BE1025643B1 - COMPOSITION COMPRISING AT LEAST ONE COMPONENT OF A PROBIOTIC BACTERIUM OF THE FIRMICUTES DIVISION AND INTENDED FOR USE IN THE TREATMENT OF UROGENITAL DISEASES - Google Patents

Abstract

The present invention relates to a composition comprising at least one component of a probiotic bacterium of the division Firmicutes and intended to be used in the preventive and / or curative treatment of urogenital diseases, in particular intended to be used in the treatment preventive and / or curative bacterial vaginosis and bacterial cystitis classified among UTI's, in particular vaginosis caused by the bacterium Gardnerella vaginalis and UTI's caused by Escherichia coli, said at least one component from the cellular content of said Probiotic bacteria of the Firmicutes Division.

Description

Composition comprising at least one component of a probiotic bacterium from the Firmicutes division and intended for use in the treatment of urogenital diseases

The present invention relates to a composition comprising at least one component of a probiotic bacterium of the Firmicutes division and being intended for use in the preventive and / or curative treatment of urogenital diseases, in particular intended for use in the treatment preventive and / or curative of bacterial vaginosis and of bacterial cystitis classified among the infections of the urinary tract (UTI - Urinary Tract Infections), in particular of the vaginosis caused by the bacterium Gardnerella vaginalis and UTI's caused by Escherichia coli.

Urogenital diseases, including non-sexually transmitted urogenital diseases, are conventionally due to a bacterial, viral, mycotic or parasitic infection. Among these, we find in particular infections due to germs like Colibacilli originating from the intestine and which can cause UTI's such as cystitis or even vaginitis caused by the yeast Candida albicans. Among the germs originating from the intestine and being responsible for the development of UTI's, 80% of the bacteria are Escherichia coli. UTI’s are acute or chronic infections and can affect multiple organs of the urinary system (bladder, kidneys, urethra, or prostate). For anatomical reasons, women are more often affected by these infections. Indeed, in women, the urinary meatus is close to the anus where the bacteria Escherichia coli are always present which can go up along the urethra towards the bladder and proliferate in the urine.

Urine is indeed a good culture medium for these germs. The body defends against urinary tract infection by emptying the bladder: a sufficient supply of water to ensure good urinary flow is therefore essential. The main causes of the appearance of a urinary tract infection in women are a lack of local hygiene,

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BE2017 / 5732 constipation and a lack of water intake. More rarely, a urinary tract infection can be caused by a malformation of the urinary tract (for example, a rise in urine from the bladder to the kidney). In humans, it is common that no cause for UTI is found. However, certain factors can favor its appearance such as an enlarged prostate (in older men) which causes a stasis of urine in the bladder, any obstacle to the flow of urine (kidney stones, bladder neck disease, narrowing of the urethra, ...) and certain neurological dysfunctions of the bladder as well as any infection of neighboring organs (gynecological apparatus or anus).

Sometimes UTI’s cause no symptoms, especially in the elderly, or result in an isolated fever. Typically, however, bladder infection, also called "cystitis", as well as a urethral infection or "urethritis" is manifested by burning during urination and frequent urination. The urine is sometimes cloudy, hemorrhagic and / or smelly. Kidney infection or "pyelonephritis" is responsible for general signs such as fever, chills, worsening of the general state and lower back pain which can be bilateral. The infection of the prostate also called "prostatitis" results in burning when urinating, frequent needs and low urine volumes as well as flu-like signs (muscle or joint pain).

Among the analyzes carried out to detect an infection of the urinary tract, the urine strip guides the diagnosis in one minute and reveals the presence of polymorphonuclear neutrophils (white blood cells) and nitrites. Cytobacteriological examination of the urine confirms the diagnosis by identifying the bacteria whose sensitivity to several antibiotics is tested. When septicemia, a generalized infection, is suspected, a blood sample is also taken.

