Preparation of deactivated saccharomyces boulardii yeast

Abstract

The present invention relates to a preparation of Saccharomyces boulardii yeast subjected to beta or gamma radiation.

Description

[0001] PREPARATION OF INACTIVATED SACCHAROMYCES BOULARDII YEAST

[0002] Field of invention

[0003] The present invention relates to a preparation of Saccharomyces boulardii yeast subjected to beta or gamma radiation and its use as a medicament. The present invention also relates to a method for inactivating a Saccharomyces boulardii yeast preparation and to a pharmaceutical composition or medical device comprising an inactivated Saccharomyces boulardii yeast preparation.

[0004] Technical background

[0005] Vulvovaginal infections, or vulvovaginitis, are common conditions in women. In fact, almost all women are affected by a vulvovaginal infection at some point in their lives, and some even experience it recurrently. Vulvovaginal infections primarily manifest as painful or bothersome physical symptoms that can affect quality of life. The most common symptoms are leukorrhea associated with various vulvovaginal manifestations such as itching, burning, and irritation.

[0006] These infections can be caused by various pathogens such as bacteria, fungi, and viruses. Bacterial vulvovaginal infections are the most common type of vulvovaginitis. They are linked to an imbalance in the vaginal microbiota characterized by the growth of anaerobic bacteria, such as Gardnerella vaginalis, Prevotella spp., and Mobiluncus spp. Vulvovaginal yeast infections, or candidiasis, are considered the second most common cause of vulvovaginitis. They are linked to the proliferation of a fungus of the genus Candida in the vagina.

[0007] Medical management of vulvovaginal infections varies depending on the pathogen causing the infection. Bacterial infections are typically treated with antibiotics, such as metronidazole and clindamycin (Wilson (2004) Sex Transm Infect, 80: 8-11). However, antibiotics can cause side effects and may contribute to antibiotic resistance. Indeed, cure rates range from 60% to 90% after 5 days of antibiotic treatment. However, approximately 30% of treated women experience a recurrence within 3 months and 66% within 6 months of treatment. Yeast infections are typically treated with topical or oral antifungal medications belonging to the azole family. However, some antifungal medications can cause unwanted side effects such as irritation, abdominal pain, or nausea.

[0008] Probiotics have also been administered to women with vulvovaginitis to maintain or restore microbiota balance. Most vaginal probiotics developed to date contain lactobacilli given the dominant presence of these microorganisms in the microbiotas of women with good gynecological health. However, although these probiotics have shown positive effects on the prevention and treatment of bacterial vulvovaginitis, they are less effective on candidiasis (Hanson et al. (2016) J Midwifery Womens Health, 61: 339-355).

[0009] Thus, in view of increasing resistance rates and high relapse rates, it is necessary to develop complementary or alternative therapeutic strategies to those existing for the management of vulvovaginal infections.

[0010] Summary of the invention

[0011] The present invention arises from the unexpected discovery by the inventors that the administration of Saccharomyces boulardii yeast inactivated by gamma or beta radiation makes it possible to treat bacterial vulvovaginal infections with Gardnerella vaginalis and to treat vulvovaginal infections with Candida albicans yeast.

[0012] Thus, the present invention relates to a preparation of Saccharomyces boulardii yeast subjected to beta or gamma radiation.

[0013] The present invention also relates to a method for inactivating the yeast preparation Saccharomyces boulardii comprising a step of subjecting the yeast preparation Saccharomyces boulardii to beta or gamma radiation.

[0014] The present invention also relates to a yeast preparation

[0015] Inactivated Saccharomyces boulardii obtainable by the process as defined above. The present invention also relates to the preparation of Saccharomyces boulardii yeast as defined above, for use as a medicament.

[0016] The present invention also relates to a pharmaceutical composition or medical device or a cosmetic product comprising a Saccharomyces boulardii yeast preparation as defined above.

[0017] The present invention also relates to a Saccharomyces boulardii yeast preparation as defined above, a pharmaceutical composition as defined above or a medical device as defined above, for use in the prevention or treatment of vulvovaginitis and / or for use in restoring vaginal flora, and / or for preventing recurrences of vulvovaginitis, and / or preventing or treating the symptoms of vulvovaginitis, in an individual.

[0018] The present invention also relates to a method for preventing or treating vulvovaginitis and / or restoring the vaginal flora, and / or preventing recurrences of vulvovaginitis, and / or preventing or treating the symptoms of vulvovaginitis, in an individual, comprising administering to said individual a Saccharomyces boulardii yeast preparation as defined above, a pharmaceutical composition as defined above, or a medical device as defined above.

[0019] The present invention also relates to the use of a Saccharomyces boulardii yeast preparation as defined above, of a pharmaceutical composition as defined above, for obtaining a medicament or a medical device intended for the prevention or treatment of vulvovaginitis and / or for the restoration of the vaginal flora, and / or for the prevention of recurrences of vulvovaginitis, and / or for the prevention or treatment of the symptoms of vulvovaginitis, in an individual.

[0020] The present invention also relates to the cosmetic use of a Saccharomyces boulardii yeast preparation as defined above or of a cosmetic product as defined above for the intimate hygiene or intimate washing of an individual.

[0021] The present invention also relates to a method of intimate hygiene or intimate washing of an individual, in which the individual is administered a preparation of Saccharomyces boulardii yeast as defined above or a cosmetic product as defined above, in particular in a cosmetically effective amount.

[0022] Detailed description of the invention

[0023] As a preliminary point, it should be recalled that the term "comprising" means "including", "containing" or "encompassing", that is to say that when an object "comprises" one or more elements, other elements than those mentioned may also be included in the object. Conversely, the expression "consisting of" means "made up of", that is to say that when an object "consists of" one or more elements, the object cannot include elements other than those mentioned.

