FIELD OF THE INVENTION

The invention relates to a biochemical composition of urogenital formulations stabilizing the acidity in the vagina and establishing therein suitable conditions for the inhibition of urogenital pathogens and the growth of lactobacilli. The invention provides compositions for vaginal or, alternatively, oral administration and to the use of such compositions for stabilizing the vaginal, or, alternatively, the intestinal acidity and establishing favorable conditions for the inhibition of pathogens and the growth of lactobacilli in the urogenital, or, alternatively, gastro-intestinal tract. These compositions are based on of chemically defined ingredients, avoiding commonly used natural or ill-defined nutritional components and compositions might be associated with human vaginal strains with specific antimicrobial and/or anti-inflammatory properties. Furthermore, attention is paid to the proper value of the redox potential following the vaginal administration of such formulation, in particular with respect to the inhibition of bacterial vaginosis (BV) -associated pathogens of the type of G. vaginalis.

Finally the formulations are also presented containing an agent to inhibit additionally the growth of opportunistic yeasts such as Candida albicans without compromising the growth of lactobacilli.

BACKGROUND TO THE INVENTION

It is well known that useful, well-tolerated non-pathogenic bacteria, the so-called human natural microbiota, predominate in the intestine, the vagina and in the urethra/bladder in the healthy state, whereas the 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 commonly used to describe, respectively, those microorganisms requiring air and those unable to grow in the presence of air.

Notably, some of the non-pathogenic and pathogenic bacteria, which can be found in the vagina, can be defined as "non-facultative anaerobe", i.e. they can grow both in the presence and in the absence of air and are basically unaffected by oxidative conditions in the growing medium. Non-limiting examples of such non-facultative anaerobic bacteria are E. coli and Staphylococcus aureus, which are known to cause a vaginal condition commonly indicated as "aerobic vaginitis".

Other microorganisms which can be found in the vagina are "obligate anaerobes", i.e. they do not grow in the presence of free oxygen and may actually be killed in the presence of oxygen or of oxidative conditions.

Anaerobic organisms like Gardnerella vaginalis, an obligate anaerobe, migrating from the perineum into the vagina, or acquired through sexual intercourse can cause a dysbiosis with few symptoms, followed by a full blown bacterial vaginosis (BV), which is the most common vaginal infection. BV constitutes a serious risk to premature labor in pregnant women and increases the risk to sexually transmitted infections (Ref. 1). In such cases of vaginal dysbiosis or vaginal infections caused by anaerobes the pH of the vaginal milieu increases from the 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 cause of the risks associated with BV. The fermentative action of the lactobacilli Doderlein microbiota on the metabolites of glycogen and other carbohydrates present in the vagina and yielding mainly lactic acid as well as other organic acids to a lesser extent and results in the low pH value of vaginal fluids. In addition, certain strains of the Gram-negative E.coli, originating from the bowel and migrating first to the vagina and, afterwards to the urethra/bladder are the most common cause of urethritis/cystitis in sexually active women. This same E.coli can also cause dysbiosis in the vagina, a condition called aerobic vaginitis, which is also accompanied by a perturbation of the vaginal microbiota, a decrease in vaginal acidity and an increase of the corresponding pH value (Ref. 2).

Another pathogen causing vaginal infections, in particular during pregnancy, is the yeast Candida that behaves opportunistically and often co-exists with lactobacilli.

Candida is the only yeast organism which behaves as a facultative anaerobe, i.e. it is basically unaffected by the presence or the absence of air and of oxidative conditions.

These infections are usually cured by anti-infective treatment directed against the pathogens that are intended to be successfully eradicated in the course of such therapy, at least to a certain degree. However, in about as much as in 70% of the cases, the patient experiences a relapse or a reinfection within 3 months after a primary treatment (Ref. 3), because either the pathogens were not adequately killed or because of the lack of protection through the restored indigenous vaginal microbiota. This microbiota is actually often damaged as a consequence of the anti-infective therapy. The indigenous vaginal microbiota, historically referred to as Doderlein microbiota, is sensitive to many anti-infective agents and in particular antibiotics. It is often perturbed or even eradicated by the anti-infective therapy and is therefore unable to inhibit the proliferation of the ever present opportunistic, often pathogenic, organisms. As a consequence, a bacterial vaginosis treated by a narrow-spectrum antibiotic specific against anaerobes like clindamycin could lead to a yeast infection, as the Lactobacillus-depleied vaginal microbiota can no longer inhibit the opportunistic yeasts.

The possibility of applying exogenous Lactobacillus directly to the vagina to treat the dysbiosis or the infection of the vagina had already been suggested by the physician who discovered them, Albert Doderlein himself, in his pioneering book in 1892 (Ref.4). This concept has since then been widely developed in the scientific and patent literature and recently comprehensively reviewed (Ref. 5). WO 2009/123982 A2 relates to methods and compositions for Lactobacillus replacement therapy, i.e. to colonize vaginal mucosa with a desired microbial species by contacting a vaginal wall with a dried formulation of live microbial cells.

WO 2016/020861 A1 relates to film or sponge compositions, and to a method for the prevention and treatment of vaginal infections, wherein said film or sponge comprises at least a mucoadhesive polumer, 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 prompted thorough investigations about the properties of previously unknown Lactobacillus strains of human vaginal origin relevant to the competition with the most common urogenital pathogens. In particular, the inventors investigated mechanisms involving the interaction of lactic acid, hydrogen peroxide and further agents, which were found to explain the exceptionally strong and specific antibiotic-like activity of, e.g. 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 as described in the mentioned papers and patents (Ref. 8) has further the advantage of using strains isolated from the same tract of perfectly healthy women for a therapeutic application targeting the urogenital tract. Thereby, possible problems with the tolerability of exogenously administered Lactobacillus strains can be to a large extent reduced in comparison to the use of such strains that are not from human origin and not from the urogenital tract of healthy young women. For strains of non-human origin, the question of the safety and tolerability in therapeutics, e.g., urogenital applications, is not clear a priori and must therefore be assessed a posteriori.

To provide suitable compositions for a pharmaceutically acceptable carrier, the inventors studied the different formulations used in probiotic products and/or described in the literature. In one published approach (Ref. 9) the inventors claim that the use of skim milk resp. skim milk preparations and derivatives called LGF (Lactobacillus growth factor) or NGF (natural growth factor) favors the growth of the desired Lactobacillus by, at the same time, inhibiting the growth of the unwanted uropathogens. Such dedicated growth factors to be used for vaginal applications were also to be found in microbiological growth media, according to the same inventors. The most used such growth medium for Lactobacillus strains is actually the MRS (DeMan-Rogosa-Sharp)-medium (Ref. 10). Besides minerals and vitamins, this broth contains also peptones, sources of carbon and especially nitrogen of chemically ill-defined composition. Other inventors as well recommend the use of complex proteinaceous substances like skim milk and albumin in the preservation matrix of a vaginal medicament containing Lactobacillus strains (Ref. 11).

In our view, while formulations containing natural nutrients such as skim milk or peptones might be useful for special applications, they are not suitable as ingredients in Lacfobac/y/us-containing pharmaceutical formulations for vaginal applications. This is because, first, they are of biological (especially animal) origin, and, second, these natural ingredients have often a chemically ill-defined composition and, thus, they carry an increased risk of causing adverse effects in patients as explained further below. Further, from the point of view of a selective efficacy, these NGF might not achieve the desired effect to inhibit the growth of uropathogens. Not only Lactobacillus strains, but also numerous pathogens can grow in such a rich ingredient (with proteins, carbohydrates, vitamins, minerals, and the like) as skim milk after having been in contact and mixed with the vaginal fluid. Pathogens such as E.coli grow sometimes quicker in milk than in a nutrient broth.

Taking 2 pathogens commonly found in the urogenital microbiota, S. aureus and E.coli, both of which also grow several logs in about 20 hours when inoculated in a medium specific for Lactobacillus such as MRS (Table 1), similar results being obtained also with skim milk (not shown in the following).

Table 1 : Growth performance of the 2 pathogens in MRS broth.