The treatment of UTI's is traditionally based on antibiotic therapy, increased drinks and the treatment of factors

2017/5732 favoring. For example, in case of cystitis, either a urinary antiseptic is administered for 10 days, or an antibiotic treatment is given for 3 days (antibiotics of the penicillin or quinolone family), or a single dose single-dose treatment is prescribed. Although these treatments seem to be effective for so-called “simple” UTIs, that is to say where the infection is not worrying and where there is no risk of complications, resistance to these treatments nevertheless appears when UTIs occur in so-called “at risk” people, namely in the elderly, in pregnant women, in children under the age of 15 or in patients suffering from other diseases. Indeed, in these so-called "at risk" people, more or less serious complications such as premature birth in pregnant women as well as recurrences are highlighted.

Among the urogenital diseases, there are also urogenital infections caused by bacteria which are part of the vaginal flora and which can sometimes, under certain conditions, become pathogenic and therefore be responsible for a disorder of the urogenital tract. This type of urogenital infection, that is to say caused by bacteria that are part of the vaginal flora, is very common and most often results in vaginosis. Vaginosis results in an imbalance in the bacterial flora of the vagina, the causes of which are multiple: excess hygiene, estrogenic deficiencies, antibiotics, etc. It is more particularly characterized by the disappearance of lactobacilli and the multiplication of anaerobic germs such as Gardnerella vaginalis. The reduction in lactobacillary activity leads to an increase in vaginal pH which exceeds 5 (therefore more basic). Thus, it is not a sexually transmitted infection, vaginosis rather testifying to an imbalance of the vaginal flora with the disappearance of the protective effect of the Döderlein bacillus.

Vaginosis is the most common specific cause of urogenital infections in women since one in five women is

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BE2017 / 5732 carrying bacterial vaginosis. It is a benign pathology, except in pregnant women for whom vaginosis is responsible, in 16 to 29% of cases, for prematurity, fetal infections, spontaneous abortions and small weights of infants to the birth.

The diagnosis of bacterial vaginosis is most often made without additional examination in front of greyish and fluid vaginal discharge and in front of a bad odor which is due to the production by anaerobic germs of odorous substances which are all the more volatile as the vaginal pH increases. If in doubt, a vaginal swab may be requested to confirm the diagnosis. A positive diagnosis results in an elevation of the pH which is then greater than 5 and by a Nugent score greater than 6. The Nugent score makes it possible to assess the quality of the vaginal bacterial ecosystem by microscopic examination by studying the presence lactobacilli, certain anaerobic germs and Gardnerella vaginalis. A Nugent score between 0 and 3 is synonymous with normal vaginal flora, a score between 4 and 6 corresponds to an intermediate flora, while a score between 7 and 10 reflects bacterial vaginosis.

As explained above, vaginosis is mainly caused by the multiplication of the Gardnerella vaginalis bacteria. The latter is the only species of the genus Gardnerella (family Bifidibacteriaceae, order Bifidobacteriales, class Actinobacteria). This bacterium is in the form of pleiomorphic rods and draws its energy from the oxidation of organic compounds and is therefore characterized as being a chemo-organotrophic bacterium. Gardnerella vaginalis is considered to be an optional anaerobic bacterium, namely a bacterium which can use dioxygen in the presence of the medium. Thus, in the presence of oxygen, this bacteria can use aerobic respiration, while in its absence, one part ferments and the other performs anaerobic respiration. The wall of the Gardnerella bacteria

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BE2017 / 5732 vaginalis resembles that of Gram positive bacteria (purple coloration under the microscope), but due to the thin wall, the colouration can however appear Gram negative or Gram variable. Gram staining is based on the membrane and wall characteristics of the bacteria and is a determining factor in bacterial taxonomy.

Treatments against vaginosis are currently known. In fact, treatments based on secnidazone (Secnol ©) in a single dose of a 2 g sachet taken orally and based on metronidazole (Flagyl ©) taken orally 1 g per day for 7 days are widely prescribed . These effective antibiotics in the short term, however, experience a surprising failure rate in the medium term with a recurrence rate reaching 80% at three months. One of the explanations for these recurrences lies in the fact that the main infectious agent involved in vaginosis, namely Gardnerella vaginalis, is resistant to these two types of antibiotics among others because they are capable of producing biofilms protecting them from action of antibiotics.

