EP4676503A1

EP4676503A1 - Compositions comprising lactobacillus crispatus, lactobacillus rhamnosus, lactobacillus jensenii, lactobacillus gasseri, and 2'-fucosyllactose

Info

Publication number: EP4676503A1
Application number: EP24707813.2A
Authority: EP
Inventors: Jutta ZONDERLAND
Original assignee: Chr Hansen AS
Current assignee: Chr Hansen AS
Priority date: 2023-03-03
Filing date: 2024-03-01
Publication date: 2026-01-14
Also published as: CN121038800A; WO2024184257A1

Abstract

The present disclosure relates to a composition comprising Lactobacillus crispatus, Lactobacillus rhamnosus, Lactobacillus jensenii, Lactobacillus gasseri, and 2'-fucosyllactose, for use as a supplement for the support of a healthy vaginal microbiome in pregnant women.

Description

COMPOSITIONS COMPRISING LACTOBACILLUS CRISPATUS, LACTOBACILLUS RHAMNOSUS, LACTOBACILLUS JENSENII, LACTOBACILLUS GASSERI, AND 2’- FUCOSYLLACTOSE

FIELD

The present disclosure relates to compositions comprising selected lactobacilli and 2’- Fucosyllactose (2’-FL). The use of the compositions in support of the vaginal microbiota, and the growth of beneficial microbiota in the vagina, is also contemplated.

BACKGROUND

It is estimated that urogenital infections (for example, urinary tract infections (UTI), bacterial vaginosis (BV), and yeast vaginitis) afflict over one billion women in the world annually. While antimicrobial agents can be effective at providing clinical remediation, the use of such agents can lead to undesirable effects such as disruption of the microbiota of the user and an increase in antibiotic-resistant pathogens.

The mode of action of urogenital pathogens is understood to involve the formation of microbiota in the intestine. Intestinal microbiota then become a reservoir for urogenital pathogens which invade the urogenital tract. Urogenital tract microbiota then become the reservoir for infection of the vagina (for example by yeast and bacteria causing vaginosis) and the urinary tract (for example by organisms causing urinary tract infections). Studies have shown that specially selected probiotic lactobacilli, provided in a pessary inserted into the vagina, can colonize (Reid, et al. 1994) and compete against colonization of enterococci and other uropathogens (Bruce & Reid, 1998). The art also describes the use of Lactobacillus to prevent and treat urogenital infections (Gardiner et al., Clin Diagn Lab Immunol. 2002 Jan; 9(1): 92-96; EP2509610). During menopause the abundance of lactobacilli in vaginal tract can decline.

Because vaginal infection is an important mechanism of disease responsible for preterm birth, maintaining the natural, healthy balance of the Lactobacillus flora in the vagina is particularly important during pregnancy. A deficiency in Lactobacilli can upset the microbial balance in the vagina, potentially resulting in the syndrome of bacterial vaginosis, which may be associated with a quantitative and qualitative shift from normally occurring Lactobacilli to a mixed flora dominated by anaerobic bacteria. Bacterial vaginosis is characterized by a loss of Lactobacilli and a concomitant increase in Gram-variable and Gram-negative rods, primary among them Gardnerella vaginalis as well as Bacteroides, Prevotella, and Mobiluncus species. The loss of vaginal Lactobacilli also leaves nonpregnant women susceptible to infection which may result in endometritis or even pelvic inflammatory disease. During menopause, there may be a depletion of Lactobacilli and increased colonization by pathogenic microorganisms associated with bacterial vaginosis and urinary tract infections. In post-menopausal women, vaginal estriol therapy reduces E. coli colonization and increases the numbers of Lactobacilli, with the result that the incidence of recurrent urinary and genital tract infections drops significantly.

Methods for selectively stimulating the beneficial microbiota are well known. For example, administering live beneficial bacteria (probiotics), administering a substrate for beneficial microbiota to grow on (prebiotics), or a combination thereof. Human milk oligosaccharides (HMOs) are non-digestible oligosaccharides (NDOs) which form a mixture of prebiotic molecules providing a substrate for beneficial microbiota. HMOs, being part of mother's milk, are relevant to help infants build the beneficial microbiota needed for optimal health. However, HMOs may also be used in adult subjects. The most abundant HMO in human milk is 2'-fucosyllactose.