Even starting at a relatively high concentration of 10 ⁸ cfu/ml these pathogens can further grow in MRS by several logs, only to be stopped after about 20 hours in the end by the low pH they generated. That is, a so-called growth medium highly specific for Lactobacillus can also promote the growth of dangerous pathogens. To inhibit the growth of such pathogens, an effective and selective inhibitor has to be added to the formulation.

Therefore, if a subject primarily harbors healthy Lactobacillus strains present in sufficient quantities in the vagina, it might be appropriate to administer LGFs, as these indigenous Lactobacillus bacteria can metabolize them. However, it will be inappropriate to administer to a dysbiotic vagina, already infected, or in deficit of beneficial Lactobacillus, LGF of any kind or such unspecific growth factors, as these might boost the growth of the pathogens present instead of that of the rare Lactobacillus strains. A dysbiosis or an infection could even get worse under such treatment with LGF, which risk should be avoided.

In conclusion: skim milk preparations or components of Lactobacillus growth media like MRS might under some circumstances sustain the growth of eubiotic, indigenous vaginal Lactobacillus. However, in general, they are not suitable as therapeutic ingredients of pharmaceutical formulations to be applied in a dysbiotic vagina, as they have low selectivity for Lactobacillus and are not well-defined chemically. The present invention is intended to overcome these drawbacks. Owing to the production of lactic and different aliphatic acids by the physiological Doderlein (i.e. vaginal) microbiota vaginal fluids exhibit an acidic pH between 3.5 and 4.5 in average, which appears to be optimum for vaginal homeostasis, as Lactobacillus spp. may still grow at this pH whereby most of the pathogens and opportunistic microbes are inhibited. Above pH 4.5, pathogens can easily grow, however below pH 3.5, the growth of the Doderlein Lactobacillus themselves becomes increasingly inhibited. This acidic vaginal fluid as brought about by the fermentation by the Doderlein microbiota of endogenous carbohydrates like the metabolites of glycogen is the very biochemical foundation of the defense mechanism of the vagina against infections and of primary importance for women's reproductive health (Ref. 12).

Considering these factors, it is not surprising that in the literature, references are found (e.g. Ref. 13) regarding the inclusion of an unspecified pH buffering agent in vaginal composition. In another application (Ref. 14) a composition is recommended that contains a buffering agent (chemically not defined in the mentioned reference) in order to stabilize the acidic degree of the vagina in the range pH 3.5 - 5.0.

However, the present inventors found that the sole acidic pH is not sufficient to prevent the growth of the pathogens in all cases. Experiments conducted in the inventors' laboratories (shown hereunder) indicate that even below pH 5, relevant pathogens like S. aureus and E.coli can rapidly grow. The medium used contained lactose as a carbon source and 15% MRS as growth-start promoter.

In conclusion: the importance of the acidic vaginal pH is unchallenged in the scientific community and several authors have recognized the usefulness of adding a buffering agent to a pharmaceutical formulation devised for vaginal application. The pH-range must thereby be clearly defined according to physiology in the range between 3.5 and 4.5. The value of pH 4.5 at the upper limit of the physiological range is deliberately chosen, as it is well tolerated by the patients. More acidic values pH 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.

Furthermore, the buffering agent chemical composition and dosages must be indicated in the pharmaceutical recipe for, e.g., a capsule. Finally, the buffer must be such as to exert its action shortly after the intravaginal administration and to last, at least, until the next administration, i.e. between 12 and 24 hours.

Besides the acidic buffer and the nutrient factors expounded in the above section there are also other factors playing a role in the design of an effective pre- or pro- or symbiotic formulation for vaginal application. One such factor is the redox potential of the vaginal fluid after application of the device.

As we shall show, this is of relevance especially in the case of bacterial vaginosis (BV) where the pathogens are obligate anaerobs (Gardnerella vaginalis, Prevotella bivia, Atopobium vaginae, ...). The redox potential can indeed influence the growth of these last-mentioned pathogens. The effect of redox potential on the growth and survival of micrororganisms in the vaginal flora is shown in Figure 3 (according to ref. 22, Eschenbach et al 1985)

Finally, another potentially dangerous microbe is the fungus Candida albicans, which is very often present as an opportunist in the vaginal flora. The authors will present a method to control the growth of the fungus without inhibiting that of the vaginal lactobacilli.

The present invention is aimed at providing a solution that addresses these objectives. SUMMARY OF THE 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, wherein the composition comprises an effective amount of each:

a) a buffering agent,

b) a preservation agent fully compatible with the vaginal microbiota,

c) a pure molecular or ionic biochemical nitrogen-source exploitable by the vaginal microbiota, d) prebiotic polysaccharide fermentable by the vaginal microbiota,

wherein the treatment or prevention comprises administering the composition intravaginally to the subject, for stabilizing the acidity of the vaginal fluid of a subject at the physiological value around pH 4.5 or lower.

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

a) a buffering agent,

b) a preservation agent fully compatible with the vaginal microbiota,

c) a pure molecular or ionic biochemical nitrogen-source exploitable by the vaginal microbiota, d) prebiotic polysaccharide fermentable by the vaginal microbiota,

wherein the treatment or prevention comprises administering the composition intravaginally to the subject.

In another embodiment, the present invention provides a method for stabilizing the acidity of the vaginal fluid of a subject at the physiological value around pH 4.5 or lower, which comprises administering intravaginally to the subject a formulation with an effective amount of each:

a) a buffering agent,

b) a preservation agent fully compatible with the vaginal microbiota,

c) a pure molecular or ionic biochemical nitrogen-source exploitable by the vaginal microbiota, d) prebiotic polysaccharide fermentable by the vaginal microbiota,

wherein the treatment or prevention comprises administering the composition intravaginally to the subject.

In another embodiment, the present invention relates to a formulation suitable for oral administration having a pH ranging from 4 to 5, wherein the composition comprises an effective amount of each:

a) a buffering agent,

b) a preservation agent fully compatible with the vaginal microbiota,

c) a pure molecular or ionic biochemical nitrogen-source exploitable by the vaginal microbiota, d) prebiotic polysaccharide fermentable by the vaginal microbiota.

FIGURES

Figure 1 : Graphic representation of the data obtained in growth experiments with Lactobacillus gasseri KS 120.1 CNCM 1-3218.

Figure 2: Graphic representation of the data obtained in growth experiments with multi-resistant S. aureus CCOS 461.

Figure 3: Schematic 2-dimensional representation of redox potential Eh and acidity pH in the the vagina as a function of the composition of the vaginal microbiota (flora)

(according to Ref. Eschenbach et al. 1985)

The present invention provides a composition for use to stabilize the pH of the vaginal fluid at the physiological value around 4.5 and optionally the redox potential of the vaginal fluid above 150 mV for a sufficient duration and to favor selectively the growth of Lactobacillus spp. against the growth of pathogens, in particular anaerobes as a method to prevent, mitigate or treat vaginal and urogenital infections such as BV caused by pathogenic micro-organisms, according to the appended claims.

The composition according to the present invention can optionally include a natural, chemically defined substance at a concentration inhibiting Candida, but not the Lactobacillus bacteria.

ACIDITY AND NUTRIENTS: FORMULATIONS AGAINST FACULTATIVE ANAEROBIC PATHOGENS This method uses specific, biochemically well-defined compositions of pharmaceutical formulations to be applied vaginally.

The composition in the context of the present invention contains the following biochemical components: a biochemical buffering agent to keep the pH of the composition (in vitro and in vivo) in the desired range of 4.0 to 5.0, preferably 4.5, following application and during up to 12-24 h thereafter;

an anti-pathogenic preservation agent of biochemical nature otherwise fully compatible with the bacterial components of the Doderlein (vaginal) microbiota;

a biological, chemically defined source of carbon chosen for selectively boosting the growth of Lactobacillus spp.;

a biologically exploitable, otherwise chemically pure source of nitrogen supporting an adequate growth and proliferation of Lactobacillus spp. (already present or administered); and optionally an adequate, chemically defined sulfur source, assimilable by the lactobacilli;

redox modulators (oxidants in combination with anti-oxidants in a proper ratio);

Chemical compounds (organic and/or inorganic) chosen for their specific and targeted anti- infective activity against pathogens of different kinds such as parasites and/or protozoa and/or bacteria and/or fungi and/or viruses and selected minerals, vitamins, and other optional components, which might be relevant for the growth of selected Lactobacillus spp. or the inhibition of unwanted pathogens. A non-limiting example of antifungal agent, particularly against Candida, is sorbate.