[0024] Saccharomyces boulardii

[0025] A "yeast" according to the invention is a fungus, preferably unicellular. The yeast according to the invention is preferably of the genus Saccharomyces, more preferably of the species Saccharomyces cerevisiae and even more preferably of the species Saccharomyces cerevisae var. boulardii. As used herein, Saccharomyces cerevisiae var. boulardii is a synonym for Saccharomyces boulardii and is well known to those skilled in the art and is notably described in Hennequin et al. (2001) J. Clin. Microbiol. 39:551-559.

[0026] Particularly preferably, Saccharomyces boulardii according to the invention is obtained from drugs of the Ultra-Levure® brand or from deposits in the American Type Culture Collection (ATCC, USA) under the reference 74012 or in the National Collection of Culture and Microorganisms (CNCM, France) under the reference 1-745. Also preferably, Saccharomyces boulardii is Saccharomyces boulardii CNCM 1-745 or Saccharomyces cerevisiae var. boulardii HANSEN CBS 5926.

[0027] Preferably, the yeast according to the invention is freeze-dried, such as Saccharomyces boulardii of the Ultra-Levure® brand.

[0028] As used herein, "freeze-drying" is a preservation method in which yeast is frozen and then subjected to sublimation of the frozen water it contains to give a lyophilizate in the form of dry yeast powder preferably containing less than 2% water and more preferably less than 1% water. Preferably, the freeze-dried yeast cells are obtained from yeast cell concentrates. Any type of yeast freeze-drying method known to those skilled in the art may be used. However, the yeast according to the invention is preferably freeze-dried using the following freeze-drying method: culturing the yeast in a liquid nutrient medium until the cells reach a stationary phase; concentrating the cultured yeast cells and freezing the concentrate; freeze-drying the concentrate.

[0029] As used herein, the term "Saccharomyces boulardii yeast preparation" means any preparation that can be obtained from Saccharomyces boulardii yeast.

[0030] Preferably, the Saccharomyces boulardii yeast preparation is obtained from yeast cells themselves or contains yeast cell secretions, yeast cell culture, yeast cell extract, yeast cell conditioned medium or yeast cell culture supernatant.

[0031] As used herein, "yeast cells" or "yeast cell culture" includes viable or dead yeast cells, intact or in the form of debris. Preferably, at least a portion of the yeast cells according to the invention or included in the yeast cell culture according to the invention are viable, in particular viable and culturable, before being subjected to beta or gamma radiation. Preferably, at least a portion of the yeast cells according to the invention or included in the yeast cell culture according to the invention, more preferably essentially all of the yeast cells according to the invention or included in the yeast cell culture according to the invention are dead or inactivated after being irradiated with beta or gamma radiation.

[0032] Yeast cell viability is defined as the ability of a yeast cell to multiply. Yeast cell viability can be determined by methylene blue staining and microscopic observation. The number of viable and culturable cells in a sample can be estimated by determining the number of Colony Forming Units (CFU) contained in the sample.

[0033] For example, the number of CFU of yeast cells in a liquid sample containing yeasts, such as a yeast cell culture, can be determined by spreading a determined volume of the sample on a solid medium, for example a gelled medium, allowing the growth of the yeasts, and incubating the solid medium for a period of time, for example 48 h, and at a temperature, for example 30°C, allowing the growth of the yeast colonies. The number of colonies relative to the volume spread on the solid medium makes it possible to determine the number of CFU contained in the sample. A detailed protocol for determining CFU according to the invention is described in particular in Toothaker and Elmer (1984) Antimicrobial Agents and Chemotherapy 26:552-556 in the paragraph “Assay for S. boulardii”.Furthermore, when the yeast sample is in the form of a solid, for example a lyophilized powder, it is preferable to determine the number of CFU contained in the sample after resuspending a determined mass of the sample in an aqueous solution, in particular distilled water or a 0.9% NaCl solution at pH 7.

[0034] Preferably, the yeast cells or a yeast cell culture according to the invention comprise 10 8 CFU / g at 10 12 CFU / g, more preferably 2.10 9 CFU / g to 2.10" CFU / g before exposure to beta or gamma radiation.

[0035] Many methods for preparing yeast cells, a yeast cell culture, a yeast cell extract, a yeast cell conditioned medium and a yeast cell culture supernatant are well known in the art and are described in particular in "Yeast Protocols" (1996) Methods in Cell and Molecular Biology, Ed. Ivor H. Evans, Humana Press.

[0036] The yeast cell culture according to the invention may be obtained by any standard method well known to those skilled in the art for the culture of yeast cells. For example, the yeast cell culture according to the invention may be obtained by inoculating a complete liquid culture medium such as yeast extract, peptone and dextrose (YEPD or YPD) and incubating the medium at 30°C - 37°C with shaking and under aerobic conditions for at least 24 hours, 36 hours or 48 hours and preferably less than 96 hours.

[0037] The yeast cells according to the invention can be obtained from a yeast cell culture medium according to the invention by sedimentation or centrifugation of the yeast cells.

[0038] The yeast cell extracts according to the invention can be obtained by any method of fragmenting yeast cells known in the state of the art applied to the culture of yeast cells or to the yeast cells according to the invention, such as autolysis, hydrolysis or autoclaving. In particular, the yeast cell extract according to the invention is chosen from the group consisting of a membrane extract, a cytoplasmic extract or a nuclear extract.

