004 with accession number CNCM I-3220. 7. Use of a composition according to any one of claims 1 to 6, wherein the composition further comprises an effective amount of a therapeutically effective anti-infective agent capable of inhibiting or killing urogenital pathogens, including parasites, protozoa, bacteria, viruses and/or fungi, preferably an anti-fungal agent capable of inhibit or kill fungi in the urogenital region, more preferably sorbic acid or its salts. 8. Use of a composition according to any one of the previous claims 1 to 7, wherein the prebiotic fiber is an indigestible carbohydrate such as: fructooligosaccharides, galactooligosaccharides, glycooligosaccharides and mixtures thereof. 9. The use of a composition according to any one of claims 1 to 8, additionally including lyophilizates: - 23 040310 i) L. gasseri 120.1 deposited by Medinova AG at the National Collection of Cultures of Microorganisms (CNCM) on June 4, 2004 with CNCM accession number I-3218, and/or ii) L. gasseri 124.3 and/or L. crispatus 119.4 deposited by Medinova AG in the National Collection of Microorganism Cultures (CNCM) on July 22, 2005 with CNCM accession number I-3484, and/or iii) L. jensenii KS 119.1 deposited by Medinova AG in the National Collection of Microorganism Cultures (CNCM) on June 4, 2004 with CNCM accession number I-3217 in total from 2 to 10 billion CFU, which is equivalent to 50 to 200 mg. 10. Prebiotic vaginal lubricant, comprising the following: i) lactic acid: 50 mg, ii) citric acid: 30 mg, iii) glycerol: 80 mg, iv) carbomer: 5 mg, v) short chain fructooligosaccharides: 50 mg, vi) thiosulfate: 10 mg, vii) glutamic acid: 10 mg, viii) NaOH 0.1 M or HCl 0.1 M to pH 4.5 lubricant. 11. The use of a symbiotic vaginal capsule, including: i) lyophilisates of vaginal strains L. gasseri 120.1 deposited by Medinova AG at the National Collection of Microorganism Cultures (CNCM) on June 4, 2004 with accession number CNCM I-3218 and L. gasseri 124.3 deposited by Medinova AG at the National Collection of Microorganism Cultures (CNCM) 4 June 2004 with CNCM accession number I-3220 at concentrations of 10 ¹¹ cfu/g: 100 mg each, ii) Ca-lactate pentahydrate: 60 mg, iii) dibasic magnesium citrate: 30 mg, iv) alanine: 40 mg, v) arabinogalactan: 100 mg, vi) dimethylsulfone (MSM): 30 mg, vii) excipients: 140 mg, for the manufacture of a medicament for the treatment of bacterial vaginosis.