Said ingredients can be alone in the composition of the present invention, or else in combination with: Probiotic Lactobacillus strains, as living cells or their lyophilisates, 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 H2O2, lactic acid as well as of relevant bacteriocins and enzymes.

The formulation itself is an appropriate galenic form in a pharmaceutically acceptable carrier or delivery system. The actual composition might be instilled in the form of a freeze-dried preparation, cream, paste, gel liquid or of a suppository, capsule or tablet/stylus for intestinal, urethral or vaginal application. Alternatively, the preparation can be a capsule or tablet or suppository, a cream or gel (lubricant) for vaginal delivery.

The probiotic Lactobacillus species named above shall be in the practice of the method administered as viable whole cells, typically in an amount between 10 ⁵ and 10 ¹⁰ cfu or as cell free supernatant of the corresponding cultures in liquid, semi-solid or lyophilized form. The 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.

On the opposite, the method according to the present invention does not comprise the use of media like natural dairy nutrients or components of animal origin as suggested previously elsewhere. In addition to the fact that the efficacy of said growth factors needs to be further confirmed in clinical studies, such nutrients and components are not suitable for a pharmaceutical intra-vaginal or intra-urethral application for the following medical reasons. They may contain known and unknown substances of proteinaceous nature with allergenic or irritative potential to the sensitive epithelia of vagina and urethra and/or bacterial spores, viruses, prions or components/metabolites thereof that could have survived sterilization and become a potential health threat to the patient.

The same considerations detailed above for the optimal composition of a probiotic formulation apply analogically to the gastro-intestinal (Gl) environment. Even though the Gl tract is, compared to the urogenital tract, richer in nutrients for the resident microbiota as well as for the pathogens residing there, the concept that the probiotic formulation should help sustaining the optimum acidity and redox state in the Gl tract and, at the same time, promoting the growth of healthy probiotic bacteria whilst inhibiting the growth of pathogens and opportunistic species remains valid.

There are, however, two main differences to consider when shifting the focus from the urogenital to the Gl tract as an ecosystem.

In first place, the acidity in the Gl tract, owing to the nature and length of the same, is variable (Ref. 17). The following pH values are typically measured in normal humans:

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 intestines varies between 6 and 7, compared to values ranging from 3.5 to 5 in the healthy vagina.

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

As a consequence, the prebiotic fibers comprised in the composition according to the invention to improve the metabolism of the strains of the Doderlein microbiota, when applied to the intestine will benefit not only the Lactobacillus spp. but also, e.g., the bifidobacteria and other resident species. Advantageously, the composition according to the invention comprises at least two different fibers acting at different levels of the intestine, as the Gl microbiota in the upper and in the lower intestine, respectively, is somehow different. The method according to the invention for the urogenital application can be translated without major change to the oral application. In fact, the optimum pH of an oral preparation containing probiotics is that of the yoghurt or of curdled milk, which is in the region around 4.5 (Ref. 19), the same pH found in the healthy vagina.

For this reason, the considerations developed above, and the experiments performed accordingly retain their validity when considering a suitable oral formulation containing probiotics. As pointed out above the main difference consists in the choice of the prebiotic fibers, as for the intestine it is preferable to include at least two fibers.

As used hereafter, the term "composition" refers to the composition for intravaginal administration as well as to the formulation for oral use, as defined in the attached claims.

The composition according to the present invention may contain the following biochemical components: a. A biochemical buffering agent to keep the pH of the composition (in vitro and as soon as dissolved in vivo) in the desired range of 4 to 5, preferably 4.5;

b. an anti-pathogenic preservation agent of biochemical nature otherwise fully compatible with the bacterial components of the intestinal lactic acid microbiota;

c. a biologically exploitable, otherwise chemically pure source of nitrogen supporting an adequate growth and proliferation of Lactobacillus spp. (already present or administered);

d. biological, chemically defined sources of carbon chosen for selectively boosting the growth of Lactobacillus spp. and being fermented in lactic acid as well as SCFA; preferably in combination with e. a biologically exploitable, otherwise chemically pure source of sulfur supporting an adequate growth and proliferation of Lactobacillus spp. (already present or administered);

f. a component modulating the redox potential in the vaginal fluid, which can be comprised in the composition in the presence or in the absence of the sulfur source,

g. selected minerals, and/or vitamins, oxidants/antioxidants and other components that might be relevant for the growth of selected Lactobacillus spp. and the inhibition of the pathogens, and h. Probiotic Lactobacillus strains as living cells (or their lyophilisates, their cell-free culture supernatants or their tyndallisates) selected for their specific properties including, but not limited to, antibiotic-like activity against urogenital and/or intestinal pathogens, adhesion to intestinal as well as vaginal/urethral epithelial cells, production of H2O2, lactic acid as well as of relevant bacteriocins and enzymes.

In the context of the present invention, the terms "chemically pure" and "chemically defined" indicate a synthetic medium or substance, in which the exact chemical composition is known (ref. Textbook "Basic microbiology" 8 ^th edition, W. A: Volk, J. C, Brown, 1997, pages 39-40).

A complex (undefined) medium is one in which the exact chemical constitution of the medium is not known. Defined media are usually composed of pure biochemicals off the shelf; 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 obviously undetermined. A defined medium is a minimal medium. The formulation itself can be an appropriate galenic formulation in a pharmaceutically acceptable carrier or delivery system. The actual composition in liquid (yoghurt drinks), semisolid (yoghurt, cheese) or solid form, the latter including tablets, capsules, sachets and sticks as well.

The following are preferred embodiments of the invention.

a. Buffering agent

The purpose of such ingredient is to help keeping the pH value of the vaginal liquid after application in the physiological range of pH 4 to 5. Acids and their salts having in solution such desired properties are e.g. organic acids like aliphatic acids: acetic, propionic, butyric a.s.o. acid, dicarboxylic acids like oxalic, malonic, succinic a.s.o. acids, hydroxy-acids like lactic acid, citric acid, dicarboxylic acids like malic acid a.s.o. Also other acids that are found in the human organism can be considered for this use, such as phosphoric acid.

b. anti-pathogenic preservation agent compatible with the Doderlein microbiota

The specificity of the metabolism of lactic acid bacteria is, as the name itself indicates, the production of lactic acid. This is the main reason for the use, in traditional as well as in industrial food preparation, of lactic acid fermentation to kill potential pathogens present in the raw nutrients and to preserve them at the end of the fermentation process through an effective amount of lactic acid (e.g. sauerkraut). As Lactobacillus spp., in particular vaginal ones, tolerate well an acidic milieu containing lactic acid, this latter compound is in fact a Doderlein-compatible preservative. Lactic acid respectively selected salts of it (calcium, magnesium, potassium, sodium, ammonium a.s.o) are included in the compositions to reach this effect.

c. biochemically exploitable source of nitrogen to support the growth of Lactobacillus spp.

Much attention is presently devoted to the properties and uses of so-called prebiotics, polysaccharides being metabolized specifically by probiotics like lactobacilli and bifidobacteria (see next point). The prebiotics are widely used in combination with probiotic bacteria to yield so-called symbiotics, which, when ingested orally, ensure a better multiplication of the probiotic bacteria in the Gl tract. However, in a vaginal environment, the availability of suitable nutrients to the already present or exogenously-administered probiotic bacteria is more limited, in particular with respect to the necessary nitrogen sources (N-sources). The addition of a suitable, organic and/or inorganic, chemically defined and pure N-source in a pharmaceutically suitable carrier appears as an essential factor allowing for a growth and proliferation of Lactobacillus spp. in the vagina.