In fact, a bacterial biolim is a structured cluster of bacterial cells coated with a polymer matrix composed of exopolysaccharides, proteins and nucleic acids and attached to a surface which, in the case of vaginosis, is biotic. Biofilm protects bacteria and allows them to survive in hostile environmental conditions. Biofilm bacteria can resist the host's immune response and are much more resistant to antibiotics and disinfectants. The presence of biofilms during infections therefore requires new methods of prevention, diagnosis and treatment.

In order to fight vaginosis more effectively, it is also recommended to combine, with antibiotics, treatments to restore the vaginal flora such as treatments based on prebiotics and / or probiotics as well as to minimize the factors

2017/5732 promoting the imbalance of the vaginal flora such as an excess of hygiene or an estrogenic deficiency.

Probiotics are living microorganisms (bacteria or yeasts) and can be, for example, in the case of treatment for vaginosis, lactobacilli which will replace the defective natural flora and then create the "ecological conditions" conducive to recolonization of the vagina by this natural flora.

In this sense, document EP1824500 discloses a probiotic bacterial strain belonging to the genus Lactobacillus which has the capacity to colonize the human vagina and to preserve or improve vaginal health when it is administered orally, rectally or vaginally. The bacterial strain according to this previous document belongs to a species chosen from the group comprising Lactobacillus plantarum, Lactobacillus crispatus and Lactobacillus gasseri. In addition, a bacterial strain according to document EP1824500 has a mannose-specific adhesin or other adhesion mechanisms allowing the bacteria to bind the internal surface of the vagina, intestinal tract or urinary bladder. This prior document describes the use of such a strain of probiotic bacteria belonging to the genus Lactobacillus for the manufacture of a medicament for the prophylactic and / or therapeutic treatment of bacterial vaginosis.

In this earlier document, the terms "probiotic bacterial strain" refer to a living microorganism which confers a benefit on the health of the host when administered in an adequate amount. In addition, still according to this prior document, when the strain is taken orally, it must survive the passage through the gastrointestinal tract. Similarly, when the bacterial strain according to this previous document is administered by vaginal or rectal view, the strain must colonize the vagina and the rectum, respectively. According to document EP1824500, the term “colonize” means that the quantity of viable Lactobacillus in the vaginal fluid and / or the faeces is at least increased

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2017/5732 after taking the bacterial strain compared to the total amount of initial Lactobacillus.

Unfortunately, even if it is true that document EP1824500 demonstrates that certain bacterial strains of Lactobacillus can survive the passage of the gastrointestinal tract and colonize the vagina, no effect of the bacterial strains according to this previous document is demonstrated on the treatment and / or prevention of vaginosis.

In addition, there are many probiotic-based products for the prophylaxis of vaginosis as well as UTI's. However, to date, there is no prophylactic and / or curative treatment which is effective against these two most common urogenital diseases.

The object of the invention is to overcome the drawbacks of the state of the art by providing a composition for the preventive and / or curative treatment of urogenital diseases which is efficient and effective in the long term, which does not exhibit resistance to biofilms, which causes little or no recurrence and which is rapid, in particular intended to be used for the preventive and / or curative treatment of bacterial vaginosis and of bacterial cystitis classified among the UTI's, in particular of the vaginosis caused by the Gardnerella bacteria vaginalis and ITUs caused by Escherichia coli.

To solve this problem, there is provided according to the invention, a composition as indicated at the beginning, characterized in that said at least one component is a component of the cellular content of said probiotic bacterium from the Firmicutes division.

By the terms "urogenital diseases" is meant in the sense of the present invention diseases and disorders of the urinary tract and female genital organs which are generally caused by infections due to germs. Urogenital diseases include non-sexually transmitted urogenital diseases which

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2017/5732 are urogenital diseases that are caused by infections caused by germs that are not sexually transmitted.