[0039] Preferably, the yeast cell-conditioned medium of the invention relates to any medium, such as a liquid cell culture medium, which has been contacted with yeast cells. Preferably, the medium has been contacted with yeast cells for a sufficient time for the yeast cells to have secreted into the medium. For example, the medium has been contacted by yeast cells for 1 to 9 days, preferably 1, 2, 3, 4, 5, 6, 7, 8 or 9 days. The conditioned medium of the invention preferably contains molecules secreted by the yeast cells such as proteins. Preferably, the conditioned medium of the invention does not contain cellular debris.

[0040] The yeast cell culture supernatant may be obtained by any method well known to those skilled in the art. The yeast cell culture supernatant according to the invention may in particular be obtained by centrifuging the yeast cell culture according to the invention and removing the supernatant portion of the centrifuged culture. The yeast cell culture supernatant according to the invention may also be obtained by filtering the yeast cell culture according to the invention through a filter retaining the yeast cells and recovering the filtrate.

[0041] The yeast cell culture, yeast cell extract, yeast cell conditioned medium and yeast cell culture supernatant according to the invention may have been subjected to at least one treatment or transformation step such as centrifugation, filtration, purification, chromatography, concentration, decantation, drying, lyophilization or distillation, in particular filtration, more particularly sterilizing filtration for example with 0.2 μm filters.

[0042] Preferably, the yeast preparation to be used according to the invention comprises:

[0043] - the collection of yeast cells,

[0044] - the culture of yeast cells,

[0045] - possibly the treatment of yeast cells,

[0046] - obtaining a yeast preparation, and

[0047] - irradiation of the yeast preparation with beta or gamma radiation.

[0048] Preferably, Saccharomyces boulardii is the only microorganism or probiotic included in the preparation, composition, in particular pharmaceutical, medicament, medical device or cosmetic product according to the invention. In other words, no other microorganism or probiotic, such as bacteria of the genus Lactobacillus, in particular L. rhamnosus, L. fermentum, L. crispatus, L jensinii or L gasseri, of the genus Enterococcus, in particular Enterococcus faecium, or yeasts of the genus Saccharomyces, in particular S. cerevisiae, is present in the preparation, composition, medicament, medical device or cosmetic product.

[0049] Inactivation of Saccharomyces boulardii

[0050] Preferably, the Saccharomyces boulardii yeast preparation according to the invention is subjected to beta or gamma radiation.

[0051] According to one embodiment of the invention, the Saccharomyces boulardii yeast preparation according to the invention is subjected to beta radiation.

[0052] According to one embodiment of the invention, the Saccharomyces boulardii yeast preparation according to the invention is subjected to gamma radiation.

[0053] Preferably, irradiation of the Saccharomyces boulardii yeast preparation according to the invention with beta or gamma radiation has the effect of inactivating the Saccharomyces boulardii yeast, i.e. the Saccharomyces boulardii yeast is incapable of growth or reproduction; we also speak of the absence of residual vitality of the Saccharomyces boulardii yeast.

[0054] Thus, preferably irradiation of the Saccharomyces boulardii yeast preparation with beta or gamma radiation results in an absence of residual vitality of the Saccharomyces boulardii yeast.

[0055] Preferably, the inactivated Saccharomyces boulardii yeast preparation according to the invention comprises essentially 0 Colony Forming Units (CFU) per gram of Saccharomyces boulardii yeast preparation. By "essentially 0 Colony Forming Units" is meant that the inactivated Saccharomyces boulardii yeast preparation does not contain living cells, or that the concentration of living cells is well below the detection limits.

[0056] Preferably, the inactivated Saccharomyces boulardii yeast preparation according to the invention comprises less than 10 3 UFC, under 10 2 CFU, less than 10 CFU, less than 1 CFU per gram of Saccharomyces boulardii yeast preparation. Preferably, the Saccharomyces boulardii yeast preparation subjected to beta or gamma radiation is in powder form, in particular dried or lyophilized.

[0057] As used herein, "kGy" is a measure of absorbed radiation dose. It stands for "kilogray," where 1 kGy is equal to 1000 grays (Gy). The gray (Gy) is the International System of Units (SI) standard unit of absorbed radiation dose, equivalent to the absorption of one joule of energy per kilogram of matter. In the context of the present invention, kGy expresses the absorbed dose of radiation, such as beta or gamma radiation, that the Saccharomyces boulardii yeast preparation received.

[0058] Preferably, the Saccharomyces boulardii yeast preparation is subjected to beta or gamma radiation of at least 10 kGy, at least 15 kGy, at least 20 kGY, at least 25 kGY.

[0059] Preferably, the Saccharomyces boulardii yeast preparation is subjected to beta or gamma radiation of at most 150 kGY, at most 100 kGy, more preferably at most 80 kGy.

[0060] Preferably, the Saccharomyces boulardii yeast preparation is subjected to beta or gamma radiation of between 10 and 150 kGy, 10 and 100 kGy, 10 and 90 kGy, 10 and 80 kGy, 10 and 70 kGy, 10 and 60 kGy, 10 and 50 kGy, 10 and 45 kGy, 10 and 40 kGy, 10 and 35 kGy, 10 and 30 kGy, 10 and 25 kGy, 10 and 20 kGy, or 10 and 15 kGy. More preferably, the Saccharomyces boulardii yeast preparation is subjected to beta or gamma radiation of between 20 and 100 kGy, 20 and 80 kGy, 25 and 80 kGy, 25 kGy and 50 kGy. Even more preferably, the Saccharomyces boulardii yeast preparation is subjected to beta or gamma radiation of about 25 kGy.

[0061] Gamma radiation treatment can be performed using any technique well known to those skilled in the art. It is generally performed using a cobalt-60 irradiator. A cobalt-60 irradiator uses a source of cobalt-60, a radioactive isotope, as the gamma ray source. The source is usually in the form of pellets or rods that are placed in the irradiator. The irradiator is designed to contain and shield the radioactive source, while allowing the gamma rays to penetrate the products to be irradiated.