Such chemically defined N-sources are e.g. ammonium salts (e.g. ammonium chloride, Ref. 15), urea, amino acids like glutamic acid and similar (arginine a.s.o.), capable of boosting the growth of lactobacilli and bifidobacteria. d. biological, chemically defined source of carbon to support the growth of lactobacilli

Obviously also a carbon source (C-source) is necessary for the growth of the vaginal lactobacilli. Preferentially, one such source that is a specific nutrient to Lactobacillus spp. rather than to opportunistic or pathogenic bacteria. Examples thereof are non-digestible carbohydrates as: fructo-oligosaccharides, galacto-oligosaccharides, glyco-oligosaccharides and others as well. The presence of these so-called prebiotics confers, in the vagina as well as in the intestine, an additional differential competitive advantage for the growth of Lactobacillus spp. with respect to that of the present pathogens or opportunists.

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

Preferably, the composition according to the present invention comprises a biologically exploitable, otherwise chemically pure source of sulfur supporting an adequate growth and proliferation of Lactobacillus spp. (already present or administered), which may be, without limitation, an organic/inorganic compound such as a sulfate, a sulfite, a sulfide, thiosulphate, a sulfur-containing aminoacid like cysteine and methionine, cysteine( the oxidized dimer of cysteine), a s.o.

Minimal, chemically defined media for the growth of different Lactobacillus spp. have been found and published (Ref. 18a). In addition to the components listed above it might be, under circumstances of a very poor availability of nutrients in the vagina, helpful to consider also the addition of the following components to the preferred formulations: metal ions: e.g. Mg ²⁺ and Mn ²⁺ , antioxidants: e.g. vitamin C, N-acetyl cysteine, thiosulphate, vitamins: e.g. riboflavin and nicotinic acid; alone or in combination with each other.

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

Preferred ingredients are also compounds with activity against pathogens (bacteria, viruses, fungi and the like), which, in the intention and understanding of this invention, are those that are typically capable of inhibiting the growth of pathogens without disturbing the metabolism and growth of the probiotic bacteria. Examples of such suitable antifungal compounds of organic or inorganic origin are cysteine, sorbate or thiosulfate, respectively.

Preferably, the composition according to the present invention comprises a biologically exploitable redox modulator, i.e. a component modulating the redox potential in a biological fluid. In the context of the present invention the term "redox modulator" indicates a substance, or at least a pair of substances, capable of reducing or enhancing the redox potential of a certain biological medium. A method for determination of the vaginal redox potential in bacterial vaginosis is indicated in Rf. 22.

Said redox modulator can be comprised in the composition of the presence invention in the presence or in the absence of the sulfur source as above defined.

When the redox modulator is present, the composition according to the present invention is particularly suitable to treat, or to prevent, infections due to obligate anaerobes, e.g. bacterial vaginosis (BV).

Preferably, the redox modulator is chosen among:

- sulphur-containing redox systems like sodium thiosulphate/bisulphite and glutathione in the reduced state (GSH) and in the 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 enhanced so as to become definitely positive (>150 mV, preferably 200-300 mV) and to inhibit anaerobes like Gardnerella.

Preferably, in the context of the present invention at least one lactobacilli strain 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 being selected from the group consisting, L. gasseri KS 114.1 deposited by Medinova AG at Collection Nationale de Cultures de Microorganismes (CNCM) on July 22, 2005 with accession number CNCM I-3482, L. crispatus 116.1 deposited by Medinova AG at Collection Nationale de Cultures de Microorganismes (CNCM) on July 22, 2005 with accession number CNCM I-3483, L. jensenii KS 119.1 deposited by Medinova AG at Collection Nationale de Cultures de Microorganismes (CNCM) on June 4, 2004 with accession number CNCM 1-3217, L crispatus 119.4 deposited by Medinova AG at Collection Nationale de Cultures de Microorganismes (CNCM) on July 22, 2005 with accession number CNCM I-

3484, L. gasseri 120.1 deposited by Medinova AG at Collection Nationale de Cultures de Microorganismes (CNCM) on June 4, 2004 with accession number CNCM 1-3218, L. jensenii KS 121.1 deposited by Medinova AG at Collection Nationale de Cultures de Microorganismes (CNCM) on June 4, 2004 with accession number CNCM 1-3219, L gasseri 123.1 deposited by Medinova AG at Collection Nationale de Cultures de Microorganismes (CNCM) on July 22, 2005 with accession number CNCM I-

3485, L. gasseri 126.2, L crispatus 127.1 deposited by Medinova AG at Collection Nationale de Cultures de Microorganismes (CNCM) on July 22, 2005 with accession number CNCM I-3486, and L. acidophilus KS 400, most preferably said Lactobacillus strain is selected from the group consisting of L. jensenii KS 119.1 CNCM 1-3217, L. crispatus 119.4 CNCM I-3484, L. gasseri 120.1 CNCM 1-3218, and L. gasseri 124.3 CNCM I-3220. The above indicated bacteria strains have been deposited under the conditions of the Budapest Treaty and are made available to the public.

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 acid, dicarboxylic acids including oxalic, malonic, succinic acid, hydroxy-acids like lactic acid, citric acid, dicarboxylic acids like malic acid.

Preferably, in the context of the present invention the preservation agent b) is lactic acid, or a derivative and/or salt thereof.

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

Preferably, in the context of the present invention the source of carbon d) is a non-digestible carbohydrate such as: fructo-oligosaccharides, galacto-oligosaccharides, glyco-oligosaccharides and mixtures thereof. Preferably, the composition according to the present invention comprises e.g. the following ingredients based on 1 ml of solution/suspension of the ingredients. The actual composition of e.g. a tablet or capsule is obtained by multiplying the quantity of ingredients by 3:

The invention comprises 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, the subjects wherein the composition comprises an effective amount of each:

a) a buffering agent,

b) a preservation agent fully compatible with the vaginal microbiota, c) a pure molecular or ionic biochemical nitrogen-source exploitable by the vaginal microbiota, d) prebiotic polysaccharide fermentable by the vaginal microbiota, and, optionally,

e) an antioxidant component,

wherein the treatment or prevention comprises administering the composition intravaginally to the subject, for stabilizing the acidity of the vaginal fluid of a subject at the physiological value around pH 4.5 or lower.

E2. The composition for use according to E1 , wherein the vaginal formulation is a lubricant gel for personal use.

E3. The composition for use according to E1 or E2, wherein the antioxidant is chosen among sulphur- containing compounds like N-acetyl cysteine, cysteine, methionine, sodium thiosulphate.

E4. The composition for use according to any one of E1-E3, wherein one or more lactobacilli strains are added to the formulation 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. The composition for use according to any one of E1-E4, wherein one or more lactobacilli strains are added to the formulation 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. The composition for use according to any one of E1-E5, in said Lactobacillus strain are selected from the group consisting of L. jensenii KS 109, L gasseri KS 114.1 deposited by Medinova AG at Collection Nationale de Cultures de Microorganismes on July 22, 2005 with accession number CNCM I-3482, L. crispatus 116.1 deposited by Medinova AG at Collection Nationale de Cultures de Microorganismes (CNCM) on July 22, 2005 with accession number CNCM I-3483, L. jensenii KS 119.1 deposited by Medinova AG at Collection Nationale de Cultures de Microorganismes (CNCM) on June 4, 2004 with accession number CNCM 1-3217, L crispatus 119.4 deposited by Medinova AG at Collection Nationale de Cultures de Microorganismes (CNCM) on July 22, 2005 with accession number CNCM I-3484, L. gasseri 120.1 deposited by Medinova AG at Collection Nationale de Cultures de Microorganismes (CNCM) on June 4, 2004 with accession number CNCM 1-3218, L. jensenii KS 121.1 deposited by Medinova AG at Collection Nationale de Cultures de Microorganismes (CNCM) on June 4, 2004 with accession number CNCM 1-3219, L. jensenii KS 122.1 , L. gasseri 123.1 deposited by Medinova AG at Collection Nationale de Cultures de Microorganismes (CNCM) on July 22, 2005 with accession number CNCM I-3485, L. gasseri 124.3 deposited by Medinova AG at Collection Nationale de Cultures de Microorganismes (CNCM) on June 4, 2004 with accession number CNCM I-3220, L. gasseri 126.2, L. crispatus ffl.\ deposited by Medinova AG at Collection Nationale de Cultures de Microorganismes (CNCM) on July 22, 2005 with accession number CNCM 1-3486, L. jensenii KS 130.1 , L. helveticus KS 300 deposited by Medinova AG at Collection Nationale de Cultures de Microorganismes (CNCM) on February 10, 2005 with accession number CNCM I-3360 and L acidophilus KS 400.