A need exists for compositions which would be useful in the support of a healthy vaginal microbiome and help provide relief from vaginal bacterial imbalance in pregnant women. Additionally, probiotic compositions which have a beneficial impact on the vaginal microbiota would be of interest.

SUMMARY

The present disclosure provides compositions, uses, methods and the like for the support of a healthy vaginal microbiome in pregnant women. In particular, the present compositions comprise Lactobacillus crispatus (DSM 22566), Lactobacillus rhamnosus (DSM 22560), Lactobacillus jensenii (DSM 22567) and Lactobacillus gasseri (DSM 22583), and 2'-fucosyllactose.

Compositions comprising Lactobacillus crispatus (DSM 22566), Lactobacillus rhamnosus (DSM 22560), Lactobacillus jensenii (DSM 22567) and Lactobacillus gasseri (DSM 22583) are known to support vaginal health and are commercially available under the brand name ASTARTE™. HMOs including 2'-fucosyllactose are known to provide support to the microbiota of infants and, in certain circumstances, adults. However, 2'-fucosyllactose is not generally thought to be a good substrate for Lactobacilli (see, for example, Salli et al., J. Agric. Food Chem. 2021 , 69, 170-182).

While not wishing to be bound by theory, it is believed that the present compositions provide pregnant women with a source of probiotics in a format that supports a healthy intestinal microbiota, leading to a healthy reservoir of microorganisms beneficial to vaginal health. The present compositions may also have utility in other populations of women such as, for example, those that suffer from repeated urogential issues/infections, taking antibiotics or other medications that disrupt the vaginal microbiota, or are menopausal/postmenopausal.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by persons skilled in the art. Although any methods and materials equivalent or similar to those described herein can be used in the practice of the present disclosure, typical methods and materials are described. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising", "having", "including" and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range including the two end values, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

As used herein, the term “and/or” is intended to mean the combined (“and”) and the exclusive (“or”) use, i.e. “A and/or B” is intended to mean “A alone, or B alone, or A and B together”.

As used herein the terms "effective amount", "effective concentration", or "effective dosage" are defined as the amount, concentration, or dosage of the bacterial strain(s) sufficient to improve the overall health of the animal and confer benefits similar to the ones demonstrated in the examples. The actual effective dosage in absolute numbers depends on factors including the state of health of the subject in question, and other ingredients present. The "effective amount", "effective concentration", or "effective dosage" of the bacterial strains may be determined by routine assays known to those skilled in the art.

As used herein the term "isolated" means that the bacterial strains described herein are in a form or environment which does not occur in nature, i.e. the strain is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature.

A bacterial “strain” as used herein refers to a bacterium which remains genetically unchanged when grown or multiplied and that originates from a single isolate or pure culture. Probiotics are classified by their genus (e.g. Bifidobacterium), species and subspecies (e.g. animalis subs p. lactis), and strains (e.g. DSM 15954 and/or BB-12®). FAO/WHO has stated that probiotic effects are strain specific and that most probiotic characteristics of a particular strain cannot therefore be extrapolated to other strains of the same species. As used herein, the term “probiotic” refers to a culture of live or freeze-dried microorganisms, dead microorganisms, fragments of microorganisms and extracts or supernatants of microorganisms which, when applied to man or animal, beneficially affects the host (Hill et al. (2014) Expert Consensus Document, The International Scientific Association for Probiotics and Prebiotics. Consensus statement on the scope and appropriate use of the term probiotic).

No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

The present invention has been described with reference to various embodiments, aspects, examples, or the like. It is not intended that these elements be read in isolation from one another. Thus, the present disclosure provides for the combination of two or more of the embodiments, aspects, examples, or the like.

All embodiments described herein are intended to be within the scope of the invention disclosed. These and other embodiments of the present invention will become readily apparent to those skilled in the art from the following detailed description of the preferred embodiments having reference to the whole description, the invention not being limited to any particular preferred embodiment(s) disclosed.