[0062] The inactivation process generally involves loading the products to be inactivated into the irradiator, activating the radioactive source, and exposing the products to gamma rays for a period of time ranging from a few seconds to several minutes. The exposure time is determined by the type of product, its density, and the desired level of inactivation. This time can be easily adapted by those skilled in the art. Advantageously, the irradiator allows the gamma rays to penetrate through the products, reaching all surfaces, crevices, and hidden areas, thus ensuring the inactivation of all microorganisms.

[0063] Beta radiation treatment can be performed using any technique well known to those skilled in the art. It is typically performed using a device called an electron beam (EB) sterilizer. EB sterilizers use a high-energy electron beam. An EB sterilizer typically consists of an electron accelerator that produces the high-energy electron beam, a series of magnets that focus and direct the beam, and a treatment chamber where the products are exposed to the beam. The accelerator is typically powered by a high-voltage power supply. The inactivation process typically involves loading the products to be inactivated into the treatment chamber, activating the electron accelerator, and exposing the products to the high-energy electron beam for a period of time, which can range from a few seconds to several minutes.The exposure time is determined by the type of product, its density and the desired level of inactivation. This time can be easily adapted by those skilled in the art.

[0064] Preferably, the method of inactivating the Saccharomyces boulardii yeast preparation comprises a step of subjecting the Saccharomyces boulardii yeast preparation to beta or gamma radiation.

[0065] Preferably, the Saccharomyces boulardii yeast preparation is in the form of a dry or freeze-dried powder before subjection to beta or gamma radiation.

[0066] By way of example, the method for inactivating the Saccharomyces boulardii yeast preparation comprises the following steps: providing a Saccharomyces boulardii yeast preparation according to the invention, in particular in powder form, in particular dried or freeze-dried; subjecting the Saccharomyces boulardii yeast preparation to beta or gamma radiation. The method may also comprise a step of verifying the inactivation of the Saccharomyces boulardii yeast preparation following the irradiation step. To do this, it is possible to measure the residual vitality of the yeast preparation, for example by determining the number of Colony Forming Units (CFU) contained in the preparation.

[0067] The method may also comprise a step of storing the inactivated Saccharomyces boulardii yeast preparation, in particular in the form of dried or freeze-dried powder.

[0068] Preferably, the irradiation dose is chosen based on the desired efficacy for inactivating the yeast and can be determined by a person skilled in the art. Preferably, the Saccharomyces boulardii yeast preparation is subjected to beta or gamma radiation of at least 10 kGy.

[0069] Wax

[0070] The preparation of Saccharomyces boulardii yeast inactivated by beta or gamma radiation can be mixed with a wax.

[0071] As used herein, the term "wax" refers to a composition based on one or more esters of an alcohol, in particular glycerol, and fatty acids, which is solid at room temperature (18-23°C). Preferably, the wax according to the invention is based on "hard fat", within the meaning of the European Pharmacopoeia (Ph. Eue), namely a mixture of triglycerides, diglycerides and monoglycerides, which may in particular be obtained either by the esterification of glycerol with fatty acids of natural origin, or by transesterification of natural fats.

[0072] The fatty acids according to the invention are saturated or unsaturated, preferably saturated. Furthermore, the fatty acids according to the invention preferably comprise at least 8 carbon atoms (C8) or at least 12 carbon atoms (C12). Also preferably, the fatty acids according to the invention comprise at most 18 carbon atoms (C18).

[0073] Preferably, the wax according to the invention comprises, or is based on, triglycerides, in particular saturated triglycerides. In particular, the wax according to the invention comprises, or is based on, saturated C8-C18 or C12-C18 triglycerides, such as, for example, C10-C18 triglycerides.

[0074] Furthermore, the wax according to the invention may comprise additives, such as at least one lubricant or at least one emulsifier, in particular selected from the group consisting of glycerol monoricinoleate, a polyethylene glycol monocetyl ether, in particular Ceteth-20, a polyethylene glycol monostearic ether, in particular Steareth-20, and lecithin.

[0075] Preferably, the wax according to the invention has a melting point above 32°C. Also preferably, the wax according to the invention has a melting point below 37°C. More preferably, the wax according to the invention has a melting point above 32°C and below 37°C.

[0076] As will be apparent to those skilled in the art, the melting point is preferably measured under normal pressure. As used herein, normal pressure is a pressure of 1 bar.

[0077] Preferably, the wax according to the invention is selected from the group consisting of SUPPOCIRE® NA PASTILLES (Gattefossé), OVUCIRE® 3460 PASTILLES (Gattefossé) and SUPPOCIRE® AML PASTILLES (Gattefossé).

[0078] For example, the wax according to the invention may comprise a mixture of polyethylene glycol (PEG) and hard fat, a mixture of hard fat, glyceryl ricinoleate and ethoxylated fatty alcohol such as polyethylene glycol hexadecyl ether, a mixture of triglycerides, diglycerides and monoglycerides, the triglyceride fraction preferably being predominant.

[0079] Preferably, the wax according to the invention has a relative humidity of 20% to 35% before mixing.

[0080] Preferably, the inactivated Saccharomyces boulardii yeast preparation according to the invention is mixed with the wax according to the invention in powder form, in particular freeze-dried.

[0081] Preferably, the mixture consists of 15 to 35% (m / m) of inactivated Saccharomyces boulardii yeast preparation according to the invention.

[0082] Preferably, the mixture of inactivated Saccharomyces boulardii yeast preparation according to the invention and a wax according to the invention has a melting point above 32°C. Also preferably, the mixture according to the invention has a melting point below 37°C. More preferably, the mixture according to the invention has a melting point above 32°C and below 37°C.