E7. The composition for use according to any one of E1-E6, wherein said Lactobacillus strain is selected from the group consisting of L. jensenii KS 119.1 deposited by Medinova AG at Collection Nationale de Cultures de Microorganismes (CNCM) on June 4, 2004 with accession number CNCM 1-3217, L. crispatus 119.4 deposited by Medinova AG at Collection Nationale de Cultures de Microorganismes (CNCM) on July 22, 2005 with accession number CNCM I-3484, L. gasseri 120.1 deposited by Medinova AG at Collection Nationale de Cultures de Microorganismes (CNCM) on June 4, 2004 with accession number CNCM I- 3218, L gasseri 124.3 deposited by Medinova AG at Collection Nationale de Cultures de Microorganismes (CNCM) on June 4, 2004 with accession number CNCM I-3220.

E8. The composition for use according to any one of E1-E7, wherein the formulation further comprises an effective amount of a therapeutically effective antimicrobial agent capable to inhibit or eradicate the urogenital pathogens.

E9. A method for the treatment or prevention of urogenital infections caused by pathogenic microorganisms in a female subject, which comprises administering intravaginally to the subject, optionally after the anti-infective treatment of a urogenital infection, a formulation with an effective amount of each: a) a buffering agent,

b) a preservation agent fully compatible with the vaginal microbiota,

c) a pure molecular or ionic biochemical nitrogen-source exploitable by the vaginal microbiota, d) prebiotic polysaccharide fermentable by the vaginal microbiota, and, optionally,

e) an antioxidant component.

E10. A method for stabilizing the acidity of the vaginal fluid of a subject at the physiological value around pH 4.5 or lower, which comprises administering intravaginally to the subject a formulation with an effective amount of each:

a) a buffering agent,

b) a preservation agent fully compatible with the vaginal microbiota,

c) a pure molecular or ionic biochemical nitrogen-source exploitable by the vaginal microbiota, d) prebiotic polysaccharide fermentable by the vaginal microbiota, and, optionally,

e) an antioxidant component. E11. The method according to claim 9 or 10, wherein 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 acid, dicarboxylic acids including oxalic, malonic, succinic acid, hydroxy-acids like lactic acid, citric acid, dicarboxylic acids like malic acid.

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

E13. The method according to E 9-E12 wherein the source of nitrogen c) is an ammonium salt, urea, a naturally-occurring or synthetic amino acid such as glutamic acid, and mixtures thereof.

E14. The method according to any one of claims E9-E13, wherein the source of carbon d) is a non- digestible carbohydrate such as: fructo-oligosaccharides, galacto-oligosaccharides, glyco- oligosaccharides and mixtures thereof.

E15. A formulation suitable for oral administration having a pH ranging from 4 to 5, wherein the composition comprises an effective amount of each:

a) a buffering agent,

b) a preservation agent fully compatible with the gastrointestinal microbiota,

c) a pure molecular or ionic biochemical nitrogen-source,

d) prebiotic polysaccharide fermentable by the gastrointestinal microbiota, and, optionally,

e) an antioxidant component.

E16. The formulation of claim E15, wherein the antioxidant e) is chosen among sulphur-containing compounds like N-acetyl cysteine, cysteine, methionine, sodium thiosulphate.

E17. The formulation of claim E15 or E16, wherein at least one lactobacilli strain is added to the formulation, 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. The formulation of any one of E15 to E17,wherein at least one lactobacilli strain is added to the formulation, said strain being selected from the group consisting of L jensenii KS 109, L gasseri KS 114.1 deposited by Medinova AG at Collection Nationale de Cultures de Microorganismes on July 22, 2005 with accession number CNCM I-3482, L crispatus 116.1 deposited by Medinova AG at Collection Nationale de Cultures de Microorganismes (CNCM) on July 22, 2005 with accession number CNCM I-3483, L. jensenii KS 119.1 deposited by Medinova AG at Collection Nationale de Cultures de Microorganismes (CNCM) on June 4, 2004 with accession number CNCM 1-3217, L. crispatus 119.4 deposited by Medinova AG at Collection Nationale de Cultures de Microorganismes (CNCM) on July 22, 2005 with accession number CNCM I-3484, L. gasseri 120.1 deposited by Medinova AG at Collection Nationale de Cultures de Microorganismes (CNCM) on June 4, 2004 with accession number CNCM 1-3218, L. jensenii KS 121.1 deposited by Medinova AG at Collection Nationale de Cultures de Microorganismes (CNCM) on June 4,

2004 with accession number CNCM 1-3219, L. jensenii KS 122.1 , L. gasseri 123.1 deposited by Medinova AG at Collection Nationale de Cultures de Microorganismes (CNCM) on July 22, 2005 with accession number CNCM I-3485, L gasseri 124.3 deposited by Medinova AG at Collection Nationale de Cultures de Microorganismes (CNCM) on June 4, 2004 with accession number CNCM I-3220, L. gasseri 126.2, L. crispatus 127.1 deposited by Medinova AG at Collection Nationale de Cultures de Microorganismes (CNCM) on July 22, 2005 with accession number CNCM I-3486, L. jensenii KS 130.1 , L. helveticus KS 300 deposited by Medinova AG at Collection Nationale de Cultures de Microorganismes (CNCM) on February 10, 2005 with accession number CNCM I-3360 and L acidophilus KS 400.

E19. The formulation of any one of E15 to E18 wherein said Lactobacillus strain are selected from the group consisting L. jensenii KS 119.1 deposited by Medinova AG at Collection Nationale de Cultures de Microorganismes (CNCM) on June 4, 2004 with accession number CNCM 1-3217, L crispatus 119.4 deposited by Medinova AG at Collection Nationale de Cultures de Microorganismes (CNCM) on July 22,

2005 with accession number CNCM I-3484, L gasseri 120.1 deposited by Medinova AG at Collection Nationale de Cultures de Microorganismes (CNCM) on June 4, 2004 with accession number CNCM I- 3218, L. gasseri 124.3 deposited by Medinova AG at Collection Nationale de Cultures de Microorganismes (CNCM) on June 4, 2004 with accession number CNCM I-3220 and L. acidophilus KS 400.

E20. The composition for use according to any one of E1-E8, or formulation according to any one of E15- E19, wherein 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 acid, dicarboxylic acids including oxalic, malonic, succinic acid, hydroxy-acids like lactic acid, citric acid, dicarboxylic acids like malic acid.

E21. The composition for use according to any one of E1-E8 or E20, or formulation according to any one of claims 15-20, wherein the preservation agent b) is lactic acid, or a derivative and/or salt thereof. E22. The composition for use according to any one of E1-E8 or E20-E21 , or formulation according to any one of claims 15-21 , wherein the source of nitrogen c) is an ammonium salt, urea, a naturally-occurring or synthetic amino acid such as glutamic acid, and mixtures thereof.

E23. The composition for use according to any one of E1-E8 or E20-E22, or formulation according to any one of E15-E22, wherein the source of carbon d) is a non-digestible carbohydrate such as: fructo- oligosaccharides, galacto-oligosaccharides, glyco-oligosaccharides and mixtures thereof.

E24. The composition for use according to any one of E1-E8 or E20-E23, or formulation according to any one of claims 15-23, having pH from 43 to 4.6, if solid after dissolution/suspension to a volume of 3 ml, and which comprises the following ingredients per single dosage:

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

Dibasic magnesium citrate: 15-45, preferably 30mg

Prebiotic fibre: 50-150, preferably 100 mg

Glutamic acid: 20-60, preferably 40 mg

Cystein/Thiosulphate: 10-50, preferably 30 mg

Excipients ad 400-1100 mg: q.s.