By the terms "cell content", it is understood in the sense of the present invention the content of a living bacterium, that is to say all of the content delimited by the bacterial wall and the cytoplasmic membrane. In particular, the bacterial cell content is equivalent to the cytoplasm, the nuclear system, and possibly plasmids.

The cytoplasm is a colloidal hydrogel comprising a dispersing phase (mineral salts and soluble compounds of lipoprotein nature) and a dispersed phase formed from nucleoproteins and lipids. The bacterial cytoplasm contains ribosomes, ribonucleic acids, reserve substances and some specialized organelles.

Unlike the nucleus of the eukaryotic cell, the bacterial nuclear system is not surrounded by a membrane and usually has only one chromosome. The nuclear apparatus is related to the mesosome where the enzymatic sites are concentrated which allow DNA to express its different functions.

Finally, some bacteria can also contain plasmids which are extrachromatic genetic elements capable of self-replication. These are small pieces of DNA (deoxyribonucleic acid) about a hundred times smaller than chromosomal DNA. One of their properties is to give bacteria resistance to antibiotics or heavy metals. Bacterial plasmids can also be responsible for the synthesis of bacteriocins, substances of a protein nature having a powerful bactericidal power. Finally, certain plasmids (called transposons) can integrate into the bacterial chromosome and thus give it the properties they carry.

In the context of the present invention, it has been surprisingly demonstrated that the cellular content of a probiotic bacterium

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2017/5732 the Firmicutes division leads to a significant reduction in the amount of Gardnerella vaginalis, the main cause of vaginosis. Indeed, the use of the cellular content of such a bacterium makes it possible to obtain superior results in comparison with those observed with compositions comprising cells of the same whole whole probiotic bacteria or even with compositions comprising the supernatant isolated from the culture. such bacteria. Indeed, it has surprisingly been observed that a composition according to the invention, that is to say one which comprises at least one component originating from the cellular content of a probiotic bacterium of the Firmicutes division, is effective and ensures inhibition of the presence of the bacterium Gardnerella vaginalis, the multiplication of which is the main cause of vaginosis, considered to be the most common non-sexually transmitted urogenital disease in women.

Specifically, in the context of the present invention, a statistically significant reduction in the number of Gardnerella vaginalis bacteria has been demonstrated when these are brought into contact with a composition according to the invention, namely with a composition comprising at least a component derived from the cellular content of a probiotic bacterium from the Firmicutes division. For example, when the Gardnerella vaginalis bacteria are brought into contact with a composition comprising at least one component of the cellular content of the probiotic bacterium Lactobacillus crispatus, this reduction is from 4 to 80 times greater than in the case where the Gardnerella vaginalis bacteria are treated (brought into contact) with compositions comprising whole live Lactobacillus crispatus cells or with compositions comprising the supernatant isolated from the culture of such bacteria.

Indeed, it has been demonstrated in the context of the present invention that the composition according to the invention, that is to say which comprises at least one component of the cellular content of a probiotic bacterium of the

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2017/5732 Firmicutes division, is more active than the same probiotic bacteria alone (living bacteria) and acts more directly than antibiotics on the germs responsible for urogenital diseases such as bacterial vaginosis and bacterial cystitis, the composition according to 5 l The invention can therefore be used as a prophylactic (preventive) treatment but also as a curative treatment for urogenital diseases such as bacterial vaginosis and bacterial cystitis.

Preferably, according to the invention, said component of the cellular content is a component derived from the cytoplasm, from the nuclear apparatus or from the plasmids of the probiotic bacterium of the Firmicutes division.

Advantageously, the probiotic bacterium of the Firmicutes division is a bacterium of the Lactobacillaceae family.

In a particularly advantageous form according to the invention, the probiotic bacterium of the Firmicutes division is a bacterium of the genus Lactobacillus.