The present disclosure provides a composition Lactobacillus crispatus (DSM 22566), Lactobacillus rhamnosus (DSM 22560), Lactobacillus jensenii (DSM 22567) and Lactobacillus gasseri (DSM 22583), and 2'-fucosyllactose.

The present compositions comprise an effective amount of probiotic. For example, each of the aforementioned Lactobacillus species is present in a concentration ranging from 0.05 x 10⁹ CFU/g to 30 x 10⁹ CFU/g, preferably from 0.5 x 10⁹ CFU/g to 25 x 10⁹ CFU/g. According to an embodiment, the present composition comprises Lactobacillus crispatus (DSM 22566) in a range of concentration of between 3 x 10⁹ and 22 x 10⁹ CFU/g, Lactobacillus rhamnosus (DSM 22560) in a range of concentration of between 3 x 10⁹ and 22 x 10⁹ CFU/g, Lactobacillus jensenii (DSM 22567) in a range of concentration of between 0.7 x 10⁹ and 6 x 10⁹ CFU/g, Lactobacillus gasseri (DSM 22583) in a range of concentration of betweenl x 10⁹ and 8 x 10⁹ CFU/g. The present compositions may comprise the ingredients as listed in the tables below: *The 2'-fucosyllactose having a particle size as measured using sieves complying with DIN ISO 3310-1 :

Percent through mesh #230 (63 pm) - <15%.

Percent through mesh #100 (150 pm) - >60%.

Percent through mesh #45 (355 pm) - >90%. Percent through mesh #20 (850 pm) - 100%.

The present compositions may comprise additional components. For example, at least one other bacterial strain, vitamins, minerals, prebiotics, fibres, or combinations thereof. The other component(s) may, for example be fructo-oligosaccharides (FOS), galacto-oligosaccharide (GOS), human milk oligosaccharides (HMO) or combinations thereof.

The present compositions may comprise biotin. For example, the compositions may comprise at least about 1 pg, at least about 2pg, at least about 5pg, at least about 10pg, at least about 15pg, at least about 17pg, biotin.

The present compositions may comprise at least one other bacterial strain, for example, Lactococcus lactis subsp. lactis biovar. diacetylactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis, any strain belonging to the genus Lactobacillus (including but not limited to Lactobacillus acidophilus, Lactobacillus easel subsp. casei, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus lactis, Lactobacillus rhamnosus, Lactobacillus salivarius), any strain belonging to the genus Bifidobacterium (including but not limited to Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium animalis subsp. lactis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium dent! urn, Bifidobacterium longum subsp. infantis, Bifidobacterium longum subsp. longum, Bifidobacterium magnum, Bifidobacterium pseudocatenulatum), or any strain from the genera of Akkermansia, Anaerostipes, Butyricicoccus, Christensenella, Clostridia, Coprococcus, Dorea, Eubacterium, Faecalibacterium or Roseburia or the family Coriobacteriaceae, as well as suitable combinations of the foregoing.

The present compositions may comprise at least one strain of a bacterium selected from the group comprising Bifidobacterium animalis subsp. lactis deposited as DSM 15954, Lactobacillus acidophilus deposited as DSM 13241 , Lactobacillus rhamnosus deposited as ATCC 53103, Lactobacillus paracasei subsp. paracasei deposited as ATCC 55544, Lactobacillus paracasei deposited as LMG-17806, Streptococcus thermophilus deposited as DSM 15957, Lactobacillus fermentum deposited as NM02/31074, Lactobacillus paracasei subsp. paracasei deposited as CCTCC M204012 and suitable combinations thereof.

The present compositions may comprise the probiotics in any suitable form for administration to the subject. In a preferred embodiment, the compositions may comprise the bacteria in dried form, which can be obtained by freeze-drying, spray-drying, lyophilization, or the like.

If the bacteria are freeze-dried, they are generally mixed with a cryoprotectant before they are freeze-dried. The term “a cryoprotectant” is used herein to refer to a substance that is able to improve the survival during freezing and/or drying and to improve the storage stability of bacteria. The cryoprotectant used herein preferably comprises a saccharide and/or a sugar alcohol such as inositol.