E25. The composition for use or formulation according to E24, further comprising:

Lyophilisates of L. gasseri 120.1 deposited by Medinova AG at Collection Nationale de Cultures de Microorganismes (CNCM) on June 4, 2004 with accession number CNCM 1-3218 and/or

L. gasseri 124.3 and/or L. crispatus 119.4 deposited by Medinova AG at Collection Nationale de

Cultures de Microorganismes (CNCM) on July 22, 2005 with accession number CNCM I-3484 and/or L. jensenii KS 119.1 deposited by Medinova AG at Collection Nationale de Cultures de

Microorganismes (CNCM) on June 4, 2004 with accession number CNCM 1-3217 for a total of 2 to 10 billion c.f.u. amounting to 50 to 200 mg.

Examples:

Example 1

Composition of different growth media and conditions of growth

Assuming a complete dissolution in the vaginal milieu (volume: 2-5 ml) a number of compositions are presented to allow for a comparative growth of Lactobacillus spp. and pathogens under test conditions: Growth control (positive) for lactobacilli: MRS

The conventional MRS medium (Biolife) without pH-adjustment allows for optimum growth of Lactobacillus spp. and is chosen for this purpose. Growth control (positive) for pathogens: TS

The conventional Tryptic Soy (TS) broth (BD, 211825) without pH adjustment was chosen.

Baseline control (negative)

A diluted solution of said MRS (15% in volume), buffered or non-buffered, is chosen to ensure a survival of the lactobacilli and pathogens tested at a low level of growth or inhibition, respectively.

Medium 17 (according to the invention):

an optimized test composition according to the following Table

and intended to stabilize the pH as well as allow the growth of Lactobacillus spp. and, at the same time, inhibition of pathogens.

Growth conditions and counting procedure

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

A first culture was prepared from material preserved at -80°C by streaking on agar plates. After incubation for no more than 24 hours, a single colony from the plate was used to inoculate 5 ml_ liquid culture and incubated for up to 18 hours and then used to inoculate 25 ml_ of each of the different media for the growth experiments. The starting cell density was adjusted to correspond to an optical density (600 nm) of 0.05.

All the experiments were performed in duplicate.

The growth of the cultures was monitored by measuring the optical density at 600 nm, the pH, by plating of serial dilutions on agar plates (cfu/mL) and by counting the total cell numbers using a haemato-cytometer (Neubauer improved). Strains tested: Vaginal Lactobacillus spp. and urogenital pathogens

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

As representative pathogens in these experiments were selected:

a) Gram-negative: uropathogenic E.coli CCOS 492, multi-resistant, human clinical isolate from University Hospital Zurich, isolated in 1976.

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

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

Comment

As mentioned in the title of this section acidifying, preserving and nutrient factors were chosen to be used against facultative anaerobic pathogens. These can produce energy with oxygen, like aerobes, or else also anaerobically (fermenting sugars to lactic a.s.o.). E. coli and S. aureus are representatives of this class of bacteria.

Comparative compositions

Hereafter are reported examples of compositions that, albeit having composition similar to Medium 17, do not equally well inhibit the growth of the above-mentioned pathogens.

Example 2a: effect of a slight increase of pH

As a first comparative example the following Medium 11 is provided:

In this medium the count evolution of the pathogens mentioned above under similar experimental conditions was studied.

E.coli CCOS 492

Medium 11 100% MRS Time (h.min) pH Count (cfu) pH Count (cfu)

00.00 5.00 1 0 ^Λ5 6.35 1 0 ^Λ5

06.10 4.98 8 0 ^Λ5 5.53 5*10 ^Λ7

20.20 4.98 5.01 >1*10 ^Λ9

S. aureus CCOS 461

Medium 11 100% MRS

Time (h.min) pH Count (cfu) pH Count (cfu)

00.00 5.00 1 0 ^Λ5 6.35 1 0 ^Λ5

06.10 4.99 1 0 ^Λ6 5.69 6 0 ^Λ7

20.20 4.98 4 0 ^Λ6 5.05 >1*10 ^Λ9

Hence, an increase of the pH of 4.5 by half a unit to a final value of 5.0 is enough to allow for the growth of pathogens for more than one log, pathogens that are otherwise inhibited at pH 4.5.

Example 2b: effect of a reduction of lactate concentration by 50% As an embodiment, the following Medium 12 was prepared and the evolution of the count of E.coli in same medium was observed.

In this medium, as well as in TSA as a positive control medium, the count evolution of pathogenic E.coli CCOS 492 mentioned above was studied under similar experimental conditions as in Example 2a.

E.coli CCOS 492

Medium 12 TSA

Time (h.min) pH Count (cfu) pH Count (cfu)

00.00 5.18 1 0 ^Λ5 6.7 1 0 ^Λ5

06.10 5.21 4 0 ^Λ6 6.1 5 0 ^Λ7

20.20 5.23 8 0 ^Λ6 5.5 >1*10 ^Λ9 It appears that the reduction in lactate concentration as in Medium 12 to half the value of Medium 17 comes along with a significant reduction of the inhibitory power with respect to the latter leading to an increase of the pathogen count of almost 2 logs.

In summary, it appears that the precise concentrations of the active principles (citrate, lactate a.s.o.) as exemplified for Medium 17 are preferable to ensure high pathogen inhibition action of the same.

Example 2: detailed discussion of the results for L gasseri 120.1 CNCM 1-3218

The in vitro model for testing the effect of the application of a pharmaceutical formulation in the vagina used in this application is that of a liquid medium containing the ingredients of the formulation at approximately the same concentration that it will have after dissolution in the vagina. The aim of the in vitro experiments to be described in the following was therefore to find a growth medium formulation that allows to install and maintain for a one-day duration a physiological pH of 4.0 - 4.5. Furthermore, said medium should allow the lactobacilli to rapidly establish themselves and to grow therein whereas at the same time the urogenital pathogens should be inhibited.

The cell counts of L. gasseri KS 120.1 CNCM 1-3218 grown for 19.4 hours on MRS medium reached 1x 10 ⁸ cfu/ml, the highest value over all of the 3 media tested. As expected, a clear pH drop from 6.4 to 4.0 was observed. This control experiment indicates the good fitness of the inoculum used and that the growth curves observed in the other media are correct.

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

Comparing the growth in Medium 17 with that in the 15% MRS medium, a 4.9 log-level reduction in the final cell counts between these media was observed. This clearly shows that the additional nutrients and components present in Medium 17 do indeed promote the growth of the tested Lactobacillus strain.

Acidity: the pH values remain remarkably stable around 4.5 from the beginning of the experiments and up to 20 hours thereafter (varying from 4.58 to 4.41), confirming thereby the adequate strength of the buffering system.

Conclusion: the presented Medium 17 formulated according to the description of the present invention induces a significant growth of the inoculated Lactobacillus gasseri, slightly less than for pure MRS, but definitely stronger than diluted (15%) MRS. The pH of the system remains constant at the optimum targeted value of approximately 4.5.

Example 3: detailed discussion of the results for S. aureus CCOS 461

The hypothesis to be tested in this case is that the chosen Medium 17 is able to inhibit the growth of the selected multi-resistant pathogen. The cell counts of S. aureus CCOS 461 grown for 21.1 hours on TS medium reached with 2x 10 ⁸ cfu/ml the highest value over all of the 3 media tested starting with a value of 9 x 10 ^Λ 6 at t = Oh. Also in this case a clear pH drop from 7.0 to 4.8 was observed. This control experiment indicates a good fitness of the inoculum used and that the growth curves observed in the other media are correct.

Comparing the evolution of the cell count obtained in Medium 17 with the TS positive control, a strong inhibition of S. aureus CCOS 461 of -5.2 logarithmic units was observed. With respect to diluted MRS the inhibition was also high, amounting to -4.4 logs.