[0083] As will be apparent to those skilled in the art, the melting point is preferably measured under normal pressure. As used herein, normal pressure is a pressure of 1 bar. Preferably, the mixture according to the invention has a liquefaction time of less than 60 min, preferably less than 50 min, less than 40 min, more preferably about 30 min in water at 37°C.

[0084] Preferably, the mixture according to the invention has a breaking strength greater than 5.4 kg at a temperature of 23°C.

[0085] Preferably, the mixture according to the invention comprises, or consists of, 15 to 35% (m / m) of Saccharomyces boulardii yeast preparation in powder form, in particular freeze-dried, inactivated and 65 to 85% (m / m) of wax according to the invention. More preferably, the mixture according to the invention comprises, or consists of, 30% (m / m) of Saccharomyces boulardii yeast preparation in powder form, in particular freeze-dried, inactivated and 70% (m / m) of wax according to the invention.

[0086] Preferably, the composition, in particular pharmaceutical, according to the invention, the medical device according to the invention, the medicament according to the invention or the cosmetic product according to the invention comprises, or consists of, the mixture in a unit mass of 1 g to 5 g, in particular of approximately or exactly 2 g.

[0087] Also preferably, the composition, in particular pharmaceutical, according to the invention, the medical device according to the invention, the medicament according to the invention or the cosmetic product according to the invention, comprises a unit mass of 0.15 g to 1.75 g, in particular 0.3 to 0.7 g, of Saccharomyces boulardii yeast preparation in powder form, in particular lyophilized, inactivated. More preferably, the composition, in particular pharmaceutical, according to the invention, the medical device according to the invention, the medicament according to the invention or the cosmetic product according to the invention, comprises a unit mass of 0.6 g, of Saccharomyces boulardii yeast preparation in powder form, in particular lyophilized, inactivated.

[0088] Administration

[0089] The composition, in particular pharmaceutical, according to the invention may comprise one or more pharmaceutically acceptable excipients or vehicles.

[0090] The composition according to the invention may be a cosmetic composition. In this case, the cosmetic composition may comprise one or more cosmetically acceptable excipients or vehicles in addition to the mixture. Similarly, the cosmetic product according to the invention may comprise one or more cosmetically acceptable excipients or vehicles in addition to the preparation or composition according to the invention.

[0091] The term "medical device" means any instrument, apparatus, equipment, software, material or other article, used alone or in combination, intended for use in humans to diagnose, prevent, control, treat or alleviate a disease and whose primary intended action in or on the human body is not achieved by pharmacological, immunological or metabolic means, but whose function can be assisted by such means. Preferably, the medical device according to the invention is a Class IIa medical device.

[0092] Preferably, the preparation of Saccharomyces boulardii yeast inactivated by a beta or gamma ray, the composition, in particular pharmaceutical, the medical device according to the invention or the cosmetic product according to the invention is administered orally, in particular in the form of a capsule or tablet, or vaginally, in particular in the form of an ovule or intravaginal capsule. More preferably, the preparation of Saccharomyces boulardii yeast inactivated according to the invention, the composition according to the invention, the medicament according to the invention, the medical device according to the invention or the cosmetic product according to the invention is administered vaginally, in particular in the form of an ovule.

[0093] Preferably, the inactivated Saccharomyces boulardii yeast preparation, the composition, in particular pharmaceutical, the medicament, the medical device according to the invention or the cosmetic product according to the invention is intended to be used as an ovule.

[0094] As used herein, the term "ovum" is considered equivalent to "vaginal ovum" or "gynecological ovum." Preferably, the ovum is a medicament, composition or medical device of hard or soft consistency and ovoid shape intended to be inserted into the vagina.

[0095] Preferably, the inactivated Saccharomyces boulardii yeast preparation according to the invention, the composition according to the invention, the medicament according to the invention, the medical device or the cosmetic product according to the invention is administered to the individual according to the invention in the form of an ovule once a day.

[0096] Preferably, the inactivated Saccharomyces boulardii yeast preparation is administered in an amount ranging from 100 to 1000 mg / day, more preferably from 200 to 800 mg / day, such as for example 300 mg / day, 400 mg / day, 500 mg / day, 600 mg / day, 700 mg / day, preferably over periods ranging from 7 to 14 days. More preferably, the inactivated Saccharomyces boulardii yeast preparation is administered in an amount of about 600 mg / day.

[0097] Applications

[0098] Preferably, the inactivated Saccharomyces boulardii yeast preparation according to the invention, the composition according to the invention, the medical device according to the invention, is administered in a prophylactically or therapeutically effective amount to prevent or treat vulvovaginitis and / or to restore the vaginal flora, and / or to prevent recurrences of vulvovaginitis, and / or to treat the symptoms of vulvovaginitis.

[0099] Vulvovaginitis is well known to those skilled in the art; it is an inflammation of the vulva and vagina. The causes of vulvovaginitis are diverse and include: infections, such as:

[0100] • Yeast infections or fungal infections, also called candidiasis. These infections, well known to those skilled in the art, are caused by a type of fungus called Candida, more specifically Candida albicans.

[0101] • Bacterial vaginosis. This infection, well known to those skilled in the art, results from a disruption in the normal balance of bacteria in the vagina. Usually, bacterial vaginosis is caused by an overgrowth of certain types of bacteria, particularly Gardnerella vaginalis, Gardnerella mobiluncus, Chlamydia trachomatis, Mycoplasma horn inis, Peptostreptococcus species, and Urea plasma urealyticum, in the vagina.

[0102] • Parasitic infections, particularly Trichomonas.