The saccharide may be a mono-, di-, oligo- or polysaccharide, or a mixture of at least two saccharides. Useful monosaccharides include, for example, glucose (also known as dextrose), fructose, ribose and galactose and useful disaccharides include, for example, sucrose, trehalose, maltose and lactose. The composition may comprise one or more mono- or disaccharides, such as one, two, or three or even more different saccharides.

The cryoprotectant may comprise a mixture of a disaccharide, such as sucrose, and a polysaccharide, such as maltodextrin.

The cryoprotectant may further comprise a peptide, protein, protein hydrolysate or a mixture thereof. Examples of peptides and proteins to be used are casein, pea, whey, albumin, glutamic acid or gelatin, and any isolate or hydrolysate thereof. Other additives, e.g. antioxidants such as sodium ascorbate, sodium citrate, trisodium citrate dihydrate and cysteine hydrochloride may also be present. Skim milk powder and yeast extract may also be ingredients.

Currently, there are approximately 200 known, structurally distinct HMOs. They can be categorized into fucosylated, sialylated, and neutral core HMOs. The composition of HMOs in breast milk is individual to each mother and varies over the period of lactation. The dominant oligosaccharide in 80% of all women is 2'-fucosyllactose, which is present in human breast milk at a concentration of approximately 2.5 g/L, other abundant oligosaccharides include lacto-N- tetraose, lacto-N-neotetraose, and lacto-N-fucopentaose. It has been found that the concentration of each individual HMO changes throughout the different periods of lactation (colostrum, transitional, mature and late milk) and depend on various factors such as the mother's genetic secretor status and length of gestation.

Generally HMOs derive from lactose, which can be decorated by four monosaccharides (N- acetyl-D-glucosamine, D-galactose, sialic acid and/or L-fucose) to form an oligosaccharide.

The present compositions comprise 2'-fucosyllactose. The compositions may be formulated as combined or as separate compositions of the probiotic strains and the HMO. The compositions may comprise other Human Milk Oligosaccharide (HMO) such as, for example, 3-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, lacto-N-tetraose, lacto-N-neotetraose, and combinations thereof. The HMOs may be in any suitable combination. For example, 2'-fu cosy I lactose may be combined with any one, two, three, four, or five of 3-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, lacto-N- tetraose, and lacto-N-neotetraose. In one embodiment, the HMO mix comprises 2'- fucosyllactose, 3-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, and lacto-N-tetraose. The present composition may comprise 2'-fucosyllactose in any suitable amount, such as, for example, at least about 0.001 g, at least about 0.01 g, at least about 0.05 g, at least about 0.1 g, at least about 0.5 g weight. The compositions may, for example, comprise less than about 20 g, less than about 10 g, less than about 5 g, less than about 4 g, less than about 3 g, less than about 2 g, less than about 1 .5 g, of 2'-fucosyllactose.

Formulating compositions with HMOs can be problematic. It has been found that a more reproducible and consistent composition can be achieved through controlling the particle size distribution (PSD) of the HMO. While not wishing to be bound by theory, it is believed that having a somewhat narrow PSD improves the flowability of the HMO enabling a more effective mixing with the other ingredients. In addition, it is believed that a PSD within a certain range provides a better solubility profile. Particle size of an HMO may be determined using a standard method, such as using a sieve tower, which separates the powder into the different fractions after a defined time with a predefined amplitude. The sieves used in such a method may be sieves which comply with DIN ISO 3310-1 .

It is preferred that the 2'-fucosyllactose (or other HMO) used in the present compositions have the following particle size characteristics:

Percent through mesh #230 (63 pm) - less than about 20%, less than about 18%, less than about 16%, less than or equal to about 15%.

Percent through mesh #100 (150 pm) - greater than about 75%, greater than about 70%, greater than about 65%, greater than or equal to about 60%.

Percent through mesh #45 (355 pm) - greater than about 95%, greater than about 92%, greater than or equal to about 90%.

Percent through mesh #20 (850 pm) - 100%.