Acidity: even in the presence of the live pathogen the pH values remain again remarkably stable around 4.5 from the beginning of the experiments and up to 20 hours thereafter with only a slight decrease (varying from 4.59 initially to 4.40 finally).

Conclusion: the presented Medium 17, formulated according to the description of the present invention, induces a significant decrease of the counts of the inoculated pathogen S. aureus CCOS 461 : with respect to the positive control medium TS but even with respect to diluted MRS. Medium 17 shows herewith by itself an anti-microbial action against this pathogen.

Example 4: summary of the experimental series

The results of the remaining strains mentioned above in Example 2 shall be presented in tabular form together with the two last examples and commented upon.

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

For all 4 Lactobacillus strains and the 2 pathogens the pH-values, designed to be buffered at a value of 4.5, the measurements confirm the excellent stability of the acidity of the medium for up to 20 hours or more of reaction time.

The pathogens S. aureus and E.coli as well were clearly not able to grow in Medium 17. The corresponding cell concentrations in this medium decreased dramatically in time.

Table Ex. 4: pH values in Medium 17 and log (reduction or increase) of cell numbers of the tested organisms grown on Medium 17 (according to the invention) against the reference media

Log change of cfu/mL on Medium 17 versus pH-value Medium 17

Strain Time 15% MRS MRS TSB initial final

KS 119.4 4.57 4.33

28.6 h 0.57 -0.49

CNCM I- 3484

KS 119.1 4.59 4.37 CNCM I- 18.6 h 1.12 -2.15

3217

KS 120.1 4.58 4.44 CNCM I- 19.4 h 4.94 -0.40

3218

KS 124.3 4.57 4.33 CNCM I- 28.6 h 0.44 -0.90

3220

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

In Table Ex. 4, the differences in log numbers at the end of the respective growth experiments are summarized. The strongest growth promoting effect was observed in Lactobacillus gasseri KS 120.1 CNCM 1-3218 with almost 5 log-levels increase compared to 15% MRS, whereas the weakest effect was found in Lactobacillus gasseri KS 124.3 CNCM I-3220, with a log-level increase of only 0.44.

Overall conclusion: During the fermentation process Medium 17 did stabilize the pH at a value between 4.5 and 4.3 for all strains tested. As to 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 of calcium-lactate as buffer substance, as well as the use of a selective carbon source (in this particular case glyco-oligosaccharide) for the probiotics proved to be sufficient to stimulate differentially the growth of the tested lactobacilli. On the other hand the 2 tested pathogens (E.coli and S. aureus) did not grow on "Medium 17" but were inhibited by the same

Example 5

Out of the systematic experiments leading to the proper media such as Medium 17 described above, the quantitative composition of a pharmaceutical dosage unit (capsule, tablet ...) to be applied intra-vaginally can be calculated. As mentioned above this requires to quantitatively take into account the volume of the vaginal fluid, a quantity that obviously varies according to the age and health conditions of the woman. Research has found that the volume of such fluid is around 3 ml on average (Ref. 16).

Table Ex. 5.1 : quantitative composition of a prebiotic vaginal capsule according to the invention

Capsule #6 Substance Amount Ca-lactate pentahydrate 60 mg

Dibasic ammonium citrate 30 mg

a-Glyco-oligosaccharide 100 mg

Target pH in vagina: Glutamic acid 40 mg

4.5 Cystein or Cystin 20 mg

Excipients as required 200 mg

Total weight 450 mg

Table Ex. 5.2: quantitative composition of vaginal capsule according to the invention

Table Ex 5.3: quantitative composition of vaginal capsule according to the invention

Tablet #9 Substance Amount

Ca-lactate pentahydrate 60 mg

Dibasic magnesium citrate 30 mg

Arabinogalactan 100 mg

Target pH in the vaginal Thiosulfate 30 mg preparation: approx. 4.5 Aspartic acid 60 mg

Excipients as required 270 mg

Total weight excipients/additives 550 mg

Lyophilisate of L gasseri KS 120.1 50 mg CNCM 1-3218

Lyophilisate of L. jensenii 119.1 CNCM 50 mg I-3484 Lyophilisate of L. crispatus KS 119.4 50 mg

CNCM 1-3217

Total weight including lyophilisates 700 mg

Table Ex. 5.4: quantitative composition of an oral symbiotic preparation (sachets) according to the invention

Table Ex. 5.5: quantitative composition of a prebiotic vaginal lubricant according to the invention

Lubricant gel # 3 Substance Amount

Lactic acid 50 mg

Citric acid 30 mg

Target pH of the Glycerol 80 mg

finished gel = 4.5 Carbomer 5 mg

Short-chain fructo-oligo-saccharides 50 mg

Thiosulphate 10 mg

Glutamic acid 10 mg

NaOH 0.1 mol or HCI 0.1 mol q.s. for pH=4.5

De-ionized water ad 1 ,000 mg

OPTIMIZING THE REDOX POTENTIAL OF THE VAGINAL FLUID

In the Example 5.1 above, the sulfur source added to M 17 is cysteine or cystin, in Example 5.2 it is sodium thiosulfate. All the above-identified 3 formulations lead e.g. to a pH value of the corresponding solution in 2-5 ml of about 4.5. From the point of view of acidity, nutrients, preservative they look very similar.

The question we raise now is whether they are equivalent w.r.t. the task assigned to them, that is to foster the growth of lactobacilli in the vagina and inhibit that of pathogens. To specify this question, we have to focus on specific pathogens; we shall choose the most frequent vaginal infection as mentioned above, the so called bacterial vaginosis or BV associated with pathogens like Gardnerella vaginalis or Prevotella bivia. In 1985 David Eschenbach and coworkers published a research paper (Ref. 22) in which they investigated the correlation between bacterial vaginosis and the vaginal redox potential respectively the pH.

On one hand they confirmed, as expected, that in the 15 patients a pH in the range 4.0 to 4.5 is mostly associated with a healthy vaginal microbiota, whereas in the range 4.5 to 5.5 the patients exhibit a strong presence of anaerobs with few lactobacilli and the symptoms of BV. The novelty was that they can also show that also the redox potential Eh in the vagina is in vivo strongly dependent on the status of the microbiota: Whereas the Eh values in the BV patients range between + 50 and - 250 mV, the Eh values of the healthy vaginas range higher between 100 and 300 mV. They also found that this is not a characteristic of the individual women, but was only dependent on the status of the microbiota. After a successful cure with metronidazole all women with a negative redox potential returned to a positive one (and also the pH returned to be more acidic).

Interestingly there have been no attempts to therapeutically use this fact to produce vaginal pharmaceutical products for treating BV, which warrant a higher redox potential.

We investigated this opportunity by the following experimental procedure:

Summary of the test procedure

A media formulation consisting of 20 mg/mL Ca-Lactate, 10 mg/mL dibasic ammonium citrate, 20 mg/mL Ecolians and 15% MRS broth was prepared (corresponding to M17 described above). After measurement of redox potential and pH several compounds (nitrogen and sulphur sources) were added to this medium and their effect on pH and redox potential was determined. Brief description of the experiments

A redox electrode (Mettler Toledo LE 501 ORP, connected to a pH-Meter MP230, Mettler Toledo ) was used to measure the redox potential of the media formulation before and after addition of the test compounds. Redox values were read after the signal was stable for at least 30 seconds. In addition the pH was measured using a standard pH Electrode connected to a MP225 pH Meter (Mettler Toledo).

Prior to each measurement series, the redox electrode was calibrated using a reference solution (Mettler Toledo, no 51350060, 220 mV, pH 7 (UH = 427 mV)).