[0103] • Sexually transmitted infections (STIs) such as herpes, chlamydia and gonorrhea.

[0104] Allergies or irritations, for example to certain soaps, detergents or creams. Hormonal changes, such as those that occur during menopause.

[0105] Skin problems, such as eczema or psoriasis.

[0106] Symptoms of vulvovaginitis may include itching, local irritation, burning, redness in the vulva and vagina, colored, excessive and / or foul-smelling vaginal discharge, pain during urination and sexual intercourse may also occur.

[0107] As used herein, “prevention or treatment of vulvovaginitis” means the relief of at least one symptom of vulvovaginitis, the control of the progression of vulvovaginitis, and / or the cure of vulvovaginitis.

[0108] As used herein, “restoration of vaginal flora” refers to the reestablishment of a normal or healthy vaginal flora, or microbiota, particularly in terms of diversity and quantity of microorganisms, particularly after treatment with a local or systemic anti-infective. Preferably, the pH of a normal or healthy vaginal flora is approximately 4.5. Preferably, the local or systemic anti-infective is selected from antibiotics, particularly from the nitroimidazole family, such as metronidazole; antifungal drugs, particularly from the imidazole family, such as econazole, clotrimazole, miconazole or fluconazole; antiseptics; antiparasitics, particularly from the nitroimidazole family, such as metronidazole.

[0109] As used here, "recurrences of vulvovaginitis" refer to the recurrence of at least one symptom of vulvovaginitis after an initial episode of this infection has been treated.

[0110] Additional compound

[0111] According to one embodiment of the invention, the inactivated Saccharomyces boulardii yeast preparation according to the invention, the composition, in particular pharmaceutical, according to the invention, the medicament according to the invention, the medical device according to the invention is administered in combination with at least one additional compound useful for the prevention or treatment of vulvovaginitis and / or for the restoration of the vaginal flora, and / or for the prevention of recurrences of vulvovaginitis.

[0112] The additional compound useful for the prevention or treatment of vulvovaginitis and / or for the restoration of vaginal flora, and or for the prevention of recurrences of vulvovaginitis may be chosen from any compound well known to those skilled in the art.

[0113] Preferably, the additional compound useful for the prevention or treatment of vulvovaginitis and / or for the restoration of vaginal flora, and / or for the prevention of recurrences of vulvovaginitis is selected from the group consisting of: antibiotics, in particular from the nitroimidazole family, such as metronidazole, or from the lincosamide family such as clindamycin; antifungal drugs, in particular from the imidazole family, such as econazole, clotrimazole, miconazole or fluconazole; antiseptics; antiparasitics, in particular from the nitroimidazole family, such as metronidazole; topical creams or ointments, in particular those containing corticosteroids and topical estrogens.

[0114] In addition, the inactivated Saccharomyces boulardii yeast preparation according to the invention, the composition according to the invention, the medicament according to the invention, the medical device according to the invention can be associated with a change in lifestyle such as, for example, having good hygiene, avoiding the use of aggressive chemicals, avoiding soaps and detergents, etc.

[0115] In one embodiment of the invention, the inactivated Saccharomyces boulardii yeast preparation according to the invention, the composition, in particular pharmaceutical, according to the invention, the medicament according to the invention, the medical device according to the invention is not administered in combination with at least one additional compound useful for the prevention or treatment of vulvovaginitis and / or for the restoration of the vaginal flora, and / or for the prevention of recurrences of vulvovaginitis, in particular an additional compound as defined above.

[0116] As used herein, "combined" or "in combination" means that the inactivated Saccharomyces boulardii yeast preparation according to the invention is administered together with an additional compound, either together, i.e., at the same administration site, or separately, or at different times, provided that the period during which the inactivated Saccharomyces boulardii yeast preparation exerts its effects on the individual and the period during which the additional compound exerts its pharmacological effects on the individual, at least partially overlap.

[0117] Individual

[0118] Preferably, the individual according to the invention is a mammal, more preferably a human, in particular of the female sex. Preferably, the individual according to the invention has at least one symptom of vulvovaginitis and / or is suffering from vulvovaginitis, and / or has a risk of recurrence of vulvovaginitis.

[0119] Preferably, the individual according to the invention has vulvovaginitis, in particular a yeast infection or candidiasis, bacterial vaginosis, in particular caused by Gardnerella vaginalis and / or Gardnerella mobiluncus.

[0120] Description of figures

[0121] Figure 1

[0122] Figure 1 represents the number of Gardnerella vaginalis cells, expressed as a percentage relative to the control, cultured on solid medium without Saccharomyces boulardii (Sb, control), and treated with 5 mg / ml of Saccharomyces boulardii yeast not subjected to gamma radiation or with 5 mg / ml of Saccharomyces boulardii yeast inactivated by 25 kGy gamma radiation.

[0123] Figure 2

[0124] Figure 2 shows the amount of Gardnerella vaginalis in CFU / ml in the liquid culture medium as a function of time elapsed without treatment with Saccharomyces boulardii (Sb, control), treated with Saccharomyces boulardii not subjected to gamma irradiation, treated with Saccharomyces boulardii inactivated by 25 kG gamma irradiation and treated with Saccharomyces boulardii inactivated by 50 kG gamma irradiation.