It is preferred that the compositions of the present disclosure are administered orally. The compositions are thus typically in a form suitable for oral administration. The composition may be a solid or a liquid composition. The composition may be in unit dosage form. For example, the composition can be a capsule, pastille, a pill, a tablet, a soft gel, a sachet, a stick, a stick powder, or in a more general composition such as oil drops, an emulsion, or a paste, or in any other suitable carrier determined by those of skill in the art to be an effective carrier for live organisms.

The compositions may be encapsulated for example using a suitable polymeric matrix to improve long-term stability and storage of the compositions. Those skilled in the art will appreciate that any suitable encapsulation material or matrix and encapsulation methods and techniques known to those skilled in the art may be used.

The composition may be included in a dietary supplement or pharmaceutical composition or may be part of a feed product or a food product such as a fermented milk product e.g. a yogurt.

Those skilled in the art will appreciate that the administration of compositions disclosed herein can be carried out with dose levels and dosing regimens as required depending on the circumstances and on the condition of the subject. Suitable dosage regimes can be determined based on the teaching of the present application. Dosage regimens may be adjusted to provide the optimal support of the vaginal microbiota of the subject. It will be appreciated that the exact amounts and rates of administration will depend on a number of factors such as the age, body weight, general health, sex, and dietary requirements of the subject. Based on the teaching herein those skilled in the art can, by routine trial and experimentation, determine suitable dosage regimes on a case-by-case basis.

In an exemplary embodiment, the composition may be administered daily for at least 1 day. Alternatively, the composition can be administered once or more daily for at least 1 day, 2 days, 4 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks or more.

The present composition may comprise 2'-fucosyllactose in any suitable amount, such as, for example, at least about 0.001 g per daily intake, at least about 0.01 g per daily intake, at least about 0.05 g per daily intake, at least about 0.1 g per daily intake, at least about 0.5 g g per daily intake. The compositions may, for example, comprise less than about 20 g per daily intake, less than about 10 g per daily intake, less than about 5 g per daily intake, less than about 4 g per daily intake, less than about 3 g per daily intake, less than about 2 g per daily intake, less than about 1.5 g per daily intake, of 2'-fucosyllactose.

The present disclosure provides a method of supporting the vaginal microbiota of a pregnant subject. Said method comprising providing to said subject an effective amount of the present compositions. For example, the present compositions may be provided to the subject once daily for one week or more, preferably one month or more.

The present disclosure provides the use of the present compositions for supporting the vaginal microbiota of a pregnant subject.

Claims

1 . A composition comprising Lactobacillus crispatus (DSM 22566), Lactobacillus rhamnosus (DSM 22560), Lactobacillus jensenii (DSM 22567), Lactobacillus gasseri (DSM 22583), and 2'-fucosyllactose, for use as a supplement for the support of a healthy vaginal microbiome in pregnant women.

2. The composition for use according to claim 1 , wherein the composition comprises at least 0.5 x 10⁹ CFU/g of Lactobacillus crispatus (DSM 22566), Lactobacillus rhamnosus (DSM 22560), Lactobacillus jensenii (DSM 22567), Lactobacillus gasseri (DSM 22583).

3. The composition for use according to claim 1 or 2, where the composition comprises at least 0.1 g of 2'-fucosyllactose.

4. The composition for use according to any preceding claim, wherein the 2'-fucosyllactose has particle size distribution, as measured using sieves complying to DIN ISO 3310-1 , of less than about 20% through mesh #230 (63 pm), greater than about 65% through mesh #100 (150 pm), greater than about 92% through mesh #45 (355 pm), and 100% through mesh #20 (850 pm).

5. The composition for use according to any preceding claim, wherein the composition is in an oral dosage form.

6. The composition for use according to any preceding claim, wherein the composition is in the form of a capsule.

7. The composition for use according to any preceding claim, wherein the composition is in the form of a dry powder.

8. The composition for use according to claim 1 or 2, wherein the composition comprises at least 0.1 g per daily intake of 2'-fucosyllactose.

9. A method of supporting the vaginal microbiota of a pregnant woman, said method comprising administering an effective amount of a composition according to any preceding claim to said woman.