Analysis results

Setup Konz. Substanz pH Redox Referenz

1 20 mg/mL Ca-Lactate 4.67 235.0 mV 236.2 mV

10 mg/mL Ammoniumcitrate, dibasic

15% MRS-Broth

20 mg/mL Ecolians

10 mg/mL L-Glutamic acid 4.48 310.8 mV

2 20 mg/mL Ca-Lactate 4.67 235.0 mV 236.2 mV

10 mg/mL Ammoniumcitrate, dibasic

15% MRS-Broth

20 mg/mL Ecolians

10 mg/mL L-Glutamic acid 4.41 265.2 mV

10 mg/mL Cystin

3 20 mg/mL Ca-Lactate 4.67 235.0 mV 236.2 mV

10 mg/mL Ammoniumcitrate, 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 Ca-Lactate 4.67 235.0 mV 233.0 mV

10 mg/mL Ammoniumcitrate, 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 Ca-Lactate 4.48 237.8 mV 234.9 mV 15 mg/mL Ammoniumcitrate, 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

Addition or not of 15% MRS to the solution has practically no effect on pH o Eh

Addition of the nitrogen source glutamic acid renders the medium slightly more acidic and more electropositive/oxydative reaching 300 mV

The sulfur compounds have, In this particular experimental series the most marked effect of the redox potential and a minor effect on the acidity: compare e.g. setup 2 and setup 3, where cysteine and cystin are interchanged. With cysteine the Eh decreases to -103.0 mV, whereas with the same amount cysteine it increases to 265.2 mV. The pH on its side varies only minimally, from 4.30 to 4.41.

Cystine (the oxidized dimeric form of cysteine) and MSM (dimethylsulfone) both maintain a high positive redox potential, whereas

Cysteine and thiosulfate cause the solution to become reducing instead of oxidizing, reaching negative values of -103 and - 36 mV

The conclusion is that adding e.g. cysteine or thiosulfate an environment favorable to the proliferation of anaerobes is obtained, whereas adding cystin or dimethylsulfone helps creating an environment favorable to the development of lactobacilli. This is of therapeutic interest as we can by such means choose not only which pH, but also which redox potential is best suited to fight locally a defined pathogen. By using a proper "redox modulator" the efficacy of the anti-BV formulation according to the invention can thus be increased.

The above considerations apply against the pathogens of BV (Gardnerella, Prevotella, Atopobium, which are by nature "obligate anaerobs", i..e microbes who cannot defend themselves against oxydants as, e.g. hydrogen peroxide or other oxidative agents. Against these pathogens increasing the redox potential is an additional therapeutic weapon. This works without damaging or inhibiting the vaginal lactobacilli: the reason being that the the lactobacilli, although being themselves anaerobs, are so-called "aero tolerants". That is, oxygen and more generally an environment with high redox potential (Eh) does not prevent them from growing. Choosing a redox modulator

Preferably, in the context of the present invention the term "redox modulator" indicates a substance, or at least a pair of substances, capable of reducing or enhancing the redox potential of a certain biological medium. Preferably, the redox modulator e) is chosen among sulphur-containing compounds the pair cysteine/cystin, like N-acetyl cysteine, cysteine, methionine, quinone/hydroquinone, selenium-based redox systems (selenite/selenate, e.g. in the sodium salt form), sodium thiosulphate/bisulphite, glutathione in the reduced state (GSH) and in the oxidized state (GSSG).. For example, if the redox potential needs to be enhanced so as to become definitely positive (>150 mV) and to inhibit anaerobs like Gardnerella, cystin (the oxidized form) will be chosen for this purpose rather than cysteine.

Concrete examples of anti-BV formulations with enhanced oydation potential

Example 6.1 : prebiotic formulation

Table Ex. 6.1 : quantitative composition of a prebiotic vaginal capsule against BV according to the invention

Example 6.2: symbiotic formulation against anaerobic vaginal infection according to the invention Table Ex. 6.2: quantitative composition of a symbiotic vaginal capsule against BV

Tablet #11 Substance Amount

Strains with proven, Lyophilisates of vaginal strains L.

pronounced activity gasseri 120.1 and 124.3 at Each 100 mg

against Gardnerella concentrations of 10 ¹¹ cfu/g

Target oxidation Ca-lactate pentahydrate 60 mg

potential in vagina:: Dibasic magnesium citrate 30 mg

> 200 mV Alanin 40 mg Arabinogalactan 100 mg

Target pH in vagina: Dimethylsulfone (MSM) 30 mg

approx.. 4.5 Excipients as required 140 mg

Total weight 600 mg

ERADICATING CANDIDA ALBICANS

As mentioned above Candida is a frequently observed microbe in the vagina, either as an opportunistic agent or as pathogen, e.g. appearing after an anti-infective treatment of vaginal infections such as bacterial vaginosis. The peculiarity of Candida is that, unlike the anaerobic pathogens involved in BV, they can co-exist with Lactobacilli, thereby exerting only an effect on the composition of the vaginal microbiota. The composition of the Doderlein flora under these 3 different states (no infection, BV or Candidiasis has been investigated recently in detail and published by G. Donders and co-workers (Ref. 23).

For the above-mentioned reasons the authors thought that it would be useful, if feasible, to shape the composition intended to foster lactobacilli but at the same time to inhibit pathogens, such as to have an inhibitory effect also on yeasts in general and Candida albicans in particular.

It would be tempting to use to this purpose a classical and safe antifungal drug like Clotrimazole, this molecule has however also a bactericidal activity against Gram-positive bacteria such as Lactobacilli are (Ref. 24"). In searching for a suitable molecule for our intended purpose we found a naturally occurring organic acid (sorbic acid) that is widely used as safe preservative agent against yeast and mould degradation in the food and pharmaceutical industry. The issue was therefore to test whether sorbic acid (usually available as sodium or potassium sorbate) was able to inhibit Candida at concentrations that were non-inhibiting for the vaginal lactobacilli. The reported optimum for the activity of sorbate is around pH 4.5, which fits perfectly in our chosen range. The concentration practically used for preservation against yeasts and molds is of 0.1 % (Ref. 25).

To this end the following experiments were performed.

Summary of the test procedure

A fresh overnight culture of the test strains was used to inoculate the test medium supplemented with different concentrations of potassium sorbate. Growth was determined by measurement of optical density and pH at start, after 6 hours and after 24 hours.

Growth conditions:

Medium for overnight culture: MRS broth with Tween

Test medium: MRS broth with Tween, diluted to 30%, pH adjusted to 4.5 with lactic acid

Testvolume: 5 ml

Incubation: 37°C, anaerob, with shaking (120 rpm)

Cell density for inoculation: approx. 10 ⁷ cfu/ml

Potassium sorbate 0.00%, 0.01%, 0.05%, 0.1%, 0.5%, 1.0%, 1.5% (weight/volume) concentrations:

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 Preparation of cultures

All cultures were re-activated from cryopreserved (-80°C) working stocks by cultivation on MRS agar for 1 to 3 days. A single colony from these plates was used to inoculate 5 mL of MRS broth. After incubation overnight, 10 ⁷ cfu/ml were used to carry out the experiments.

Analysis results

111;: 0.67 I 1.24 I 1.15 I 4.97 I 4.73 I 4.77

1.00% 0.65 1 13 0.96 58 501 508

50% 0.64; 099 0 76 5 32 5.2 526

Interpretation: 0: no inhibition, 1 : slight inhibition, 2: strong inhibition

Conclusions

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

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

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

Transformed into a formula of a capsule that will be dissolved in approximately 3 ml of vaginal fluid this corresponds to the addition of 3 to 6 mg potassium sorbate to the composition (see below).

Example 7.1 : Formulation against obligate anaerobic pathogens with anti-Candida activity as well

Table Ex. 7.1 : quantitative composition of prebiotic vaginal capsule according to the invention without lyophilisates and containing also sorbate as antifungal ingredient

Capsule #12 Substance Amount

Ca-lactate pentahydrate 60 mg

Dibasic magnesium citrate 30 mg

a-Glyco-oligosaccharide 100 mg

Target pH in vagina: Glutamic acid 40 mg

4.5 Cystin 30 mg

K-sorbate 5 mg

Excipients as required 185 mg

Total weight 450 mg Table Ex. 7.2: quantitative composition of vaginal capsule according to the invention containing lyophilisates and also potassium sorbate as antifungal agent

Example 7.3: Lubricant prebiotic formulation with activity against BV and Candida

Table Ex. 7.3: quantitative composition of a prebiotic vaginal lubricant according to the invention

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