[0125] Figure 3

[0126] Figure 3 represents the percentage of Candida albicans (Ca) cell attachment to VK2 vaginal epithelial cells as a function of the amount of unirradiated and irradiated lyophilized Saccharomyces boulardii (SBL) used to treat VK2 cells. Light gray bars correspond to 0 kGy irradiation, black bars correspond to 3 kGy irradiation, white bars correspond to 10 kGy irradiation, white bars with black horizontal stripes correspond to 25 kGy irradiation, white bars with black dots correspond to 50 kGy irradiation, and white bars with black vertical stripes correspond to 80 kGy irradiation. Figure 4

[0127] Figure 4 shows the vaginolysin / GAPDH signal ratio of untreated G. vaginalis-infected VK2 cell cultures treated with lyophilized Saccharomyces (SBL) (10 mg / ml) and lyophilized Saccharomyces boulardii irradiated with 25 kGy gamma radiation (10 mg / ml) as a function of time.

[0128] Figure 5

[0129] Figure 5 shows the percentage of trypan blue permeability of untreated G. vaginalis-infected VK2 cells, G. vaginalis-infected VK2 cells treated with lyophilized Saccharomyces boulardii (SBL), G. vaginalis-infected VK2 cells treated with lyophilized Saccharomyces boulardii irradiated with 25 kGy gamma radiation, uninfected VK2 cells, uninfected VK2 cells treated with lyophilized Saccharomyces boulardii, and uninfected VK2 cells treated with lyophilized Saccharomyces boulardii irradiated with 25 kGy gamma radiation.

[0130] EXAMPLES

[0131] EXAMPLE 1

[0132] The inventors evaluated the effect of inactivated Saccharomyces boulardii on Gardnerella vaginalis.

[0133] 1. On solid support

[0134] The effect of 5 mg / ml of lyophilized Saccharomyces boulardii CNCM 1-745 yeast preparation inactivated by 25 kGy gamma radiation was tested on the growth of Gardnerella vaginalis cultured on agar medium. The effect of the inactivated Saccharomyces boulardii yeast preparation was compared with the effect of 5 mg / ml of lyophilized Saccharomyces boulardii CNCM 1-745 not subjected to gamma radiation.

[0135] The results show that the yeast preparation Saccharomyces boulardii irradiated with 25 kGy gamma radiation inhibits the growth of Gardnerella vaginalis similarly to Saccharomyces boulardii not subjected to gamma radiation (see Figure 1).

[0136] 2. In liquid medium

[0137] The effect of two inactivated lyophilized Saccharomyces boulardii CNCM 1-745 yeast preparations was tested on the growth of Gardnerella vaginalis in liquid medium. The effect of the two inactivated Saccharomyces boulardii yeast preparations was compared with the effect of 5 mg / ml of non-gamma-irradiated Saccharomyces boulardii CNCM 1-745.

[0138] The inactivated Saccharomyces boulardii yeast preparations used are:

[0139] 5 mg / ml of lyophilized Saccharomyces boulardii CNCM 1-745 yeast preparation inactivated by 25 kGy gamma radiation;

[0140] 5 mg / ml of lyophilized Saccharomyces boulardii CNCM 1-745 yeast preparation inactivated by 50 kGy gamma radiation. The results show that the growth of Gardnerella vaginalis is inhibited by the gamma-irradiated Saccharomyces boulardii yeast preparations (see Figure 2). The results further show that the inhibition of Gardnerella vaginalis growth achieved by the inactivated Saccharomyces boulardii yeast preparations is at least equal to that achieved with treatment with non-inactivated Saccharomyces boulardii.

[0141] EXAMPLE 2

[0142] The inventors evaluated the effect of inactivated Saccharomyces boulardii in the treatment and prevention of vaginal candidiasis, particularly on the attachment of Candida albicans to vaginal epithelial cells after adapting the protocol described by Yang et al. (2018) Biomed Environ Sci, 31: 816-828.

[0143] VK2 vaginal epithelial cells were cultured in six-well plates and used to assess the adhesion capacity of C. albicans cells (standard strain SC5314).

[0144] VK2 vaginal epithelial cells were infected with C. albicans at 2 x 10 2 yeast cells per well in Keratinocyte Medium. VK2 vaginal epithelial cells were treated with:

[0145] Saccharomyces boulardii CNCM 1-745 lyophilized non-irradiated in quantities of 1 mg / ml, 3.16 mg / ml, and 31.62 mg / ml;

[0146] Lyophilized Saccharomyces boulardii CNCM 1-745 irradiated with 3 KGy gamma radiation in quantities of 1 mg / ml, 3.16 mg / ml, and 31.62 mg / ml;

[0147] Lyophilized Saccharomyces boulardii CNCM 1-745 irradiated with 10 KGy gamma radiation in quantities of 1 mg / ml, 3.16 mg / ml, and

[0148] 31.62 mg / ml;

[0149] Lyophilized Saccharomyces boulardii CNCM 1-745 irradiated with 25 KGy gamma radiation in quantities of 1 mg / ml, 3.16 mg / ml, and

[0150] 31.62 mg / ml;

[0151] Lyophilized Saccharomyces boulardii CNCM 1-745 irradiated with 50 KGy gamma radiation in quantities of 1 mg / ml, 3.16 mg / ml, and

[0152] 31.62 mg / ml; Saccharomyces boulardii CNCM 1-745 lyophilized irradiated with 80 KGy gamma radiation in quantities of 1 mg / ml, 3.16 mg / ml, and 31.62 mg / ml.

[0153] The number of adherent cells was verified by colony counting after 1 h of incubation. After 1 h of incubation, dissociated yeast cells were removed by rinsing the wells with PBS. The wells were then covered with agar, and the number of adherent cells was quantified by colony counting. Adhesion efficiency is expressed as the ratio of the number of adherent cells treated with Saccharomyces boulardii to that of cells cultured in DMEM alone.

[0154] The results show that treatment with Saccharomyces boulardii reduces the adhesion of C. albicans to vaginal cells. The inhibitory effect on the adhesion of C. albincans cells to vaginal cells is dose-dependent.