In addition, in a particular form according to the invention, the probiotic bacterium of the Firmicutes division is chosen from the group consisting of Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus iners, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus plantarum, and their mixtures.

Advantageously, according to the invention, said at least one component is a component of the cellular content of the strain Lactobacillus crispatus LMG 29995.

Advantageously, the composition according to the invention is in the form of a powder. In fact, the composition according to the invention is dried by lyophilization, drying in a fluid bed or any other suitable process allowing a powder to be obtained. This dry powder 30 can then be used in all possible formulations.

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Preferably, the composition according to the invention also comprises at least one excipient.

Preferably, according to the invention, the composition is administered vaginally.

In addition, in a particular form, the composition according to the invention is administered in the form of vaginal tablets, vaginal capsules, creams, gels, foams, ointments, applicators, vaginal devices, tampons , or any other form that can be administered through the vagina.

Preferably, according to the invention, the composition further comprises a carrier material.

In a particularly advantageous form according to the invention, the composition is in the form of a pharmaceutical composition.

In addition, in a particular form, the composition according to the invention is in the form of a hygiene product.

Preferably, according to the invention, the composition is in the form of a medical device.

Advantageously, according to the invention, said vehicle material is at least one pharmaceutically acceptable vehicle.

Other embodiments of a composition according to the invention are indicated in the appended claims.

Example

Evaluation of the inhibitory effect of the Lactobacillus crispatus bacteria on the Gardnerella vaginalis bacteria via the agar disc diffusion method

The LMG 7832 strain of the Gardnerella vaginalis bacteria in lyophilized form was cultured in a culture medium (with

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2017/5732 sheep blood) and purity was assessed by depositing the strain on Columbia-type blood agar. This made it possible to demonstrate that this strain of Gardnerella vaginalis bacteria grows better under anaerobic conditions rather than aerobic conditions, which demonstrates that the evaluation of the final inhibition must be carried out under anaerobic conditions.

It was then necessary to select the appropriate agar medium to carry out the inhibition test, this medium having to allow the growth of the strain of the bacteria Garnerella vaginalis and of the bacteria Lactobacillus crispatus. For this purpose, the two types of bacteria were cultivated in a culture medium and deposited on an agar of different media:

- MRS agar (optimal medium for Lactobacillus crispatus); and

- MH agar (Mueller-Hinton) which is the standard medium for carrying out inhibition studies when different types of bacteria are used.

The results demonstrated that both types of bacteria grow on HD agar, which is why it was decided to do the inhibition test with HD agar.

For the actual inhibition test, both types of bacteria were grown under optimal conditions. After a night of growth:

- the Gardnerella vaginalis bacterium was centrifuged, the bacterium was then resuspended in PBS (phosphate buffered saline) to obtain a concentration of 10 ⁸ CFU / ml and the resulting suspension was deposited on 22 HD agar discs to allow the strain growth; and

- the bacterium Lactobacillus crispatus has been divided into different aliquots (each corresponding to a different condition):

• centrifuged supernatant and filtered as is;

• centrifuged and filtered supernatant for which the pH has been corrected to 6.5;

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2017/5732 • sonicated cells; and • living cells centrifuged and filtered.

Sonication has the effect of releasing the contents of the bacteria, that is, releasing the cytoplasm, nuclear system, and plasmids from the bacteria. Of course, any other technique allowing the content of the bacteria to be released is envisaged in the context of the present invention.

In a preferred embodiment of the invention, the sonication process uses ultrasound to obtain a complete lysate of living bacteria.

The antimicrobial effect of different dilutions of the composition comprising Lactobacillus crispatus for each of the four conditions was tested, namely no dilution, 1/2 dilution, 1/10 dilution, 1/100 dilution and 1/1000 dilution. Each dilution of each condition was deposited (at a point 10 mm in diameter) in the center of an HD agar disc which was completely covered with the Gardnerella vaginalis suspension mentioned above. Two MH agar discs were used as negative controls. The results are presented in Table 1 below.