[0155] EXAMPLE 3

[0156] The inventors evaluated the effect of inactivated Saccharomyces boulardii on vaginolysin which is responsible for the lysis of vaginal epithelial cells and is therefore a carbon source for Gardnerella vaginalis.

[0157] Monolayers of VK2 cells cultured in six-well plates in the presence of medium comprising 10% fetal calf serum are infected with G. vaginalis at a rate of 2 * 10 4 cells per well.

[0158] 3 groups were formed: a group of VK2 vaginal epithelial cells infected with G. vaginalis and untreated; a group of VK2 vaginal epithelial cells infected with G. vaginalis and treated with 10 mg / ml of lyophilized Saccharomyces boulardii CNCM 1-745; a group of VK2 vaginal epithelial cells infected with G. vaginalis and treated with 10 mg / ml of lyophilized Saccharomyces boulardii CNCM 1-745 irradiated with 25 kGy gamma radiation. After incubation and homogenization, the wells were washed with PBS.

[0159] VK2 cells are lysed and the amount of vaginolysin in the cell lysate is analyzed.

[0160] The results show that at 24h and 48h after G. vaginalis infection, a large amount of vaginolysin is detected in the lysates of VK2 cells in the absence of treatment (untreated group) (see Figure 4).

[0161] The results show that treatment with lyophilized Saccharomyces boulardii at 1 0mg / ml reduces the amount of vaginolysin associated with VK2 cell lysates.

[0162] Furthermore, treatment with irradiated lyophilized Saccharomyces boulardii shows increased efficacy in inhibiting vaginolysin activity. Irradiated lyophilized Saccharomyces boulardii acts more effectively on G. vaginalis-induced vaginolysin than non-irradiated lyophilized Saccharomyces boulardii.

[0163] EXAMPLE 4

[0164] The inventors evaluated the effect of treatment with inactivated Saccharomyces boulardii on the membrane permeability of VK2 cells infected with G. vaginalis.

[0165] Monolayers of VK2 cells were cultured in six-well plates in the presence of a medium comprising 10% fetal calf serum and were infected with G. vaginalis at a rate of 2 * 10 6 cells per well.

[0166] 3 groups are formed: a group of VK2 vaginal epithelial cells infected with G. vaginalis and untreated; a group of VK2 vaginal epithelial cells infected with G. vaginalis and treated with 10 mg / ml of lyophilized Saccharomyces boulardii CNCM 1-745; a group of VK2 vaginal epithelial cells infected with G. vaginalis and treated with 10 mg / ml of lyophilized Saccharomyces boulardii CNCM 1-745 irradiated with 25 kGy gamma radiation. After incubation, trypan blue is added to the wells. When the integrity of the cell plasma membrane is impaired, trypan blue will penetrate the cells and mark them in blue.

[0167] After incubation, the wells are washed with PBS. Trypan blue is eluted and membrane permeability is quantified.

[0168] The results show that infection of VK2 cells with G. vaginalis results in a massive increase in membrane permeability (see Figure 5, untreated group).

[0169] Treatment with Saccharomyces boulardii, without prior infection with G. vaginalis, had no effect on the membrane permeability of VK2 cells.

[0170] Treatment with lyophilized Saccharomyces boulardii in an amount of 10 mg / ml reduces by approximately 70% the increase in membrane permeability induced by G. vaginalis infection of cells.

[0171] Furthermore, treatment with lyophilized Saccharomyces boulardii irradiated with gamma radiation in an amount of 10 mg / ml completely abolished the increase in membrane permeability induced by G. vaginalis infection. Thus, irradiated lyophilized Saccharomyces boulardii completely abolished infection-related cytotoxicity.

Claims

Claims 1. Preparation of Saccharomyces boulardii yeast subjected to beta or gamma radiation.

2. Preparation according to claim 1, wherein the Saccharomyces boulardii yeast preparation is subjected to beta or gamma radiation of at least 10 kGy.

3. Preparation according to claim 1 or 2, in which the Saccharomyces boulardii yeast preparation is in powder form, in particular dried or freeze-dried.

4. Preparation according to any one of claims 1 to 3, comprising essentially 0 Colony Forming Units (CFU) per gram of Saccharomyces boulardii yeast preparation.

5. A method of inactivating a Saccharomyces boulardii yeast preparation comprising a step of subjecting the Saccharomyces boulardii yeast preparation to beta or gamma radiation.

6. The method of claim 5, wherein the Saccharomyces boulardii yeast preparation is subjected to beta or gamma radiation of at least 10 kGy.

7. Method according to claim 5 or 6, in which the Saccharomyces boulardii yeast preparation is in powder form, in particular dried or freeze-dried.

8. Preparation of inactivated Saccharomyces boulardii yeast obtainable by the process as defined in any one of claims 5 to 7.

9. Preparation as defined in any one of claims 1 to 4 and 8, for use as a medicament.

10. Pharmaceutical composition or medical device comprising a preparation as defined in any one of claims 1 to 4 and 8.

11. Pharmaceutical composition or medical device according to claim 10, further comprising a wax.

12. Pharmaceutical composition or medical device according to claim 10 or 11, for use as an ovum.

13. A preparation as defined in any one of claims 1 to 4 and 8, a pharmaceutical composition or a medical device as defined in any one of claims 10 to 12, for use in the prevention or treatment of vulvovaginitis and / or for use in restoring vaginal flora, and / or for preventing recurrences of vulvovaginitis, and / or preventing or treating symptoms of vulvovaginitis, in an individual.

14. Cosmetic product comprising a preparation as defined in any one of claims 1 to 4 and 8. CORRECTED SHEET (RULE 91) ISA / EP

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