First, the results demonstrate that the disks corresponding to the negative controls are completely covered with the Gardnerella vaginalis bacteria. In addition, a reduction in the number of CFUs (colony-forming units) is demonstrated for all conditions derived from Lactobacillus crispatus. According to the various conditions tested, it is demonstrated that the greatest reduction is observed with the sonicated cells of Lactobacillus crispatus compared to the living cells (whole) and compared to the supernatant of these, this being able to be the result of the release of 'internalized antimicrobial agents.

These different results are shown in Table 1.

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Table 1: Number of Gardnerella vaginalis (CFU) cells in a 30 mm diameter circle around the 10 mm point containing the different types of Lactobacillus crispatus suspensions.

Condition Dilution CFU supernatant Not corrected No dilution 2160 1/2 dilution 3240 1/10 dilution 3240 1/100 dilution 4325 1/1000 dilution 4325 Corrected to pH 6.5 No dilution 109 1/2 dilution 800 1/10 dilution 450 1/100 dilution 225 1/1000 dilution 500 Sonicated Bacillus crispatus cells No dilution 22 1/2 dilution 51 1/10 dilution 228 1/100 dilution 250 1/1000 dilution 250 Living cells of Bacillus crispatus washed No dilution 1800 1/2 dilution 650 1/10 dilution 1800 1/100 dilution 2500 1/1000 dilution 1800 Negative controls AT / > 5000 B / > 5000

It is understood that the present invention is in no way limited to the embodiments described above and that many

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BE2017 / 5732 modifications can be made without departing from the scope of the appended claims.

Claims (14)

1. Composition comprising at least one component of a probiotic bacterium from the Firmicutes division and being intended to be used in the preventive and / or curative treatment of urogenital diseases, in particular intended to be used in the preventive and / or curative treatment of bacterial vaginosis and bacterial cystitis classified among the UTI's, in particular vaginosis caused by the Gardnerella vaginalis bacterium and UTI's caused by Escherichia coli, said composition being characterized in that said at least one component is a component of the content cell released from said Firmicutes division probiotic bacteria. 2. Composition intended to be used according to claim 1, said component of the cellular content being a component derived from the cytoplasm, from the nuclear apparatus or from the plasmids of said probiotic bacterium of the Firmicutes division. 3. Composition intended to be used according to claim 1 or 2, said cellular content being released from said probiotic bacterium of the Firmicutes division by sonication. 4. Composition intended for use according to any one of the preceding claims, characterized in that said probiotic bacterium of the Firmicutes division is a bacterium of the family of Lactobacillaceae. 5. Composition intended for use according to any one of the preceding claims, characterized in that said probiotic bacterium of the Firmicutes division is a bacterium of the genus Lactobacillus. 6. Composition intended to be used according to any one of the preceding claims, characterized in that said probiotic bacterium of the Firmicutes division is chosen from the group BE2017 / 5732 consisting of Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus inners, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus plantarum. 7. Composition intended for use according to any one of the preceding claims, said composition being characterized in that said at least one component is a component of the cellular content of the strain Lactobacillus crispatus LMG 29995. 8. Composition intended to be used according to any one of the preceding claims, characterized in that it is in the form of a powder. 9. Composition intended to be used according to any one of the preceding claims, characterized in that it also comprises at least one excipient. 10. Composition intended for use according to any one of the preceding claims, characterized in that it is administered vaginally. 11. Composition for use according to any one of the preceding claims, which is administered in the form of vaginal tablets, vaginal capsules, creams, gels, foams, ointments, applicators, vaginal devices , tampons, or any other form that can be administered through the vagina. 12. Composition intended to be used according to any one of claims 1 to 10, characterized in that it is in the form of a pharmaceutical composition. 13. Composition intended for use according to any one of claims 1 to 10, characterized in that it is in the form of a hygiene product. 14. Composition intended to be used according to any one of claims 1 to 10, characterized in that it is in the form of a medical device. PATENT COOPERATION TREATY